VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS
Pyriproxyfen and House Flies (Diptera: Muscidae): Effects of Direct
Exposure and Autodissemination to Larval Habitats
CHRISTOPHER J. GEDEN1
AND
GREGOR J. DEVINE2
J. Med. Entomol. 49(3): 606Ð613 (2012); DOI: http://dx.doi.org/10.1603/ME11226
ABSTRACT Pyriproxyfen is an insect growth regulator with juvenile hormone-like activity that has
potential uses for dipterans that are difÞcult to manage with conventional insecticides, such as house
ßies (Musca domestica L.). The objectives of this study were to determine the efÞcacy of this insect
growth regulator against house ßies using variety of delivery systems and target life stages, including
an evaluation of the potential for autodissemination by female ßies to larval development sites. Adult
female house ßies exposed to Þlter paper (3.75% active ingredient) or sugar treated with pyriproxyfen
(0.01Ð 0.1%) produced signiÞcantly fewer F1 pupae than untreated ßies. Adult emergence from pupae
was unaffected. In contrast, treatment of larval rearing medium with 0.35 ml/cm2 of a 12 mg
pyriproxyfen/liter preparation had no effect on the number of pupae developing from eggs but
markedly inhibited adult emergence from those pupae. There was little difference in susceptibility
between an insecticide-susceptible and a wild strain of house ßy. The LC50 for inhibiting ßy emergence
of dust formulations in diatomaceous earth incorporating commercial pyriproxyfen products ranged
from 8 to 26 mg/liter, with little difference among products. Compared with untreated ßies, significantly fewer pupae were produced at concentrations ⬎0.5% and no adults were produced at
concentrations ⬎0.05% pyriproxyfen. When gravid females were exposed for 1 h to treated fabric (6
mg pyriproxyfen/cm2) and allowed to oviposit in rearing media containing eggs, sufÞcient pyriproxyfen was autodisseminated to reduce adult emergence from those eggs by ⬎99%. Intermittent contact
with treated fabric over 2 d reduced adult emergence by 63Ð76%.
KEY WORDS house ßy, Musca domestica, pyriproxyfen, autodissemination
House ßies (Musca domestica L.) remain a major agricultural and public health pest throughout the world.
Efforts to control them are compromised by their
ability to develop resistance to new chemical control
agents. Resistance to organophosphate, carbamate,
and pyrethroid insecticides was observed soon after
their introduction (Boxler and Campbell 1983, Plapp
1984, Scott and Georghiou 1986, Scott et al. 1989,
Kaufman et al. 2001, Butler et al. 2007, Kozaki et al.
2009). Resistance to spinosad was documented almost
immediately after market entry (Shono and Scott
2003, Deacutis et al. 2006) and the neonicotinoid imidacloprid, which provided such superior performance
that it quickly dominated the ßy control market in the
early 2000s, is now facing product failure (Kaufman et
al. 2010a,b; Memmi 2010).
Most research on chemical control of the house ßy
has concentrated on the adult stage using space sprays,
residual treatments, and baits. Few materials have
been developed that target the immatures. The insect
growth regulator (IGR) cyromazine was used in the
1980s, but excessive reliance on this material as a
1 Corresponding author: USDAÐARS, Center for Medical, Agricultural and Veterinary Entomology, 1600 SW 23rd Drive, Gainesville, FL
32608 (e-mail: [email protected]).
2 Public Health Unit, Queensland Health, Cairns, Queensland 4870,
Australia.
feed-through product in the poultry industry led to
overwhelming resistance in just a few years (Bloomcamp et al. 1987, Sheppard et al. 1990, Sheni and Plapp,
1990).
Pyriproxyfen mimics juvenile hormone, inhibiting
pupal-adult metamorphosis (Invest and Lucas 2008,
Seng et al. 2008). It has high activity against immature
dipterans including house ßies (Hatakoshi et al. 1987,
Kawada et al. 1987, Bull and Meola 1994a). House ßies
are several orders of magnitude more susceptible to
pyriproxyfen than are the parasitoids that attack the
ßy in the pupal stage (Shono et al. 1993). This suggests
that pyriproxfen would be an attractive tool for use in
integrated pest management (IPM) programs for ßies
that include the use of parasitoids (Geden et al. 1992,
Crespo et al. 1998). Recently it has been shown that
pyriproxyfen can be disseminated to the aquatic habitats of mosquitoes by the adult females themselves;
both in the laboratory (Gaugler et al. 2011) and Þeld
(Devine et al. 2009). This prompted a reexamination
of its activity against house ßies. In this study we
examine 1) the effects of pyriproxyfen on the F1 progeny of females exposed to treated surfaces and baits,
2) impact of different formulations as surface treatments, and 3) the use of adult female house ßies to
autodisseminate pyriproxfen to larval development
sites.
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GEDEN AND DEVINE: AUTODISSEMINATION OF PYRIPROXYFEN BY HOUSE FLIES
Materials and Methods
Insect Rearing and Test Conditions. Unless otherwise stated, house ßies were from an insecticide-susceptible colony established in the late 1950s from ßies
collected near Orlando, FL. In some tests, ßies from
the Center for Medical, Agricultural and Veterinary
Entomology (CMAVE) wild-type colony were used.
This colony is restarted periodically from ßies collected from local dairy farms (Gilchrist County, FL)
and is maintained so that the colony is ⬍6 generations
removed from the Þeld. These populations are moderately resistant to many of the insecticides commonly
used for ßy control such as permethrin and imidacloprid (Deacutis et al. 2006, Rinkevich et al. 2007, Kaufman et al. 2010a). The colonies are maintained using
standard rearing procedures (Hogsette 1992). All assays were conducted at 27⬚C, ⬇70% RH and constant
(24-h) light conditions. Liquid, emulsiÞable concentrates of pyriproxyfen were used: Nyguard IGR (10%
active ingredient [a.i.]), Knack (11.23% a.i.), and
Sumilarv (2% a.i.). These formulations were produced
by MGK, Minneapolis, MN; Valent Biosciences Co.,
Walnut Creek, CA; and Sumitomo Chemical Australia,
Chatswood, NSW, Australia, respectively.
