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Biochemical Genetics, Vol. 42, Nos. 3/4, April 2004 (°

C 2004)

Variation of Genetic Expression During

Development, Revealed by Esterase Patterns
in Aedes aegypti (Diptera, Culicidae)
Alba Regina de Abreu Lima-Catelani,1 Carlos Roberto Ceron,2
and Hermione E. Melara de Campos Bicudo1,3

Received 8 May 2002—Final 12 February 2003

Polyacrylamide gel electrophoresis was used to analyze esterase patterns during

development of Aedes aegypti from the cities of Marı́lia and São José do Rio Preto
(SJRP), Brazil. The zymograms showed a total of 23 esterase bands, 22 of which
were in the specimens from Marı́lia and 19 in those from SJRP. These esterase
bands were considered to be the product of 23 alleles distributed tentatively in
eight genetic loci. Most of the alleles were developmentally regulated. The larval
stage expressed the greatest number of them (19 alleles, from the eight loci, in
Marı́lia; and 17 alleles, from seven loci, in SJRP). The pupal stage expressed 10
alleles from seven loci, in both populations, and the adult stage expressed 8 alleles
from five and six loci in SJRP and Marı́lia, respectively. Some alleles that were
active in every stage were developmentally controlled at the level of expression
(amount of product). A single allele was constitutively and highly expressed, in
larvae, pupae, and adults, in both populations. Differences in esterase synthesis
among stages are probably due to regulatory mechanisms acting in agreement
with the requirements of a variable number of processes in which esterases are
involved. The larval stage is the most active in developmental processes and shows
very intense intake of food and very high mobility. These features may demand
increased esterase production at that stage. Comparison of the two populations
examined showed (besides the existence of alleles that they do not share) that they
exhibit differences in the control of expression of other alleles. Such findings may

1 Departamento de Biologia, IBILCE, Universidade Estadual Paulista-UNESP, 15054-000 São José do

Rio Preto, SP, Brazil.
2 Departamento de Quimica e Geociências, IBILCE, Universidade Estadual Paulista-UNESP, 15054-
000 São José do Rio Preto, SP, Brazil.
3 To whom correspondence should be addressed; e-mail: bicudo@bio.ibilce.unesp.br.

69
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C 2004 Plenum Publishing Corporation
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70 Lima-Catelani, Ceron, and Bicudo

reflect genetic differences between founders in each population, but the possibility
of involvement of the intensive use of insecticides in SJRP is also discussed.

KEY WORDS: Aedes aegypti; esterases; development; expression variation.

INTRODUCTION
Esterases are highly variable and multifunctional hydrolytic enzymes (Miller and
Novak, 1983; Wagner et al., 2002). In insects they take part in biological processes
such as regulation of juvenile hormone (Dekort and Granger, 1981; Shanmugavelu
et al., 2000), digestion (Jones and Brancoft, 1986; Kapin and Ahmad, 1980),
reproduction (Mane et al., 1983; Richmond et al., 1980), and insecticide resis-
tance (Fournier et al., 1993; Karunaratne and Hemingway, 1996; Motoyama and
Dauterman, 1974; Perez-Mendoza et al., 2000; Siegfried and Ono, 1993).
Esterase patterns have been shown to vary among populations and species
and are an important tool for analysis of genetic differentiation and evolution-
ary relationships in insects (e.g., in Haematobia irritans, Castiglioni-Ruiz et al.,
1997; in Drosophila, Lapenta et al., 1995, 1998; Nascimento and Bicudo, 2002).
Stage-specific and tissue-specific expression patterns have also been observed in
studies of esterase bands or isozymes (Arnason and Chambers, 1984; Brady and
Richmond, 1990; Pasteur et al., 2001; Sergeev et al., 1995).
We examined esterase patterns in Aedes aegypti, an important vector of yellow
fever, hemorrhagic fever, and dengue, during development, in two populations.
The knowledge of such patterns is relevant, not only because variation in genetic
expression among stages and differences among populations may be detected but
also because esterases are involved in the development of resistance to insecticides
that are presently the major control method for those mosquitoes throughout the
world. In this study, mosquito samples for analysis were collected in São José
do Rio Preto and Marı́lia (State of São Paulo, Brazil). An important difference
between these populations is that during this study, São José do Rio Preto (but not
Marı́lia) was submitted to frequent insecticide applications by sanitary authorities
for Aedes population size control. The finding of esterase differences between
them might be the basis for another study dealing with insecticide resistance.

