Ontogenetic development of Anastrepha fraterculus (Diptera: Tephritidae): Isoenzyme patterns of isocitrate and alcohol dehydrogenases

Comp. Biochem. Physiol. Vol. 118A, NO. 3, pp. 847-854, 1997 Copyright 0 1997 Elsevier Science Inc. All rights reserved.

ISSN 0300.9629/97/$17.00 PII SO300-9629(97)00038-8

ELSEVIER

Ontogenetic Development of Anastregha fraterculus (Diptera: Tephritidae): Isoenzyme Patterns of Isocitrate and Alcohol Dehydrogenases lurema Cruz do Nascimento and Alice Kalisx de Oliveira DEPARTAMENTODE GEN~TICA, INSTITUTODE BIOCI~NCIAS,UNIVERSIDADEFEDERALDO RIO GRANDE DO SUL, PO Box 15053, CEP: 91501-970, PORTO ALECRE RS, BRAZIL

ABSTRACT. Anastrephafraterculuswas analysed by electrophoresis during the whole development period. Two Isocitrate dehydrogenase (IDH) 1oci were found, one of them expressed from first-stage to 100sday-old adults with varied intensity of staming; the other one was detected only from third-stage larvae until 216-hr pupae. Three presumptive Alcohol dehydrogenase (ADH) 1oci and I4 electromorphs were detected. Isopropanol was the best ADH substrate for adults and both isopropanol and ethanol were similar as good substrates for thirdstage larvae. The ADH-1 locus presented two to four electromorphs and its product was better visualized in starch gel; ADH-2 showed three electromorphs and ADH-3 seven electromorphs; polyacrylamide gel was better for the latter isoenzymes. The three loci were activated in third-stage larvae, pupal stage, and adult period; ADH-1 and ADH-3 were present in the second-stage larvae, and no ADH activity was detected during the embryonic period and first-stage larvae. Males and females (more than 30 days old) showed higher ADH activity; the ADH of females was more acrive in 100-day-old flies. A specific Iocus for Octanol dehydrogenase was not detected. Third-stage larvae reared in papaya, guava, and banana show identical ADH patterns. No activity of this enzyme was detected when larvae were grown in kiwi. COMPBIOCHEMPHYSIOL118A;3:847-854, 1997. 0 1997 Elsevier Science Inc. KEY WORDS.

A. fraterculus, dehydrogenase

INTRODUCTION Anastrepha is a neotropical flies with about

genus of fruit-infesting

193 currently

recognized

tephritid

species,

78 of

which occur in Brazil (24). Six species are considered major economic

pests in the American

several others are potentially

tropics and subtropics and

serious pests. Anastrepha fru-

terculus is highly polyphagous and known to attack dozens of cultivated

and wild fruits (17).

There are a great number of taxonomic genus Anastrepha, especially

problems in the

in South American

(3 1). The species is morphologically described under nine different

fruit-flies

variable and has been

names (31,33).

A variable

characteristic, for instance, is the alar pattern. Three patterns can be recognized, each one associated with a different geographic region of Brazil and others regions of South and Central

America.

Based on this evidence,

Stone (31) pro-

posed that the species could have many geographic

races.

However, he also suggests the possibility that populations considered as the same species are really a complex of species undergoing evolution. Address reprrnt requests to:Alice Kalisz de Olivelra, Department0 de G&tica, lnstituto de Bioci@ncias, Universldade Federal do Rio Grande do Sul, PO Box 15053, CEP: 91501-970, Porte Alegre RS. Brazil. Tel. (051) 316 6717; Fax (051) 319 2011. Received 25 July 1996; revised 19 March 1997; accepted 4 April 1997.

The study of the alloenzymatic variation of 11 A. fruterculus populations collected from eight different hosts in the same orchard, tance;

showed very low levels of genetic

the low values of heterozygosity

11 being monomorphic dependent

genetic

dis-

with 6 loci out of

also provided no evidence of host-

differentiation

performed in eight populations

(16).

