Fournet, Florence

Monteny, Nicole

Sannier, Christiane

Horizon / Pleins textes

Jaurna! c$the American Mosquito Control Association, 9(4):426-430, 1993 
Copyright O 1993 by the American Mosquito Control Association, Inc. 
EFFECTS OF THE INSECT GROWTH REGULATORS 
OMS 2017 AND DIFLUBENZURON ON THE 
REPRODUCTIVE POTENTIAL OF AEDES AEGYPTI 
F. FOURNET, C. SANNIER AND N. MONTENY 
Laboratory of Medical Entomology, ORSTOM, 
70 Route d'Aulnay, 93 140 Bondy, France 
ABSTRACT. The effects of 2 benzoylphenylurea insect growth regulators, OMS 2017 and difluben- 
zuron, on 4th instar larvae ofAedes aegypti were investigated in the laboratory. Apart from delayed lethal 
action, possible effects on the reproductive potential were also studied. A baseline concentration of each 
compound causing 30% emergence inhibition was determined. At this concentration, OMS 201 7 affected 
fecundity of the mosquito but diflubenzuron did not. The fertility of females that survived OMS 2017 
exposure also was decreased; 23% of the eggs reached the 4th larval instar. Diflubenzuron did not cause 
any reduction in fertility. The basal follicle number showed great variability after both treatments as it 
was alternatively equal, lower, or higher than in the control. 
INTRODUCTION 
Insect growth regulators (IGRs) are considered 
a new generation of insecticides having great 
prospects for insect control. These compounds 
are specific in activity against arthropods, par- 
ticularly insects. They are biodegradable and thus 
do not accumulate in environment. They are safe 
to a large number of nontarget organisms in- 
cluding mammals (Beadles et al. 1975, Apperson 
et al. 1978). 
Best control of insects through the use of IGRs 
is possible because of their interactions with hor- 
monal control of growth and development. Tar- 
get tissues for these compounds are the same as 
for juvenile hormones or ecdysones. The IGRs 
that seem to act on ecdysone-dependent mech- 
anisms, such as diflubenzuron, are listed as in- 
hibitors of insect molting. Several hypotheses 
have been proposed to explain their mode of 
action, such as inhibition of chitin synthesis (Post 
et al. 1974) by inhibiting chitine synthetase (Van 
Eck 1979), but also through increase of chitinase 
activity (Ishaaya and Casida 1974). However, 
the mode of action of IGRS appears more com- 
plex because they probably act on several phys- 
iological systems and also differently depending 
on the compound. 
The activity of IGRs generally results in the 
reduction of adult emergence of the target insect. 
However, they seem to have side effects, partic- 
ularly on female reproduction, as indicated by 
several studies utilizing molt inhibitors as well 
as juvenile hormone mimics, the other main 
group of IGRs (Arias and Mulla 1975). For ex- 
ample, effects on fecundity (increase or dimi- 
nution of the number of eggs laid) and on fertility 
(reduction of hatchability or viability of eggs) of 
mosquitoes following larval IGR treatments were 
documented (Miura et al. 1976, Kelada et al. 
198 1). 
This study investigated effects of 2 benzoyl- 
phenylurea molt inhibitors, OMS 2017 and di- 
flubenzuron, on fecundity and fertility of females 
of Aedes aegypti (Linn.) that survived larval ex- 
posure to the IGRs. The effects of both com- 
pounds on the basal follicle number developed 
by the surviving females was also studied to un- 
derstand disturbances of fecundity, 
MATERIALS AND METHODS 
Mosquito treatment: The IGRs, OMS 2017 
WP 25%, [ 1-(3,5-dichloro-4-(2,2-dichlorocyclo- 
propyl-methoxy)-phenyl)-3-(2,6 difluoroben- 
zoyl) urea] and diflubenzuron (DFB) WP 25%, 
[ 1 -(4-~hlorophenyl)-3-(2,6 difluorobenzoyl) urea], 
used in the present study were provided by World 
Health Organization. The bioassays were con- 
ducted on the GKEP strain of Ae. aegypti, orig- 
inally collected from Ghana and maintained since 
1985. The larval colonies, kept at 28 rt l'e, were 
fed a mouse biscuit powder. Adults were main- 
tained at 26 & 1°C and 65% RH; they were pro- 
vided a 10% glucose solution and females a pe- 
riodic blood meal on a guinea pig. 
