VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

Assessing Insecticide Susceptibility of Laboratory Lutzomyia

longipalpis and Phlebotomus papatasi Sand Flies (Diptera:
Psychodidae: Phlebotominae)
DAVID S. DENLINGER,1 SAUL LOZANO-FUENTES,2 PHILLIP G. LAWYER,3
WILLIAM C. BLACK IV,2 AND SCOTT A. BERNHARDT1,4

J. Med. Entomol. 52(5): 1003–1012 (2015); DOI: 10.1093/jme/tjv091

ABSTRACT Chemical insecticides are effective for controlling Lutzomyia and Phlebotomus sand fly
(Diptera: Psychodidae) vectors of Leishmania parasites. However, repeated use of certain insecticides
has led to tolerance and resistance. The objective of this study was to determine lethal concentrations
(LCs) and lethal exposure times (LTs) to assess levels of susceptibility of laboratory Lutzomyia longipal-
pis (Lutz and Nieva) and Phlebotomus papatasi (Scopoli) to 10 insecticides using a modified version of
the World Health Organization (WHO) exposure kit assay and Centers for Disease Control and Preven-
tion (CDC) bottle bioassay. Sand flies were exposed to insecticides coated on the interior of 0.5-gallon
and 1,000-ml glass bottles. Following exposure, the flies were allowed to recover for 24 h, after which
mortality was recorded. From dose–response survival curves for L. longipalpis and P. papatasi generated
with the QCal software, LCs causing 50, 90, and 95% mortality were determined for each insecticide.
The LCs and LTs from this study will be useful as baseline reference points for future studies using the
CDC bottle bioassays to assess insecticide susceptibility of sand fly populations in the field. There is a
need for a larger repository of sand fly insecticide susceptibility data from the CDC bottle bioassays, in-
cluding a range of LCs and LTs for more sand fly species with more insecticides. Such a repository would
be a valuable tool for vector management.

KEY WORDS Phlebotomus papatasi, Lutzomyia longipalpis, insecticide resistance, CDC bottle
bioassay, WHO

Since their introduction in the 1940s, synthetic chemi- insensitivity and metabolic detoxification resistance to
cal insecticides remain an effective tool for controlling all classes of synthetic insecticides in all major vector
insects that are vectors of disease agents (Hemingway species (Nauen 2007, Rivero et al. 2010). Acquiring
and Ranson 2000, World Health Organization [WHO] data on vector species’ susceptibility to insecticides will
2006). Unfortunately, insecticides have been used indis- support the strategies directed at effectively managing
criminately, exerting tremendous selective pressure for these vector populations (Surendran et al. 2005). The
insecticide resistance (Feyereisen 1995, WHO 2006). following two techniques are commonly used to mea-
The insecticide resistance phenotype is defined as a sure a vector species’ susceptibility to insecticides: 1)
heritable, genetic change in response to insecticide ex- the WHO exposure kit bioassay and 2) the Centers for
posure (Feyereisen 1995, Scott 1999, Hemingway et al. Disease Control and Prevention (CDC) bottle bioassay
2002). Increasing the insecticide dosage in response to (CDC 2010, WHO 2013).
resistance only exacerbates the problems of resistance The WHO exposure kit bioassay is widely accepted
by increasing the frequency of the genetic trait(s) in a because it can measure insecticide susceptibility in
vector population (Feyereisen 1995). Two resistance many species of insect vectors worldwide (Braverman
phenotypes observed in the field are target site insensi- et al. 2004, Ocampo et al. 2011, Faraj et al. 2012, Aı̈zoun
tivity and metabolic detoxification resistance (Mallet et al. 2013, Chen et al. 2013). The assays can be run
1989, Brogdon and McAllister 1998a, Rivero et al. with live insects collected in the field or with their prog-
2010). Today, there is evidence of target site eny reared in the laboratory. The WHO bioassay is a
standardized protocol that consists of an exposure kit
containing tubes lined with filter papers that are impreg-
1
Department of Biology, Utah State University, Logan, Utah. nated with a specific concentration of an insecticide
2
Department of Microbiology, Immunology, and Pathology, Colo- (WHO 1998, 2013). Despite its accepted use, the WHO
rado State University, Fort Collins, Colorado, Saul.
3
bioassay is expensive, filter papers are not available for
Laboratory of Parasitic Diseases, Intracellular Parasite Biology some insecticides, and there is a limited range of con-
Section, National Institute of Allergy and Infectious Disease, National
Institutes of Health, Bethesda, MD. centrations that can be purchased for some insecticides
4
Corresponding author, e-mail: [email protected]. (Perea et al. 2009, Aı̈zoun et al. 2013).
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
V
All rights reserved. For Permissions, please email: [email protected]
1004 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 5