Similarly,

studies

of different host plants of

A. fraterculus collected only at Itaquera (Sao Paula, SP, Brazil) and investigated for 12 enzymatic loci (eight enzymatic systems), also showed low genetic authors attributed

variability

the results obtained

(21).

These

to the “generalist”

status of the species favouring fixation of the more flexible alleles whose products could react with most substrates. The same low genetic variation was found at 11 presumptive loci in natural populations of 10 Anastrepha species collected from nine different graphical proportion

regions

(number

of polymorphic

hosts and in 11 different of alleles per locus

=

geo1.31;

loci = 0.21; and mean number

of heterozygotes per locus per individual = 0.040) (14,15). However, isoenzyme analysis of eight populations of the South America fruit fly, A. fiaterculw, from a large portion of its geographic range (from Mexico to S5o Paula) revealed genetic discontinuities among populations for 18 enzymatic systems (30). Populations from northern Brazil, coastal Venezuela,

Costa Rica, and Mexico

were all very similar.

848

J. Cm: cio Nascimento

Populations

from Southern

Peru were genetically each

other

as well. The

order of magnitude

between

reported

probably

in other

result

from

the

stand

of

and are comparable

to

species

All these

(30) are higher

(15,22).

resolution

studies

on enzyme

focused

on structural

those affecting ance during

These

cycle

their phenotype

performed. genetic

ond stage,

and inheritance

it is important

of several

how enzymatic

less attention, are gener-

Thus, direct experimental tests of the of regulatory variation have not been

So, initially

pattern

whereas

or the time of appear-

received

systems

choosing

systems

with

morphism, cently,

can explore

by apples

causing

we analyse

nase nicotinamide

adenine

dent (IDH-NADP), tanol

alcohol

dehydrogenase.

seventeen

embryonic

cal needle,

as previously

homogenized,

ahsorbed

per (4 mm X 2 mm) and stored ontogenetic

stages, stages,

sufticient

in 3MM Whatman

activity.

stage, we analysed

in acrylic plate-excavations

of: 8 g agar, 200 ml grape juice,

acid. Just enclosed

to ohtain

pupae

which

were collected

patterns,

this ap-

of hosts;

re-

losses. Thus,

in this paper

of isocitrate

dinucleotide

dehydnqe-

(ADH)

at -20°C

at 4, 15, 30, 60, and 1OL7days

until

analysis.

larvae.

used as control Isoenzyme genase

Oregon-R

patterns

(Cyanogum,

Drosophikz

for electrophoretic

were studied

of IDH, ADH,

and removes

toxic metabolites

6%) using a discontinuous

buffer system:

hydroxide)

for IDH a continuous

and oc-

system

NADH

such as eth-

or benzyl

study of the ontogenetic

alcohol

pattern

as substrates

of these

(2). The

isoenzymatic

sys-

tems

in A. fraterczdus

is very important,

not only because

they

respond

to environmental

changes,

quickly

but alsc)

because if they show dynamic expression throughout devrlopment; this knowledge may contribute to the understanding of physiological, regulatory, ecological and evolutionary relationships in this species and at the same time provide insight

for its control

MATERIAL

AND

and management.

METHODS

The population of A. fruterculr4.s was ohtained from Infested fruits collected at the city of Pelotas (State of Rio Grande do Sul, Brazil) and maintained under laboratory conditions

0.076

M Tris-citrate buffer, pH 8.65, containing 0.005M citric ,Icid (26). The bridge buffer was horate, pH 8.6 (0.34 M boric acid and 0.05 M sodium

anol (8). The last is an enzyme similar to ADH, but with different substrate specificities using longer chain alcohols such as I-octanol

dehydro-

tanol

the oxidative

was

gel electrophoresis

dehydrogenase,

whereas

8.6 for cubes, starch

PC.

for ADH

was used: 0.34 M Tris and 0.078 M citric and 0.038 M Tris and 0.0025

HCl, pH 8.5 in the gel). Electrophoresis

reduced),

to nhtain

melanogaster

and octanol

on polyacrylamide

M sodium borate,

(NAD

first-stage

migration.

dinucleotide

adenine aldehyde

Further

chinensis Planch.)

