416
JounNar, oF THE AnroRlctr.tMoseurro CoNrnor, AssocIATroN
VoL. 8, No. 4
REDUCTION IN THE SUSCEPTIBILITY OF AEDES AEGYPTI TO
BRUGIA MALAYI INFECTION AFTER TREATMENT WITH ETHYL
METHANESULFONATE
PAUL H. RODRIGUEZ, CATHERINE LAZARO eNn RICHARD CASTILLON
Diuision of Life Sciences,Uniuersity of Texasat San Antonio, San Antonio, TX 78249
ABSTRACT. The susceptibility to Brugia malayi infections of Fz and Fa progeniesof Aedesaegypti
(Black Eye strain) treated with ethyl methanesulfonate(EMS) was tested. Both 3-day-old males and
females were treated with 0.025,0.050, 0.075 and 0.10% EMS. Control and treated females were then
rnated with normal or treated males to recover F1 progeny. F2 offspring were derived from sibling
intercrosses,and 3 lines were establishedby further intercross matings to generatethe Fa.Susceptibility
"fhe 0.025%
in the 0.025,0.050,0.075and0.10%EMS Fzswasreducedby L3,12,4 and,25%,respectively.
and 0.050%EMS F, femalesshoweda 29 to 39Vodecreasein mean Ls numbers.At 0.075 and 0.L0%
EMS, mean L3 numbers decreasedby 0.8 and 1lVo, rcspectively. The Fa populations gave overall
infectionsof 65, 56 and 22% for the control, 0.25 and 0.10%EMS lines, respectively.Mean L:s were
reducedby 24 and 77%,respectively,in the 0.025and 0.10%Fa EMS-selectedpopulations.
ln Aedesaegypti(Linn.) susceptibilityto the
filarial nematode,Brugia malayi (Brug), is controlled by a sex-linkedrecessivegenenamed/(Macdonald 1962b)."Ihe f^ gene (Macdonald
and Ramachandran1965)also controls the development of the human parasite, Wuchereria
bantofti (Cobbold),and the animal filarid, B.
pahnngi (Buckleyand Edeson).
Treatment with ethyl methanesulfonate
(EMS) changedthe susceptibility to B. pahangi
in the Fr progeny of a moderately susceptible
Ae. aegypti strain (PUGU) (Rodriguez 1985).
The present study was conducted to determine
whether treatrnent with EMS alters the expression of the f^ gene for susceptibility to the
human filarid. B. malayi. The Fz progeny, derived from EMS-treated parental males and females, and three.Fa populations, established
through sibling intercrossmatings, of Ae. aegypti
(Black Eye strain) were tested.
Aedesaegyptj(Black Eye strain) was obtained
from GeorgeB. Craig Jr., Vector Biology Laboratory, University of Notre Dame. Mosquitoes
were reared, and females infected were maintained in a reach-in incubator at 27 + 1"C and
85 + 10% RH. The infection techniquesused
weredescribedpreviously(Rodriguez1973).
Four groups of 50 males and 50 females were
allowed to feed freely for 5 days on sugar cubes
treatedwith 0.025,0.050,0.075and 0.10%EMS
(Sigma Laboratories, St. Louis, MO) as described earlier (Rodriguez 1985). After treatment with EMS, 2 replicatesof 5 groups (1
control and 4 treated) of 10 mosquitoesof each
sex were maintained in l.9-liter (1/2-gal) mosquito cages.The Fz and F4progeny were derived
from sibling F1 and F3 matings, respectively.
Five groupsof 10-pair matings with one replicate
eachwere maintained for the F2 progeny. Three
groups of 10-pair matings (control, 0.025 and
0.10%EMS) and one replicateeachwere made
to form the Fapopulations.To ensuremaximum
eggproduction,femalemosquitoeswere given 2
bloodmealsbeforemating and at 3-dayintervals
after mating, on anesthetizedmice. Eggs were
collectedat7-day intervalsovera 21 day period.
Surviving F2 and Fa adult females collected
from each of the 7-day intervals were tested for
changes in filarial nematode susceptibility.
Briefly, 3-day-old Fz and Fa females from each
of the 5 or 3 groups were starved for 24 h then
fed simultaneously on 2 jirds (Meriones unguicuLatus)infected with Brugia malayi having a
microfilarialdensityof 108per 20 mm3of blood.
