Electrophoretic Esterase Patterns in Insecticide-Resistant and Susceptible Mosquitoes1,2 GEORGEP. GEORGHIOU' and NICOLEPASTEUR' ABSTRACT The esterase patterns of insecticide-resistant and susby DEF® (S,S,S-tributyl phosphorotrithioate) is most ceptible strains of Culex pipiens fatigans Wiedemann, probably associated with organophosphorus multiresisCulex pipiens pipiens L., C. tarsalis Coquillett, and tance in C. p. fatigans from California, Esterase B2 has Anopheles albimanus Wiedemann, were investigated by no equivalent in chlorpyrifos-resistant C. p. pipiens from starch gel electrophoresis. At least one highly active France. Interspecific and interstrain differences in esterase esterase is present in every organophosphate-resistant patterns and their relationship to resistance are disstrain of Culex spp. A highly active esterase B2, catalyzing cussed. the hydrolysis of p-naphthylacetate and being suppressible
1 Diplera: Culicidae. ~ Received for publication Nov. 8, 1977. • Division of Toxicology and Physiology, Department of Entomology. University of California, RIverside 92521. • Laboratoire c\'Ecologie Medicale et de Pathologie Parasitaire, Faculte de Medecine, 34000 Montpellier, France.
fasciatus) Wiedemann and 4 G. tarsalis Coquillett strains of California origin, and 4 AnoPheles albimanus Wiedemann strains of Central American origin were studied by starch gel electrophoresis. In addition, each gel induded samples of Iyophilyzed homogenates of 3 strains of C. p. PiPiens L. from southern France for comparison (Table 1). Esterases and acetylcholinesterases (AChE) were studied after electrophoresis with Poulik (1957) buffer systems or with Tris-citrate buffers described by Ayala et aL (1972) under the name of system C. Esterases were revealed according to the technique used by de Stordeur (1976). AChE activity was demonstrated by soaking the gels for 20-25 min in sodium phosphate buffer (pH 7.5) at 5°C and subsequently transferring them to 100 ml of fresh buffer containing 8 ml acetylthiocholine (0.05 M) for 30 min at room temperature. AChE activity was revealed as yellow spots within one h following the addition of 4 ml of 0.01 M DTNB (dithiobisnitrobenzoate) . The effect of various inhibitors on esterase and AChE activities was assayed by the addition of the required amount of inhibitor in the buffers during· staining. EDT A (ethylenediamine tetraacetic acid, disodium salt) action was studied by adding 2mM of EDTA/liter in both the gel and the electrode buffers. For ease of comparison, we will name esterase-A those esterases using a-naphthylacetate and staining blue, and esterase-B those using p-naphthylacetate and staining red in the presence of both substrates. Some esterases were found to be completely inhibited in the absence of EDT A and they will be named esterases N or B' according to the substrate they hydrolyze. RESULTS AND DISCUSSIoN.-Comparison of Esterase Patterns in C. p. pipiens and C. p. fatigans.- Two esterase systems can be recognized in the strains of C. p. fatigans studied, one of type A and one of type B. In the absence of genetic data, it is difficult to attribute esterases to given loci on the basis of differences in electrophoretic mobility alone. However, because of extreme differences in activity (or staining intensity), we have divided esterases B in 2 groups, Bl with low activity and B2 with an extremely high activity (Fig. I). In C. p. pipiens, de 201
©1978 Entomological
Society of America
0022-0493(78(0071-0201$00.75(0
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Among the mechanisms responsible for resistance to insecticides, enzyme modifications play a very important role. Thus, dehydrochlorinase is a major factor in DDT resistance, carboxylesterases, phosphotriesterases, acetylcholinesterases, glutathion-dependent transferases, and mixed function oxidases (MFO) are involved in organophosphorus (OP) resistance. MFO's are also important in carbamate resistance (reviews by Openoorth and Welling 1976, Plapp 1976). O-demethylase was recently shown to be the major detoxication enzyme in methopreneresistant house flies, Musca domestica L. (Hammock et aL 1977). Since these enzymes have undergone biochemical modifications, it is reasonable to assume that, at least in some cases, they will be electrophoretically different from normal enzymes. Until recently, very few studies of insecticide resistance coupled with electrophoresis analyses have been reported, but the few available have been extremely encouraging, though a direct cause-effect relationship has not always been ascertained (Krimbas and Tsakas 1971, Pasteur and Sinegre 1975, 1977, Tobgy et al. 1976, Beranek and Oppenoorth 1977). It has become apparent, however, that electrophoresis techniques hold considerable promise in relating specific est erases to resistance, and thus are a means of identifying resistant genotypes rather than phenotypes in single insects. Insecticide resistance is especially serious in disease vector and nuisance mosquitoes, occurring in at least 83 anopheline and culicine species (Anon. 1976, Georghiou and Taylor 1976). The availability in our respective laboratories of several strains of mosquitoes of known origin and resistance characteristics has made possible the present search of interstrain and interspecific similarities or differences in the electrophoretic patterns of their esterases related to resistance. MATERIALSAND METHODS.-Enzymes of single adult mosquitoes of 8 Culex PiPiens fatigans (= quinque-
202
JOURNAL
OF ECONOMIC
A_--
Vol. 71, no. 2
ENTOMOLOGY
DDB
D
D
•.•••
••..
a:
B c:::::J c:::::J
>-
I-
B=
....J III
o
~
BI= -A AB=
Bc:=:J
= BIc:::::J
DB2
55
• •
C.p. pipiens (Fr'ance)
F4
FI,F2,F3
C. p. fatigans
F6,F8 (Calif.)
-
a:
_A'
TI
C. tarsa/is
0
J: Q.
0
a::
A'-
I
U
Il£J
•
l-
u 0
l£J ....J l£J
T2 (Calif.)