Early 4th instar larvae were exposed to a range 
of concentrations of OMS 2017 and difluben- 
zuron according to the procedure of Mulla et al. 
(1 974). Four replicates of 25 larvae for each con- 
centration and corresponding controls were 
maintained for each IGR evaluated on 3 different 
occasions. During the exposure period of 24 h, 
larvae were not supplied with food. After 24-h 
exposure, larvae were washed thoroughly with 
tap water and transferred to a pan containing 2 
liters of tap water. Each pan, one per concentra- 
tion, contained 100 larvae. Pupae, when ap- 
peared, were caged for emergence. Dead indi- 
viduals (larvae, pupae, and adults) were recorded 
daily. Concentrations that induced 30% emer- 
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426 ORSTOM Fonds Documentaire I 
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~ECEMB~BR 1993 DECREASE OF REPRODUCTIVE POTENTIAL FTER IGR LARVAL TREATMENT 427 
Table 1. Concentrations (mdliter) of the IGRs OMS 20 17 and diflubenzuron inducing 30, 50, 
and 90% of emergence inhibition (EI) of Aedes aegypti in the laboratory. 
Compound E130 EISO EI90 
OMS 2017 0.0029 0.0078 0.085 
Diflubenzuron 0.00026 0.0005 0.0035 
(0.00 19-O.004)’ (0.0058-0.0 104) (0.045-0.259) 
(0.000 19-0.00033) (0.00042-0.0006 1) (0.0026-0.005) 
95% CI in parentheses. 
gence reduction (EI30) were then determined for 
each compound by probit analysis. Subsequent- 
ly, 4th instar larvae were exposed to these con- 
centrations of each compound and surviving 
adults fiom each treatment were collected to 
evaluate the effects on fecundity, fertility, and 
basal follicle number. 
Fecundity and fertility of surviving females: 
Four to 5 days after emergence, surviving fe- 
males were fed on a guinea pig. They were then 
placed individually in vials for oviposition, which 
occurred 48 h after the blood meal. The mean 
number of eggs laid was noted. Eight to 10 days 
later, 100 eggs were placed in water to evaluate 
the impact of treatments on the egg hatch. 
Basal follicle number ìn surviving females: 
Ovaries of surviving females were dissected un- 
der a stereoscopic microscope. The last abdom- 
inal segment was pulled away with the ovaries 
attached to determine the number of basal fol- 
licles. Dissections were made in saline solution. 
Ovaries were transferred in a clear droplet of 
saline solution on the same slide. Adhering tis- 
sues were removed, ovarioles were separated, and 
the basal follicle number was determined. Suth- 
erland et al. (1967) stated that the basal follicle 
number is numerically equal to the “ovariole 
number” provided each ovariole contains a basal 
follicle. 
The OMS 20 17-treated females were dissected 
at different periods: 24 h after emergence, 24 h 
after a blood meal that occurred 4-5 days after 
emergence, and 5 days after oviposition. For each 
period, larval treatments were repeated several 
times. Dissections of DFB-treated females oc- 
curred only 24 h after a blood meal with 2 rep- 
licates. Differences between treated and untreat- 
ed females were analyzed by a Student t-test. 
RESULTS AND DISCUSSION 
Diflubenzuron caused higher larval and pupal 
mortality than OMS 2017, which induced more 
delayed effects. We observed 5% larval and pupal 
mortality at 0.001 mg/liter of OMS 2017; the 
same concentration of DFB induced 17% larval 
and 2 1% pupal mortalities. Laboratory bioassays 
revealed that diflubenzuron was more effective 
than OMS 2017 (Table 1). Concentrations cho- 
sen for next treatments with both IGRs were 
those that induced 30% emergence inhibition 
Eflects on fecundity and fertility: Females that 
survived the OMS 20 17 treatment laid 30% less 
eggs compared to controls and this difference was 
statistically significant (P c 0.001). Our results 
were based only on the surviving population and 
so displayed a real reduction of the reproductive 
capacity after the OMS 2017 application. Fe- 
cundity of DFB-treated females was not affected. 