The CDC bottle bioassay is an inexpensive and por- malathion and propoxur resistance. Both of these insec-
table alternative to the WHO bioassay, especially in re- ticides have been extensively used in this region as part
gions where there is little money to implement the of the antimalaria mosquito control program (Hassan
WHO bioassay (Perea et al. 2009, Aı̈zoun et al. 2013). et al. 2012).
The CDC bottle bioassay requires fewer test insects Many of the examples demonstrating reduced insec-
than the WHO bioassay (Aı̈zoun et al. 2013). The pro- ticide susceptibility in sand flies have been determined
tocol consists of coating the interior of a glass bottle using the WHO bioassay. However, a few studies have
with an insecticide that has been diluted in a solvent. used the CDC bottle bioassay to measure the suscepti-
The solvent is then allowed to evaporate, leaving the in- bility status of sand fly populations to insecticides (San-
secticide coated to the glass surface. Once the bottles tamarı́a et al. 2003, Alexander et al. 2009, Henriquez
are treated, insects are introduced into the bottles and et al. 2009). These studies have been completed en-
exposed to the insecticide for a specified amount of tirely in the New World. The CDC bottle bioassay is
time (Brogdon and McAllister 1998b, CDC 2010, preferred over the WHO bioassay because the suscep-
Aı̈zoun et al. 2013). Insect mortality can be scored at tibility results can be generated quickly, the bottles can
distinct time intervals during the exposure test (e.g., ev- be prepared with any insecticide, the results are repro-
ery 15 min for 1-h), and percent mortality at each time ducible with fewer insects and fewer replicates, and
interval is plotted (Brogdon and McAllister 1998b). the results allow one to infer the detoxification mecha-
The CDC bottle bioassay can also be used as an end- nism conferring resistance (Santamarı́a et al. 2003).
point assay where mortality is only measured at the end It is imperative to develop expansive baseline suscep-
of the exposure test. Susceptibility is measured by sim- tibility data to different insecticides in different sand fly
ply comparing mortality rates between insect popula- species and in flies from different geographic regions
tions (Perea et al. 2009). (CDC 2010). In addition, these bioassays require base-
Sand flies (Diptera: Psychodidae: Phlebotominae) line data from known susceptible sand fly populations
are among the insect vectors that require resistance to assess insecticide-susceptibility in field populations
monitoring because they have been actively targeted and for the calculation of relative risk ratios (e.g., lethal
with insecticides. Many sand fly species in the genera concentration causing 50% mortality [LC50] in a field
Lutzomyia and Phlebotomus are capable of vectoring population / LC50 control population). These data will
Leishmania parasites, infection with which causes leish- provide vector management programs the information
maniasis, a disease currently infecting millions of peo- necessary to ensure appropriate and effective insecti-
ple worldwide (Guerin et al. 2002, Rutledge and Gupta cide application (Maharaj 2011). Potentially, the CDC
2009). To control sand flies, populations around the bottle bioassay is one tool that could be incorporated
world have been exposed to the four main classes of in- into sand fly surveillance programs to a greater extent
secticides— 1) organochlorines, 2) organophosphates, worldwide, especially in regions where Leishmania
3) carbamates, and 4) pyrethroids—via residual spray- transmission is a concern.
ing, ultra-low volume spraying, insecticide-treated The objective of this study was to quantify, using a
clothing, and insecticide-treated nets. These exposures modified version of the WHO exposure kit assay and
are either intentional in directed vector control efforts the CDC bottle bioassay, the susceptibility of laboratory
or are inadvertent as part of vector control efforts tar- L. longipalpis and P. papatasi to 10 insecticides that are
geted against other insect vectors (Alexander and Mar- incorporated globally in vector control efforts. Specifi-
oli 2003, Surendran et al. 2005, Alexander et al. 2009, cally, for each insecticide, a dose–response survival
Henriquez et al. 2009, Rutledge and Gupta 2009, curve was produced. From each curve, LC50, LC90,
Dinesh et al. 2010, Faraj et al. 2012, Hassan et al. and LC95 values were determined. These doses can
2012, Saeidi et al. 2012). now be used for comparison in future studies to assess
Some sand fly populations have been found to be sand fly susceptibility to insecticides.
tolerant or resistant to the insecticides used in the Mid-
dle East, southern Asia, and South America. In Montes
Claros, Brazil, 29 of 80 (36.3%) Lutzomyia longipalpis
Materials and Methods
(Lutz and Nieva) survived a 0.05% deltamethrin expo-
sure (Alexander et al. 2009). In a Delft Island popula- Sand Flies. Insecticide-susceptible L. longipalpis
tion from Sri Lanka, 11 of 80 Phlebotomus argentipes and P. papatasi sand fly colonies at Utah State Univer-
Annandale & Brunetti (14%) had insensitive acetylcho- sity (USU) were derived from long-established colonies
linesterase, and 20 (25%) had elevated esterases, of maintained at the Walter Reed Army Institute of
which both of these findings are associated with resis- Research (Silver Spring, MD). The original colonies
tance to malathion (Surendran et al. 2005). P. argen- are >30 years old and have never been exposed to
tipes was found to be DDT-resistant throughout the insecticides. All life stages were reared at USU at 25 C,
Muzaffarpur, Vaishali, and Patna districts of the Bihar 85% relative humidity, and a photoperiod of 16:8 (L:D)
state, India, and in the Amahibelha village of the Sun- h according to methods described by Lawyer et al.
sari district, Nepal, as only 43 and 62% of populations (1991) and Modi and Rowton (1999). Larvae were fed
died from DDT exposure, respectively (Dinesh et al. a composted 1:1 mixture of rabbit feces and rabbit
2010). In the Surogia village of Khartoum State, Sudan, food (Young et al. 1981; Volf and Volfova 2011). Adults
51 Phlebotomus papatasi (Scopoli) (79.7%) had insensi- were provided 30% sucrose–water solution daily on
tive acetylcholinesterase, which is associated with saturated cotton balls, and adult female L. longipalpis
September 2015 DENLINGER ET AL.: SAND FLY INSECTICIDE SUSCEPTIBILITY 1005