(MUX spp.), and kiwi (Actinidia third-stage

and

appropriate

soil

at 24, 120, and 216

depen-

phosphate

dehydrogenase

hr. The adults were collected

samples

on garden

larvae were also grown in guava (Psidium guujapia L.) banana

number

of nicotinamide

were then larval stage

( 144 hr) and third larval stage (3 12 hr). Additional of the end of this last stage were also placed

concurrent

the

larvae

the second

me-

and 1 ml

In a set-

The first one catalyses

reduction

containing

first instar

to papaya fruits to obtain

pH 8.6 for gel ( 11). ADH

to yield

10 samples. The tly eggs

were also placed

decarboxylation and recycles NADPH (NADP reduced), the second catalyses the oxidation of an alcohol and the (NAD)

For the re-

we used 10 males and 10 females.

transferred

two

was used in each sam-

dium consisting propionic

pa-

For these

were used in each sam-

enzyme

only one individual

pie. For each developmental

(23). The

with a histologi-

at -20°C.

150 individuals

ple in order to reach maining

described

stages were collected

at

and tirst-

or repressed. varied

pattern

stage larvae were obtained

fruit feeding,

Embryos

old and stored

such a broad

great tinancial

L.) as larval

to verify

began to infest a new host represented

the isoenzymatic

papaya

the onto-

by which the species, in spite of being when analysed as to its structural pnly-

A. fraterculus

(Car&

1“C and a 12-hr photoperiod.

systems

proach could be used to study geographically different populations, growing in varied hosts, for the purpose of looking for the mechanism genetically similar

t

to analyse

isoenzymatic

are activated

usmg papaya 25°C

For adults,

and its adapti\re

polymorphisms,

of enzymes

largely because

allocn-

systems.

variation

the amount

than

differences

of additional

the ontogenetic

ally more complex. adaptive significance

of Rhugoletrs.

per locus, and percentage

zymes, as well as a set of new isoenzymatic significance

out as an

populations

by Steck

studies

and from

distances

alleles

loci found

Venezuela, and possibly

seen among

recognized

for heterozygosity,

of polymorphic those

genetic

larger than

distances

Values

from them

or Rhagoletis species

other Anasrrrphu genetic

Brazil, Andean

distinct

anJ A. K. de Oliveira

gel (12%)

enzyme

dehydrogenase

on (0.30

and 0.0 1 M Tris-

was applied


Isocitrate

acid,

was performed front,

hrom<)phenol (0.1%) dissolved in ethanol some samples, was 9 cm from the origin. Staining:

M citric

buffer system

pH 8.1, at the electrode, field of 10 V/cm

buffer acid, pH

was also analysed

using a discontinuous

An electric

and oc-

(IDH):

at

across

the

identified

by

and applied

to

50 ml of Tris

HCI 0.05 M, pH 8.5, 0.010 g (0.25 mM) of nitrobluetetrazolium (NBT),

0.010 g (0.26 mM) of nicotinamide

adenine

dinucleotide phosphate (NADP), 0.075 g (5.81 mM) of isocitric acid, 0.020 g of manganese chloride (M&l:)

( IO%), and 0.005 g (0.33 tnM) of phenazine

methasulfate

(EMS), and incubated at 38°C for approximately 2 hr in the staining mixture (in the dark) (5). Alcohol dehydrogenase (.At)H): 100 ml of Tris HCl 0.1 M, pH 8.5 buffer, 5 ml of ethylic

alcohol

95% or 5 ml of isopropanol,

0.025 g (0.38

mM) of NAD, 0.025 g (0.3 1 mM) of NBT, 7 drops ot 0.5% methylene-hlue, 0.005 g (0.16 mM) of PMS, and incubated at 38°C for approximately 3 hr in the staining mixture (in the dark).