FIG. I.-Esterase patterns observed in single mosquitoes of various Culex species and strains. A = a-naphthylacetate specific. B= p-naphthylacetate specific. ·Pattern obtained in presence of EDTA. Rf value of 100 given arbitrarily to fastest esterase of T2 strain.'
Stordeur (1976), Pasteur and Sinegre (1975), and Pasteur (1977)' have demonstrated 3 esterase-coding loci, Est-1, Est-2, and Est-3, coding for esterases of types B, A, and N, respectively. Both Est-2 and Est-] polymorphisms are associated with OP (chlorpyrifos) resistance, but recent studies have shown that if OP resistance is the result of a specific esterase allozyme, it is probably one coded by Est-]. This locus is homozygous for a null allele (Est_JNull) in every susceptible mosquito, while the resistant individuals bear the Est-]'" active allele in single or double dose. Esterase B2 of the California C. p. fatigans has no equivalent in the French C. p. pipiensj it is an enzyme with a very strong staining intensity which diffuses rapidly and blurs out any adjacent esterase system. It is not known at present if esterase B2 is an allozyme of the locus coding esterases B1 of C. p. fatigans or of Est-2 of C. p. pipiens. The absence in C. p. fatigans of an esterase N similar to C. p. PiPiens Est-] shows that if OP resistance is due to an esterase in both subspecies different loci are involved. Comparison of Esterase Patterns among C. p. fatigans Strains.-One of the most striking features of C. p. fatigans esterase patterns is the presence or absence of the highly active esterase B2 (Fig. 1). This esterase was found in every individual of the temephos-resistant F6 and F8 strains in which OP resistance is completely suppressible by the synergist • Pasteur, N. 1977. Recherches de genetique chez Culex piPiens Pipiens L. Polymorphisme enzymatique, autogenese et resistance aux insecticides organophosphores. These de Doctorat d'Etat, mention Sciences. Universite des Sciences. Montpellier, France.
DEF® (S,S,S-tributyl phosphorotrithioate) (Georghiou et al. 1975, L. E. Ranasinghe and G. P. Georghiou, unpublished) while it is absent from the susceptible Fl strain, and from strains F2, F3, F4, and F7 in which resistance is not affected, or only slightly so, by DEF (Table I). These observations indicate that in California C. p. fatigans OP resistance is most probably associated with esterase B2. The observed patterns also indicate that permethrin resistance (strains F3, F4) is independent of esterase B2. How do the esterases of the permethrin-resistant strains differ from those of other strains lacking esterase B2? A and Bl esterases in strain F3 (resistant to d-trans permethrin) have an electrophoretic mobility identical to that of the Fl susceptible strain and of the F2 and F7 strains in which resistance is based on MFO. The F4 strain (resistant to d-cis permethrin) shows a distinctly different electrophoretic pattern; esterases A are electrophoretically slower and esterase Bl is faster than in any other strain (Fig. 1). Whether these variations are relevant to pyrethroid resistance remains to be determined, but it is quite clear that the esterases of the 2 strains, selected in the presence of different isomers, are not identical although they are issued from the same parental stock (F5). A further point concerning C. p. fatigans must be emphasized; strains F3, F4, F6, F7, and F8 originate from the same parental strain (F5) (Table 1) which is composed of some 89% mosquitoes with esterase B2, although it has been maintained in the laboratory free of insecticidal exposure for ca. 26 mo. Therefore, it is clear that, depending on the type of selection pressure applied, some strains evolved
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57/5G S5
.A•
A-
-A-
D
on
APril 1978 Table
I.-Strains
Species Culex p. pipiens (France) C. p. fatigans (California)
C. tarsa/is
of mosquitoes investigated
for esterase activity and their insecticide resistance characteristics. Resistance status
Resistance mechanism
S5
field field
suscepti b Ie chlorpyri fos-R'
esterase (?)
Fl F2 F3
Bakersfield Coachella F5
susceptible propoxur-Rb d-trans permethrin-Rb
MFO
F4
F5
d-cis permethrin-Rb
(?)
F5 F6
Hanford F5
field 1975 (OP-R) temephos-Rb
esterase esterase, MFO
F7
F5
F8
F5
Tl
Bakersfield
susceptible
T2
Coachella
methyl parathion-Rb
T3
Coachella
methoprene- R b,.
(?)
T4
Coachella
diflu benzuron -selected b
(?)
Al A2 A3
Panama EI Salvador EI Salvador
Rd Chlorphoxim-selectedb
AChE
A4
EI Salvador
field (OP ICarb.-R)
AChE (?)
S7/SG
+ DEF-Rb temephos + pb-Rb
temephos
(?)
MFO esterase
esterase (?)
susceptible
o P I Carbamate-
(?)