and P. papatasi were blood-fed on anesthetized mice insecticide by swirling the acetone:insecticide solution
placed inside holding cages twice weekly. on the bottom, on the sides, and on the lid. The bottle
Insecticides. Ten technical-grade insecticides were was then placed on a mechanical bottle roller under a
used in this study: four pyrethroids [cypermethrin chemical hood for 30 min to dry. During this time, the
(Sigma-Aldrich, St. Louis, MO), deltamethrin (Sigma- lids were slowly loosened to allow the acetone to evapo-
Aldrich, St. Louis, MO), lambda(k)-cyhalothrin (Sigma- rate. After 30 min, the caps were removed, and the bot-
Aldrich, St. Louis, MO), and permethrin (Chem tles were rolled until all of the acetone had evaporated.
Service, Inc., West Chester, PA)]; three organophos- The bottles were then left open to dry overnight. For
phates [chlorpyrifos (Sigma-Aldrich, St. Louis, MO), each test replicate, one bottle serving as a control was
fenitrothion (Sigma-Aldrich, St. Louis, MO), and mala- coated with either 7.57 or 4.0 ml of acetone, depending
thion (Chem Service, Inc., West Chester, PA)]; two car- on its volume. All bottles were reused throughout the
bamates [bendiocarb (Sigma-Aldrich, St. Louis, MO), duration of the experiment. To clean a bottle with
and propoxur (Sigma-Aldrich, St. Louis, MO)]; and the residual insecticide, the bottle and lid was first triple-
organochlorine DDT (Sigma-Aldrich, St. Louis, MO). rinsed with acetone; filled with warm, soapy water;
The concentrations of each insecticide to which L. drained; rinsed and filled with cold water; drained; and
longipalpis and P. papatasi were exposed are provided autoclaved for at least 20 min. After being autoclaved,
in Table 1. The diagnostic doses for Anopheles and the bottles were left to dry for at least one day before
Aedes mosquitoes were used as starting reference being used again (CDC 2010). Each cleaned bottle also
points for initial insecticide exposure (CDC 2010). underwent testing to determine the presence of resid-
Concentrations higher and lower than these diagnostic ual insecticide. Ten sand flies were aspirated into each
doses were determined to derive the dose–response bottle and were left in the bottle for at least 3 h. If no
survival curves for the two sand fly species. All insecti- mortality was observed at the end of the 3 h, the bottles
cide dilutions were prepared in acetone, stored in glass were cleared and allowed to be reused. If mortality was
bottles, wrapped in aluminum foil, and refrigerated observed, the bottles were cleaned again and retested
while not being used (CDC 2010). until no mortality was observed.
Preparation of Exposure Bottles. On the day Insecticide Exposure Tests. Approximately 12 h
prior to exposing the sand flies, 0.5-gallon glass bottles after the bottles were prepared with insecticide, adult
(1,892.5 ml) (unknown maker) or 1,000-ml glass bottles sand flies at least 2 d posteclosion were aspirated from
(Fisher Scientific, Pittsburgh, PA) were prepared by the main colony and gently blown into each bottle: 40–
coating them with insecticide. For both bottle sizes, the 50 flies into each 0.5-gallon bottle and 20–30 flies into
concentration of insecticide in each bottle was deter- each 1,000-ml bottle. Approximately equal numbers of
mined to be X mg per bottle (CDC 2010). For a 250-ml un-fed female and male flies were used for each repli-
bottle, 1 ml of insecticide at 10 mg insecticide/ml ace- cate. At least three replicates were completed for each
tone gives a concentration of 10 mg/250 ml bottle. To concentration of every insecticide.
maintain an equivalence of 10 mg insecticide/250 ml Both species were exposed for the same length of
bottle to compensate for the larger bottle sizes, 4.0 ml time to each insecticide. In preliminary tests, exposure
of 10 mg insecticide/ml acetone is needed to coat the time for all 10 insecticides was 60 min, but it was soon
interior of the 1,000-ml bottle, and 7.57 ml of 10 mg discovered that for some insecticides, 60 min of expo-
insecticide/ml acetone is needed to coat the interior of sure was either too short or too long because sand fly
the 0.5-gallon bottle. The bottles were coated with survival was nearly 0 or 100% for most of the

Table 1. Concentrations of 10 insecticides used in the CDC bottle bioassays to expose L. longipalpis and P. papatasi sand flies

Insecticide class Insecticide Species Concentration (mg insecticide per bottle)

Pyrethroid Cypermethrin L. longipalpis 1, 5, 10, 25, 50, 100, 125, 150

P. papatasi 0.5, 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250
Deltamethrin L. longipalpis 0.01, 0.1, 1, 5, 10, 25, 50, 75
P. papatasi 5, 10, 25, 50, 100, 125, 150, 200
k-Cyhalothrin L. longipalpis 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 75
P. papatasi 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50
Permethrin L. longipalpis 1, 5, 10, 25, 50, 75, 100
P. papatasi 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250
Organophosphate Chlorpyrifos L. longipalpis 0.01, 0.05, 0.1, 0.5, 1, 5, 10
P. papatasi 0.001, 0.01, 0.05, 0.1, 0.5, 1, 5, 10
Fenitrothion L. longipalpis 0.1, 0.5, 1, 3, 5, 10, 25, 50
P. papatasi 0.1, 0.5, 1, 3, 5, 10, 25, 50
Malathion L. longipalpis 5, 8, 10, 12, 15, 25, 50, 100
P. papatasi 10, 25, 50, 100, 125, 150
Carbamate Bendiocarb L. longipalpis 0.01, 0.1, 1, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200
P. papatasi 0.01, 0.1, 0.5, 1, 5, 10, 25
Propoxur L. longipalpis 0.01, 0.1, 0.5, 1, 5, 10, 25, 50, 75, 100
P. papatasi 0.01, 0.1, 0.5, 1, 5, 10, 25, 50, 75
Organochlorine DDT L. longipalpis 10, 25, 50, 75, 100, 150, 200, 300, 350, 450
P. papatasi 5, 10, 25, 50, 75, 100, 150, 200, 300, 350
1006 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 5