MATERIALS AND METHODS

Mosquito Origin and Developmental Stages
Esterase patterns were studied by polyacrylamide gel electrophoresis in larvae
(L2, L3, and L4 stages), pupae (P), and adult males (M) and females (F) of Aedes
aegypti from Marı́lia and São José do Rio Preto (SJRP). Both are cities in the State
of São Paulo, Brazil, located 200-km away from each other.
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Variation of Genetic Expression During Development 71

Mosquito Samples
The mosquitoes from SJRP were collected at different times and sites from ar-
tificial containers such as tires and bottles by the SUCEN (Supervising Group
for the Control of Endemic Diseases in the State of São Paulo). Mosquito eggs
from Marı́lia were obtained from frequently renewed cultures maintained in the
Laboratory of SUCEN in that city. In both cases, the mosquitoes were frozen in
the desired stages and used within a month. The number of mosquitoes analyzed
in each developmental stage of both populations was the following: from SJRP:
L2 = 255, L3 = 190, L4 = 162, P = 120, adult females = 61, adult males =
58, total = 846; from Marı́lia: L2 = 439, L3 = 274, L4 = 239, P = 323, adult
females = 153, adult males = 57, total = 1585.

Control of Aedes aegypti in SJRP

Aedes aegypti population size in SJRP has been monitored by SUCEN and con-
trolled by putting granulated organophosphorous insecticides into breeding sites
(tires and other man-made containers) for larva control, and by using vaporization
of insecticides in the streets for adult control. During the present study (1993–
1995), insecticide vaporization (mainly with pyrethroids but also with organophos-
phates) was carried out as follows: (1) 4 weeks of daily insecticide spraying be-
tween June 21 and July 16, in 1993; (2) 8 weeks of daily spraying from March 28
to May 27, in 1994; (3) 22 weeks of daily spraying from January 16 to May 29, in
1995. Granulated insecticides were used systematically in potential breeding sites.

Sample Preparation for Electrophoresis

Five second-instar larvae or three third-instar larvae, or individually fourth-instar
larvae, pupae, and male and female adults were homogenized at 0◦ C, in 25 µL
of buffer solution (0.1 M Tris-HCl plus 10% glycerol at pH 8.8). Homogenates
were centrifuged at 3000 rpm for 3 min. After the sample application (10 µL),
the gels (8% polyacrylamide) were submitted to a constant 200 V, using a buffer
system at room temperature (∼25◦ C). The running time was 2.5 h. Variations of
this procedure, including changes in gel concentration and running time, were also
tested, but the best results, i.e., clear bands for analysis, were obtained with the
technique used.

Identification of Esterases in the Gels

Esterases were identified in the gels following basically the technique described
by Johnson et al. (1966) and Steiner and Johnson (1973), by using the α- and
β-naphthyl acetates as substrates. The technique involved gel preincubation for
1 h, at room temperature (∼25◦ C), in 50 mL 0.1 M sodium phosphate at pH 6.2,
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72 Lima-Catelani, Ceron, and Bicudo

and staining reaction in the dark (1 h) with a solution containing 30 mg of α- and

15 mg of β-naphthyl acetates, 60 mg of fast blue ruthenium red, and 5 mL of
n-propanol in 50 mL of sodium phosphate buffer solution. When only α-naphthyl
acetate was hydrolyzed, the bands in the gels became black and were named
α-esterases. When only β-naphthyl acetate was hydrolyzed, the bands became red
and were named β-esterases. The gels were destained during 24–48 h in a solution
containing ethyl alcohol, acetic acid, and water in a proportion of 2:1:8. They were
then air dried at room temperature by placing gels between gelatine and cellophane
membranes in an embroidering hoop (Ceron et al., 1992).