Reference de Stordeur (1976) Pasteur and Sinegre (1977) Georghiou et a1. (1966) Shrivastava et a1. (1970) Priester and Georghiou (1978) Priester and Georghiou (1978) Georghiou et a1. (1975) Ranasinghe and Georghiou (unpublished) Ranasinghe and Georghiou (unpublished) Ranasinghe and Georghiou (unpublished) Apperson and Georghiou (1974) Apperson and Georghiou (1975b) Georghiou and C. S. Lin (unpublished) Georghiou and C. S. Lin (unpublished) Georghiou (1972) Ayad and Georghiou (1975) Georghiou and M. K. Hawley (unpublished) Georghiou and M. K. Hawley (unpublished)
• Selected on the basis of its esterase electrophoretic pattern and subsequently found resistant to chlorpyrifos. b Selectedin laboratory by chemicalindicated. • 437.5-foldresistanceto methoprene at ED50. d Selectedby propoxur and subsequentlyparathion. for the presence of esterase B2 and others for its absence. Thus, selection by temephos alone or in combination with piperonyl butoxide (pb) , an inhibitor of MFO, resulted in strains (F6 and F8) having esterase B2 as one would have expected if this enzyme locus, or one closely linked to it, plays a role in temephos resistance. But the total absence of esterase B2 from strains F3, F4, and F7 indicates that esterase B2 was actively selected against since it is unlikely that it could have disappeared in all 3 strains by chance alone while it has persisted in the parental (F5) strain. Culex tarsa/is.- Two esterase systems (A' and B) could be demonstrated in the susceptible (Tl) as well as in the methyl parathion-selected (T2) strain. Both esterases show electrophoretic mobility differences within and between strains, denoting a polymorphism of the loci coding them. But in the OPresistant strain, both esterases have a much stronger staining intensity. Mosquitoes of the methoprene-resistant (T3) and diflubenzuron-selected (T4) strains possess esterases A' and B with staining intensities comparable to those found in the Tl susceptible strain. But, in addition, these mosquitoes have supernumerary esterases which are not found in the other strains. Although the resistance mechanism of the methopreneR strain has not yet been investigated, the extreme
variability observed in the esterase pattern among individuals of this strain might suggest that esterase detoxication does not play an important role in its resistance. In methoprene-resistant house flies, 0demethylation by MFO enzymes was shown to be the primary detoxication mechanism (Hammock et a1. 1977). A most interesting finding of this study is the extremely strong staining intensity of esterase A' and esterase B in OP-resistant Culex mosquitoes. This denotes either a higher specific activity or a higher titer of the enzyme, and suggests that in Culex at least 2 different esterase genes may be involved independently or jointly in resistance. The biochemical differences between these esterases, e.g., substrate specificities and EDTA action, exclude that they can be allozymes coded by the same locus. Thus, in C. pipiens (strain 55) where only one of the 2 esterases is present, resistance has been shown to be monofactorial (Pasteur and 5inegre 1977). In contrast, in C. tarsa/is (T2) in which both esterases are present, resistance was found to be polyfactorial (Apperson and Georghiou 1975c) although bifactorial inheritance cannot be excluded (see their Fig. 5) . Furthermore, if the 2 esterase genes confer different biochemical properties, it may be expected that the resistance spectrum should be different according to the gene or genes involved. The data in Table 2
or
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Origin or parent
Strain code
(California)
Anopheles albimanlls (Central America)
203
GEORGHIOUANDPASTEUR: EsTERASESIN MOSQUITOES
204
JOURNAL
Table 2.-Interspecific
and interstrain
OF ECONOMIC
Vol. 71, no. 2
ENTOMOLOGY
diJferences in esterases of OP·resistant and susceptible patterns of the strains.
C. p. pifJiens, C.
p.
totigons, and C. tof'sa.lis, and the cross resistance and inheritance
c. p. Esterases'
Resist.
5usc.
Resist.
5usc.
Resist.
(55)
(FI)
(F5,F6,F8)
(TI)
(T2)
0
+++ + +
0
0
+
+++
+ +
+ +++
0
0
+
+++
of R
monofactorial (Ranasinghe & Georghiou unpublished)
polyfactorial (Apperson & Georghiou 1975c)
LCoo 55 strain
LC•• F5
LC•• T2
LCoo 57 strain
LC•• FI
LC•• TI
750.0b 48.3 69.2 230.8 41.2 114.9 62.1 (Ranasinghe & Georghiou unpublished)
13.6 96.5b 29.6 253.8 49.4 76.8 10.9 (Apperson & Georghiou 1975a)
temephos methyl parathion chlorpyrifos chlorpyrifos.meth yl feni trothion femhion malathion
9.3 8.2 21.4 15 5.9 7.3 3.3 (5inegre et a!. 1976) =
none,
+
=
low,
++ +
=
very high.
OP resistance in the French C. p. 55 (with esterase A') increased 21.4fold for chlorpYTi£os but only 9.3-fold for temephos (Sinegre et a1. 1976), whereas in the Californian C. p. fatigans strain F6 (with esterase B) OP resistance increased 750-fold for temephos but only 69.2fold for chlorpyrifos (L. E. Ranasinghe and G. P. Georghiou, unpublished). Moreover, C. tarsalis (with indicate
that
pipiens strain
Est. A
Est. a
l
both esterases A' and B) demonstrates 13.6-fold reo sistance to temephos and 29.6-fold resistance to chlorpyri£os but considerably higher resistance (253.8fold) to chlorpYTifos-methy1. AnoPheles albimanus.-Esterases A and B were found in 4 strains of A. albimanus of different geographic origin (Fig. 2). These show variations in electrophoretic mobility which can be attributed to
-
-- -----
~
~
---
o $? •••
.•.. a:
>-
~ c:::::::I
I-
c=::1
c:::::::J
c::=J
c::::::J c:::::J
c=::1
c:::::J c::::::J
.J CD
Est.A
--~
~ ~
~
~
~
~
t;;mm
~ ~
t;;mm
0
."
u IUI
a: 0
:I:
C!:!:m1
Q.
0
~
cmsm
cmm2
0
:e
a:
I-
c:::::::J c:::::J
Est.a
c:::::::J c::::::J c:::::J c::::::::J c:::::J
u
c:::::J c:::::J c:::::J 0
AI
A2
A4
UI .J UI
A3
patterns observed in single mosquitoes of various AnoPheles albimanus strains. A= a-naphthylFIG. 2.-Esterase acetate specific. B= ,B-naphthylacetate specific. Black, striped. and white rectangles designate decreasing staining intensities.
Downloaded from http://jee.oxfordjournals.org/ by guest on April 13, 2016
monofactorial (Pasteur & 5inegre 1977)
Cross Resistance
0
C. tarsalis (Calif.)
5usc.
+ +
• Esterase activity rating: b Selecting insecticide.
