Persistence and inheritance of costs of resistance to imidacloprid in Colorado potato beetle.
ABSTRACT Reduced fitness among resistant versus susceptible individuals slows resistance evolution and makes it easier to manage. A loss of resistance costs could indicate novel adaptations or mutations contributing to resistance. We measured costs of resistance to imidacloprid in a Massachusetts resistant population compared with a Massachusetts susceptible population in 1999 in terms of fecundity, hatching success, egg development time, and sprint speed. Resistance was additive and seemed to be polygenic with high heritability. The fecundity cost appeared overdominant in 1999, and the hatch rate cost was partly recessive in 1999, but neither was significantly different from dominant or recessive. In 2004, we repeated our measures of resistance costs in Massachusetts in terms of fecundity and hatching success, and we added a new resistant population from Maine. In 2005, we compared development time of Maine resistant and the laboratory susceptible colony eggs. Significant fecundity costs of resistance were found in both population in both 1999 and 2004, and significant egg developmental time costs were found in 1999 and 2005. However, the hatching success costs of resistance were significant in 1999 and not apparent in 2004, suggesting some modification or replacement of the resistance genes in the intervening time.
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ABSTRACT: Select populations of Colorado potato beetle, Leptinotarsa decemlineata, in Wisconsin have recently become resistant to soil-applied neonicotinoids in potato. Sub-lethal insecticide concentrations persisting in foliage through the growing season may select for resistance over successive years of use. Over the two years of this study, our aim was to document the in-plant insecticide concentrations over time resulting from four different types of soil-applied insecticide delivery for thiamethoxam and imidacloprid in potato and to measure the impact upon L. decemlineata populations following treatments. After plant emergence, insect life stages were counted and plant tissue was assayed weekly for nine consecutive weeks using ELISA. Peak concentration of both imidacloprid and thiamethoxam occurred in the first sample week following plant emergence. Average concentration of both insecticides dissipated sharply over time as the plant canopy expanded 50 days after planting in all delivery treatments. Both insecticides were detected at low levels during the later weeks of the study. Among plant concentrations of both neonicotinoids were highly variable throughout the season. Populations of L. decemlineata continued to develop and reproduce throughout the period of declining insecticide concentrations. Sub-lethal, chronic exposure to soil-applied systemic insecticides resulting from these delivery methods may accelerate selection for resistant insects in potato.Pest Management Science 01/2014; · 2.74 Impact Factor
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ABSTRACT: Originally designed to reconcile the insecticide applications with biological control, the concept of integrated pest management (IPM) developed into the systems-based judicious and coordinated use of multiple control techniques aimed at reducing pest damage to economically tolerable levels. Chemical control, with scheduled treatments, was the starting point for most management systems in the 1950s. Although chemical control is philosophically compatible with IPM practices as a whole, reduction in pesticide use has been historically one of the main goals of IPM practitioners. In the absence of IPM, excessive reliance on pesticides has led to repeated control failures due to the evolution of resistance by pest populations. This creates the need to constantly replace failed chemicals with new compounds known as “the insecticide treadmill." In evolutionary biology, a similar phenomenon is known as The Red Queen Principle – continuing change is needed for a population to persevere because its competitors undergo constant evolutionary adaptation. The Colorado potato beetle, Leptinotarsa decemlineata (Say), is an insect defoliator of potatoes that is notorious for its ability to develop insecticide resistance. In the present article, we review four case studies from across the United States to demonstrate the importance of using IPM for sustainable management of a highly adaptable insect pest. Excessive reliance on often-indiscriminate insecticide applications and inadequate use of alternative control methods, such as crop rotation, appear to expedite evolution of insecticide resistance in its populations. Resistance to IPM would involve synchronized adaptations to multiple unfavorable factors, requiring statistically unlikely genetic changes. Therefore, integrating different techniques is likely to reduce the need to constantly replace failed chemicals with new ones.Pest Management Science 05/2014; · 2.74 Impact Factor