0ctanol

dehydrogenase:

100 ml of Tris HCI 0.1

A. fwterculus Dehydrogenase

849

lsoenzyme Patterns

TABLE 1. IDH activation pattern during the ontogenetic

of Anastrepha fi-aterculus

development

STAGES Adults Females

Males Pupal

Larval Isoenzymes

IDH-2 0.40 0.38 0.35 IDH-1 0.08

120 hr

2 16 hr

4 days

++++ _

++++ ++ ++

++++ _ _

24 hr

First

Second

Third

+ _ _

+++ _ _

++++ _ _

+++++ _ _

_

_

++

++

+

+

_

15-30 days old

++++ ++++ ++++ _

60-100 days old

++++ _ _ _

4 days

+++ _ _ _

15-30 days old

++++ _ _ _

60-100 days old

++++ _ _ _

(+ + + + +) Highest to (+) lowest isoenzyme expression; (-) not detected.

M, pH 8.5, 0.005 g (0.16 mM) of PMS, 0.025 g (0.31 mM) of NBT, 0.025 g (0.38 mM) of NAD, 5 ml of 1-Octanol for approximately 3 hr at 38°C in the staining mixture (in the dark).

The

reagents

were obtained

Co. (St Louis, MO, U.S.A.). In the nomenclature used named The

according

RM values

to rising

from

here,

the

relative

are the means

Sigma

Chemical

isoenzymes

mobility

of several

(RM)

were values.

estimations.

En-

zyme activity measurements were semiquantitative and the following scale was used: - absent or not detected, + = very

weak,

strong

++

= weak,

and +++++

+++

=

median,

++++

=

= very strong.

REGULTS Isocitrate

Dehydrogenase

Four electromorphs

(IDH)

were identified

during

development

of A. fraterculus

(Table

not detected

in the embryonic

stage.

Idh 0.40, with very weak intensity, stage larvae,

but showed

larvae and strong enzyme pupae,

median

activity

pae, the activity

during

was strong.

4 to 100 days old presented

Adult

in first-

larvae. This isostages.

In 24-hr

in 120- and 216-hr males and females

this electromorph

an allele

whose

was

in second-stage

all pupal

was very strong;

to strong activity. Idh 0.35 is probably

was observed

intensity

in the third-stage

was also observed the activity

the ontogenetic

1). This enzyme

FIG. 1. Isocitrate dehydrogenase electrophoretic pattern during the development of A. f%aterculm (2-4) embryonic period; (6-S) first-stage larvae; (10-13) second-stage larvae; (14-16) third-stage larvae; (17-19) 24ehr pupae; (20-21) 120 hr pupae; (22-25) 216 hr pupae; (1 and 26) D. melanogaster as control.

product

pufrom

with median associated

to the product of Idh 0.40 producing the Zdh electromorph 0.38; this is suggested because 0.35 and 0.38 electromorphs were found

only

under

heterozygous

isoenzyme is dimeric. Idh 0.08 was observed

condition;

only from third-stage

thus, larvae

this until

the 216-hr stage of pupae. Therefore, two loci can be inferred for the IDH system and the isoenzymes are differentially expressed during ontogenetic

development,

(Figs. 1 and 2).

both qualitatively

and quantitatively

FIG. 2. Isocitrate dehydrogenase electrophoretic pattern during the adult stage of A. ~atercdus: males (l-2, 4 days old; 3-4, 15 days old; 5-6,30 days old; 7-8,60 days old; 910, 100 days old) and females (ll-12,4 days old; 13-14, 15 days old; 15-16,30 days old; 17-18,60 days old; 19-20,100 days old).

850

J. Guz do Nascimento

Alcohol

Dehydrogenase

Fourteen

ADH electromorphs

velopment

were identitied

of A. fruterculus

This enzyme nol. The alcohols

(ADH)

reacted

(Table

with isopropanol,

same RM values

during the de-

2 ).

were used as substrate loci of the ADH

togenetic

development

and ADH-3).