C. p. fatigans (Calif.)
(57,56)
A' A B Inheritance
pipiens (France)
April 1978
GEORGHIOU AND PASTEUR:
REFERENCES CITED Anonymous. 1976. Resistance of vectors and reservoirs of disease to pesticides. WHO Tech. Rep. Ser. 585. 88 pp. Apperson, C. S., and G. P. Georghiou. 1974. Comparative resistance to insecticides in populations of three sympatric species of mosquitoes in the Coachella Valley of California. J. Med. Entomo!. 11: 57H. 1975a. Changes in cross-resistance spectrum resulting from methyl-parathion selection of Culex tarsalis Coq. Am. J. Trop. Med. Hyg., 24:. 698-703. 1975b. Mechanisms of resistance to organophosphorus insecticides in Culex tarsalis. J. Econ. Entomo!. 68: 153-7. 1975c. Inheritance of resistance to organophosphorus insecticides in Culex tarsalis CoquilJett. Bull. WHO 52: 97-100. Ayad, H., and G. P. Georghiou. 1975. Resistance to organophosphates and carbamates in Anopheles alb imanus based on reduced sensitivity of acetylcholines. terase. J. Econ. Entomol. 63: 295-7. Ayala, F. S., J. R. Powell, M. L. Tracey, C. A. Mourao, and S. Perez·Salas. 1972. Enzyme variability in the Drosophila willis toni group. IV. Genic variation in natural populations of Drosophila willis toni. Genetics 70: 113-39. Beranek, A. P., and F. J. Oppenoorth. 1977. Evidence that the elevated carboxylesterase (Esterase 2) in organophosphorus resistant Myzus persicae (Sulz.) is identical with the organophosphate hydrolyzing enzyme. Pestic. Biochem. Physiol. 7: H~20.
205
Georghiou, G. P. 1972. Studies on resistance to carba· mate and organophosphorus insecticides in Anopheles albimanus. Am. J. Trop. Med. Hyg. 21: 797-806. Georghiou, G. P., and C. E. Taylor. 1976. Pesticide resistance as an evolutionary phenomenon. Proc. XV Int. Congr. Entomo!., Washington, D. C., p. 759-85. Georghiou, G. P., R. L. Metcalf, and F. E. Gidden. 1966. Carbamate resistance in mosquitoes. Bull. WHO 35: 691-708. Georghiou, G. P., V. Ariaratnam, M. E. Pasternak, and C. S. Lin. 1975. Organophosphorus multiresistance in Culex pipiens quinquejasciatus in California. J. Econ. Entomol. 68: 461-7. Hammock, B. D., S. M. Mumby, and P. W. Lee. 1977. Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica. Pestic. Biochem. Physio!. 7: 261-72., Krimbas, C. B., and S. Tsakas. 1971. The genetics of Dacus oleae. V. Changes of esterase polymorphism in a natural population following insecticide con· trol·selection or drift. Evolution 25: 454-60. Oppenoorth, F. J., and W. Welling. 1976. Biochemistry and physiology of resistance. P. 507-51. In C. F. Wilkinson, [ed.] Insecticide Biochemistry and Physiology. Plenum Press, N. Y. 768 pp. Pasteur, N., and G. Sinegre. 1975. Esterase polymorphism and sensitivity to Dursban organophosphorus insecticide in Culex pipiens pipiens populations. Biochem. Genet. 13(11/12): 789-803. 1977. Chlorpyrifos resistance in Culex pipiens pipiens L. from Southern France: inheritance and linkage. Experientia (In press) . Plapp, F. W. 1976. Biochemical genetics of insecticide resistance. Annu. Rev. Entomol. 21: 179-97. Poulik, M. D. 1957. Starch gel electrophoresis in a discontinuous system of buffers. Nature (London) 180: 1477-9. Priester, T., and G. P. Georghiou. 1978. Induction of high resistance to permethrin in Culex pipiens quinquefasciatus. J. Econ. Entomo!. 71: 197-200. Shrivastava, S. P., G. P. Georghiou, R. L. Metcalf, and T. R. Fukuto. 1970. Carbamate resistance in mos· quitoes. II. The metabolism of propoxur by sus· ceptible and resistant Culex pipiens jatigans Wied. Bull. WHO 49: 932-42. Sinegre, G., B. Gaven, and J. L. Jullien. 1976. Acti· vite comparee de 31 insecticides sur des larves de Culex pipiens (L.) sensibles et resistantes au chlor· pyrifos dans Ie midi de la France. Document No. 32, Entente Interdepartementale pour la Demoustication du Littoral Mediterraneen, Montpellier. 12 pp. Stordeur, E. de. 1976. Esterases in the mosquito Culex p. pipiens: formal genetics and polymorphism of adult esterases. Biochem. Genet. 14: 481-93. Tobgy, A. H., G. E. Nasrat, H. Nafei, and A. Z. EI· Abidin Salam. 1976. Insecticide resistance. VI. The inheritance of parathion resistance in DrosoPhila melanogaster strains with special reference to esterases. Egypt. J. Genet. Cyto!. 5: 300-12.
Downloaded from http://jee.oxfordjournals.org/ by guest on April 13, 2016
different polymorph isms. No differences are observ· able in their staining intensities, thus no suggestion can be made that they are associated with OP reo sistance. Since resistance in strain A2 is due to lower sensitivity of AChE to inhibition by organophosphates and carbamates (Ayad and Georghiou 1975) this enzyme was examined by electrophoresis as previous. ly mentioned in strains of A. albimanus and C. p. fatigans. The latter demonstrated no variation in electrophoretic mobility nor in staining intensity of AChE. However, in A. albimanus, AChE exhibited the same electrophoretic mobility in all strains, but· important variations in staining intensities. Thus, AChE of all susceptible mosquitoes (AI) shows strong staining intensity (high activity) while that of every OP-resistant mosquito (A2) shows low staining in· tensity. The parental field strain (A4) and the recently initiated chlorphoxim strain (A3) are composed of mosquitoes with one or the other type of AChE. Inhibition studies showed that AChE with high activity is inhibited by lO-'M methyl paraoxon while that with low activity is largely unaffected, and this is in agreement with the results of Ayad and Georghiou (1975).