Impact on the F1 Progeny of Exposed Females.
Filter paper disks were treated with 1 ml of pyriproxyfen (Nyguard IGR) diluted in distilled water to make
six solutions in a Þvefold dilution series ranging from
3.75 to 0.0012% a.i. Control papers were treated with
distilled water alone. Papers were allowed to dry and
groups of 10 female, 1-d-old ßies were exposed for 24 h
in petri dishes lined with treated paper. Flies were
provided with 10% sucrose on cotton balls placed over
a hole in the lid of the dishes. After 24 h, the exposed
females were transferred to untreated cages with 10
untreated male ßies. Five female ßies were chosen
randomly from each cage on day 7 and allowed to
oviposit for 24 h on a 30 cm3 ball of spent larval rearing
medium (medium that had been used previously to
rear ßy larvae). This oviposition substrate and eggs
were then transferred to a cup containing ⬇400 cm3
of fresh larval rearing medium. The pupae that developed were separated from the medium 7 d after transfer, counted, and held in cups at 27⬚C for adult emergence. Three sets of ßies were tested for each
concentration per test date, and the experiment was
repeated on three test dates with different cohorts of
ßies.
In a second contact method, which allowed ßies to
display more natural resting behaviors, groups of 10
male and 10 female 1-d-old ßies were placed in 17 ⫻
17 ⫻ 17 cm cages and provided with food and water.
A disk of pyriproxyfen-treated Þlter paper (diameter,
9 cm) was suspended over the food supply; control
cages received water-treated papers. Flies visited and
rested on the suspended papers repeatedly. After 7 d,
Þve females were removed from each cage and allowed to oviposit for 24 h. As in the previous test,
pupae were collected, counted, and held for emergence. Treatment effects on pupal and adult production in both tests were analyzed by one-way analysis
607
of variance (ANOVA) using the General Linear Models Procedure (PROC GLM) of the Statistical Analysis
System (SAS Institute 1992), and differences among
means were compared using the Means/Tukey statement of PROC GLM.
In tests that exposed females to treated food baits,
pyriproxyfen was diluted in acetone and mixed with
sugar to give, after evaporation, a series of Þve threefold dilutions from 0.001 to 0.1% wt:wt pyriproxyfen.
Sugar treated with acetone but without pyriproxyfen
served as controls. Groups of 10 male and 10 female
1-d-old ßies were given water and the treated sugar in
small cages for 3 d, after which they were given standard food for a further 4 d. On day 7, Þve female ßies
were removed from each cage and allowed to oviposit
for 24 h as before; pupae were collected, counted and
held for emergence as in the contact assays, and data
analyzed by one-way ANOVA as before.
Evaluation of Pyriproxyfen Formulations as Surface Treatments. Eggs of an insecticide-susceptible
laboratory strain (500 eggs) or the wild-type strain
(350 eggs) were placed on 400 cm3 of larval medium
in 500-cm3 cups (surface area, 86 cm2) and lightly
covered with ⬇5 cm3 of medium. Fewer eggs were
used for the wild-type strain because it showed elongated development times at egg loads of ⬎350 in this
volume of medium. The surface of the medium was
evenly treated with 3 ml of pyriproxyfen (Nyguard
IGR) diluted in distilled water to make six solutions in
a Þvefold dilution series ranging from 3.75 to 0.0012%
a.i. Pupae were separated 9 d later, counted, and held
for adult emergence; data were analyzed with
ANOVA as in previous tests.
In a second test, dust formulations were made by
mixing diatomaceous earth powder (Safer Brand Ant
and Crawling Insect Killer, 78% SiO2, Woodstream,
Lititz, PA) with three liquid pyriproxyfen concentrates: Nyguard IGR, Knack, and Sumilarv. After drying, these preparations contained between 0.0005% (5
␮g/g) and 0.5% pyriproxyfen (wt:wt). Five hundred
eggs of susceptible ßies were pipetted onto 86 cm2 of
rearing medium (volume, 450 cm3) and lightly covered with medium. The surface of the medium was
then treated evenly with 0.5 g of dust. Controls were
treated with 0.5 g of dust containing no pyriproxyfen.
Pupae were separated 9 d later, counted, and held for
adult emergence.
For all evaluations, three sets of ßy eggs were used
to test each concentration and the control. The entire
experiment was repeated three times. The results
were subject to probit analyses (PROC PROBIT of
SAS) to identify differences in the inhibition of
adult ßy emergence. The pyriproxifen concentration
(LC50) that resulted in 50% suppression of emergence
of adult ßies was calculated after adjusting for control
mortality by AbbottÕs formula.
Autodissemination by Female Flies. Groups of Þve
6-d-old gravid females were gently shaken for 30 s in
a 60-ml cup with 0.5 g of pyriproxyfen dust containing
0, 0.05, 0.5, 2.5, 5, and 10% (wt:wt) pyriproxyfen
(Nyguard IGR) in diatomaceous earth. Flies were
chilled after the exposure, removed from the cups,
608
JOURNAL OF MEDICAL ENTOMOLOGY
tapped lightly to remove any excess or caked dust, and
transferred (in groups of Þve) to screen-covered 500cm3 cups containing 400 cm3 of ßy larval rearing medium. Flies were allowed to oviposit for 24 h then
removed. Pupae were collected 7 d later, counted, and
held until emergence as in previous tests, and data
were analyzed by one-way ANOVA as before.
In the next tests, dusts containing 0, 0.05, 0.5, and 5%
pyriproxyfen were prepared from both the Nyguard
and Knack liquid concentrates using diatomaceous
earth as before and applied to cotton fabric. Cotton
muslin (104 cm2) was used to line the sides of 60-ml
plastic cups, 0.5 g of dust was added, and the cups were
covered and shaken for 30 s. Excess dust was removed
from the fabric by inverting the cup and tapping it
lightly. This resulted in an application rate of 6 mg of
dust/cm2.