Biochemical Characterization With Inhibitors

Inhibition tests for biochemical characterization of esterases involved the use of
malathion (0.4 mM solution), eserine sulphate (1 mM solution), phenylmethyl-
sulphonyl fluoride—PMSF (1 mM solution), iodoacetic acid (1 mM solution),
β-mercaptoethanol (10 mM solution), and sodium fluoride (5 mM solution), in-
dividually included in the preincubation and stain solutions. Esterase bands from
fourth-instar larvae, pupae, and adults were used in the inhibition experiments.
Two mosquitoes at each of these stages were crushed to prepare the samples.
Following the classification of Oakeshott et al. (1993), esterases inhibited by
organophosphate (malathion) and carbamate (eserine sulphate) are classified as
cholinesterases, esterases inhibited only by organophosphate (malathion) are clas-
sifed as carboxylesterases, and esterases that are inhibited only by sulphydryl
reagents (PMSF) are arylesterases. Sekar and Hageman (1979) described PMSF
as an inhibitor of serine-esterases, which are enzymes that carry a serine in their
activity core. Acetylesterases are esterases not inhibited by any of the inhibitors
used. NaF is an enzyme inhibitor. It provides an additional means of identifying
specific cell types. For example, unspecific esterase activity in human monocytes
and platelets is inhibited by sodium fluoride while in granulocytes it remains un-
affected by this substance (Dufer et al., 1984).

Statistical Analysis
For comparison among developmental stages, in each population, analysis of
dependence (ANADEP) was used (Cordeiro, 1987; Lapenta et al., 1995). The
ANADEP is a geometrical method used in the study of association between lines
and columns, in a table of cross-classification. In the present case, lines and columns
are represented by esterase bands and developmental stages, respectively, and the
table of cross-classification is that of percentages of detection of each esterase
band, in L4, pupae, and male and female adults. Coefficients of codependence (δ)
vary from −1 < δ ≤ 1. Coefficient values closer to 1 mean greater similarity and
closer to −1 indicate greater difference between the features compared.
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Variation of Genetic Expression During Development 73

For comparison between populations in each stage (L4, pupae, and male and
female adults), the chi-square test was used.

RESULTS
Characterization of Esterase Bands by Substrate Preference
and Inhibition Pattern
A total of 23 esterase bands was detected in developmental stages examined in
A. aegypti from SJRP and Marı́lia (Figs. 1 and 2). They were designated EST-1 to
EST-23 from the less anodic to the more anodic ends of the gel. Using α- and β-
naphthyl acetates as substrates, the band staining patterns revealed 18 α-esterases
(EST-1–EST-16, EST-22, and EST-23) and 5 β-esterases (EST-17–EST-21). The
α-esterases were able to hydrolyze β-naphthyl acetate in the absence of α-naphthyl
acetate, but the reverse was not true, indicating that β-esterases are more specific
for the substrate.
The results of inhibitor applications for biochemical classification of esterase
bands detected in the different stages were the same in both populations (Table I).
In the concentration used, PMSF inhibited esterases completely in bands EST-1

Fig. 1. Esterase patterns of A. aegypti in developmental stages from Marı́lia (A) and São José do Rio
Preto (B). Solid lines = α-esterases; dotted lines = β-esterases. Larvae (L2–L4), pupae (P), and adults
(M = males; F = females).
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74 Lima-Catelani, Ceron, and Bicudo

Fig. 2. Polyacrylamide gels showing esterase bands of A. aegypti specimens from Marı́lia (A) and
São José do Rio Preto (B). Larvae (L2–L4), pupae (P), and adults (M = males; F = females).