EsTERASES IN MOSQUITOES
1 Diplera: Culicidae. ~ Received for publication Nov. 8, 1977. • Division of Toxicology and Physiology, Department of Entomology. University of California, RIverside 92521. • Laboratoire c\'Ecologie Medicale et de Pathologie Parasitaire, Faculte de Medecine, 34000 Montpellier, France.
fasciatus) Wiedemann and 4 G. tarsalis Coquillett strains of California origin, and 4 AnoPheles albimanus Wiedemann strains of Central American origin were studied by starch gel electrophoresis. In addition, each gel induded samples of Iyophilyzed homogenates of 3 strains of C. p. PiPiens L. from southern France for comparison (Table 1). Esterases and acetylcholinesterases (AChE) were studied after electrophoresis with Poulik (1957) buffer systems or with Tris-citrate buffers described by Ayala et aL (1972) under the name of system C. Esterases were revealed according to the technique used by de Stordeur (1976). AChE activity was demonstrated by soaking the gels for 20-25 min in sodium phosphate buffer (pH 7.5) at 5°C and subsequently transferring them to 100 ml of fresh buffer containing 8 ml acetylthiocholine (0.05 M) for 30 min at room temperature. AChE activity was revealed as yellow spots within one h following the addition of 4 ml of 0.01 M DTNB (dithiobisnitrobenzoate) . The effect of various inhibitors on esterase and AChE activities was assayed by the addition of the required amount of inhibitor in the buffers during· staining. EDT A (ethylenediamine tetraacetic acid, disodium salt) action was studied by adding 2mM of EDTA/liter in both the gel and the electrode buffers. For ease of comparison, we will name esterase-A those esterases using a-naphthylacetate and staining blue, and esterase-B those using p-naphthylacetate and staining red in the presence of both substrates. Some esterases were found to be completely inhibited in the absence of EDT A and they will be named esterases N or B' according to the substrate they hydrolyze. RESULTS AND DISCUSSIoN.-Comparison of Esterase Patterns in C. p. pipiens and C. p. fatigans.- Two esterase systems can be recognized in the strains of C. p. fatigans studied, one of type A and one of type B. In the absence of genetic data, it is difficult to attribute esterases to given loci on the basis of differences in electrophoretic mobility alone. However, because of extreme differences in activity (or staining intensity), we have divided esterases B in 2 groups, Bl with low activity and B2 with an extremely high activity (Fig. I). In C. p. pipiens, de 201
©1978 Entomological
Society of America
0022-0493(78(0071-0201$00.75(0
Downloaded from http://jee.oxfordjournals.org/ by guest on April 13, 2016
Among the mechanisms responsible for resistance to insecticides, enzyme modifications play a very important role. Thus, dehydrochlorinase is a major factor in DDT resistance, carboxylesterases, phosphotriesterases, acetylcholinesterases, glutathion-dependent transferases, and mixed function oxidases (MFO) are involved in organophosphorus (OP) resistance. MFO's are also important in carbamate resistance (reviews by Openoorth and Welling 1976, Plapp 1976). O-demethylase was recently shown to be the major detoxication enzyme in methopreneresistant house flies, Musca domestica L. (Hammock et aL 1977). Since these enzymes have undergone biochemical modifications, it is reasonable to assume that, at least in some cases, they will be electrophoretically different from normal enzymes. Until recently, very few studies of insecticide resistance coupled with electrophoresis analyses have been reported, but the few available have been extremely encouraging, though a direct cause-effect relationship has not always been ascertained (Krimbas and Tsakas 1971, Pasteur and Sinegre 1975, 1977, Tobgy et al. 1976, Beranek and Oppenoorth 1977). It has become apparent, however, that electrophoresis techniques hold considerable promise in relating specific est erases to resistance, and thus are a means of identifying resistant genotypes rather than phenotypes in single insects. Insecticide resistance is especially serious in disease vector and nuisance mosquitoes, occurring in at least 83 anopheline and culicine species (Anon. 1976, Georghiou and Taylor 1976). The availability in our respective laboratories of several strains of mosquitoes of known origin and resistance characteristics has made possible the present search of interstrain and interspecific similarities or differences in the electrophoretic patterns of their esterases related to resistance. MATERIALSAND METHODS.-Enzymes of single adult mosquitoes of 8 Culex PiPiens fatigans (= quinque-
202
JOURNAL
OF ECONOMIC
A_--
Vol. 71, no. 2
ENTOMOLOGY
DDB
D
D
•.•••
••..
a:
B c:::::J c:::::J
>-
I-
B=
....J III
o
~
BI= -A AB=
Bc:=:J
= BIc:::::J
DB2
55
• •
C.p. pipiens (Fr'ance)
F4
FI,F2,F3
C. p. fatigans
F6,F8 (Calif.)
-
a:
_A'
TI
C. tarsa/is
0
J: Q.
0
a::
A'-
I
U
Il£J
•
l-
u 0
l£J ....J l£J
T2 (Calif.)
FIG. I.-Esterase patterns observed in single mosquitoes of various Culex species and strains. A = a-naphthylacetate specific. B= p-naphthylacetate specific. ·Pattern obtained in presence of EDTA. Rf value of 100 given arbitrarily to fastest esterase of T2 strain.'
