Development of a Larval Bioassay for Susceptibility of Cat Fleas
(Siphonaptera: Pulicidae) to Imidacloprid
M. K. RUST,1M. WAGGONER,1N. C. HINKLE,1,2N. MENCKE,3O. HANSEN,3M. VAUGHN,4
M. W. DRYDEN,5P. PAYNE,5B. L. BLAGBURN,6D. E. JACOBS,7T. BACH,3D. BLEDSOE,4
T. HOPKINS,8H. MEHLHORN,9AND I. DENHOLM10
J. Med. Entomol. 39(4): 671Ð674 (2002)
dramatic changes in the past 5 yr. With the advent of on-animal treatments with residual activity the
potential for the development of insecticide resistance increases. A larval bioassay was developed to
determine the baseline susceptibility of Þeld-collected strains of cat ßeas to imidacloprid. All four
laboratory strains tested showed a similar level of susceptibility to imidacloprid. Advantages of this
bioassay are that smaller numbers of ßeas are required because ßea eggs are collected for the test.
Insect growth regulators and other novel insecticides can also be evaluated. Using a discriminating
dose, the detection of reduced susceptibility in Þeld strains can be determined with as few as 40 eggs.
cat ßea, Ctenocephalides felis felis, larval bioassay, ßea eggs, imidacloprid
INTEGRATED PEST MANAGEMENT(IPM)approachestocat
and Dryden 1997) with a view to conserving the new
efÞcacy but prevent new ßea infestations from estab-
lishing for a period of weeks. It is important in the
management of ßea bite hypersensitivity that a high
level of ßea control is maintained because no other
effective therapeutic approaches are currently avail-
able (Carlotti and Jacobs 2000). Thus, it is likely that
ßea control will continue to be largely dependent on
the intelligent use of these new chemical classes. One
important aspect of conserving this valuable asset is
to these insecticides and to determine when alterna-
tive treatments may be necessary. Insecticide resis-
tance has typically been determined with adult ßeas
using various modiÞcations of the World Health Or-
1986, El-Gazzar et al. 1986, Rust and Dryden 1997,
reviewed by Bossard et al. 1998) and recently, by
topically applying insecticides (Moyses and Gfeller
2001). Many factors may affect contact exposure as-
says including relative humidity (Rust 1993), solvents
(Rust 1993), CO2exposure (El-Gazzar et al. 1988),
Þeld sites. To conduct adult bioassays it is generally
necessary to maintain the ßea strain on artiÞcial feed-
ing systems or live hosts such as cats until there are
sufÞcient numbers to test. With adult bioassays large
numbers of adult ßeas are required to obtain satisfac-
tory probit lines. Certain insecticides such as insect
against adult cat ßeas in tests such as the WHO bio-
eggs can be easily collected and shipped, insecticides
that are active against adults and larvae can be eval-
uated and sufÞcient numbers of eggs can be collected
from Þeld infestations, thereby avoiding laboratory
of late instar oriental rat ßea, Xenopsylla cheopis
(Rothschild), to various organophosphate, carbamate
and potential insect growth regulator (IGR) com-
pounds. Zakson-Aiken et al. (2000) exposed cat ßea
larvae to pretreated media. These techniques offer a
Use of Laboratory Animals” promulgated by the Committee on Care
and Use of Laboratory Animals of the Institute of Laboratory Animal
1Department of Entomology, University of California, Riverside,
Riverside, CA 92521Ð0314.
2Current address: Department of Entomology, 463 Biological Sci-
ences Building, University of Georgia, Athens, GA 30602.
3Bayer Animal Health, Agricultural Center Monheim, D-51368
4Bayer Corporation, Animal Health, Kansas City, KS 64120.
5Department of Diagnostic Medicine, Kansas State University,
Manhattan, KS 66502.
6Department of Pathobiology, Auburn University, Auburn, AL
7Department of Pathology and Infectious Diseases, The Royal
Veterinary College, North Mymms, HatÞeld, Herts AL9 7TA, UK.
8Bayer Australia, Limited, Beenleigh 4207 Queensland, Australia.
9Heinrich-Heine-University, Institute of Parasitology, 40225 Dus-
10Department of Biological & Ecological Chemistry, IACR-Roth-
amsted, Harpenden, Hertfordshire, AL5 2JQ, England.
0022-2585/02/0671Ð0674$02.00/0 ? 2002 Entomological Society of America
promising alternative to testing cat ßea adult suscep-
The objective of this study was to develop a larval
bioassay that could be used to determine the sensi-
ber of insects, and eliminating the need to maintain
Materials and Methods
Insects. Four laboratory strains of cat ßeas, Cteno-
cephalides felis felis (Bouche ´) (UCR, KSU, Auburn,
Monheim) were maintained on separate cats accord-
ing to a procedure modiÞed from Metzger and Rust
(1996). UCR is the ßea strain maintained for about 19
laboratory strains include KSU (Kansas State Univer-
sity), Auburn (Auburn University), and Monheim
(Bayer Animal Health laboratories in Monheim, Ger-
many). Cat ßea eggs were collected from trays un-
derneath cats supporting each strain. The eggs and
debris were passed through a series of four sieves, 10,
16, 20, and 60 mesh, with the eggs being retained on
the 60-mesh screen.