and EST-2, but affected less strongly those of bands EST-12–EST-14 and EST-18;
it inhibited still less strongly the esterase in band EST-20, and very weakly the
esterases of bands EST-3–EST-7.
(For purposes of simplification, esterases will henceforth be referred to by
the band designation to which they correspond.)
Eserine sulphate showed complete inhibition of esterases EST-12–EST-14 but
affected slightly EST-1–EST-7, EST-18, and EST-20. Malathion inhibited com-
pletely EST-1–EST-7, EST-18, and EST-20, and almost completely EST-12–EST-
14 (in pupae EST-13 was weakly inhibited). NaF inhibited completely EST-18
and EST-20, moderately EST-1 and EST-2, and weakly EST-12–EST-14; esterases
EST-3–EST-7 were not inhibited by NaF. Iodoacetic acid inhibited a single es-
terase, EST-20, but only in pupae, and β-mercaptoethanol inhibited completely all
the esterases. Some bands (EST-8–EST-11, EST-15–EST-17, EST-19, and EST-
21–EST-23) were not examined as to inhibition patterns because they were absent
in the treated gels.
The results of inhibition tests suggested that esterases in bands EST-1–EST-7
and in EST-18 and EST-20 are carboxylesterases, while those in bands EST-12 and
EST-14 are cholinesterases. EST-13 is probably also a cholinesterase. The complete
inhibition of all esterases by β-mercaptoethanol and the absence of effects by
iodoacetic acid on all of them except EST-20 in pupae indicate that there are no
arylesterases among the bands examined. All were inhibited in varying degrees
by PMSF, suggesting that at least those more strongly inhibited (EST-1, EST-2,
EST-12–EST-14, and EST-18) are serine-esterases. Since every band was inhibited
by one or more of the inhibitors used, the present results indicate that none of the
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Biochemical Genetics [bigi]

Table I. Effect of Inhibitors on the Esterase Bands of A. aegypti Specimens From Both Cities
PP1109-478129-01

Esterase bands
Inhibitors Concentration 1–2 3–7 12 13 14 18 20

PMSF 1 mM + + ++ + +++ +++ +++ +++ ++

Eserine 1 mM + + + + ++ + + ++ + + ++ + +
Malathion 0.4 mM + + ++ + + ++ +++ + + +a +++ + + ++ + + ++
Variation of Genetic Expression During Development

Iodoacetic acid 1 mM — — — — — — + + ++b

March 16, 2004

Sodium fluoride 5 mM ++ – + + + + + ++ + + ++
β-Mercaptoethanol 10 mM + + ++ + + ++ + + ++ + + ++ + + ++ + + ++ + + ++
18:29

Note. − = No inhibition; +, ++, + + +, and + + ++ = inhibition degrees in increasing order.

a Weak inhibition was found in pupae.
b Effect observed only in pupae.
75
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76 Lima-Catelani, Ceron, and Bicudo

Table II. The Eight Loci Tentatively Proposed for the 23 Esterase Bands in A. aegypti

Loci Bands Substrate affinity Biochemical classification

Est 1 EST-1–EST-2 α Carboxylesterases

Est 2 EST-3–EST-7 α Carboxylesterases
Est 3 EST-8–EST-11 α —
Est 4 EST-12–EST-13 α Cholinesterases
Est 5 EST-14 α Cholinesterase
Est 6 EST-15–EST-16 α —
Est 7 EST-17–EST-21 β Carboxylesterases
Est 8 EST-22–EST-23 α —

Note. Based on substrate affinity, biochemical classification by inhibition patterns and pres-
ence (simultaneous or not) in the individuals. — = not classified bands.

esterases can be classified as an acetylesterase. The NaF inhibition pattern was

important in reinforcing differences among esterases whose corresponding alleles
were included in separate loci.

Tentative Loci Assignment of Esterases

On the basis of location in the gels, substrate preference, inhibition patterns, and
presence (simultaneous or not) in each mosquito, the 23 bands, i.e., the alleles that
express the esterases observed as 23 bands in the gels, were distributed tentatively
in eight loci (Table II) as follows: locus 1 (Est 1), including alleles responsible for
esterases in bands EST-1 and EST-2; locus 2 (Est 2), alleles expressing esterases
EST-3–EST-7; locus 3 (Est 3), alleles coding for EST-8–EST-11; locus 4 (Est 4),
alleles for EST-12 and EST-13; locus 5 (Est 5), allele for EST-14; locus 6 (Est 6),
alleles for EST-15 and EST-16; locus 7 (Est 7), alleles for EST-17–EST 21; and
locus 8 (Est 8), alleles for EST-22 and EST-23 (Table III). In spite of the fact that
EST-12, EST-13, and EST-14 share several characteristics, EST-14 was included
in a separate locus because simultaneous presence of that esterase with the other
two was observed in two females and four males from SJRP.