Stordeur (1976), Pasteur and Sinegre (1975), and Pasteur (1977)' have demonstrated 3 esterase-coding loci, Est-1, Est-2, and Est-3, coding for esterases of types B, A, and N, respectively. Both Est-2 and Est-] polymorphisms are associated with OP (chlorpyrifos) resistance, but recent studies have shown that if OP resistance is the result of a specific esterase allozyme, it is probably one coded by Est-]. This locus is homozygous for a null allele (Est_JNull) in every susceptible mosquito, while the resistant individuals bear the Est-]'" active allele in single or double dose. Esterase B2 of the California C. p. fatigans has no equivalent in the French C. p. pipiensj it is an enzyme with a very strong staining intensity which diffuses rapidly and blurs out any adjacent esterase system. It is not known at present if esterase B2 is an allozyme of the locus coding esterases B1 of C. p. fatigans or of Est-2 of C. p. pipiens. The absence in C. p. fatigans of an esterase N similar to C. p. PiPiens Est-] shows that if OP resistance is due to an esterase in both subspecies different loci are involved. Comparison of Esterase Patterns among C. p. fatigans Strains.-One of the most striking features of C. p. fatigans esterase patterns is the presence or absence of the highly active esterase B2 (Fig. 1). This esterase was found in every individual of the temephos-resistant F6 and F8 strains in which OP resistance is completely suppressible by the synergist • Pasteur, N. 1977. Recherches de genetique chez Culex piPiens Pipiens L. Polymorphisme enzymatique, autogenese et resistance aux insecticides organophosphores. These de Doctorat d'Etat, mention Sciences. Universite des Sciences. Montpellier, France.
DEF® (S,S,S-tributyl phosphorotrithioate) (Georghiou et al. 1975, L. E. Ranasinghe and G. P. Georghiou, unpublished) while it is absent from the susceptible Fl strain, and from strains F2, F3, F4, and F7 in which resistance is not affected, or only slightly so, by DEF (Table I). These observations indicate that in California C. p. fatigans OP resistance is most probably associated with esterase B2. The observed patterns also indicate that permethrin resistance (strains F3, F4) is independent of esterase B2. How do the esterases of the permethrin-resistant strains differ from those of other strains lacking esterase B2? A and Bl esterases in strain F3 (resistant to d-trans permethrin) have an electrophoretic mobility identical to that of the Fl susceptible strain and of the F2 and F7 strains in which resistance is based on MFO. The F4 strain (resistant to d-cis permethrin) shows a distinctly different electrophoretic pattern; esterases A are electrophoretically slower and esterase Bl is faster than in any other strain (Fig. 1). Whether these variations are relevant to pyrethroid resistance remains to be determined, but it is quite clear that the esterases of the 2 strains, selected in the presence of different isomers, are not identical although they are issued from the same parental stock (F5). A further point concerning C. p. fatigans must be emphasized; strains F3, F4, F6, F7, and F8 originate from the same parental strain (F5) (Table 1) which is composed of some 89% mosquitoes with esterase B2, although it has been maintained in the laboratory free of insecticidal exposure for ca. 26 mo. Therefore, it is clear that, depending on the type of selection pressure applied, some strains evolved
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57/5G S5
.A•
A-
-A-
D
on
APril 1978 Table
I.-Strains
Species Culex p. pipiens (France) C. p. fatigans (California)
C. tarsa/is
of mosquitoes investigated
for esterase activity and their insecticide resistance characteristics. Resistance status
Resistance mechanism
S5
field field
suscepti b Ie chlorpyri fos-R'
esterase (?)
Fl F2 F3
Bakersfield Coachella F5
susceptible propoxur-Rb d-trans permethrin-Rb
MFO
F4
F5
d-cis permethrin-Rb
(?)
F5 F6
Hanford F5
field 1975 (OP-R) temephos-Rb
esterase esterase, MFO
F7
F5
F8
F5
Tl
Bakersfield
susceptible
T2
Coachella
methyl parathion-Rb
T3
Coachella
methoprene- R b,.
(?)
T4
Coachella
diflu benzuron -selected b
(?)
Al A2 A3
Panama EI Salvador EI Salvador
Rd Chlorphoxim-selectedb
AChE
A4
EI Salvador
field (OP ICarb.-R)
AChE (?)
S7/SG
+ DEF-Rb temephos + pb-Rb
temephos
(?)
MFO esterase
esterase (?)
susceptible
o P I Carbamate-
(?)