The eggs were placed on UCR larval ßea rearing
medium (1 part nutritive medium [0.15 dried beef
blood (AmericaÕs Laboratories, # NK3027034 SD He-
moglobin Powder, Omaha, NE)] to 0.75 ground dog
Bio Products ÐNuttrex 55, Milwaukee, WI]) to three
parts silica sand [30 mesh] by volume) and held at
80%RH and 26?C. Larvae of all four strains completed
development in 11Ð13 d and the cocoons and larval
medium were passed through a 16-mesh sieve to sep-
arate the cocoons. Adults began emerging 16Ð18 d
after egg collection. To maintain the strains on each
every 2 wk.
Larval Media Studies. To determine if solvents af-
fected the larval rearing media, 0.2 ml aliquots of
various solvents were applied to 2 g of larval rearing
media in 76 by 20 mm plastic vials (Sarstedt, Newton,
NC) and allowed to dry for at least 4 h before being
transferred to glass petri dishes (5 cm diameter by 1.5
cm). Twenty cat ßea eggs (UCR strain) were placed
on the media and the dishes and eggs were placed in
a chamber maintained at 80% RH and 26 ? 2?C. After
28 d, the number of adults that emerged was deter-
mined. The data were analyzed with a one-way anal-
ysis of variance (ANOVA) and means separated with
TukeyÕs honestly signiÞcant difference (HSD) (Jan-
Larval Bioassays. To determine the activity of imi-
dacloprid against larval cat ßeas, immature ßeas were
exposed to larval rearing media treated with serial
dilutions of imidacloprid (99.9% technical, Bayer An-
imal Health, Monheim, Germany). Two grams of me-
dia were placed in the bottom of each plastic Sarstedt
vial and treated with 2 ml of the serial dilutions of
0.0005, 0.0003, 0.0001, 0.00005, 0.00001, and 0.000005%),
providing treated media ranging from 30 to 0.05 ppm.
glass petri dishes (5 cm diameter by 1.5 cm); it is
important to use glass petri dishes instead of plastic to
reduce static and prevent eggs and media from ßying
out of or clinging to the sides of the petri dishes.
To determine the number of ßea eggs that hatched,
eggs were cemented to the upper inner surface of the
petri dish. A thin streak of glue (UHUStic, Saunders,
Winthrop, ME) was applied to the glass with a moist-
ened paint brush. Eggs and debris were put into a
the dried fecal blood and other debris. Eggs were
Attachment of the eggs was veriÞed by touching the
eggs with a Þne camelÕs-hair brush. Once the glue
This method (the so called ÔAdvantage Monitoring
methodÕ) permitted an accurate count of the number
of eggs that hatched and separated the eggs from the
adult fecal blood collected under the cats, ensuring
that immatures fed only on treated media. The petri
dish lids were transferred over the media and placed
into incubators maintained at 26 ? 2?C and 80% RH.
The number of hatched eggs was counted at day 5.
The media and cocoons were passed through a 16-
mesh screen at day 12 and the number of cocoons
counted. The cocoons were placed in a 2.5 cm diam-
eter by 4.5 cm plastic snap cap vial and a 5.5 cm
diameter disk of Whatman Þlter paper (Whatman,
Hillsboro, OR) was placed over the top and secured
with a snap cap lid. The vials and cocoons were re-
turned to a chamber maintained at 26 ? 2?C and 80%
All larval bioassays were conducted at UC River-
side. The number of adults that developed was
counted on day 28. Adult emergence data were ana-
lyzed by probit analysis with POLO program (Rob-
ertson and Preisler 1992).
Results and Discussion
Of the solvents applied to larval rearing media, ac-
etone, water, hexane, and methylene chloride pro-
duced the least amount of larval mortality (Fig. 1).
Larval rearing media treated with 100% EtOH or
MeOH produced signiÞcant larval kill. It is likely that
alcohols affect the nutritional quality of the yeast be-
cause larvae did develop on dried fecal blood treated
with alcohols (M.K.R., unpublished data). Silverman
component of larval rearing medium, increasing the
number of larvae maturing and decreasing the devel-
opmental time. Acetone was selected as a convenient
solvent in this study.
Four laboratory strains of cat ßeas (Auburn, Mon-
heim, KSU and UCR) were tested (Table 1). The
LD50s ranged from 0.21 to 0.70 ppm, but none of the
672JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 39, no. 4
strains had the same slope and intercept. The Mon-
heim, UCR, and KSU strains had parallel slopes (2.2Ð
3.1), whereas the Auburn strainÕs slope was about
twice as steep (4.9). The Auburn strain had consid-
erably less variability in response to imidacloprid.