In third-stage

larvae,

for f&t and slow developmental

specificity

the way to investigate

broad

in the

(ADH-

I, ADH-2, and adult

high activity

and the

ADH- 1 was very weak. This enzyme was not detected embryonic

stage and in first-stage

vae and ADH-3

when

starch

adults

anol

and

was better

for ALIH-2

while in third-stage

isopropanol

in the adult,

5), whereas

the polyacrylamide

On the other hand,

were

good

this was shown

related

since

resistance

In A. frntrrculus

probable

ADH

tcrculus development,

lar-

While

and

stage larvae, ADH-2

AL>H-1

and

ADH-3

were

larvae, both cth-

ubliq~,

and A. bisrriguta show three

conclude

that

during

at different

and

observed

A. fm-

moments.

from

secc&-

were found frotn the third-stage

A. striata, and A. fraterc&s,

(Figs 6

only a single

was detected

loci, observed

,pdis,

substrates,

and detoxit;ca-

(30). In our population, in the anodal IDH zone

were activated

gel was better.

as enzymatic

on

in adults. three

until the adult period.

only with isopropanol

data clear of NADPH

adults,

of activity

characterized as a dimeric molecule we’ also observed a dimeric pattern detected

rrsisselected

role of this enzyme

regeneration

to free radicals

(1,29).

IDH zone with varied patterns

larvae (Fig. 3).

gel was used for both third-stage

(Figs 4 and

loci products

in the

has been

herbicide)

population>

time (3). These

the possible

in A. fruterculus

tion pathways

contact

in D. tnelanogclstel

longevity

The

that the ADH- I locus product

It was observed visualized

INI-

while during the second-

showed

(a non-selective

and octa-

pupal stage,

period, the three loci were activated; stage larvae, the locus ADH-3

and longevity

the three

were detected

of A. f&r&us

parayuat

when

suggesting

enzyme

both ethanol,

of this enzyme. Three

energy at the beginning of adult life. Higher amounts of IDH-NADP allele products can play an important role in tance

were observed

nnd A. K. de 0livena

Two ADH

tephritid

species

larvae

loci were detected

in A.

while A. serprntina, loci (20). These

vary in the number

and 7). The ADH-1 (cathodic locus) presented two to four electromorphs; the largest number was observed in the pupal

and population genetic structure of A. fraterculus and related species, also detected only two ADH loci in adult indi-

stages

(A&

0.11, Adh 0.17, Adh 0.22, and Adh

0.27). The Adh 0.11 was a primary ers were secondary The ADH-2

isoenzymes

locus showed

three

Adh 0.36, and Adh 0.41), while found

with seven

isoenzyme,

and the oth-

(post-translational

electromorphs

changes).

electromorphs ADH-3

(anodic

locus) was Adh and

isoenzymes.

ADH

activity

in both

30 days old. Females

isoenzymes

At least three

alleles

at different

ages,

males and females

at 100 days old showed

showed ADH

activity than males of the same age (Figs. 5 and 7). Electrophoresis carried out using octanol as an enzyme substrate

showed

observed

when

Third-stage and banana

an isoenzymatic ethylic

alcohol

pattern

showed

similar

activity

patterns

in larvae

similar

to those

fed in papaya, for ADH.

guava,

We did not

fed in kiwi fruit (Fig. 8).

can

gel as migration

occurred

tinding

be accounted

support;

in a geographically

In the case of the second

of this

for by two

technical

gel, whereas

in A.

intermediate

when compared

The

1) the use of differential

used starch event5

approaches

the other authors

and isolated

2) duplication A. fratercldus

hypothesis,

investiga-

tions can he performed. The activation niftcantly

pattern

variable

very important

and intensity

(Table

2). The

role during

of bands

ADH

enzyme

the larvae-pupae

were bigplayed

transition

a

and

during the whole pupal period. These facts could be attributed to physiological larvae conditions inside fruits where the

was used.