Reference de Stordeur (1976) Pasteur and Sinegre (1977) Georghiou et a1. (1966) Shrivastava et a1. (1970) Priester and Georghiou (1978) Priester and Georghiou (1978) Georghiou et a1. (1975) Ranasinghe and Georghiou (unpublished) Ranasinghe and Georghiou (unpublished) Ranasinghe and Georghiou (unpublished) Apperson and Georghiou (1974) Apperson and Georghiou (1975b) Georghiou and C. S. Lin (unpublished) Georghiou and C. S. Lin (unpublished) Georghiou (1972) Ayad and Georghiou (1975) Georghiou and M. K. Hawley (unpublished) Georghiou and M. K. Hawley (unpublished)
• Selected on the basis of its esterase electrophoretic pattern and subsequently found resistant to chlorpyrifos. b Selectedin laboratory by chemicalindicated. • 437.5-foldresistanceto methoprene at ED50. d Selectedby propoxur and subsequentlyparathion. for the presence of esterase B2 and others for its absence. Thus, selection by temephos alone or in combination with piperonyl butoxide (pb) , an inhibitor of MFO, resulted in strains (F6 and F8) having esterase B2 as one would have expected if this enzyme locus, or one closely linked to it, plays a role in temephos resistance. But the total absence of esterase B2 from strains F3, F4, and F7 indicates that esterase B2 was actively selected against since it is unlikely that it could have disappeared in all 3 strains by chance alone while it has persisted in the parental (F5) strain. Culex tarsa/is.- Two esterase systems (A' and B) could be demonstrated in the susceptible (Tl) as well as in the methyl parathion-selected (T2) strain. Both esterases show electrophoretic mobility differences within and between strains, denoting a polymorphism of the loci coding them. But in the OPresistant strain, both esterases have a much stronger staining intensity. Mosquitoes of the methoprene-resistant (T3) and diflubenzuron-selected (T4) strains possess esterases A' and B with staining intensities comparable to those found in the Tl susceptible strain. But, in addition, these mosquitoes have supernumerary esterases which are not found in the other strains. Although the resistance mechanism of the methopreneR strain has not yet been investigated, the extreme
variability observed in the esterase pattern among individuals of this strain might suggest that esterase detoxication does not play an important role in its resistance. In methoprene-resistant house flies, 0demethylation by MFO enzymes was shown to be the primary detoxication mechanism (Hammock et a1. 1977). A most interesting finding of this study is the extremely strong staining intensity of esterase A' and esterase B in OP-resistant Culex mosquitoes. This denotes either a higher specific activity or a higher titer of the enzyme, and suggests that in Culex at least 2 different esterase genes may be involved independently or jointly in resistance. The biochemical differences between these esterases, e.g., substrate specificities and EDTA action, exclude that they can be allozymes coded by the same locus. Thus, in C. pipiens (strain 55) where only one of the 2 esterases is present, resistance has been shown to be monofactorial (Pasteur and 5inegre 1977). In contrast, in C. tarsa/is (T2) in which both esterases are present, resistance was found to be polyfactorial (Apperson and Georghiou 1975c) although bifactorial inheritance cannot be excluded (see their Fig. 5) . Furthermore, if the 2 esterase genes confer different biochemical properties, it may be expected that the resistance spectrum should be different according to the gene or genes involved. The data in Table 2
or
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Origin or parent
Strain code
(California)
Anopheles albimanlls (Central America)
203
GEORGHIOUANDPASTEUR: EsTERASESIN MOSQUITOES
204
JOURNAL
Table 2.-Interspecific
and interstrain
OF ECONOMIC
Vol. 71, no. 2
ENTOMOLOGY
diJferences in esterases of OP·resistant and susceptible patterns of the strains.
C. p. pifJiens, C.
p.
totigons, and C. tof'sa.lis, and the cross resistance and inheritance
c. p. Esterases'
Resist.
5usc.
Resist.
5usc.
Resist.
(55)
(FI)
(F5,F6,F8)
(TI)
(T2)
0
+++ + +
0
0
+
+++
+ +
+ +++
0
0
+
+++
of R
monofactorial (Ranasinghe & Georghiou unpublished)
polyfactorial (Apperson & Georghiou 1975c)
LCoo 55 strain
LC•• F5
LC•• T2
LCoo 57 strain
LC•• FI
LC•• TI
750.0b 48.3 69.2 230.8 41.2 114.9 62.1 (Ranasinghe & Georghiou unpublished)
13.6 96.5b 29.6 253.8 49.4 76.8 10.9 (Apperson & Georghiou 1975a)
temephos methyl parathion chlorpyrifos chlorpyrifos.meth yl feni trothion femhion malathion
9.3 8.2 21.4 15 5.9 7.3 3.3 (5inegre et a!. 1976) =
none,
+
=
low,
++ +
=
very high.
OP resistance in the French C. p. 55 (with esterase A') increased 21.4fold for chlorpYTi£os but only 9.3-fold for temephos (Sinegre et a1. 1976), whereas in the Californian C. p. fatigans strain F6 (with esterase B) OP resistance increased 750-fold for temephos but only 69.2fold for chlorpyrifos (L. E. Ranasinghe and G. P. Georghiou, unpublished). Moreover, C. tarsalis (with indicate
that
pipiens strain
Est. A
Est. a
l
both esterases A' and B) demonstrates 13.6-fold reo sistance to temephos and 29.6-fold resistance to chlorpyri£os but considerably higher resistance (253.8fold) to chlorpYTifos-methy1. AnoPheles albimanus.-Esterases A and B were found in 4 strains of A. albimanus of different geographic origin (Fig. 2). These show variations in electrophoretic mobility which can be attributed to
-
-- -----
~
~
---
o $? •••
.•.. a:
>-
~ c:::::::I
I-
c=::1
c:::::::J
c::=J
c::::::J c:::::J
c=::1
c:::::J c::::::J
.J CD
Est.A
--~
~ ~
~
~
~
~
t;;mm
~ ~
t;;mm
0
."
u IUI
a: 0
:I:
C!:!:m1
Q.
0
~
cmsm
cmm2
0
:e
a:
I-
c:::::::J c:::::J
Est.a
c:::::::J c::::::J c:::::J c::::::::J c:::::J
u
c:::::J c:::::J c:::::J 0
AI
A2
A4
UI .J UI
A3
patterns observed in single mosquitoes of various AnoPheles albimanus strains. A= a-naphthylFIG. 2.-Esterase acetate specific. B= ,B-naphthylacetate specific. Black, striped. and white rectangles designate decreasing staining intensities.
Downloaded from http://jee.oxfordjournals.org/ by guest on April 13, 2016
monofactorial (Pasteur & 5inegre 1977)
Cross Resistance
0
C. tarsalis (Calif.)
5usc.
+ +
• Esterase activity rating: b Selecting insecticide.