Groups of Þve 5-d-old gravid female ßies were exposed to the treated fabric in two ways. In the Þrst test
(forced contact), ßies were chilled and placed on the
bottom of the cup. After recovering, the ßies were
allowed to walk on the treated inner lining of the cup
for 1 h then transferred to cages with larval rearing
medium as before. In a second test, the bottom was cut
from the treated cup that was then placed over the
food dish in a cage. This arrangement created a subconical cylindrical treated surface surrounding the
food. Flies made repeated contacts with pyriproxyfen
as they alighted on their way to and from the food, as
well as from contact with dust that fell onto the food
surface. After 2 d the treated fabric was removed and
rearing medium was placed in the cages for oviposition.
In both methods, the rearing medium was removed
from the cages after 24 h and held to monitor pupal and
adult production as before (n ⫽ 3 replications of three
cages for each product and concentration). Data on
pupal and adult production were analyzed by two-way
ANOVA using dust concentration and product (Nyguard vs. Knack) as Þxed main effects, plus the concentration-product interaction.
Two additional tests were conducted to determine
whether reductions in adult production seen in the F1
generation resulted from the autodissemination of material by the parent and not from direct impacts of
pyriproxyfen on fecundity or fertility. The Þrst of
these tests was designed to examine the immediate
impact of treatment on the number and viability of
eggs produced. In this test, groups of 50 6-d-old gravid
females were shaken with dusts containing 0, 0.5, or 5%
pyriproxyfen. Dusted ßies were transferred to cages,
provided with food and water, and allowed to oviposit
for 6 h on an oviposition pad made by wrapping a ball
of spent larval rearing medium in black cloth. These
eggs were rinsed from the oviposition pads and
counted. One set of 200 eggs from each cage was then
held on moist blotting paper and assessed for fertility
by counting unhatched eggs after 24 h. Another set of
200 eggs was placed on larval rearing medium and
allowed to develop normally. Pupae were collected
and counted after 7 d and held for adult emergence.
Three sets of 50 ßies were tested for each concentra-
Vol. 49, no. 3
tion. Treatment effects on egg deposition and survivorship to larval, pupal, and adult stages were analyzed
by one-way ANOVA.
The second test was designed to examine the effect
of treating adult ßies on the development of immatures from untreated ßies. In this test, groups of Þve
6-d-old gravid females were Þrst dusted as above and
transferred to cages with food and water (n ⫽ 3 cages
per concentration). These ßies were presented with
cups of 400 cm3 of ßy larval rearing medium that also
contained 500 eggs that had been collected from untreated ßies and pipetted onto the medium surface 1 h
previously. Treated ßies were allowed to oviposit in
(and transfer pyriproxyfen to) that medium for 12 h,
after which the medium was removed. Additional
fresh medium was added 3 d later if crowding levels
warranted, and pupal production and adult emergence was determined and analyzed as in previous
tests. Not included in the experimental design and
analysis were additional cups that were set up for
quality control to determine ßy production from cups
that received only the 500 untreated eggs or the 12-h
oviposition event by Þve gravid females.
Results
Impact on the F1 Progeny of Exposed Females.
When young adult female ßies were exposed to pyriproxyfen in forced-contact assays there was no signiÞcant effect on the number of pupae or adults produced (Table 1). However, when female ßies were
allowed to make voluntary contacts with treated papers for 7 d, there was a signiÞcant effect on pupal
production at the higher rate of 3.75% pyriproxyfen
(38% of control productivity) but little effect on adult
emergence success (80 Ð90% of control emergence;
Table 1).
Female ßies that were allowed to feed on treated
sugar (0.01 and 0.33% pyriproxyfen) for 3 d produced
⬇35% as many pupae as the controls. Pupal production
at the higher concentration of 0.1% pyriproxifen was
only 14% of the controls (Table 2). Again, adult emergence success from the pupae that did develop was
⬇90% across all baited sugar treatments.
Evaluation of Pyriproxyfen Formulations as Surface Treatments. When pyriproxyfen was applied as
an aqueous solution to rearing medium containing
eggs, there was no effect on the numbers of pupae
produced at any dose for either the insecticide-susceptible or wild colony ßies (Table 3). In contrast,
adult emergence was almost 100% inhibited at all
doses. Pyriproxyfen applied as dusts to rearing media
containing eggs at the rate of 0.5 g/86 cm2 was also
effective at inhibiting adult emergence. Dusts prepared from three commercial concentrates had similar
effects on adult emergence, with LC50s of 8 Ð26 mg/
liter (Table 4).
Autodissemination by Female Flies. Gravid females
that were shaken with pyriproxyfen (Nyguard) dust at
concentrations ⬎0.05% produced eggs that did not
yield any adults. At pyriproxyfen concentrations
⬎0.5%, pupal production was also affected (Fig. 1).
May 2012
GEDEN AND DEVINE: AUTODISSEMINATION OF PYRIPROXYFEN BY HOUSE FLIES
609
Table 1. Pupal and adult progeny produced by groups of five 1-d-old female house flies that were exposed to pyriproxyfen-treated
filter paper (aqueous dilution of Nyguard 10) for either 1 or 7 d
Pyriproxifen
conc. (% a.i.)