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ABSTRACT: CHAPTER 1 Striped cucumber beetle, Acalymma vittatum F., is the primary insect pest of cucurbit crops in the Northeastern United States. Adult beetles colonize squash crops from field borders, causing feeding damage at the seedling stage and transmitting the bacteria Erwinia tracheiphila Hauben et al. Conventional control methods rely on insecticide applications to the entire field, but surrounding main crops with a more attractive perimeter could reduce reliance on insecticides. Acalymma vittatum demonstrates a marked preference for Blue Hubbard squash (Cucurbita maxima Duchesne) over butternut squash (C. moschata Duchesne). Given this preference, Blue Hubbard squash has the potential to be an effective perimeter trap crop. We evaluated this system in commercial butternut fields in 2003 and 2004, comparing fields using perimeter trap cropping with Blue Hubbard to conventionally managed fields. In 2003 we used a foliar insecticide to control beetles in the trap crop borders, and in 2004 we compared systemic and foliar insecticide treatments for the trap crop borders. We found that using a trap crop system reduced or eliminated the need to spray the main crop area, reducing insecticide use by up to 94% compared to conventional control methods, with no increase in herbivory or beetle numbers. We also surveyed the growers who participated in these experiments and found a high level of satisfaction with the effectiveness and simplicity of the system. These results suggest that this method of pest control is both effective and simple enough in its implementation to have high potential for adoption amongst growers. ABSTRACT CHAPTER 2 Winter squash is a vital agricultural commodity in many parts of the world. In the Northeastern United States, the primary insect pest of these crops is the striped cucumber beetle, Acalymma vittatum F, which has traditionally been controlled with multiple full field pesticide applications. Recent studies have indicated that using a Blue Hubbard squash perimeter trap crop system (PTC hereafter) can reduce insecticide use by >90% in butternut squash, the primary winter squash grown in this region. This method involves dedicating a portion of the field to the trap crop. Despite the savings in insecticide costs, growers may be reluctant to give up field space for Blue Hubbard squash, which has a limited market. Finding a more marketable trap crop than Blue Hubbard would lower the barrier for adoption of this system. We tested eight varieties of three species of cucurbits for attractiveness to beetles relative to Blue Hubbard and butternut squash, and chose buttercup squash as the most promising replacement for Blue Hubbard. We compared the effect of a buttercup border, Blue Hubbard border, or control (no border) on beetle numbers, herbivory, and insecticide use. We found that buttercup squash performed equally well as Blue Hubbard as a trap crop, with up to 97% reduction in the total field area requiring insecticide compared to control fields. This study confirms the effectiveness of PTC systems and offers growers a more marketable trap crop.
INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT
Persistence and Inheritance of Costs of Resistance to Imidacloprid in
Colorado Potato Beetle
MITCHELL B. BAKER,1,2,3ANDREI ALYOKHIN,4ADAM H. PORTER,3DAVID N. FERRO,3
SHANA R. DASTUR,5AND NEHAL GALAL1
J. Econ. Entomol. 100(6): 1871Ð1879 (2007)
Reduced Þtness among resistant versus susceptible individuals slows resistance evo-
lution and makes it easier to manage. A loss of resistance costs could indicate novel adaptations or
mutations contributing to resistance. We measured costs of resistance to imidacloprid in a Massa-
chusetts resistant population compared with a Massachusetts susceptible population in 1999 in terms
of fecundity, hatching success, egg development time, and sprint speed. Resistance was additive and
the hatch rate cost was partly recessive in 1999, but neither was signiÞcantly different from dominant
or recessive. In 2004, we repeated our measures of resistance costs in Massachusetts in terms of
fecundity and hatching success, and we added a new resistant population from Maine. In 2005, we
fecundity costs of resistance were found in both population in both 1999 and 2004, and signiÞcant egg
developmental time costs were found in 1999 and 2005. However, the hatching success costs of
resistance were signiÞcant in 1999 and not apparent in 2004, suggesting some modiÞcation or re-
placement of the resistance genes in the intervening time.