Table 2. Length of exposure of L. longipalpis and P. papatasi cyhalothrin, and permethrin during and after exposure.
to 10 insecticides with the CDC bottle bioassay This was observed predominantly at the higher concen-
trations of each insecticide. Neither species shed its
Insecticide Exposure time (Min) legs when exposed to organophosphates, carbamates,
Cypermethrin 60 or DDT. In addition, for the pyrethroids, both L. longi-
Deltamethrin 60 palpis and P. papatasi experienced the “knockdown
k-cyhalothrin 60 effect,” evident by involuntary movements and muscle
Permethrin 60 spasms, during insecticide exposure and during the ini-
Chlorpyrifos 60
Fenitrothion 30 tial recovery time in the holding containers (Martins
Malathion 60 et al. 2009). At lower concentrations of the four pyreth-
Bendiocarb 30 roids, many sand flies were able to recover from the
Propoxur 30 knockdown (no convulsions or erratic movements) by
DDT 120
the completion of the 24-h holding period. At higher
pyrethroid concentrations, sand flies succumbed to
insecticide concentrations (Brogdon and McAllister muscle spasms, convulsions, and paralysis.
1998b). Therefore, the range of exposure times was It was also observed that the time required for the
adjusted to 30 or to 120 min, depending on unexpected carbamates, organophosphates, and organochlorine
and actual sand fly survival rates (Table 2) (CDC (DDT) to cause mortality differed. The carbamates
2010). were lethal very quickly, causing death only a few
The sand flies were captured after insecticide expo- minutes after the sand flies were aspirated into the bot-
sure via mechanical aspiration, released into 1-pint tles. This quick lethality necessitated a reduction in the
cardboard containers with a fine mesh screen top, and exposure time of both sand fly species to the carba-
kept under the same temperature, light, and humidity mates (Table 2). On the other hand, the three organo-
environment as the main untreated colonies. A cotton phosphates and DDT caused delayed mortality. Many
ball saturated with 30% sugar–water was placed on the sand flies appeared physically healthy after exposure to
top of each container as an energy/water source. Using these insecticides, but died during the 24-h holding
procedures established for mosquitoes, sand flies were period.
held in these containers for 24 h prior to mortality Survival Curves. A dose–response survival regres-
being recorded (Saavedra-Rodriguez et al. 2008). Mor- sion analysis was performed for L. longipalpis and
tality was scored as a complete cessation of movement P. papatasi to estimate LC50, LC90, and LC95 for all 10
(Perea et al. 2009). A 24-h holding period was used insecticides. Figure 1 shows each species’ survival curve
because in some preliminary experiments, many of the for cypermethrin (pyrethroid), chlorpyrifos (organo-
sand flies that appeared physically affected, and would phosphate), propoxur (carbamate), and DDT (organo-
have been scored as dead at the end of a 30-, 60-, or chlorine). These graphs were produced in GraphPad
120-min exposure period as described in Brogdon and Prism (version 6.0, GraphPad Software Inc., San
McAllister (1998b), recovered after this 24-h period. Diego, CA). Table 3 shows the QCal logistic regression
If mortality in the control group ranged between 5 parameters and the extrapolated LC50, LC90, and LC95
and 20%, mortalities in the experimental bottles of that values for each insecticide for both species. For many
test group were corrected using Abbott’s formula insecticides, the LC95 was substantially greater than the
(CDC 2010). Abbott’s formula was not used to correct LC90 (e.g., P. papatasi’s LC90 for cypermethrin was
experimental mortalities if the control group mortality 73.279 mg cypermethrin per bottle, while its LC95 for
was <5%. If control group mortalities exceeded 20%, cypermethrin was 150.010 mg cypermethrin per bottle),
the entire testing replicate was not used (Saeidi et al. which may be attributed to the sigmoidal shape of the
2012). logistic curve, where it takes much higher doses to
Survival Curves. Using the QCal software, a dose– reach a smaller percentage change in mortality (i.e.,
response survival curve was created for each insecticide LC90 to LC95) nearing the 100% mortality asymptote.
(Lozano-Fuentes et al. 2012). This software can be Pyrethroids. L. longipalpis and P. papatasi have very
used for any insect vector with data from insecticide similar LC50’s for cypermethrin, roughly 9.0 mg cyper-
bioassays. The QCal software also uses a logistic regres- methrin per bottle; however, P. papatasi has an LC95
sion model to generate LC50s, LC90s, and LC95s for more than twice as large as L. longipalpis (Table 3).
each insecticide. Mortalities corrected with Abbott’s For deltamethrin, L. longipalpis has a 10-fold lower
formula were rounded to the nearest whole fly. For LC50 than P. papatasi (Fig. 2A) and a much lower LC90
example, a cohort of 30 flies had an empirical mortality and LC95 than P. papatasi (Fig. 2B; Table 3). L. longi-
of 80% (24 flies died). If 80% was Abbott’s-corrected to palpis and P. papatasi have very similar lethal concen-
78.1% mortality, then 23.43 flies died. In QCal, a mor- tration values for lambda-cyhalothrin, and both species
tality of 23 flies of 30 was recorded. are very susceptible as their LC50, LC90, and LC95 val-
ues are <20.0 mg lambda-cyhalothrin per bottle, which
are the lowest LC95 values for of the four pyrethroid
Results
insecticides (Table 3). For permethrin, P. papatasi has
Physical Observations. Both L. longipalpis and a LC50, LC90, and LC95 that are at least twice as
P. papatasi sand fly species shed their legs when large compared with those same LC values of
exposed to cypermethrin, deltamethrin, lambda- L. longipalpis.
September 2015 DENLINGER ET AL.: SAND FLY INSECTICIDE SUSCEPTIBILITY 1007