Control
0.0012
0.006
0.03
0.15
0.75
3.75
ANOVA F6,14b 1.3 ns
a
b
Mean (SE) no. pupae
Mean (SE) no. adults
Forced
contact (1 d)
Voluntary
contact (7 d)
Forced
contact (1 d)
Voluntary
contact (7 d)
492.1 (21.1)
489.6 (95.1)
561.8 (18.0)
542.9 (63.2)
441.8 (27.6)
475.3 (46.0)
461.9 (77.6)
7.46**
413.2 (48.4)aa
542.6 (54.3)a
516.8 (9.4)a
521.6 (80.0)a
542.4 (29.3)a
551.9 (69.0)a
158.7 (40.8)b
0.57 ns
387.7 (73.8)
454.7 (68.9)
529.7 (30.3)
519.7 (75.5)
412.3 (32.6)
379.0 (45.3)
438.0 (65.0)
9.57**
400.3 (45.6)a
510.3 (57.0)a
481.3 (11.0)a
490.3 (73.7)a
453.7 (38.7)a
520.3 (37.9)a
129.7 (34.0)b
Means within columns followed by the same letter are not signiÞcantly different (TukeyÕs method; P ⱕ 0.05).
**P ⬍ 0.01; ns P ⬎ 0.05.
When ßies were conÞned on treated fabric for 1 h
(forced tarsal contact), pupal production was significantly affected by the concentration of the dusts
(F3,16 ⫽ 5.77; P ⬍ 0.01) but not by the product (Nyguard vs. Knack) (F1,16 ⫽ 1.64; P ⬎ 0.05) or the concentration-product interaction (F3,16 ⫽ 1.94; P ⬎ 0.05;
Table 5). Effects on pupal production were restricted
to 30% reductions in pupal numbers in ßies treated
with the highest concentration of dust prepared from
Nyguard. Adult production was signiÞcantly affected
by concentration (F3,16 ⫽ 48.59; P ⬍ 0.01) but not
product (F1,16 ⫽ 0.41; P ⬎ 0.05) or the interaction
(F3,16 ⫽ 3.10; P ⬎ 0.05). The strongest effect on adult
production was observed in ßies treated with dust
made with Knack at 5%, where treatments produced
just 8% of the ßies seen in the controls.
When gravid females were allowed to make intermittent contact with treated fabric over 2 d there was
no effect on pupal production because of product
(Nyguard vs. Knack, F1,16 ⫽ 0.47; P ⬎ 0.05), concentration (F3,16 ⫽ 2.92; P ⬎ 0.05), or the interaction of
product and concentration (F3,16 ⫽ 1.44; P ⬎ 0.05;
Table 5). Adult production was signiÞcantly affected
by concentration (F3,16 ⫽ 37.03; P ⬍ 0.01) but not
product (F1,16 ⫽ 1.43; P ⬎ 0.05) or the interaction
(F3,16 ⫽ 1.01; P ⬎ 0.05). Adult production among ßies
treated at the highest concentration was 24 and 36% of
control values for Nyguard and Knack, respectively.
Table 2. Pupal and adult progeny produced by groups of five
female flies fed pyriproxyfen-treated sugar
Pyriproxyfen
conc. (% a.i.)
Mean (SE)
no. pupae
Mean (SE)
no. adults
0
0.001
0.003
0.010
0.033
0.100
ANOVA F5,12b
322.3 (19.5)aba
358.7 (16.8)a
288.7 (19.2)b
118.7 (6.6)c
117.0 (5.8)c
45.7 (12.2)d
80.9**
297.0 (18.8)ab
334.3 (20.9)a
268.3 (15.9)b
110.0 (6.1)c
101.3 (6.4)c
40.7 (13.2)d
69.65**
Flies were allowed to feed for 3 d after emergence, then given
untreated food and allowed to oviposit on day 7.
a
Means within columns followed by the same letter are not signiÞcantly different (TukeyÕs method; P ⱕ 0.05).
b
**P ⬍ 0.01; ns, P ⬎ 0.05.
The fecundity of gravid female ßies was unaffected
by treatment with pyriproxyfen dust. Treated and
untreated ßies laid 154 Ð180 eggs per female during a
6-h oviposition window after treatment (Table 6).
Eggs from treated ßies were as viable as controls
(overall hatch rate, 92Ð97%), and there were no signiÞcant differences in survival to the pupal or adult
stage of progeny from treated ßies that were reared in
untreated medium (Table 6).
However, when gravid females dusted with the
higher treatment concentration (5% a.i.) were allowed to disseminate pyriproxyfen to media already
containing the eggs of untreated ßies, almost no adult
ßies emerged (Table 7). There was no signiÞcant effect on adult emergence at the lower concentration
(0.5% a.i.), or on pupal production at either concentration.
Discussion
Pyriproxyfen is an IGR whose activity mimics that
of juvenile hormone and has its primary effect on the
target by blocking development from the pupal to
adult stages, although embyrogenic and reproductive
effects are known as well (Invest and Lucas 2008).
Mosquitoes are highly sensitive to this IGR, and longlasting control of species of Culex, Aedes, and Anopheles has been reported after treatment of larval habitats (Lee 2002, Yapabandara and Curtis 2004,
Seccacini et al. 2008). Pyriproxyfen is compatible with
other control agents, and combinations with Bacillus
thuringiensis, permethrin, and spinosad have been
used successfully, with evidence of strong synergism
in the latter combination (Lee et al. 2005, Darriet and
Corbel 2006, Lucia et al. 2009, Darriett et al. 2010).
Comparatively, little is known about the activity of
pyriproxyfen on muscoid ßies, although its activity
against house ßies was recognized at the time of its
discovery (Hatakoshi et al. 1987; Kawada et al. 1987,
1992). In one of these initial reports, LC50s of 30 Ð270
␮g/liter were observed when the IGR was incorporated into larval rearing medium or poultry manure
(Hatakoshi et al. 1987). In another study, lower LC50s
(31Ð 48 ␮g/liter) were observed for larvae in treated
rearing medium than for larvae dipped in the same
610
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 49, no. 3
Table 3. F1 pupal production and adult emergence of two strains of house flies (lab and wild-type) reared in medium treated topically
with 3 ml of pyriproxyfen (Nyguard)
Mean (SE) no. pupae
Mean (SE) no. adults
Pyriproxyfen
conc. (% a.i.)