The variability of the effects of IGRs on repro- 
duction was confirmed by different authors. Ke- 
(EI30). 
Table 2. Effects of the IGRs OMS 20 17 at 0.0029 mg/literl and diflubenzuron at 0.00026 mg/ 
liter’ on fecundity and fertility of female Aedes aegypti surviving larval treatments in the 
laboratory. 
Emergence No. of Average no. of FI 4th instar 
Compound inhibition (Yo) fed females eggs laid f SD larvae (Yo) 
OMS 2017 
Control 8.6 32 80 k 37 65 
Treated 32.8 25 56 k 232 23 
Control 11.0 28 68 -+ 21 86 
Treated 24.0 21 68 k 303 83 
Concentration producing 30% emergence inhibition (E13J. 
Diflubenzuron 
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Significant difference (P < 0.00 1) 
No significant difference. 
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* -  I . 
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428 JOURNAL OF THE AMERICAN MOSQ~ITO CONTROL ASSOCIATION VOL. 9, NO. 4 - 
Table 3. Effects of the IGR OMS 2017 on the basal follicle number developed by female Aedes 
aegypti that survived several larval treatments. 
Time of dissection 
24 h after 24 h after 5 days after 
emergence blood meal oviposition 
EI' (O/O) B W  (4SD) EI' (Oh) BFW (fSD) EI' (Yo) BFN2 (fSD) 
Control 4.7 94 f 14 6 93 k 22 8.7 90 f 20 
Treated 33.5 99 f 233 56 107 f 234 41.5 91 4 143 
Control 8 96 f 19 8.8 82 f 19 7.4 91 4 19 
Control 3.4 109 f 16 3.3 84 +- 13 6 88 f 15 
Treated 14.5 96 f 234 13.7 78 4 134 32.4 93 f 163 
Control 11.4 115 f 16 8.8 103 f 14 
1 reated í4.7 93 * 14" 28.Ï 104 -+ i j '  
Treated 73 100 f 233 60.5 82 f 193 36.4 80 f 124 
- 
I Emergence inhibition. 
3 No significant difference. 
Mean of basal follicle number. 
Significant difference (P 5 0.0 1). 
lada et al. (1 98 1) tested several IGRs, including 
methoprene (Altosid@) and diflubenzuron 
(TH6040), against Culexpipiens Linn. larvae for 
their effects on the reproductive potential of the 
surviving adults and one following generation. 
Results varied according to the compound and 
the applied concentration. The average number 
of eggs deposited was affected by treatment. The 
numbers increased with methoprene in both gen- 
erations. After treatment with diflubenzuron, fe- 
cundity increased only in the second generation. 
Diflubenzuron had no effect upon the treated 
generation and caused an increase or a decrease 
that was not dose-dependent. 
OMS 2017 caused a significant reduction of 
fertility of surviving females (P I 0.000 1): only 
23% of the eggs reached the 4th instar (Table 2). 
Fifty-eight percent of the treated eggs had still 
not hatched 12 h after the immersion vs. only 
36% in the control. This difference was statisti- 
cally significant (P 1 0.0001). The presence of 
an embryo had been observed and so there was 
probably additional larval mortality during the 
first stages. Diflubenzuron had no effect on the 
fertility of the surviving females. 
Eflects on the development of ovarioles: The 
basal follicle number showed wide variability 
from the effects of both IGRs (Tables 3 and 4). 
In surviving females, it was equal, lower, or high- 
er than that observed in the control. In the assays, 
the emergence inhibition was not always at 30% 
as expected; it varied from 73 to 15% and 23 to 
39% for OMS 2017- and DFB-treated adults, 
respectively. It was then possible to define a cor- 
relation between the basal follicle number and 
the emergence inhibition. With OMS 20 17 treat- 
ment (Table 3), when the emergence inhibition 
was lower than 30%, the basal follicle number 
tended to be lower than the control: we observed 
that with 14.7% EI, treated females dissected 24 
h after emergence had 93 ovarioles and the con- 
trol had 115 ovarioles. With diflubenzuron, re- 
sults were the same (Table 4); when EI was 23%, 
treated females had significantly fewer ovarioles 
than the control (86 and 11 1, respectively). In 
Cx. pipiens females that survived larval treat- 
ment by methoprene and diflubenzuron, the basal 
follicle number increased (Gaaboub 1976) 
without any dose-dependent effect. This vari- 
ability might be based either on the actual in- 
gested amount of compound, possibly evaluated 
by the emergence inhibition, or on the physio- 
logical state of larvae at the time of treatments, 
which was difficult to determine because syn- 
chronously reared larvae were difficult to obtain. 