C. p. fatigans (Calif.)
(57,56)
A' A B Inheritance
pipiens (France)
April 1978
GEORGHIOU AND PASTEUR:
REFERENCES CITED Anonymous. 1976. Resistance of vectors and reservoirs of disease to pesticides. WHO Tech. Rep. Ser. 585. 88 pp. Apperson, C. S., and G. P. Georghiou. 1974. Comparative resistance to insecticides in populations of three sympatric species of mosquitoes in the Coachella Valley of California. J. Med. Entomo!. 11: 57H. 1975a. Changes in cross-resistance spectrum resulting from methyl-parathion selection of Culex tarsalis Coq. Am. J. Trop. Med. Hyg., 24:. 698-703. 1975b. Mechanisms of resistance to organophosphorus insecticides in Culex tarsalis. J. Econ. Entomo!. 68: 153-7. 1975c. Inheritance of resistance to organophosphorus insecticides in Culex tarsalis CoquilJett. Bull. WHO 52: 97-100. Ayad, H., and G. P. Georghiou. 1975. Resistance to organophosphates and carbamates in Anopheles alb imanus based on reduced sensitivity of acetylcholines. terase. J. Econ. Entomol. 63: 295-7. Ayala, F. S., J. R. Powell, M. L. Tracey, C. A. Mourao, and S. Perez·Salas. 1972. Enzyme variability in the Drosophila willis toni group. IV. Genic variation in natural populations of Drosophila willis toni. Genetics 70: 113-39. Beranek, A. P., and F. J. Oppenoorth. 1977. Evidence that the elevated carboxylesterase (Esterase 2) in organophosphorus resistant Myzus persicae (Sulz.) is identical with the organophosphate hydrolyzing enzyme. Pestic. Biochem. Physiol. 7: H~20.
205
Georghiou, G. P. 1972. Studies on resistance to carba· mate and organophosphorus insecticides in Anopheles albimanus. Am. J. Trop. Med. Hyg. 21: 797-806. Georghiou, G. P., and C. E. Taylor. 1976. Pesticide resistance as an evolutionary phenomenon. Proc. XV Int. Congr. Entomo!., Washington, D. C., p. 759-85. Georghiou, G. P., R. L. Metcalf, and F. E. Gidden. 1966. Carbamate resistance in mosquitoes. Bull. WHO 35: 691-708. Georghiou, G. P., V. Ariaratnam, M. E. Pasternak, and C. S. Lin. 1975. Organophosphorus multiresistance in Culex pipiens quinquejasciatus in California. J. Econ. Entomol. 68: 461-7. Hammock, B. D., S. M. Mumby, and P. W. Lee. 1977. Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica. Pestic. Biochem. Physio!. 7: 261-72., Krimbas, C. B., and S. Tsakas. 1971. The genetics of Dacus oleae. V. Changes of esterase polymorphism in a natural population following insecticide con· trol·selection or drift. Evolution 25: 454-60. Oppenoorth, F. J., and W. Welling. 1976. Biochemistry and physiology of resistance. P. 507-51. In C. F. Wilkinson, [ed.] Insecticide Biochemistry and Physiology. Plenum Press, N. Y. 768 pp. Pasteur, N., and G. Sinegre. 1975. Esterase polymorphism and sensitivity to Dursban organophosphorus insecticide in Culex pipiens pipiens populations. Biochem. Genet. 13(11/12): 789-803. 1977. Chlorpyrifos resistance in Culex pipiens pipiens L. from Southern France: inheritance and linkage. Experientia (In press) . Plapp, F. W. 1976. Biochemical genetics of insecticide resistance. Annu. Rev. Entomol. 21: 179-97. Poulik, M. D. 1957. Starch gel electrophoresis in a discontinuous system of buffers. Nature (London) 180: 1477-9. Priester, T., and G. P. Georghiou. 1978. Induction of high resistance to permethrin in Culex pipiens quinquefasciatus. J. Econ. Entomo!. 71: 197-200. Shrivastava, S. P., G. P. Georghiou, R. L. Metcalf, and T. R. Fukuto. 1970. Carbamate resistance in mos· quitoes. II. The metabolism of propoxur by sus· ceptible and resistant Culex pipiens jatigans Wied. Bull. WHO 49: 932-42. Sinegre, G., B. Gaven, and J. L. Jullien. 1976. Acti· vite comparee de 31 insecticides sur des larves de Culex pipiens (L.) sensibles et resistantes au chlor· pyrifos dans Ie midi de la France. Document No. 32, Entente Interdepartementale pour la Demoustication du Littoral Mediterraneen, Montpellier. 12 pp. Stordeur, E. de. 1976. Esterases in the mosquito Culex p. pipiens: formal genetics and polymorphism of adult esterases. Biochem. Genet. 14: 481-93. Tobgy, A. H., G. E. Nasrat, H. Nafei, and A. Z. EI· Abidin Salam. 1976. Insecticide resistance. VI. The inheritance of parathion resistance in DrosoPhila melanogaster strains with special reference to esterases. Egypt. J. Genet. Cyto!. 5: 300-12.
Downloaded from http://jee.oxfordjournals.org/ by guest on April 13, 2016
different polymorph isms. No differences are observ· able in their staining intensities, thus no suggestion can be made that they are associated with OP reo sistance. Since resistance in strain A2 is due to lower sensitivity of AChE to inhibition by organophosphates and carbamates (Ayad and Georghiou 1975) this enzyme was examined by electrophoresis as previous. ly mentioned in strains of A. albimanus and C. p. fatigans. The latter demonstrated no variation in electrophoretic mobility nor in staining intensity of AChE. However, in A. albimanus, AChE exhibited the same electrophoretic mobility in all strains, but· important variations in staining intensities. Thus, AChE of all susceptible mosquitoes (AI) shows strong staining intensity (high activity) while that of every OP-resistant mosquito (A2) shows low staining in· tensity. The parental field strain (A4) and the recently initiated chlorphoxim strain (A3) are composed of mosquitoes with one or the other type of AChE. Inhibition studies showed that AChE with high activity is inhibited by lO-'M methyl paraoxon while that with low activity is largely unaffected, and this is in agreement with the results of Ayad and Georghiou (1975).
EsTERASES IN MOSQUITOES