Lab
Wild
Lab
Wild
Control
0.0012
0.006
0.03
0.15
0.75
3.75
ANOVA F6,14a
475.5 (21.3)
435.4 (10.6)
453.1 (35.7)
482.5 (20.4)
472.2 (19.4)
487.1 (16.5)
551.5 (36.0)
2.2ns
305.0 (13.7)
299.3 (7.5)
284.0 (10.1)
306.3 (12.3)
293.3 (11.9)
333.7 (28.1)
308.0 (18.6)
0.95ns
434.3 (23.6)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
287.0 (17.7)
2.3 (1.5)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
0.0 (0.0)
There were 500 eggs from untreated female ßies placed on the medium on the day of treatment.
a
ns, P ⬎ 0.05.
solutions (Kawada et al. 1987). Bull and Meola (1994a)
observed that ßies exposed to glass treated with up to
142.9 ␮g/cm2 of pyriproxyfen for 5 d produced signiÞcantly fewer pupae after exposure than untreated
ßies. These ßies were conÞned in glass jars so that
contact was essentially continuous throughout the 5-d
exposure. The same authors found that stable ßies
(Stomoxys calcitrans L.) were also sensitive to pyriproxyfen (Bull and Meola 1994b). In our tests, a 1-h
exposure of adults to treated Þlter paper did not result
in reduction of F1 pupal production at any dose (Table
1). However, ßies that made frequent contact with
treated papers suspended in their rearing cages produced fewer pupae, although only at the high rate of
3.75% a.i. Presumably, ßies in the latter group acquired
a cumulatively higher dose over time than did ßies that
were conÞned on a treated surface for a short time.
Adult emergence from surviving pupae was similar to
emergence from untreated controls. This was also
observed by Bull and Meola (1994a). A much stronger
effect was observed in ßies that ingested pyriproxyfentreated sugar for 3d after emergence. Both pupal and
adult production in the F1 were affected when the
parent ßies were fed 0.01% a.i. IGR solutions (Table 2).
Only pupal production, and not fecundity, egg, or
larval mortality was measured in these tests. Therefore, the exact stage at which effects were manifest is
unknown, but Bull and Meola (1994a) reported that
the primary cause of reduced pupal production was
because of embryogenic effects and nonviable eggs.
Although such direct effects are interesting, the rates
required to treat adults with sufÞcient material to have
substantial embryogenic impacts on F1 progeny are
quite high.
Treatment of larval rearing medium with dust formulations made from liquid commercial pyriproxyfen
products resulted in LC50s of 8 Ð26 ␮g/g (Table 4).
These rates appear at Þrst glance to be several orders
of magnitude higher than those reported by Bull and
Meola (1994a) (8 ␮g/liter), but in the latter study the
dose was expressed as the concentration of pyriproxyfen in water that was incorporated into dry ingredients
to prepare the diet. In contrast, our doses reßect the
concentration used in a dust that was applied to already-prepared diet at a rate of 0.5 g/assay cup. Our
results are comparable to the earlier studies after accounting for these differences in approach. Taken
together, the data indicate that surface treatment with
0.03Ð 0.35 ␮g/cm2 is required to ensure inhibition of
adult house ßy emergence.
The status of cross-resistance to pyriproxyfen is
uncertain. Tolerance to pyriproxyfen has been noted
in a number of strains resistant to other insecticides
(Kawada et al. 1987, Bull and Meola 1994a, Londershausen et al. 1996). In our tests, a few wild-type ßies
emerged from medium treated at the lowest dose (12
mg/liter), no ßies from either strain emerged from
medium treated with higher concentrations. House
ßies are notorious for their ability to develop resistance to insecticides and IGRs, especially when used
in continuous delivery systems (Sheppard et al. 1990).
Were pyriproxyfen ever to be used ubiquitously and
continuously [i.e., incorporation into animal feeds to
provide continuous treatment of manure (Miller and
Miller 1994)], it would likely result in the rapid development of resistance problems. Therefore, pyriproxyfen must be used as part of integrated resistance
and pest management programs.
Table 4. Dose response values of Musca domestica to three pyriproxyfen dusts made by mixing liquid concentrates of three commercial
products with diatomaceous earth
Dust
Intercept
␹2
Slope (SE)
␹2
LC50a
(95% CL)
Nyguard
Sumilarv
Knack
⫺2.4 (7.32)
⫺1.8 (0.41)
⫺1.9 (0.20)
10.91**
18.44
92.88**
1.72 (0.38)
1.95 (0.32)
1.80 (0.13)
20.68**
37.85**
189.31**
25.8 (0.1Ð66.8)
8.1 (1.0Ð20.5)
12.0 (0.4Ð14.8)
a
␮g/g pyriproxyfen.
** P ⬍ 0.01.
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GEDEN AND DEVINE: AUTODISSEMINATION OF PYRIPROXYFEN BY HOUSE FLIES
611
Table 6. Egg deposition by female house flies after treatment
with pyriproxyfen dust and development success of progeny of
treated flies
Mean (SE)a
no. eggs
deposited
per female
Hatched
Pupated
Emerged
adults
0
0.5
5
ANOVA F1,7a
154.3 (16.5)
177.4 (27.1)
179.8 (16.3)
0.15ns
196.7 (0.2)
195.3 (1.9)
197.0 (0.7)
0.09ns
147.0 (5.4)
140.7 (12.2)
159.3 (10.5)
0.23ns
140.3 (5.9)
136.0 (12.0)
150.7 (11.4)
0.35ns
a
Fig. 1. Mean (SE) pupal and adult progeny produced by
groups of Þve gravid female house ßies after being shaken in
a container with pyriproxyfen dust. Means on bars with the
same letters (pupae) are not signiÞcantly different (ANOVA
F ⫽ TukeyÕs method, P ⱕ 0.05; overall ANOVA F5,12 ⫽ 18.73).
ANOVA was not performed on adult data because no ßies
were produced in four of the treatments (zero variance).