Insecticide resistance is a major problem facing most
pest control programs (Denholm and Rowland 1992,
proposed strategy to delay the evolution of resistance
is to provide untreated plots within or among Þelds
(McGaughey and Whalon 1992; Tabashnik 1994;
Caprio 1998, Carrie `re and Tabashnik 2001). Suscep-
tible populations persist within these plots and mate
with any newly resistant genotypes that arise in ad-
jacent treated areas. However, a number of assump-
tions need to be met for this refuge-based strategy to
succeed. First, resistant alleles should be recessive, so
that insecticides kill heterozygotes. Second, in the
absence of pesticide, resistant alleles should be asso-
ciated with the decreased Þtness of resistant individ-
uals, so that they are at a selective disadvantage. Pref-
erably, such Þtness costs are themselves genetically
dominant, so that selection acts efÞciently against
both hetero- and homozygotes (Carrie `re and Tabas-
hnik 2001). Finally, there should be sufÞcient gene
ßow between resistant and susceptible populations,
curtailing production of resistant homozygotes.
expected to maintain pest populations susceptible to
insecticides (Bauer 1995, Carrie `re and Tabashnik
2001). However, it is important to remember that
resistance development is, by deÞnition, a dynamic
evolutionary process that could be likened to an arms
race between humans coming up with new toxins and
ways of their deployment and pests developing new
adaptations (Denholm and Rowland 1992). ModiÞca-
tion of Þtness costs and their inheritance, changes in
populations, and competition between resistance
genes may present additional obstacles to otherwise
sound resistance management strategies (McKenzie
and OÕFarrell 1993, Charpentier and Fournier 2001,
Therefore, sustainable use of chemical control
depends on a good understanding of resistance de-
velopment as an evolutionary process, not just on
characterization of resistant individuals and their
Colorado potato beetle, Leptinotarsa decemlineata
(Say), is a major pest in most potato (Solanum tubero-
sum L.)-growing areas of the world. This species has
a truly remarkable ability to develop resistance to a
variety of insecticides. The Þrst case of resistance was
beetle has lost susceptibility to a wide range of chem-
1Biology Department, Queens College of CUNY, 65-30 Kissena
Boulevard, Flushing, NY 11367.
2Corresponding author, e-mail: email@example.com.
versity of Massachusetts, Amherst, MA 01003.
4Department of Biological Sciences, 5722 Deering Hall, University
of Maine, Orono, ME 04469.
5Department of Biology, Franklin and Marshall College, P.O. Box
3003, Lancaster, PA 17604.
0022-0493/07/1871Ð1879$04.00/0 ? 2007 Entomological Society of America
icals, including the arsenicals, organochlorines, car-
bamates, organophosphates, and pyrethroids (For-
gash 1985; Ioannidis et al. 1991; Stewart et al. 1997).
Some of them failed after 1 yr (e.g., endrin) or even
during the Þrst year of use (e.g., oxamyl) (Forgash
In-furrow systemic applications of imidacloprid are
widely used for the Colorado potato beetle control.
Such applications maximize plant coverage and sig-
niÞcantly increase insecticide persistence in potato
foliage. Unfortunately, they also create strong selec-
tion pressure on beetle populations toward develop-
ing resistance to this compound (Olson et al. 2000,
efÞcient against Colorado potato beetles in most
places, isolated cases of its failure have been reported
States (Alyokhin et al. 2006, 2007; Mota-Sanchez et al.
onset of imidacloprid resistance in the Colorado po-
On one hand, decreased relative Þtness of resistant
genotypes in insecticide-free environments was ob-
(Argentine et al. 1989b; Trisyono and Whalon 1997;
Alyokhin and Ferro 1999a), although it has not yet
been documented for imidacloprid-resistant beetles.
However, resistance to a variety of insecticides in this
pest is inherited as a suite of incompletely dominant
traits (Argentine et al. 1989a, 1989b; Ioannidis et al.