Comparing the Two Populations

Locus and Allele Sharing
Products of alleles from loci Est 1 to Est 7 were detected in gels of both populations,
but products of alleles from Est 8 (esterases EST-22 and EST-23) were exclusively
observed in mosquitoes from Marı́lia.
Some loci expressed in both populations included alleles apparently specific
to a single population since their products were not observed in gels of the other
population. This is the case for esterase EST-10, from locus Est 3, and esterase
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Variation of Genetic Expression During Development 77

Table III. Percentage of Mosquitoes Bearing the Esterase Bands Expressed in Each Locus, Detected
in A. aegypti From Marı́lia and São José do Rio Preto (SJRP), in Each Developmental Stage

L4 Pupae Females Males

Band
Loci (allele) Marı́lia SJRP Marı́lia SJRP Marı́lia SJRP Marı́lia SJRP

Est 8 23 2.09 — — — — — — —
22 2.09 — — — — — — —
Est 7 21 5.44 0.62 — — — — — —
20 79.50 82.72 2.78 10.00 — — — —
19 14.22 0.62 — — — — — —
18 11.29 1.85 — — — — — —
17 — — — — 20.26 — 8.28 —
Est 6 16 43.10 12.96 70.28 55.83 58.82 8.19 57.32 15.51
15 — — — 2.50 52.29 22.96 61.78 8.62
Est 5 14 53.14 51.85 37.77 49.17 88.23 75.41 87.90 48.27
Est 4 13 44.77 52.47 52.01 60.00 53.59 60.65 33.12 77.58
12 42.68 53.70 47.37 50.00 — 13.11 — 46.55
Est 3 11 0.42 — 1.55 0.83 — — — —
10 — — 0.31 — — — — —
9 20.92 50.00 — — — — — —
8 — 24.69 — — — — — —
Est 2 7 56.90 64.20 29.10 1.33 — 44.26 17.20 13.79
6 29.29 40.12 — — — — — —
5 63.60 53.09 — — — — — —
4 88.70 85.80 — — — — — —
3 56.48 86.10 — — — — — —
Est 1 2 6.70 6.79 2.17 2.50 5.23 26.23 5.09 34.48
1 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

EST-17, from Est 7, exclusively found in Marı́lia, and EST-8, from Est 3, exclu-
sively found in SJRP.

Esterase Patterns and Band Frequencies During Development

The band frequencies in gels were computed for L4, pupae, and adults in both
populations because developmental stages anterior to L4 involved the crushing of
three–five larvae for preparing the samples for electrophoresis and thus their data
are not individual.
As shown in Fig. 1 and Table III, some alleles were expressed in every de-
velopmental stage, in both populations, specifically, those for esterases EST-1,
present in 100% of gels of both populations, and EST-2 (both from locus Est 1),
EST-13 (Est 4), EST-14 (Est 5), and EST-16 (Est 6). The alleles for EST-7 (Est 2)
and for EST-12 (Est 4) were also present in every developmental stage, in SJRP;
however, in Marı́lia the first esterase was not detected in females and the second
was detected in neither males nor females.
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78 Lima-Catelani, Ceron, and Bicudo