Perhaps the most exciting potential for pyriproxyfen use lies in the autodissemination approach, in
which adult females treat themselves at bait stations
then transfer the IGR to oviposition sites. This approach was Þrst suggested for mosquitoes by Itoh
(1994) in laboratory tests with Aedes aegypti L., and
later by Chism and Apperson (2003) for Ae. albopictus
(Skuse) and Ae. triseriatus (Say). Devine et al. (2009)
demonstrated the feasibility of the autodissemination
method in the Þeld, using treat-and-disseminate stations to treat gravid Ae. aegypti that then carried pyriproxyfen to larval breeding sites (cemetery vases) in
a Peruvian cemetery. Simulation models indicate that
this approach might even be effective against malaria
vectors (Devine and Killeen 2010), and a new formulation and autodissemination station was described
recently for use against Ae. albopictus (Gaugler et al.
2011).
Can a parallel approach be developed for muscoid
ßies? Hargrove and Langeley (1990, 1993) observed
Mean (SE) no. (out of 200 eggs)
developing to stage
Pyriproxyfen
conc. (% a.i.)
ns, P ⬎ 0.05.
signiÞcant reductions in tsetse populations in Zimbabwe after deployment of traps or visual targets for
adult ßies that were treated with pyriproxyfen at 2
mg/cm2 of surface area. In the tests reported in Fig. 1
and Table 5 we exposed gravid females brießy to dusts
and then allowed them to oviposit, resulting in reduced emergence of adult progeny. This was unlikely
to be the result of direct embryogenic effects on females, as these were small and required long exposure
times (Tables 1 and 2). In further tests we showed
unequivocally that the treatment of gravid females
had no immediate impact on fecundity or fertility of
treated ßies (Table 6) but that the eggs of untreated
ßies failed to develop to the adult stage when their
rearing substrate was visited by treated ßies during
oviposition (Table 7). For these reasons, reductions in
ßy production in the autodissemination tests can be
attributed to the morphogenic effects of the ßy-transferred pyriproxyfen on ßy immatures in the oviposition or larval development medium. Results presented
in Fig. 1 and Table 7 demonstrate that house ßies are
capable of carrying a large enough quantity of pyriproxyfen to transfer a sufÞcient amount to block ßy
development at the site where eggs are deposited.
Male ßies could also transfer material during visits to
larval habitats, and treated males might even facilitate
the venereal transfer of pyriproxyfen to females during mating and impact parental fecundity and fertility.
This has been shown in mosquitoes (Gaugler et al.
Table 5. Autodissemination test: Pupal and adult progeny produced by groups of five gravid female flies exposed to pyriproxyfen dust
for either 1 h (forced tarsal contact) or 2 d (self-application by walking on stations near the food supply), then allowed to oviposit for 24 h
Mean (SE) no. pupae
Mean (SE) no. adults
Pyriproxyfen conc. (% a.i.)
Forced
contact (1 h)
Voluntary
contact (2 d)
Forced
contact (1 h)
Voluntary
contact (2 d)
Control
Dust made from Nyguard concentrate
0.05
0.5
5
ANOVA F4,8b
Dust made from Knack concentrate
0.05
0.5
5
ANOVA F4,8b
651.0 (17.6)aa
669.0 (62.5)a
523.2 (28.2)a
542.3 (36.8)a
628.7 (42.4)a
761.7 (45.9)a
471.3 (69.0)b
6.4*
640.0 (58.5)a
563.7 (86.6)a
530.0 (80.4)a
0.8ns
443.7 (27.6)a
163.0 (37.3)b
162.7 (95.1)b
12.7**
552.6 (38.3)a
497.0 (77.0)a
131.3 (19.4)b
17.4
751.7 (37.8)a
525.0 (49.2)a
274.0 (214.1)a
3.4ns
627.7 (54.0)a
770.0 (28.9)a
476.0 (56.6)b
5.5*
519.3 (14.3)a
284.0 (20.4)b
42.3 (31.1)c
92.2**
498.0 (56.6)a
635.0 (33.1)a
197.7 (63.5)b
14.9**
a
Means within columns under the same subheading (Nyguard, Knack) followed by the same letter are not signiÞcantly different (TukeyÕs
method; P ⱕ 0.05).
b
** P ⬍ 0.01; ns, P ⬎ 0.05.
612
JOURNAL OF MEDICAL ENTOMOLOGY
Table 7. Pupal and adult progeny from rearing medium with
500 eggs from untreated flies and eggs deposited by groups of five
gravid female flies after treatment with pyriproxyfen dust
Pyriproxyfen
conc. (% a.i.)
Mean (SE)
no. pupae
Mean (SE)
no. adults
0 (controls)
0.5
5.0
ANOVA F1,16b
858.2 (57.5)aa
876.0 (55.5)a
893.2 (60.8)a
0.09ns
832.3 (53.4)a
692.5 (56.0)a
0.3 (0.2)b
99.5**
Not shown in body of table: ßy production from cups that only
received 500 eggs was 429.7 ⫹ 21.9 pupae and 419.7 ⫹ 21.4 adults;
production from cups that only received oviposition by Þve ßies was
518.3 ⫹ 29.9 pupae and 499.1 ⫹ 22.2 pupae.
a
Means within columns followed by the same letter are not signiÞcantly different (TukeyÕs method; P ⱕ 0.05).
b
ns, P ⬎ 0.05; **P ⬍ 0.01.
2011), house ßies (Kawada et al. 1992), and tsetse ßies
(Langely et al. 1990). Our results with self-treatment
methods involving brief and intermittent visits to
treated fabric were encouraging, but high doses were
necessary (Table 5). A high-potency formulation that
would adhere readily to the ßy at the time of pickup,
remain adhered after acquisition, and detach on contact with manure would be ideal. Further research is
needed to identify those formulations and to design
exposure tools and autodissemination station designs
that might make this approach an operational option
for house ßies.
Acknowledgments
We thank M. Doyle and H. Mckeithen for providing ßies
and assisting with the bioassays. This research was supported
in part by the Deployed War Fighter Protection Research
Program of the Armed Forces Pest Management Board.