1992; Rahardja and Whalon 1995). This includes one
population from Long Island, NY, that is resistant to
imidacloprid (Zhao et al. 2000a). In this study, we
investigate the inheritance of traits associated with
to wild-type, susceptible alleles, as measured by two
Þtness components, fecundity and egg-hatching suc-
cess, and whether those costs themselves seem to be
evolving. The presence of resistance costs will greatly
enhance the probability of success for the high-doseÐ
refuge approach to resistance management.
Materials and Methods
Susceptible Populations. Colorado potato beetles
susceptible to imidacloprid were taken from a labo-
ratory colony of 150Ð300 beetles originally collected
from a commercial Þeld treated with imidacloprid for
in group cages (65 by 48 by 48 cm) at 25Ð27?C and a
year, 30Ð50% of the colony was replaced with Þeld-
collected beetles from the least resistance wild pop-
ulation found in western Massachusetts to reduce in-
breeding and adaptation to cage conditions in
Resistant Populations. 1999 Experiments. The bee-
from an imidacloprid-treated Þeld in Hadley, MA, in
1999. Alternating generations of their progeny were
second instars with a 1-?l drop of 1.4 ? 10?5g/ml
imidacloprid dissolved in acetone, a dose that corre-
sponded roughly to the LD50of the colony. That
(measured by LD50) as the susceptible colony.
2004 Experiments. The beetles resistant to imida-
cloprid were collected as eggs from two Þelds in Had-
and four adjoining Þelds in Fryeburg, ME. The exper-
imental adults were drawn at random from ?200
adults from each location that had survived exposure
as second instars to a 1-?l drop of either 5 ? 10?5or
1 ? 10?4g/ml imidacloprid dissolved in acetone.
2005 Experiment. The beetles resistant to imidaclo-
prid were collected as eggs from the same four Þelds
in Fryeburg as the previous yearÕs study. The adults
used were drawn at random from ?200 adults from
2004 described above.
Crosses. A series of reciprocal crosses within and
between resistant and susceptible populations were
conducted to measure inheritance and Þtness costs of
1999 Experiments.Twenty-Þve males and 25 fe-
males were drawn randomly from the resistant and
and isolated by sex to preserve their virginity
(Alyokhin and Ferro 1999b). Twelve resistant virgin
females were individually paired with Þve resistant
virgin females were mated individually to eight resis-
tant and Þve susceptible males. All survived to pro-
were maintained in individual vented Nalgene boxes
26 ? 1?C and a photoperiod of 16:8 (L:D) h. The
beetles were provided with excised potato foliage
daily and any eggs were collected. A second genera-
offspring from 22 families from both parental and
2004 Experiments. Offspring of the Massachusetts
hood and isolated by sex upon emergence as adults.
each resistant population and the laboratory-suscep-
for up to 22 d (12Ð22), depending on when laying
primary means for measuring population- and family-
level resistance. Second instars weighing 6Ð8.5 mg
were assayed by direct topical application of a 1-?l
1872JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 100, no. 6
acetone on the abdomen. Seven concentrations rang-
ing from 1.6 ? 10?6to 1 ? 10?4g/ml plus an acetone
control were tested. Larvae were scored 24 h after
application, with mortality deÞned as failure to move
a leg for 10 s after the larvae is placed on its back.
DoseÐmortality curves were analyzed using Polo-PC
included the LD50results if the index of signiÞcance
for potency estimation, g (Finney 1971), was ?0.7 at
the 0.95 conÞdence level.