larvae of A. fraterculus

fmd any ADH

hypotheses:

more than

higher

product

l~ocus

specificity

loci products.

since we used polyacrylamide

population.

were detected at this locus. Adult individuals, analysed higher

but the same substrate two ADH

pattern

loci for ADH

migration, additional

(Adh 0.50, Adh 0.54, Adh

three

the new locus show lower activity,

(Adh 0.30,

0.59, Adh 0.63, Adh 0.67, Adh 0.72, and Adh 0.76). were secondary

In our work we found

enzymatic

fraterculus;

to the other

0.50, Adh 0.59, and Adh 0.67 were primary the others

viduals.

studying

of loci

c:oding for AL>H. Steck

and adult

(30),

A.

authors

fermentation

which

process

were ingested

released

and stored

alcoholic

compounds,

by larvae as a source

of cn-

ergy in fatty cells. In the

adult

period,

individuals

over

30 days old had

higher ADH activity. Furth ermore, lOO-day-old females had higher activity than males of the same age. ADH activity of Drosophila

DISCUSSION The occurrence

of an additional

IDH band,

specifically

in

third larval and pupal stages in A. fraterctdus, could be related to increase regeneration of NADPH, which would be used in several metabolic pathways, especially lipid synthcsis. The increased activity served in D. melanogustrr high energy consumption,

appears adequate because, as ob(4), the metatnorphosis requires and lipids are the main source of

populations

at 30 days old was higher

for

the long-lived Oregon (OR) strain than for the short-lived Canton Special (CS) (6). Taking into account that the mean aging of A. fraterculus females and males is 87.8 days old and 156.6 days old, respectively (la), our females at 100 ilay> old may be considered long-lived and also showed high ADH activity. However, a possible correlation between AL)H and aging is very complex (13). In this sense, more data need to be obtained regarding this question.

++ ++++ ++++ ++-t+ _ _

_ _

_ +

_ _

_ _ _

Second

_ _ -

Fit

+++ +++ ++++ ++++

++ +++

+ ++ ++++ ++++ ++t ++

Third

+++ +++ +++ ++++

++ ++ +++

+++ ++++ ++++ ++++ t+t ++t

24 hr

(-)

not detected.

_ ++ ++++ ++++

-

+4

++ ++ ++++ ++++

++ ++

+++ ++++ ++++ ++++

2 16 hr

++ +++

+++ +++ +t++ ++++

120 hr

Puval

+-I+++ +++

+

_

++ ++t +t +++ +++

_

4 days

+ ++ +++ +++

_ -

_ +++ +++ +t +t

15 days

+ ++ +++ +++

_

-

_

++ ++++ ++++ ++++ ++

30 days

Males

+ ++ +++ tt+

+ + +

60 days

+ +-iffff +++

+ + +

4

++-I++++ ++++ ++i++

+

100 days

t ++ +++ +++

++ ++ ++

+ +++ +++ ++

4 days

Adults

of Anastrepha thtercuhs

STAGES

activation pattern during the ontogenetic development

( + + + +) Highest to (+) lowest isoenzyme expression;

ADH-3 0.76 0.72 0.67 0.63 0.59 0.54 0.50 ADH-2 0.41 0.36 0.30 ADH-1 0.27 0.22 0.17 0.11

Isoenzymes

ADH

Larval

TABLE 2. Electrophoretic

+ ++ +++ ++t

_ _ -

_ + ++ ++ ++ _

15 days

+ ++ +++ +++

-

4-f -i-f +++ +t+ -

_

30 days

Females

++ +++ ++++ ++++

+ + +

_ ++ ++++ +++ + +

60 days

++ +++ ++++ ++++

++ ++ ++

_ +++ ++++ ++++ _ _

100 days

G:

852

+

m It I

-Cl

E 1

Lt

1

L2

1

L3

1 P24

iP120i

P216

ic

FIG. 3. Alcohol dehydrogenase electrophoretic pattern during the pre-imaginal development of A. fi-atercufus analysed in polyacrylamide gel: (2-4) embryonic period-E; (6-8) first-stage larvae-Ll; (10-12) second.stage larvae-12; (1315) third-stage larvae-W; (17-19) 24Mhr pupae-P24; (2022) 120.hr pupae-P120; (23-25) 216.hr pupaeJ’216; (1 and 26) D. mefanogaster as controlC.