References Cited
Bloomcamp, C. L., R. S. Patterson, and P. G. Koehler. 1987.
Cyromazine resistance in the houseßy (Diptera: Muscidae). J. Econ. Entomol. 80: 352Ð357.
Boxler, D. J., and J. B. Campbell. 1983. Survey of resistance
by houseßy, Musca domestica (L.) (Diptera: Muscidae),
to dichlorvos in Nebraska feedlots. J. Kans. Entomol. Soc.
56: 159Ð163.
Bull, D. L., and R. W. Meola. 1994a. EfÞcacy and toxicodynamics of pyriproxyfen after treatment of insecticidesusceptible and -resistant strains of the house ßy (Diptera: Muscidae). J. Econ. Entomol. 87: 1407Ð1415.
Bull, D. L., and R. W. Meola. 1994b. Interactions of the
insect growth regulator pyriproxyfen with immature and
adult stages of the stable ßy. Southwest. Entomol. 19:
257Ð263.
Butler, S. M., A. C. Gerry, and B. A. Mullens. 2007. House
ßy (Diptera: Muscidae) activity near baits containing
(Z)-9-tricosene and efÞcacy of commercial toxic ßy baits
on a southern California dairy. J. Econ. Entomol. 100:
1489 Ð1495.
Chism, B. D., and C. S. Apperson. 2003. Horizontal transfer
of the insect growth regulator pyriproxyfen to larval microcosms by gravid Aedes albopictus and Ochlerotatus
triseriatus mosquitoes in the laboratory. Med. Vet. Entomol. 17: 211Ð220.
Vol. 49, no. 3
Crespo, D. C., R. E. Lecuona, and J. A. Hogsette. 1998. Biological control: an important component in integrated
management of Musca domestica (Diptera : Muscidae) in
caged-layer poultry houses in Buenos Aires, Argentina.
Biol. Control. 13: 16 Ð24.
Darriet, F., and V. Corbel. 2006. Laboratory evaluation of
pyriproxyfen and spinosad, alone and in combination,
against Aedes aegypti larvae. J. Med. Entomol. 43: 1190 Ð
1194.
Darriet, F., S. Macombe, M. Etienne, A. Yebakima, P. Agnew,
M. M. Yp-Tcha, and V. Corbel. 2010. Field evaluation of
pyriproxyfen and spinosad mixture for the control of
insecticide resistant Aedes aegypti in Martinique (French
West Indies). Parasit. Vect. 3: 88.
Deacutis, J. M., C. A. Leichter, A. C. Gerry, D. A. Rutz, W. D.
Watson, C. J. Geden, and J. G. Scott. 2006. Susceptibility
of Þeld collected house ßies to spinosad before and after
a season of use. J. Agric. Urban Entomol. 23: 105Ð110.
Devine, G. J., and G. F. Killeen. 2010. The potential of a new
larviciding method for the control of malaria vectors.
Malaria J. 9: 142 (doi: http://dx.doi.org/10.1186/1475Ð
2875-9 Ð142).
Devine, G. J., E. Z. Perea, G. F. Killeen, J. D. Stancil, S. J.
Clark, S. J., and A. C. Morrison. 2009. Using adult mosquitoes to transfer insecticides to Aedes aegypti larval
habitats. Proc. Natl. Acad. Sci. U.S.A. 106: 11530 Ð11534.
Geden, C. J., D. C. Steinkraus, R. W. Miller, and D. A. Rutz.
1992. Suppression of house ßies on New York and Maryland dairies using Muscidifurax raptor in an integrated
management program. Environ. Entomol. 21: 1419 Ð1426.
Gaugler, R., D. Suman, and Y. Wang. 2011. An autodissemination station for the transfer of an insect growth
regulator to mosquito oviposition sites. Med. Vet. Entomol. (doi: http://dx.doi.org/10.1111/j.1365Ð2915.2011.
00970.x).
Hargrove, J. W., and P. A. Langley. 1990. Sterilizing tsetse
(Diptera: Glossinidae) in the Þeld: a successful trial. Bull.
Entomol. Res. 80: 397Ð 403.
Hargrove, J. W., and P. A. Langley. 1993. A Þeld trial of
pyriproxyfen-treated targets as an alternative method for
controlling tsetse (Diptera: Glossinidae). Bull. Entomol.
Res. 83: 361Ð368.
Hatakoshi, M., H. Kawada, S. Nishida, H. Kisida, and I. Nakayama. 1987. Laboratory evaluation of 2-[1-methyl-2(4-phenoxyphenoxy)-ethoxy] pyridine against larvae of
mosquitoes and houseßy. Jpn. J. Sanit. Zool. 38: 271Ð274.
Hogsette, J. A. 1992. New diets for production of house ßies
and stable ßies (Diptera: Muscidae) in the laboratory. J.
Econ. Entomol. 85: 2291Ð2294.
Invest, J. F., and J. R. Lucas. 2008. Pyriproxyfen as a mosquito larvicide, pp. 239 Ð245. In W. H. Robinson and D.
Bajomi (eds.), Proceedings of the Sixth International
Conference on Urban Pests. Veszprem, Hungary.
Itoh, T. 1994. Utilization of blood-fed females of Aedes aegypti as a vehicle for the transfer of the insect growth
regulator, pyriproxyfen, to larval habitats. Trop. Med. 36:
243Ð248.
Kaufman, P. E., J. G. Scott, and D. A. Rutz. 2001. Monitoring
insecticide resistance in house ßies (Diptera: Muscidae)
from New York dairies. Pest Manage. Sci. 57: 514 Ð521.
Kaufman, P. E., S. Nunez, R. S. Mann, C. J. Geden, and M. E.
Scharf. 2010a. Nicotinoid and pyrethroid insecticide resistance in house ßies (Diptera: Muscidae) collected from
Florida dairies. Pest Manage. Sci. 66: 290 Ð294.