Baseline resistance was determined separately for
Þeld-collected and susceptible populations. Larval as-
says were carried out as above and LD50values and
95% conÞdence intervals (CIs) estimated using like-
lihood ratios in Polo-PC (LeOra Software 1987). In
addition, knockdown assays were carried out on the
adult beetles used in crosses in 2004 to compare rel-
ative resistance of Massachusetts, Maine, and suscep-
tible strains. Adults were given a single dose of 1 ?l of
imidacloprid dissolved in acetone at a concentration
of 1 ? 10?3g/ml. Beetles were considered knocked
being ßipped over in a petri dish lined with Þlter
were observed every 15 min for 8 h and then one Þnal
time after 24 h since application. After 24 h, beetles
were scored as alive or knocked down as described
Strain differences in resistance were tested using
analysis of variance (ANOVA) of offspring LD50val-
ues and planned orthogonal contrasts between each
as in Stone (1968) on a ?1 to 1 scale:
D ? (2RS ? RR ? SS)/(RR ? SS)
Standard errors were calculated after Preisler et al.
(1990). The 95% conÞdence interval for D is ?2 SE.
single-locus Mendelian inheritance were compared
expected mortality was the average of the hybrid and
parental LD50values (but see Preisler et al. 1990). In
regressed against the midpoint of the LD50values of
the parentsÕ families to measure heritability of resis-
tance. This is a standard midparent-offspring regres-
sion, but in the context of our resistance assays, it is
actually carried out between LD50values of the prog-
eny of grandparents and parents. Population shifts
from 1999 to 2004 were analyzed by ANOVA.
Fecundity, Developmental Time, and
1999 Experiments. All eggs from each day were
pooled for that pair and kept in a single 35-mm-diam-
eter plastic petri dish, held in the same incubator at
25?C and a photoperiod of 16:8 (L:D) h. In the Þrst
generation, eggs were collected until the female died,
from 8 to 42 d of egg laying in this sample. In the
second generation, eggs were collected for up to 21 d,
so that average rather than total fecundity was ana-
upon hatching, larvae were removed and the remain-
ing eggs were checked for up to three more days for
further hatching. The number of days to hatching for
eggs from each clutch for each pair was recorded in
the Þrst generation, and the mean, weighted by the
number hatching in each clutch, was calculated per
pair. In the second generation, fecundity (deÞned as
a total number of eggs laid) but not hatching success
and hatching success was calculated using the same
This is mathematically equivalent to the index of het-
of resistance cost (Roux et al. 2004) except that the
scale differs, varying from ?1 to 1, rather than from 0
2004 Experiments. Virgin adult offspring of resis-
tant, Þeld-collected Maine and Massachusetts beetles
virgin susceptible beetles. Pairs were housed as de-
scribed above, and eggs were collected daily for up to
21 d. All eggs from each day were pooled by pair and
kept in a single 35-mm-diameter plastic petri dish and
held in the same incubator at 25?C and a photoperiod
of 16:8 (L:D) h. After 20 d, the adults (aged 24Ð27 d)
were assayed using the knockdown trial described
above. Hatchlings were counted and removed twice a
day to reduce cannibalism. Hatch rates were arcsine
transformed to improve normality. Costs of resistance
in Massachusetts and Maine populations in 2004 were
compared using ANOVA and StudentÕs t-test to look
for signiÞcant differences between individual means.
To compare fecundity costs of resistance within Mas-
sachusetts populations among years, a subset of the
1999 clutches were used including only those laid in
the Þrst 20 d after pairing, to remove potential con-
founding effects of including older femalesÕ fecundity
from 1999. Persistence of the costs of resistance vis-
cess was analyzed using ANOVA of effects of year,
parental resistance, and their interaction.
2005 Experiment. Egg developmental time was
compared between resistant and susceptible popula-
tions from Maine. Five females from each population
were mated with a single male from the same popu-
lation. Egg masses were collected two to three times
per day, kept in single 35-mm-diameter plastic petri
dishes, and they were held in the same incubator at
observed two to four times per day, all hatchlings
removed, and the weighted average of the number of
hours to hatch calculated per pair. Development time
to hatching was compared using values from resistant
and susceptible populations from Massachusetts col-
lected in 1999.