Fig. 1. L. longipalpis and P. papatasi dose–response survival curves to cypermethrin (pyrethroid), chlorpyrifos
(organophosphate), propoxur (carbamate), and DDT (organochlorine).

Table 3. QCal logistic regression parameters and lethal concentration (LC) values causing 50, 90, and 100% mortality in L. longipal-
pis and P. papatasi exposure to 10 insecticides with the CDC bottle bioassay

Insecticide Species LC50 (mg insecticide LC90 (mg insecticide per bottle) LC95 (mg insecticide per bottle)
per bottle) [LL, UL]* [LL, UL]* [LL, UL]*

Cypermethrin L. longipalpis 8.955 [7.888, 10.167] 41.851 [35.499, 49.338] 70.704 [57.530, 86.886]
P. papatasi 8.897 [7.499, 10.556] 73.279 [61.313, 87.584] 150.010 [120.265, 187.354]
Deltamethrin L. longipalpis 0.922 [0.637, 1.334] 28.707 [18.291, 45.056] 92.434 [51.594, 165.571]
P. papatasi 9.907 [8.165, 12.020] 90.244 [67.938, 119.869] 191.290 [130.804, 279.779]
k-Cyhalothrin L. longipalpis 0.232 [0.189, 0.284] 5.001 [3.627, 6.895] 14.215 [9.487, 21.298]
P. papatasi 0.269 [0.217, 0.334] 3.654 [2.625, 5.087] 8.873 [5.863, 13.430]
Permethrin L. longipalpis 17.069 [14.889, 19.570] 82.402 [65.957, 102.946] 140.752 [105.890, 187.073]
P. papatasi 41.344 [37.233, 45.906] 188.579 [162.796, 218, 438] 315.955 [261.648, 381.572]
Chlorpyrifos L. longipalpis 0.458 [0.377, 0.557] 5.734 [4.058, 8.099] 13.538 [11.695, 29.020]
P. papatasi 0.327 [0.256, 0.419] 6.417 [4.102, 10.037] 17.653 [10.135, 30.774]
Fenitrothion L. longipalpis 0.347 [0.277, 0.434] 2.655 [1.933, 3.647] 5.306 [3.549, 7.934]
P. papatasi 1.368 [1.173, 1.595] 7.334 [5.684, 9.489] 13.007 [9.478, 17.850]
Malathion L. longipalpis 8.432 [8.004, 8.883] 13.815 [12.914, 14.779] 16.340 [14.957, 17.852]
P. papatasi 20.011 [17.277, 23.176] 77.008 [63.459, 93.447] 121.778 [94.869, 156.319]
Bendiocarb L. longipalpis 0.986 [0.737, 1.318] 38.961 [29.312, 52.159] 136.047 [94.292, 196.311]
P. papatasi 0.289 [0.232, 0.359] 2.507 [1.875, 3.353] 5.229 [3.632, 7.529]
Propoxur L. longipalpis 3.837 [2.860, 5.148] 75.446 [46.150, 123.347] 207.763 [112.101, 385.060]
P. papatasi 5.502 [4.524, 6.692] 39.135 [28.126, 54.451] 76.264 [50.729, 114.652]
DDT L. longipalpis 28.364 [23.173, 34.716] 218.581 [166.052, 287.724] 437.685 [303.506, 631.249]
P. papatasi 15.047 [11.321, 19.997] 295.979 [196.684, 445.412] 815.173 [463.219, 1434.541]
*LL, lower 95% confidence limit; UL, upper 95% confidence limit.

Organophosphates. Both sand fly species are highly the LC50’s for both species to chlorpyrifos are <0.5 mg
susceptible to chlorpyrifos and fenitrothion. The LC95’s chlorpyrifos per bottle. Like chlorpyrifos, both L. longi-
for both L. longipalpis and P. papatasi are <20.0 mg per palpis and P. papatasi are highly susceptible to fenitro-
bottle. Besides P. papatasi’s LC95 for bendiocarb and thion (Table 3) even with exposure times of 30 min.
both species LC95’s for lambda-cyhalothrin, these are the P. papatasi has a LC95 malathion that is approximately
lowest LC95’s for all 10 insecticides (Table 3). In addition, eight times larger than L. longipalpis’ LC95 for malathion.
1008 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 5

50
45 L. longipalpis
40 P. papatasi
μg inseccide / bole

35
30
25
20
15
10
5
0
Cypermethrin Deltamethrin λ-cyhalothrin Permethrin Chlorpyrifos Fenitrothion Malathion Bendiocarb Propoxur DDT
(P) (P) (P) (P) (OP) (OP) (OP) (C) (C) (OC)
500
450 L. longipalpis
400 P. papatasi
μg inseccide / bole

350
300
250
200
150
100
50
0
Cypermethrin Deltamethrin λ-cyhalothrin Permethrin Chlorpyrifos Fenitrothion Malathion Bendiocarb Propoxur DDT
(P) (P) (P) (P) (OP) (OP) (OP) (C) (C) (OC)

Fig. 2. Bar graphs of L. longipalpis and P. papatasi lethal concentrations causing 50% mortality (LC50) (2A) and 90%
mortality (LC90) (2B) for 10 insecticides. Error bars represent the lower and upper 95% CIs, as determined by QCal. Letters
in parentheses below each insecticide represent the insecticide class: P, pyrethroid; OP, organophosphate; C, carbamate; OC,
organochlorine.