A total of 125 Fz femalesper group from each
week (1, 2 and 3) wereexposedto infectedjirds.
From these only 50 (week 1), 60 (week 2) and
75 (week 3) engorgedfemales per group and
week were selected,placedin different 3.8-liter
(l-gal) mosquitocages,and held in an incubator
L0-14 days for dissection.With the F+ females
only 100 per group were infectedand 60 of the
engorgedfemalesper group (control, 0.025and
0.10%EMs-tested) were tested for susceptibility. All Fr and Fr infectedfemalesthat remained
alive after this 10- to 14-dayperiod were dissectedin 2-3 dropsofAedesphysiologicalsaline.
Mosquitoeswere classified as susceptibleif one
or more L3sper female were present (Rodriguez
and Craig 1973).The number of 3rd-stagelarvae
(Lr) per femalefor eachtreatment was compared
statisticallyby analysisof variance(ANOVA).
Table 1 summarizes results from 3 separate
experiments wherein F2 females were infected
with B. malayi to test for changes in filarial
susceptibilityover a 3-wk period.
Mosquito mortality during the first week was
low. The control group only showed an ll%
mortality. The 0.075%EMS-treated group had
DECEMBER 1992
OppnettoNlr,
AND SCIENTIFIc NorES
4t7
of 56Vo,
EMS-selectedline gavea slight decrease
while the 0.10%EMS-selectedexperimentalline
was significantly reducedby 43% (x2 -- 28.41;df
:3:P<0.001).
The mean number of Lgs suPPortedper Fr
Females dissected for Lg
female in EMS-selected populations was also
larvae
significantlyreduced(F : 13.377;df = 2.357;P
DoseEMS
% lnfected
<
0.00001).The control line showedan average
(%\
(n)
Mean + SE
Week
3.82Lss during 3 wk while the 0.10%EMS line
2.86+ 0.59
0
1
54 (43)
was decreasedsignificantly by 76% (X L' at
2.83+ 0.58
2
42 (60)
0.10%EMS : 0.91;t : 5.333;df : 258;P <
t.75 + 0.45
s2 (75)
0.001). Two-sample f-tests also showed mean
1.38+ 0.43
I
24 \45)
0.025
number Lg differences between control and
+
2.08 0.51
2
37 (60)
0.10%EMs-selectedpopulationsduring weeks
1.07r 0.26
27(75)
1 , 2 , a n d 3 ( t : 5 . 6 6 1d, f : 1 1 8 ,P < 0 . 0 0 1 t; :
2.03+ 0.53
1
35(40)
0.050
+
(60)
2.07
0.43
5
. 0 5 5d, f : 1 1 8 ,P < 0 . 0 0 1t;: 5 . 1 5 0d, f : 1 1 8 ,
2
33
1.20+ 0.30
25(75)
P < 0.001,respectively).
2.4t + 0.60
0.075
I
43 (37)
Although previous studies with Aedesaegypti
2.43+ 0.57
2
37 (60)
have been conducted on the susceptibility to
2.53+ 0.50
36 (75)
vertebrate pathogens,Plasmodium gallinaceum
0.83+ 0.32
0.10
I
15(48)
(Kilama and Craig 1969)and the filarial nema2
22 (60)
0.82+ 0.26
todes,Brugia malayi (Macdonald 1962a,1962b;
0.52+ 0.77
15(75)
Macdonald and Ramachandran1965) and B.
pahangi (Rodri guez1973, Rodriguez 1975, P aige
and Craig 1975,Rodriguezand Craig 1973)there
13%,while at 0.025,0.050and 0.10%EMS only have been relatively few reports on the genetic
gave5, 10 and llVo mortality, respectively.
effects of chemically (EMS) induced changeson
Susceptibility in the control population over the susceptibility and development of a pathoa 3-wk test period was 437o.At 0.075%EMS gen in a vector host (Rodriguez 1985). Genetic
susceptibility was 397o. The 0.025% EMS- (Macdonald 1962b) and environmental factors,
treated group showed a 297o susceptibility. At
such as radiation (Richey and Rodriguez 1976)
0.050%EMS susceptibility was reducedto 317o, and temperature (Rodriguez 7975), have been
while at 0.10%EMS susceptibilitydecreasedto shown to affect filarial del'eloprnent, with the
I7%. Only the 0.10% EMS-treated group latter possibly modifying.the:,expressionof the
showed a statistically significant decreasein
f^ gene.