FIG. 6. Alcohol dehydrogenase electrophoretic pattern larval stage of A. fiatercufus analysed in polyacrylamide and using ethanol as substrate (Ll = first-stage larvae, = seconddstage larvae, L3 = third*stage larvae, and C = melanogaster as control).

in gel L2

D.

f

+

FIG. 4. Alcohol dehydrogenase electrophoretic pattern in third-stage larvae of A, fiatercufus (2-19) analysed in starch gel and using isopropanol as substrate (1 and 20) D. melanogaster as control.

FIG. 7. Alcohol dehydrogenase electrophoretic pattern during the adult-stage of A. fiaterculus: males (l-2,4 days old; 3-4, 15 days old; 5-6, 30 days old; 7-8, 60 days old; 9-10, 100 days old) and females (11-12, 4 days old; 13-14, 15 daysold; 15-16,30daysold; 17-18,60daysold; 19-20, 100 days old) analysed in polyacrylamide gel and using isopropano1 as substrate.

+ +

FIG. 5. Alcohol dehydrogenase electrophoretic pattern during the adult-stage of A. fiatercuhs.: males ( l-2,4 days old; 3-4, 15 days old; 5-6, 30 days old; 7-8, 60 days old; 9-10, 100 days old) and females (11-12, 4 days old; 13-14, 15 days old; 15-16, 30 days old; 17-18, 60 days old; 19-20, 100 days old) analysed in starch gel and using isopropanol as substrate.

FIG. 8. Alcohol dehydrogenase electrophoretic pattern in third&age larvae fed different fruits: P (papaya), G (guava), B (banana), and K (kiwi), analysed in polyacrylamide gel, and using ethanol as substrate ( 1 = D. melanogaster as control).

A. fraterculus Dehydrogenase

The ADH

activities

used as substrate,

Isoenzyme Patterns

differed depending

but in general,

853

on the alcohol

the intensities

detected

be due either to structural differences influencing

the cata-

lytic properties, or to regulatory differences controlling

the

with primary alcohols were highest. As the same migration

amount of each isoenzyme. Multiple forms of ADH in Dro-

pattern of the isoenzymes was observed when ethanol,

sophila may represent enzyme configurations present in viva and may be related to the developmental stage; multiple

propanol,

and octanol

iso-

were used, we suggest that A. fra-

terculw did not present a specific locus to octanol dehydro-

forms could function

genase. In our enzyme resolution

toxify low levels of toxic ketocompounds

polyacrylamide loci (ADH-3

as a buffering system to bind and de(9).

we observed

that the use of

Further, in our study, the ADH pattern of third-stage lar-

improves the visualization

of most anodal

vae of A. fraterculus grown in different fruits showed that

and ADH-2)

products. When starch gel was

used, the opposed was observed good resolution)

(ADH-1

while among papaya, guava, and banana no substantial dif-

products

have

ference was found, at least at visual inspection,

but the bands formed by the ADH-2

locus

ity was not detected

ADH activ-

in larvae fed in kiwi fruit. These data

stayed near the slot line and joined with the products of the

suggest differential

ADH- 1 locus. This may account for the difference observed

vouring a possible adaptive role in the detoxification

concerning

mentation products. A preliminary analysis of approximately

the number of loci.