Carbamates. L. longipalpis has a smaller LC95 than bottle for L. longipalpis and P. papatasi, respectively).
P. papatasi to all of the pyrethroids and to all of the These are the highest LC95’s for any of the 10 insecti-
organophosphates except lambda-cyhalothrin. For the cides evaluated in this study (Table 2).
carbamates, P. papatasi is more susceptible than L.
longipalpis to bendiocarb and propoxur. The exposure
Discussion
time for both species is 30 min. In preliminary tests for
the carbamates, 100% mortality was observed for all The objective of this study was to quantify insecti-
of the insecticide doses with a 60-min exposure time. cide susceptibility in laboratory L. longipalpis and
Therefore, the duration of exposure was reduced to 30 P. papatasi to 10 insecticides comprising four chemical
min, which was a sufficient amount of time to obtain classes using a modified version of the CDC bottle bio-
50, 90, and 95% mortality (Table 3). P. papatasi has a assay. It was demonstrated that this modified CDC bot-
LC95 for bendiocarb that is 26 times lower than L. tle bioassay is an effective tool for measuring the
longipalpis’ bendiocarb LC95 (Table 3), and P. papatasi susceptibility of these two sand fly species to pyreth-
has a LC90 of bendiocarb that is 15 times lower than roid, organophosphate, carbamate, and organochlorine
L. longipalpis’ bendiocarb LC90 (Fig. 2B). Both species insecticides.
have a LC50 <1.0 mg bendiocarb per bottle (Fig. 2A). P. One important observation of this study was that dif-
papatasi has a much lower LC95 for propoxur ferent insecticide classes have different LTs. Organo-
(LC95 ¼ 76.264 mg propoxur per bottle) than L. longi- phosphate insecticides caused delayed mortality, while
palpis (LC95 ¼ 207.763 mg propoxur per bottle). How- carbamate insecticides caused mortality extremely
ever, P. papatasi does have a greater LC50 to propoxur quickly, although both insecticide classes have similar
than does L. longipalpis (Fig. 2A). modes of action: inhibiting the acetylcholinesterase
Organochlorine. In preliminary tests with DDT, 60 enzyme from hydrolyzing acetylcholine (Fukuto 1990).
min was insufficient to quantify 50, 90, and 95% mor- Despite the differences in kill rates for carbamates and
tality with all of the insecticide doses. Therefore, the organophosphates, L. longipalpis and P. papatasi are
duration of exposure was increased to 120 min to allow most susceptible to the carbamates bendiocarb and
sufficient time to obtain these values for both L. Longi- propoxur and to the organophosphate fenitrothion. A
palpis and P. papatasi. Even with this extended expo- 30-min exposure to these insecticides is sufficient to
sure period, both species have very high LC95’s cause 100% mortality in these sand fly species. Aedes
(437.729 mg DDT per bottle and 815.173 mg DDT per and Anopheles mosquitoes both have diagnostic LTs of
September 2015 DENLINGER ET AL.: SAND FLY INSECTICIDE SUSCEPTIBILITY 1009