susceptibility when compared to the control
The present study indicated ,that EMS-in-:
(Table li x" 12.54;df 4; P < 0.05).
ducedmutations affectedthe,f- Iocus for filarial
The mean number of 3rd-stagelarvae (La) susceptibility in Fz and F+progenyof Ae. aegypti.
presentper F2 female in EMS-treated populations was also significantly reduced with inTable 2. Susceptibility and variation to Brugia
creaseddosesof EMS (Table l; F : 2.746;df :
rnalayi infections in female Fa Aedes aegypti
4,883; P < 0.002). The control population
(BLACK EYE) selected from control and EMSshowedan averageof 1.75Lgsafter 3 weeks.At
treated lines.'
0.10% EMS, the mean La number was significantly reduced(71% decrease,P < 0.05).TwoFemales dissected for Lg
sample t-tests also indicate that differences in
larvae
mean L3 numbers between control and 0.10%
DoseEMS
% lnfected
EMS-treated populations occurred after weeks
(%)
(n)
Week
Mean + SE
:
1 , 2 a n d3 ( t : 3 . 0 2 5 , d f
8 9 ,P < 0 . 0 1 t; :
(60)
1
67
4.67+ 0.55
Control
3 . 1 6 2 , d: f1 1 8 , P < 0 . 0 1 ; t : 2 . 5 5 7 , d f : 1 4 8 , P
(60)
2
67
4.10+ 0.53
< 0.05;respectively).During week 1, the 0.0257o
2.70+ 0.40
60 (60)
EMS F, females also had a 527o decreasein
I
55 (60)
3.27+ 0.53
0.025
:
rlean LsS,which proved significant (t
2.027,
z
62 (60)
3.15+ 0.46
df:86.P<0.05).
50 (60)
2.28+ 0.39
Table 2 summarizesthe results obtained from
23 (60)
1.15+ 0.29
0.10
1
the 3 lines (control, 0.025 and 0.10% EMS1.0?+ 0.28
2
25 (60)
18(60)
0.50+ 0.15
treated) of Fr femaleswhich were only infected
" Derived from Fzand adult male and femaleparents
with B. malayi over a 3-wk period.
Susceptibility in the F+ control population originallyexposedorally to 0, 0.025,and 0.10%EMS,
over the 3-wk test period was 65Vo.The 0.025% respectively.
Table 1. Susceptibility and variation to Brugia
malayi in female Fz Aed.es aegypti (BLACK EYE)
derived from male and female parents treated orally
with EMS.
418
JounNar,oF THEAurRrcex Mosqurro CoNrnor,Assocrn.rrot
Alternatively, the mutations induced by EMS
couldhavealteredmodifier genesor specificIoci
that are associatedwith the f ^ gene.This genetic
changewas manifest by both a reducedsusceptibility and number of infective larvae in F2
females derived from adult parental males and
femalesexposedto EMS.
Other experimentscompletedin our laboratory with B pahangi infections and the same
BLACK EYE strain of Ae. aegyptl have given
similar results in F2 progeny.Adult F2 females
derived from female pupae exposed,to 0.025%
EMS showed a 2l% decteasein filarial nematode susceptibility and 25Vofewer Lrs. Although
susceptibilitywas reducedby 18% in 0.075%
EMS-treatedpopulations,no significant reductions in meanL3 numberswereobtained(Larson
1986).1Too few F2 progeny were recovered in
0.10%EMs-treated populationsto permit any
filarial susceptibility experiments.
This study was supported in part by NIH
Grant GM-08194-11from the Minority Biomedical ResearchSupport Program, National Institute of GeneralMedical Sciences,with co-funding through the National Institute of Allergy
and Infectious Disease.The clawed jirds, Merionesunguiculotus,infected with Brugia malayi,
were obtained from John McCall, School of Veterinary Medicine, University of Georgia,
through a National Institute of Allergy and Infectious Diseases supply contract (AI-02642),
U.S.-JapanCooperativeMedical ScienceProgram. We also thank Lois Tews for typing the
manuscript.
I Larson, G. A. 1986. Genetic
fitness and filarial
susceptibility
effects of ethyl methanesulfonate
(EMS) and thiotepa on populations of Aedes azgypti.
M.S. thesis, University of Texas at San Antonio.
Vor,.8, No. 4
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