The alleles for esterases EST-3–EST-6 (Est 2) were exclusively expressed in

larvae, in both populations. These bands were observed in high percentages of gels
from L4. EST-9 (from Est 3) was another allele coding esterases detected only in
larval stage, in both populations. EST-18, EST-19, and EST-21 (from Est 7) were
also observed only in gels from L4, with low frequencies in both populations, but
lower in SJRP than in Marı́lia. The bands EST-8, from Est 3, and EST-22 and
EST-23, from Est 8, were exclusively detected in gels from L4, with very low
frequencies and in a single population (the first in SJRP and the second and third
in Marı́lia).
The allele for esterase EST-17 (Est 7), exclusively detected in Marı́lia, was
expressed only in adults. EST-15 (Est 6) was expressed exclusively in adults from
Marı́lia and almost exclusively in adults from SJRP (where it was also detected in
pupae, with low frequency).
Among the esterases that showed great differences in expression frequency
between Marı́lia and SJRP are EST-16 (higher frequencies in Marı́lia than in SJRP),
mainly in L4 and adult stages; EST-2 (higher in adults from SJRP than in those
from Marı́lia); and EST-15 (much lower frequencies in SJRP than in Marı́lia), in
the adult stage.
Differences between the two populations as to the frequency of mosquitoes
expressing the esterase bands in L4, pupae, males and females, evaluated by con-
tingency chi-square, were highly significant in every comparison (P < 0.000 for
L4 and adult stages, and P < 0.009 for pupal stage).
Codependent coefficients among stages in each population, computed by
ANADEP, showed negative association of L4 with pupal and adult stages in SJRP
and Marı́lia. Pupal stage showed positive association with adult stage, and the
association was stronger in SJRP than in Marı́lia. Males and females showed high
positive codependence coefficients in both populations (Table IV).

Table IV. Coefficients of Codependence Among Stages in Each A. aegypti

Population

Stages
Population Stages L4 P F M

Marı́lia L4 0.1 −0.1612 −0.7780 −0.7804

P −0.1612 1.0 0.2947 0.3653
F −0.7780 0.2947 1.0 0.9145
M −0.7804 0.3653 0.9145 1.0
SJRP L4 1.0 −0.6473 −0.7553 −0.8191
P −0.6473 1.0 0.5184 0.7398
F −0.7553 0.5184 1.0 0.8633
M −0.8191 0.7398 0.8633 1.0

Note. Larvae (L4), pupae (P) and adult females (F) and males (M).
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Variation of Genetic Expression During Development 79

Enzyme Activity
The thickness and the staining degree of some bands varied in different develop-
mental stages. This observation is important because high degrees of staining and
thickness are indicative of great enzymatic activity. For example, band EST-2 was
thin in L2 and L3 larval stages and thick and highly stained in L4, pupae, and
adults; band EST-7 was thick and highly stained in larvae and thin in pupae and
adults; and band EST-16 was thin in larvae and pupae and thick and highly stained
in adults. These observations were the same in both populations.

DISCUSSION
In the last two decades, A. aegypti has reinfested several regions of tropical and
subtropical zones in the Americas, including some that had successfully eradi-
cated these mosquitoes in the past, like Brazil. Concern about diseases transmitted
by these mosquitoes (mainly dengue and hemorragic fever, for which there is no
vaccine yet), as well as coordinated actions for eliminating their breeding sites,
has increased the interest in research efforts to develop genetic knowledge about
the mosquitoes, aiming at their control. In this study, polyacrylamide gel elec-
trophoresis made it possible to study genetic variability involved in the esterase
patterns of two mosquito populations of A. aegypti from Brazil and to analyze the
pattern of gene expression during the species development. The main aspects of
the findings are discussed.

Housekeeping Esterases
Constitutively expressed enzymes (i.e., those enzymes expressed in most
mosquitoes, during the entire development) are considered to be involved in es-
sential physiological activity and to be produced by housekeeping genes. In the
present study, this seems to be the case of the allele for the α-esterase EST-1, aris-
ing from the locus Est 1. This esterase is remarkable: it is present in gels of every
mosquito from every stage, and it exhibits the greatest thickness and highest de-
gree of staining among all bands, denoting constant and high gene activity during
development and in adult life. Trebatoski and Craig (1969) and Townson (1972)
describe these characteristics for esterase bands from A. aegypti detected by starch
gels and polyacrylamide gels, respectively. The authors named them EST-6 and
EST-α, respectively. Townson (1972) considered EST-6 and EST-α to be derived
from the same locus. Its high polymorphic state was later verified in geographic
strains (Mani et al., 1986; Saul et al., 1976). Saul et al. (1976) found at least 14
alleles in this locus, but the pattern of each mosquito showed single, double, or
null bands. The alleles for esterases EST-1 and EST-2 (both from locus Est 1),
detected in the present study, are probably part of that polymorphic locus.
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80 Lima-Catelani, Ceron, and Bicudo