December 2007BAKER ET AL.: IMIDACLOPRID RESISTANCE COSTS
This study was conducted in 2002. We adapted a
sprint-speed assay commonly used as a measure of
general physiological condition in vertebrates (Gar-
land 1994) for the Colorado potato beetle. Our
method relied on high temperature as an irritant to
stimulate locomotion. Individual 5Ð10-d-old beetles
were placed in the center of a paper disc set on a
Sybron Thermocline hot-plate. Because experiments
using locomotor performance often suffer from difÞ-
culty distinguishing behavioral motivation from abil-
ity, we conducted preliminary assays to Þnd appro-
not walk or waited before walking, and above that
temperature some beetles lost coordination to walk;
therefore, surface temperature was maintained at
60?C. Beetles were timed as they walked 7 cm from
thirty-six resistant and 105 susceptible beetles of both
sexes were tested. After log10transformation, walking
speeds were not signiÞcantly different from normal
(ShapiroÐWilk W ? 0.980, P ? 0.258, N ? 266). Walk-
Baseline Resistance of Tested Populations. LD50
values for Þeld-collected beetles were 5.2Ð21.8 times
(Table 1). Maine Þeld populations were signiÞcantly
in 2004, with no overlap between the 95% CIs of their
LD50values. There was also no overlap between 95%
CIs of Massachusetts resistant and susceptible beetles
no overlap between Maine and susceptible-strain CIs
of LD50in 2004 or 2005.
Knockdown trials of adults used in 2004 crosses
conÞrmed signiÞcant differences in resistance among
all three populations (Fig. 1). Mean knockdown time
varied among populations (ANOVA: F ? 42.312; df ?
2, 91; P ? 0.0001). Using TukeyÕs honestly signiÞcant
difference (HSD) test, all three populations were sig-
niÞcantly different from the other two in knockdown
time, with Massachusetts population being interme-
diate between Maine and susceptible populations.
Inheritance of Resistance. We assayed 4863 second
instars from 26 families in the Þrst 1999 generation.
Because LD50values of hybrid offspring were not
affected by the sex of the resistant parent (using pair-
wise StudentÕs t-test comparisons), they were pooled
into a single hybrid cross category. The mean resis-
tance (LD50) for each mating combination in the Þrst
generation is presented in Fig. 2. We assayed 5,329
individuals in 21 families in the second 1999 genera-
and susceptible laboratory-colony adults used in 2004
crosses. The box encloses the 25Ð75% quartiles, with the
median line drawn within, and the outlier whiskers include
the outer points up to 1.5 * interquartile range. There is no
box for Maine because all adults were not knocked down at
24 h. Similar letters denote means that are not signiÞcantly
different at ? ? 0.05 by using TukeyÕs HSD test.
0.05 by using TukeyÕs HSD test.
Box plots of LD50values of 1999 crosses. Similar
Table 1.Second instar LD50values of field and laboratory populations in micrograms per gram
Yr Strainn LD50
Massachusetts resistant Þeld
Massachusetts resistant Þeld
Maine resistant Þeld
Maine resistant Þeld
RR, resistance ratio.
1874JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 100, no. 6
tion. When the observed mortalities of the RS ? SS
backcross lines were compared with the expected
means of R ? S and S ? S lines for each dose, Þve of
seven single-dose chi-square tests were signiÞcant,
and the combined test of predicted values from a
single major gene was P(?2
itability of resistance estimated as the slope of the
regression of offspring with the midparental values
was h2? 0.86 (Fig. 3) (F ? 33.86; df ? 1, 19; P ?
The mean resistance (LD50) for each mating com-
bination in 2004 is presented in Fig. 4. We assayed
13,202 second instars from 59 families. The ln-trans-
formed LD50values differed signiÞcantly among pa-
not affected by the sex of the resistant parent (using
pair wise StudentÕs t-test comparisons), they were
pooled into a single hybrid cross category for each
geographical strain. Resistant pairsÕ offspring from
both Massachusetts and Maine had signiÞcantly
higher LD50values than the hybrid offspring, and
hybrids were signiÞcantly more resistant than off-
8)?0.0001 (Table 2). Her-
test. However, resistant pairs from Maine and Massa-
chusetts were not signiÞcantly different from each
other, although there was a slight trend for higher
resistance in Maine offspring.