The three ADH

loci in A. fiaterculus produce dimeric

isoenzymes. In the ADH-3

locus, three alleles seem to occur

host-related

species revealed no correlation

enzymatic

fa-

of fer-

40 Drosophila

between feeding habits and

but this suggestion needs to be confirmed by the analysis of

ADH or ODH content,

a higher sample, and crosses among strains homozygous for

with regard to biogeographic

each of these alleles. In heterozygotes,

ethanol content of the guava fruits (Psi&m

seven bands occur;

regulation

but such correlations

were found

origin (25). Furthermore,

the

guajaua, Myrta-

as the secondary and principal bands both heterodimerize,

ceae)

and as previously observed in D. melanogaster, it can be a

ceae) infested with A. fruterculus was measured by gas-liquid

modified form of the principal band. This phenomenon

chromatography

been explained

by the non covalent

carbonyl compound A distinctive

has

binding of a NAD-

to the ADH protein (27).

feature of fruit fly isoenzymes,

and red “mombim”

tion between

(19).

(Spondias purpurea,

Anacardia-

They also did not find any correla-

fruit ethanol

content

and ADH

activity of

larvae that were living in these fruits. However, in a later compared

paper, specific activity of larval ADH and ethanol content

with D. melanoguster and also with ADH from other Dro-

of these and other additional host fruits (pumpkins

so@a

curbita pepo, Cucurbitaceae;

species, is the substrate preference

for primary alco-

hols. D. melanogaster adults presented higher ADH activity for isopropanol(10,28),

whereas for D. ananassae, D. m&r-

= Cu-

star fruit = Averhoa carambola, = Spondias tuberosa, Anacardia-

Oxalidaceae; “tapereb6” ceae; “abric6 da praia” = Manilkara zapotilla, Sapotaceae),

kotiliana, and D. bipectinata (12), no difference was detected

were determined

in the activation

be positively correlated (20). From the results shown in this paper, we can conclude

pattern for ethanol

and isopropanol.

In

C. capitata larvae, the better substrate is ethanol (7), whereas adults did not present a visible difference in ADH

that important

and these two parameters were found to

regulatory mechanisms

activity with ethanol and isopropanol as substrates (data not

tively and also quantitatively

published). Concerning

during the ontogenetic

A. jiaterculus third-stage larvae, our

produce a qualita-

different isoenzymatic pattern

development

of A. fruterculus. This

results showed that both ethanol and isopropanol were good

effect, added to different ecological

ADH substrates. The difference

(as observed in the breeding of A. fiaterculus in different fruits), could be used by this species as a strategy to survive

of these species in regard

to ADH specificity could be related to a difference ecological

niche

of these dipteran

breeding on fermenting

in the

with Drosophila larvae

plant material, and C. ca@~ta and

A. fruterculus breeding on ripening fruits. When

niches and host plants

in such a great spectrum of hosts and geographically

wide

distribution

simi-

in spite of its relatively

high structural

larity.

adult individuals of different ages were analysed,

we observed a higher specificity to isopropanol when compared to ethalnol and octanol as substrates. These results corroborate

data previously observed by us that showed a

higher mortality of A. fraterculus adults with a topical application of ethanol. Furthermore, to organophosphorous

synergism of ethanol added

insecticides

may have a strong effect

on A. fiaterculus survival and, therefore, its management. As in most species of Drosophila

could be used in

(32), enzyme activity in

the med fly and in the South American fly is higher in larvae than in adults. In our work, ADH-3 always has higher specific activity than ADH-1 and ADH-2.

These results can

We are a grateful to Dra. Suzanu Cavalli-Molinu, Ivana Da Cruz and Gilson Da Cunhu for suggestions, and to Murtinu du Silva for laboratory help. Also, thanks are due to CiVPq (Conselho N&owl de Desenvolvimento Cientifco e Tecnol6gico) , FAPERGS (Fundu@o de Ampare i Pesquisa do Es& do Rio Grunde do Sul), PROPESP-UFRGS (Pr&Reitoriu de Pesquisa e Pds-Grudua@o du Universidade Federal do Rio Grunde do Sul) and FINEP (Financiudoru de Estudos e Projetos) for grants and fellowships.

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