Alleles for esterases in bands EST-13 and EST-14 were also expressed
throughout development with frequencies that were relatively high in the two
A. aegypti populations. EST-16 was also expressed throughout development, with
frequencies higher in Marı́lia than in SJRP.

Pattern Changes During Development

Differences in esterase patterns among stages reflect gene regulation of protein
production during development, coordinating the synthesis as required. In the
present study, five of the eight loci proposed were active throughout development
of mosquitoes in both analyzed populations: Est 1, Est 2, Est 4, Est 5, and Est 6.
The most active stage in synthesis of esterases was the larval one. Products of
alleles of Est 3, Est 7, and Est 8 were detected only in larval stages. Some of these
alleles showed high frequency of expression and high level of synthesis, such as
those for EST-3, EST-4, and EST-5. Even the five loci that are active throughout
development exhibited greater polymorphism in larval stages (for example, of the
five esterases produced by alleles of locus Est 2 in larvae, only one was expressed
in pupal and adult stages). Perhaps expression of the allele for EST-20, from locus
Est 7, could also be considered limited to the larval stage, since it was found in
a high number of larvae and in a small number of pupae in both populations. In
mosquitoes from Marı́lia, another band, EST-12, showed more than 40% frequency
in larvae and pupae, indicating that the correspondent esterase is important for both
stages. In the adult stage, the least active in esterase synthesis, eight bands produced
by alleles from Est 1, Est 2, Est 4, Est 5, and Est 6 were expressed in SJRP, and eight
bands produced by alleles from the same loci plus Est 7 were expressed in Marı́lia.
Analysis of dependence confirmed for both populations that L4 is significantly
different from pupae and adults, while the last two stages did not differ.
It is reasonable to think that esterase requirements in larval stages are greater
than in the other stages due to high level of food ingestion, juvenile hormone
metabolism, and specific developmental processes (e.g., high mitotic index). In the
pupal stage, the food intake ceases, and in adults, development is completed. The
esterase enzymes in this last stage are probably involved mainly with reproduction,
digestion, and nervous system physiology.
Different levels of esterase band activity, denoted by different thicknesses and
degrees of staining, were another kind of variation observed among developmental
stages in the present study. It shows that even esterases that are necessary in
different stages may be under control, increasing or decreasing their expression
level during development. This is the case for bands EST-2, EST-7, and EST-16.
The observations related to these three bands were the same in both populations,
suggesting that this is part of the developmental program of the species.
Sex-specific bands as well as bands showing sex differences in frequency and
activity degree are apparently submitted to a regulatory, sex-dependent control.
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Variation of Genetic Expression During Development 81

Sex-specific bands have been detected in some Drosophila species, such as

D. aldrichi, in which Kambysellis et al. (1968) described esterase-F as the product
of an autosomic gene, with sex-limited expression. In the present study, greater dif-
ferences in the frequency of bands were detected between sexes for bands EST-13
and EST-17 in Marı́lia and for bands EST-7 and EST-12 in SJRP. Overall, however,
dependence analysis showed that males and females do not differ significantly in
each population.