30 min for bendiocarb and fenitrothion using the CDC infection, which probe the skin of a vertebrate host,
bottle bioassay (CDC 2010). For vector control pro- have also been shown to transmit Leishmania parasites
grams aimed at targeting sand flies with synthetic insec- without a complete blood-meal. During probing, Leish-
ticides, bendiocarb, propoxur, and fenitrothion deserve mania metacyclic promastigotes are regurgitated in
attention for their efficacy. attempt of the female sand fly to clear her alimentary
Conversely, of the 10 insecticides tested, both L. canal of the Leishmania-secreted promastigote secre-
longipalpis and P. papatasi are least susceptible to tory gel (PSG) (“blocked-fly hypothesis”) (Bates 2007).
DDT. Even with an exposure time of 120 min, the lon- One future study could quantify and evaluate the
gest exposure time of the 10 insecticides, both species’ ability of surviving sand flies, with shed legs that have
LC95’s are very large: at least 400 mg DDT per bottle. been routinely exposed to pyrethroids or DDT, to per-
Unlike pyrethroids, which inhibit the sodium channels sist with probing vertebrate hosts. Rogers and Bates
involved in action potential propagation in the central (2007) demonstrated that female sand flies infected
nervous system and in the peripheral nervous system, with Leishmania metacyclic promastigotes are manipu-
DDT only blocks the sodium channels in the peripheral lated by the Leishmania to increase their biting persis-
nervous system (Davies et al. 2007). Only affecting the tence, leading to an increase in the number of parasites
peripheral nervous system requires more time and transmitted to the vertebrate host. We have observed
higher doses to cause excitatory paralysis that leads to that a loss of legs is a potential physical challenge for
death (Davies et al. 2007). Similar results have been the female sand fly. When other sand flies are in the
found in insecticide-susceptible Italian P. perniciosus vicinity of the female with shed legs during a blood
and P. papatasi, where the LT50’s and LT90’s for DDT feeding event, the female with shed legs would often
were longer compared with permethrin and lambda- lose her balance and would need to relocate to find a
cyhalothrin (Maroli et al. 2002). Also, Saeidi et al. suitable position to probe and blood-feed. Increased
(2012) found both insecticide-susceptible male and probing because of a physical challenge, in combination
female P. papatasi to have much longer LT50’s and with Leishmania manipulation, could theoretically
LT90’s to DDT than to permethrin, deltamethrin, cyflu- increase probing and the number of parasites vectored
thrin, and lambda-cyhalothrin. to a host. These hypothetical scenarios apply to pyreth-
For many years, DDT has been used worldwide to roid and DDT insecticides. Future studies with organo-
control sand flies by direct intervention or inadvertently phosphate and carbamate insecticides, which do not
as a collateral benefit of antimalaria campaigns (Kaul cause sand flies to shed their legs, and their effect on
et al. 1994, Alexander and Maroli 2003, Surendran surviving flies’ ability to probe and transmit Leishmania
et al. 2005, Kishore et al. 2006, Dinesh et al. 2010, warrant investigation as well.
Afshar et al. 2011, Faraj et al. 2012, Saeidi et al. 2012). Another observation of this study is the difference
Our results suggest that laboratory colonies of insecti- between the LC values of the Type I and Type II pyr-
cide-susceptible sand flies are not very susceptible to ethroid insecticides. Type I pyrethroids, including per-
DDT. Despite reports of sand fly tolerance and resist- methrin, have been described to cause sodium channel
ance to DDT in India, Iran, Nepal, and Turkey (WHO modifications that can last up to tens of milliseconds
1986, Kaul et al. 1994, Yaghoobi-Ershadi and Javadian and are better at causing knockdown in insects.
1995, Dinesh et al. 2010, Afshar et al. 2011), DDT’s Whereas Type II pyrethroids, including cypermethrin,
use for indoor residual spraying is still permitted deltamethrin, and lambda-cyhalothrin, cause sodium
(WHO 2007). The data from this study suggest that channel modifications that can last for many seconds
large doses of DDT are required, which may produce and are better at causing mortality in insects (Davies
strong selection pressure for resistance if it not applied et al. 2007). In this study, permethrin LC50’s for both
correctly or at appropriate times (Maharaj 2011). Com- L. longipalpis and P. papatasi were greater than cyper-
pounded with years of DDT use, and the potential for methrin, deltamethrin, and lambda-cyhalothrin LC50’s
underlying low levels of tolerance and resistance, field (Fig. 2A; Table 3). These findings at the LC50 support
populations of sand flies may be able to develop resist- previous research and are consistent with the physio-
ance to DDT more quickly than to other insecticides. logical differences between the two types of pyreth-
The shedding of legs in response to exposure to the roids in that it takes a higher concentrations of
four pyrethroids and DDT used in this study was evi- permethrin (Type I pyrethroid) to cause 50% mortality
dent for both L. longipalpis and P. papatasi. A similar than it does cypermethrin, deltamethrin, or lambda-
phenomenon was observed in L. longipalpis from Bra- cyhalothrin (Type II pyrethroids; Fletcher and Axtell
zil when exposed to permethrin, deltamethrin, and 1993, Jirakanjanakit et al. 2007).
lambda-cyhalothrin (Alexander et al. 2009). It is sug- One potential limitation of this study is that we used
gested that sand flies lacking one or more legs will be well-established, laboratory-adapted strains of L. longi-
unable to blood-feed effectively, which could subse- palpis and P. papatasi. All the female sand flies used in
quently reduce the potential to vector Leishmania par- this experiment were nulliparous. Comparisons of the
asites (Alexander et al. 2009). However, we have efficacy of the 10 insecticides between parous and nul-
consistently observed that laboratory L. longipalpis and liparous females would be extremely difficult. Through
P. papatasi exposed to pyrethroids that have shed one several years of laboratory observation, the percent sur-
or more legs are still capable of blood-feeding on anes- vival of gravid females after oviposition is extremely
thetized mice (unpublished data). Female sand flies low. This low survivorship presents a challenge to repli-
with shed legs, and with a mature Leishmania cate this experiment in parous females. In addition,
1010 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 52, no. 5