Monheim and UCR were the most susceptible to imi-
dacloprid. The LD95ratios compared with the UCR
strain ranged from 1.08 to 1.81, indicating that there
were differences among these laboratory strains but
no signiÞcant tolerance or insecticide resistance. All
strains were susceptible to imidacloprid.
The larval bioassay provided a convenient method
of determining susceptibility of C. felis to imidaclo-
prid. By counting the number of hatched eggs an
accurate count of larvae being exposed to the treated
as 30% of the eggs to fail to hatch even in laboratory
2000). Consequently, it becomes important to accu-
rately determine the number of eggs failing to hatch
eggs. The difference of just several eggs can have a
hatch within 1Ð2 d and larvae fall into the media
reducing the likelihood of cannibalism by late instars.
Þeld-collected strains. Eggs can be collected off pets
by grooming them over large trays. Aliquots of 10Ð20
dose of imidacloprid such as 3 ppm to determine
susceptibility, eliminating the need to maintain and
rear cat ßea strains in the laboratory. Strains of ßeas
that survived exposure to 3 ppm imidacloprid would
be considered suspicious and the adults from the un-
treated control would be transferred to hosts for ad-
We thank Jody Hampton-Beesley and Kris Gilbert (UC
Riverside), Tracy Land (Auburn University), and Vicky
Smith (Kansas State University) for assisting in the care and
maintenance of the cat ßea strains. The study was supported
in part by Bayer Animal Health.
Bossard, R. L., N. C. Hinkle, and M. K. Rust. 1998. Review
of insecticide resistance in cat ßeas (Siphonaptera: Puli-
cidae). J. Med. Entomol. 35: 415Ð422.
Carlotti,D.N.,andD.E.Jacobs. 2000. Therapy,controland
Dermatol. 11: 83Ð98.
Development of cat ßea larvae in media treated with various solvents (x ? ? SEM).
fleas, C. f. felis, to larval media treated with imidacloprid
Susceptibility profiles of four laboratory strains of cat
StrainsnSlope ? SE LD50(95% CI)
4.9 ? 1.13
2.3 ? 0.30
3.1 ? 0.64
2.2 ? 0.33
0.70 (0.508Ð0.844) 1.50 (1.207Ð2.371)
0.40 (0.208Ð0.578) 2.04 (1.254Ð6.132)
0.36 (0.185Ð0.481) 1.22 (0.899Ð2.672)
0.21 (0.127Ð0.286) 1.13 (0.795Ð2.007)352
July 2002RUST ET AL.: LARVAL BIOASSAY FOR IMIDACLOPRID
Chamberlain, W. F., J. Maciejewska, and J. J. Matter. 1988.
Response of the larvae and pupae of the oriental rat ßea
(Siphonaptera: Pulicidae) to chemicals of different
chemical types. J. Med. Entomol. 81: 1420Ð1425.
Collart, M. G., and W. F. Hink. 1986. Development of re-
sistance to malathion in cat ßea (Siphonaptera: Pulici-
dae). J. Econ. Entomol. 79: 1570Ð1572.
El-Gazzar, L. M., J. Milio, P. G. Koehler, and R. S. Patterson.
Pulicidae). J. Econ. Entomol. 79: 132Ð134.
El-Gazzar, L. M., P. G. Koehler, and R. S. Patterson. 1988.
Factors affecting the susceptibility of the cat ßea, Cteno-
Jandel. 1995. SigmaStatforWindows95.Jandel,SanRafael,
Lawrence, W., and L. D. Foil. 2000. The effects of ßea egg
consumption on larval cat ßea (Siphonaptera: Pulicidae)
development. J. Vector Ecol. 25: 98Ð101.
Metzger, M. E., and M. K. Rust. 1996. Egg production and
emergence of adult cat ßeas (Siphonaptera: Pulicidae)
exposed to different photoperiods. J. Med. Entomol. 33:
Moyses, E. W., and F. J. Gfeller. 2001. Topical applications
as a method for comparing the effectiveness of insecti-
cides against cat ßeas (Siphonaptera: Pulicidae). J. Med.
Entomol. 38: 193Ð195.
Robertson, J. L., and H. K. Preisler. 1992. Pesticide bioas-
says with arthropods. CRC, Boca Raton, FL.
Rust,M.K. 1993. Insecticideresistanceinßeas,pp.18Ð26.In
Proceedings of International Symposium on Ectopara-
sites of Pets, Lexington, KY. Univ. Kentucky, Lexington.
Rust, M. K., and M. W. Dryden. 1997. The biology, ecology,
and management of the cat ßea. Annu. Rev. Entomol. 42:
Silverman, J., and A. G. Appel. 1994. Adult cat ßea (Sipho-
naptera: Pulicidae) excretion of host blood proteins in
relation to larval nutrition. J. Med. Entomol. 31: 265Ð271.
Zakson-Aiken, M., L. M. Gregory, and W. L. Schoop. 2000.
against larvae of the cat ßea (Siphonaptera: Pulicidae).
J. Med. Entomol. 37: 571Ð574.
Received for publication 22 October 2001; accepted 28 Jan-
674JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 39, no. 4