Our results led to the hypothesis that, if treat- 
ment induced higher mortality, weaker individ- 
uals were eliminated and survivors were those 
Table 4. Effects of the IGR diflubenzuron on 
the basal follicle number developed by 
surviving female Aedes aegypti, dissected 24 h 
after the blood meal. 
Emergence 
inhibition Mean basal 
(Yo) follicle no. f SD 
Control 2.5 99 4 14 
Treated 39.3 103 f lo1 
Control 9.5 111 f 15 
Treated 23.0 86 f 16' 
' No significant difference. 
* Significant difference (P -= 0.00 1). 
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DECEIp&ER 1993 DECREASE OF REPRODUCTIVE POTENTIAL FTER IGR LARVAL TREATMENT 429 
Fig. 1. Aedes aegypti follicles at 24 h post-blood meal. A. Normal untreated follicles (100 x); B. OMS 2017- 
treated follicles showing degeneration (d) and nonmatured follicle (n-m) (1 O0 x). 
that presented a higher reproductive capacity than 
the control. On the other hand, with a lower 
pressure, the decrease of the reproductive ca- 
pacity showed the physiological effect of the 
treatments. According to Gaaboub (1 976), treat- 
ment may cause a stimulus before or at the time 
the number of ovarioles is determined. Rudi- 
ments of ovaries appear in mosquito females at 
the 3rd larval stage (Parks 1955 l )  and if the or- 
ganization of the gonads becomes obvious at the 
beginning of the 4th larval stage, ovarioles only 
differentiate at the pupal stage. Then stimulus of 
the treatment could induce the maturation of 
some rudimentary ovarioles or the inhibition of 
other ones. According to the last hypothesis, OMS 
20 17 treatment could inhibit the development 
of some ovarioles in larvae. 
Degenerating and nonmatured follicles were 
observed during dissections (Fig. 1). They were 
more abundant in DFB-treated females than in 
OMS 2017-treated ones (P < 0.02). As the re- 
productive potential of surviving females was 
less affected after diflubenzuron treatment, it 
* Parks, J. J. 1955. An anatomical and histological 
study of the female reproductive system and follicular 
development in Aedes aegypti (L.). M.Sc. thesis. Uni- 
versity of Minnesota, St. Paul. 
suggests that diflubenzuron could act later than 
OMS 20 17, during oogenesis in adults. This hy- 
pothesis agrees with the observations of Behan 
and Hagedorn (1 978) showing that larval fat body 
is not transformed at the time of metamorphosis. 
If diflubenzuron has an effect on the larval fat 
body, some of the consequences might be ob- 
served at the adult stage during vitellogenin syn- 
thesis. The IGRs used might have dual effects 
on the determination of the basal follicle num- 
ber, which occurred during the larval life, and 
the ovarial maturation after the blood meal. 
The decrease of fecundity observed after a sub- 
lethal larval treatment by OMS 2017 may be 
explained by a decrease of the basal follicle num- 
ber developed. Larval treatments often have de- 
layed effects and epidermal cells and reproduc- 
tive tissues can be considered possible targets of 
this compound. Results obtained with difluben- 
zuron show that the mode of action of this com- 
pound can be different or that, at the applied 
concentration and at the time of the treatment, 
ovaries of Ae. aegypti are more susceptible to 
OMS 20 17 than to diflubenzuron. 
The entire results obtained with OMS 2017 
showed that even if the treatment did not cause 
100% adult reduction, the size of the residual 
cage population was reduced. Field studies are 
needed to confirm these observations. 
430 JOURNAL OF THE AMERICAN h'~OSQUIT0 CONTROL ASSOCIATION VOL. 9, No. 4 1 
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Effects of the insect growth regulators OMS 2017 and diflubenzuron on the reproductive potential of Aedes aegypti

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