Kaufman, P. E., S. C. Nunez, C. J. Geden, and M. E. Scharf.
2010b. Selection for resistance to imidacloprid in the
house ßy (Diptera: Muscidae). J. Econ. Entomol. 103:
1937Ð1942.
May 2012
GEDEN AND DEVINE: AUTODISSEMINATION OF PYRIPROXYFEN BY HOUSE FLIES
Kawada, H., K. Dohara, and G. Shinjo. 1987. Evaluation of
larvicidal potency of insect growth regulator, 2Ð1-methyl2-(4-phenoxyphenoxy)ethoxy pyridine, against the
houseßy, Musca domestica. Jpn. J. Sanit. Zool. 38: 317Ð322.
Kawada, H., S. Senbo, and Y. Abe. 1992. Effects of pyriproxyfen on the reproduction of the houseßy, Musca
domestica, and the German cockroach, Blattella germanica. Jpn. J. Sanit. Zool. 43: 169 Ð175.
Kozaki, T., S. G. Brady, and J. G. Scott. 2009. Frequencies
and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and Þeld populations of
the house ßy, Musca domestica L. Pest. Biochem. Physiol.
95: 6 Ð11.
Langley, P. A., J. W. Hargrove, B. Mauchamp, C. Royer, and
H. Oouchi. 1990. Prospects for using pyroproxyfentreated targets for tsetse control . Entomol. Exp. Appl. 66:
153Ð159.
Lee, D-K. 2002. Mosquito control evaluation of an insect
growth regulator, pyriproxyfen against Culex pipiens pallens (Diptera: Culicidae) larvae in marsh area, Korea.
Korean J. Entomol. 32: 37Ð 41.
Lee, Y. W., J. Zairi, H. H. Yap, and C. R. Adanan. 2005.
Integration of Bacillus thuringiensis H-14 formulations
and pyriproxyfen for the control of larvae of Aedes aegypti
and Aedes albopictus. J. Am. Mosq. Control Assoc. 21:
84 Ð 89.
Londershausen, M., B. Alig, R. Popsischil, and A. Turberg.
1996. Activity of novel juvenoids on arthropods of veterinary importance. Arch. Insect Biochem. Physiol. 32:
651Ð 658.
Lucia, A., L. Harburguer, S. Licastro, E. Zerba, and H. Masuh.
2009. EfÞcacy of a new combined larvicidal-adulticidal
ultralow volume formulation against Aedes aegypti (Diptera: Culicidae), vector of dengue. Parasitol. Res. 104:
1101Ð1107.
Memmi, B. K. 2010. Mortality and knockdown effects of
imidacloprid and methomyl in house ßy (Musca domestica L., Diptera: Muscidae) populations. J. Vector Ecol. 35:
144 Ð148.
Miller, R. W., and J. A. Miller. 1994. Pyriproxyfen bolus for
control of ßy larvae. J. Agric. Entomol. 11: 39 Ð 44.
Plapp, F. W. 1984. The genetic basis of insecticide resistance in the house ßy: evidence that a single locus plays
a major role in metabolic resistance to insecticides. Pestic.
Biochem. Physiol. 22: 194 Ð201.
613
Rinkevich, F. D., R. L. Hamm, C. J. Geden, and J. G. Scott.
2007. Dynamics of insecticide resistance alleles in house
ßy populations from New York and Florida. Insect
Biochem. Mol. Biol. 37: 550 Ð558.
SAS Institute. 1992. SAS users guide: statistics. SAS Institute, Cary, NC.
Scott, J. G., and G. P. Georghiou. 1986. Mechanisms responsible for high levels of permethrin resistance in the houseßy. Pestic. Sci. 17: 195Ð206.
Scott, J. G., R. T. Roush, and D. A. Rutz. 1989. Insecticide
resistance of house ßies (Diptera: Muscidae) from New
York USA dairies. J. Agric. Entomol. 6: 53Ð 64.
Seccacini, E., A. Lucia, L. Harburguer, E. Zerba, S. Licastro,
and H. Masuh. 2008. Effectiveness of pyriproxyfen and
dißubenzuron formulations as larvicides against Aedes
aegypti. J. Am. Mosq. Control Assoc. 24: 398 Ð 403.
Seng, C., T. Setha, J. Nealon, D. Socheat, and M. B. Nathan.
2008. Six months of Aedes aegypti control with a novel
controlled-release formulation of pyriproxyfen in domestic water storage containers in Cambodia. Southeast
Asian J. Trop. Med. Public Health 39: 822Ð 826.
Sheni, J., and F. W. Plapp, Jr. 1990. Cryomazine resistance
in the house ßy (Diptera: Muscidae): Genetics and crossresistance to dißubenzuron. J. Econ. Entomol. 83: 1689 Ð
1697.
Sheppard, D. C., N. C. Hinkle, J. S. Hunter, and D. G.
Gaydon. 1990. Resistance in constant exposure livestock
insect control systems: a partial review with some original
Þndings on cyromazine resistance in house ßies. Fla.
Entomol. 72: 360 Ð369.
Shono, T., and J. G. Scott. 2003. Spinosad resistance in the
houseßy, Musca domestica, is due to a recessive factor on
autosome 1. Pestic. Biochem. Physiol. 75: 1Ð7.
Shono, Y., R. Arakawa, M. Hirano, and Y. Abe. 1993. Effect
of pyriproxyfen, fenitrothion and permethrin on the ßy
pupal parasitoids, Spalangia endius and Nasonia vitripennis. Jpn. J. Sanit. Zool. 44: 29 Ð32.
Yapabandara, A.M.G.M., and C. F. Curtis. 2004. Control of
vectors and incidence of malaria in an irrigated settlement scheme in Sri Lanka by using the insect growth
regulator pyriproxyfen. J. Am. Mosq. Control Assoc. 20:
395Ð 400.
Received 13 October 2011; accepted 10 January 2012.