lethal concentrations and lethal times from insecticide- mortality curves (CDC 2010). To assess an insect popu-
susceptible laboratory and field-collected sand flies lations’ insecticide susceptibility status, diagnostic con-
may differ. This is why determining LCs and LTs for centrations and diagnostic times are used (CDC 2010).
susceptible laboratory strains are imperative for using a A diagnostic dose is the dose of an insecticide that kills
bioassay on field populations. Due to the highly varia- 100% of susceptible insects within a given time, the
ble conditions in nature, wild sand flies may exhibit dif- diagnostic time. Because we used our assays to produce
ferent development times, body sizes, longevity, dose–response survival curves, we were insufficiently
behaviors, and physiologies that make them more or able to determine diagnostic doses and diagnostic
less susceptible to insecticides (Rivero et al. 2010). times, even though doses causing 100% mortality were
In the initial development of the bottle bioassay by discovered. QCal cannot determine LC100 values (diag-
Brogdon and McAllister (1998b), 250-ml Wheaton bot- nostic doses) because an insecticide concentration
tles were used. These sized glass bottles are now rec- causing empirical 100% mortality cannot be deter-
ommended for all bottle assays (CDC 2010), although mined with a logistic regression because 100% mortal-
Alexander et al. (2009) used 200-ml Wheaton glass bot- ity is the upper asymptote. When put into the model,
tles. Another potential limitation of this study is that doses causing 100% mortality empirically are adjusted
owing to availability, 0.5-gallon and 1,000-ml glass bot- to causes mortality <100%. In time–response mortality
tles were used. For both L. longipalpis and P. papatasi, curves, mortality from an insecticide dose is measured
the deltamethrin, fenitrothion, chlorpyrifos, propoxur, at distinct time intervals during the exposure test. Per-
and DDT exposure trials were completed using both cent mortality is then plotted at each time interval
the 0.5-gallon and the 1,000-ml bottles. In these situa- (Brogdon and McAllister 1998b). A time–response
tions, when the bottles of one size were temporarily diagnostic dose is the lowest concentration of insecti-
unavailable (e.g., being cleaned for reuse), the other cide that causes 100% mortality in a specified exposure
size bottles were used. Therefore, the survival curves time period, between 30 and 60 min (CDC 2010). A
for these insecticides were generated by combining the diagnostic dose and diagnostic time can both serve as
mortalities from the 0.5-gallon and from the 1,000-ml reference points to understand the insecticide suscepti-
bottles. Comparatively, the mortalities between the bot- bility of a population of insects (WHO 1998).
tle sizes were similar, but often the percent mortality The baseline LCs and LTs for each insecticide were
was higher in the smaller 1,000-ml bottles than in the determined for laboratory L. longipalpis and P. papa-
0.5-gallon bottles. Despite an equal concentration of tasi and can now be incorporated as comparative refer-
insecticide and the even coating of insecticide, an ence points in field assays measuring the insecticide
unequal density of sand flies exposed, 20–30 and 40–50 susceptibility of sand flies. The CDC recommends
in the 1,000-ml and 0.5-gallon bottles, respectively, or determining diagnostic doses and diagnostic times for
potential differences in air volume to bottle surface an insecticide for each vector species in a specific geo-
area may explain the differing mortalities. graphic region (CDC 2010). Similarly, the LCs and LTs
Using a modified bioassay that combines aspects of from this experiment should not be considered univer-
the CDC bottle bioassay and the WHO exposure kit sal for L. longipalpis or P. papatasi. The data from this
bioassay allowed us to manipulate insecticide concen- study should be used only as a reference point for
trations to collect dose–response survival curve data future determinations of diagnostic doses and diagnos-
and to determine LCs and LTs. In our experiments, to tic times for different populations of Phlebotomus and
determine LCs and LTs, a 24-h holding period was Lutzomyia around the world.
incorporated for all 10 insecticides after insecticide Insecticide resistance management requires control
exposure (Saavedra-Rodriguez et al. 2008, Norris and programs to monitor for resistance (Surendran et al.
Norris 2011). A 24-h holding period was used because 2005, Badolo et al. 2012). Insecticide resistance result-
many of the sand flies that scored as dead following the ing from poor timing of insecticide application or from
insecticide exposure were able to completely recover. incorrect dosage applications can lead to ineffective
We suggest that the additional 24 h of recovery time vector control programs. Where insecticides are used,
provided more precise susceptibility data than seen resistance monitoring will ensure that appropriate
immediately at the end of the insecticide exposure insecticides and dosages are applied at times when they
period. Using the data from this study, a future direc- will most effectively control the target vectors (Maharaj
tion could still be to determine diagnostic doses and 2011). This modified version of the CDC bottle bioas-
diagnostic times for L. longipalpis and P. papatasi using say and the WHO exposure kit assay can help to inform
the CDC bottle bioassay for these same 10 insecticides. researchers and epidemiologists of sand fly populations
With these future data, researchers and public health that are resistant to specific insecticides or to entire
administrators will have diagnostic doses and diagnostic insecticide classes. It is vital to continue to further
times comparable with what is available for Aedes and develop integrated public health management pro-
Anopheles mosquitoes (CDC 2010). Having diagnostic grams that include effective vector surveillance and
doses and diagnostic times for phlebotomine sand flies control.
will enable field researchers to assess the insecticide
susceptibility status of sand fly populations in the wild
using the CDC bottle bioassay. Acknowledgments
The CDC recommends determining diagnostic con- We are grateful to J.C. McAllister and W.G. Brogdon
centrations and diagnostic times from time–response (CDC) for their careful review and constructive critique of
September 2015 DENLINGER ET AL.: SAND FLY INSECTICIDE SUSCEPTIBILITY 1011

the manuscript. We thank the many undergraduate research susceptibility of Phlebotomus (Paraphlebotomus) sergenti and
students in the Bernhardt laboratory for their assistance with Phlebotomus (Phlebotomus) papatasi in endemic foci of cuta-
maintaining and rearing the sand fly colonies. The mainte- neous leishmaniasis in Morocco. Parasit. Vectors 5: 51.
nance of SKH1 hairless mice (Charles River, Wilmington Feyereisen, R. 1995. Molecular biology of insecticide resis-
MA) and the experimental animal-use protocol was approved tance. Toxicol. Lett. 82/83: 83–90.
by Utah State University’s Institutional Animal-Care and Use Fletcher, M. G., and R. C. Axtell. 1993. Susceptibility of the
Committee. This work was supported by Utah State Univer- bedbug, Cimex lectularis, to selected insecticides and various
sity’s Office of Research and Graduate Studies. treated surfaces. Med. Vet. Entomol. 7: 69–72.
Fukuto, T. R. 1990. Mechanism of action of organophosphorus
and carbamate insecticides. Environ. Health Perspect. 87:
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