Band Differences Between SJRP and Marı́lia Populations

Of the eight loci to which the esterase bands detected in the present study were
tentatively assigned, all except Est 8 were present in both populations. The locus
Est 8 includes the alleles for larval α-esterase bands EST-22 and EST-23, and it was
found only in mosquitoes from Marı́lia. In addition, one pupal band (α-esterase
EST-10, from locus Est 3) and one adult band (β-esterase EST-17, from Est 7) were
found exclusively in Marı́lia. In turn, the mosquitoes from SJRP showed a single
band not found in Marı́lia, the larval α-esterase EST-8 (locus Est 3). However,
only EST-17 from Marı́lia and EST-8 from SJRP showed frequencies that may
be considered meaningful (about 20% in females and 8% in males for EST-17
and 25% for EST-8). By sampling error, bands with low frequency may remain
undetected.
Another difference between populations involved esterase EST-12 (locus
Est 4). In Marı́lia it was expressed only in larvae and pupae with relatively high
frequencies, whereas in SJRP it was found in larvae, pupae, and adults with fre-
quencies of about 13% for females and 47% for males.
The difference between the two populations involving the esterase EST-2
(Est 4) also seems meaningful. This band showed low frequency in larvae and
pupae in both populations. In adults from Marı́lia its frequency remained low
(about 5% in both males and females), but in SJRP it increased to 26% for females
and 34% for males, indicating a high level of activation (or derepression) in the
second population. Statistical analysis confirmed that the differences in esterase
frequencies between populations were significant in every stage.
Because the mosquitoes from SJRP were exposed for long periods to in-
secticides, before and during the present study, and chromosome studies of the
same populations had shown a higher frequency of chromosome changes (lag-
ging chromosomes, polyploidy, and chromosome losses) in SJRP than in Marı́lia
(Lima-Catelani and Bicudo, 1994), we wonder if some of the differences observed
between populations might be related to insecticides. The degree of activity of
esterases has been related to resistance to the organophosphate and carbamate in-
secticides in several insects (in Aedes, Chen and Sudderunddin, 1978; Field et al.,
1984; Field and Hitchen, 1981). Field and Hitchen (1981) and Field et al. (1984)
showed that the degree of resistance to malathion (organophosphate) in A. aegypti
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82 Lima-Catelani, Ceron, and Bicudo

is correlated with the degree of staining of the products (bands) of alleles in the
locus Est 6 (described by Trebatoski and Craig, 1969), to which we believe that
the gene for EST-2 also belongs.
The inhibition tests performed in the present study indicated that EST-12
and EST-2 are a cholinesterase and a carboxylesterase, respectively. Both kinds
of esterases are involved in resistance. Organophosphate and carbamate insecti-
cides kill the insects by inhibiting irreversibly the acetylcholinesterases (Aldridge,
1953; Pralavorio and Fournier, 1992), and carboxylesterases were considered to be
involved in insecticide resistance in more than 30 species of medically and agro-
nomically important insects, being the main mechanism for organophosphorous
insecticide resistance in mosquitoes (Karunaratne and Hemingway, 1996; Mourya
et al., 1993). For carboxylesterases and acetylcholinesterases, gene mutation and
increased degree of synthesis were found in resistant insects (Karunaratne and
Hemingway, 1996; Mourya et al., 1993; Mutero et al., 1994; Vaughan et al., 1997;
Zhu and Gao, 1999).
In the present study, the major differences detected between populations were
related to esterases EST-12 and EST-2 and might be considered indicative of in-
volvement with resistance to the insecticides used in SJRP. The alleles for these
esterases are present in both populations, but as mentioned, they exhibited dif-
ferent activation patterns during development, increasing in expression time and
frequency, in SJRP. The possibility that the distinctive characteristics detected be-
tween these A. aegypti populations reflect only the genetic differences of founders
or geographic divergence, without effect of transitory environmental changes, still
remains. In light of the 20-year struggle by sanitary authorities in SJRP to control
these mosquitoes with intensive use of insecticides, however, we cannot discard the
possibility that the esterase patterns of mosquitoes in this city are being affected.
Other studies will be performed in order to test this hypothesis.

ACKNOWLEDGMENTS
Thanks are due to SUCEN (Superintendência de Controle de Endemias do Estado
de São Paulo) for providing the mosquitoes, to CNPq for a fellowship given to A.
R. A. L. C. and to Dr Peter James Harris for corrections made in our English text.

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