Resistance in 2004 was similar and incompletely
dominant in Massachusetts and Maine populations,
and between 1999 and 2004 Massachusetts popula-
tions. In 1999, the degree of dominance in the Mas-
sachusetts population was D ? SE ? 0.18 ? 0.35. This
value is not signiÞcantly different from exactly addi-
tive (D ? 0), and the 95% CI excludes complete
dominance or recessiveness. In 2004, the degree of
dominance in the Massachusetts population was D ?
population was D ? 0.16 ? 0.35. Similarly to 1999, the
0, but exclude complete dominance or recessiveness,
but all values tend to slight dominance of LD50.
Fitness Costs of Resistance. Sprinting Speed. There
was no difference between sexes in walking speed
speed on resistance and sex (Parameter test for sex:
F ? 0.0623; df ? 1, 237; P ? 0.8), nor was there any
signiÞcant interaction between sex and resistance.
LD50values from 1999. The slope? SE is 0.82 ? 0.14 and the
95% CI for heritability is 0.55Ð1.12.
0.05 by using TukeyÕs HSD test.
Box plots of LD50values of 2004 crosses. Similar letters denote means that are not signiÞcantly different at ? ?
The chi-square tests the difference of observed and expected re-
sponse from the F1? S backcross.
December 2007BAKER ET AL.: IMIDACLOPRID RESISTANCE COSTS
but signiÞcant difference between resistant and sus-
ceptible beetles (F ? 6.57; df ? 1,239; P ? 0.011.
Fecundity. Total fecundity varied among crosses in
1999 (ANOVA: F ? 3.162; df ? 3, 21; P ? 0.046) with
at ? ? 0.05 (Fig. 5). Massachusetts female parental
population had a signiÞcant effect on fecundity in
both years (1999: ANOVA; F ? 8.764; df ? 1, 23; P ?
0.007, 2004: ANOVA; F ? 10.709; df ? 2, 38; P ?
0.0002), and Maine and Massachusetts resistant fe-
males did not differ in mean fecundity by using pair-
cost of resistance was stable within Massachusetts be-
tween 1999 and 2004. The ANOVA results of average
daily fecundity on year, resistance, and their interac-
tion are presented in Table 3. There were no signiÞ-
In the second 1999 generation, female genotype
again had a signiÞcant effect on fecundity (ANOVA:
F ? 10.394; df ? 2, 19; P ? 0.0009) (Fig. 5). The
estimated dominance, D, was 2.04, suggesting strong
underdominance of the Þtness cost. However, the
high variance of D, 6.02, cannot signiÞcantly distin-
guish among any levels of dominance. Furthermore,
pairwise comparisons using StudentÕs t-test suggest
that the mean fecundities of heterozygotes and sus-
ceptible females were not different.
Hatching Success. Arcsine-transformed hatching
success varied signiÞcantly among cross types in 1999
(ANOVA: F ? 3.094; df ? 3, 21; P ? 0.023) (Fig. 6).
Resistant clutches had signiÞcantly lower hatch rates
than susceptible ones, using TukeyÕs HSD test for
pairwise comparison, but hybrid eggs had hatch rates
not signiÞcantly different from each other or from
either pure strain. The LS means for hatch rates, SS ?
0.82, SR ? 0.63, and RR ? 0.35, suggest a somewhat
recessive hatching cost, with an estimate of h ? 0.
In 2004, there were no signiÞcant differences in
brid clutches (ANOVA: F ? 0.698; df ? 6, 34; P ?
0.653). Within Massachusetts, the hatching success
cost seems to have been lost between 1999 and 2004.
The ANOVA of arcsine transformed hatch rates on
year, cross, and their interaction are presented in
Table 3, and a plot of the LS means from the ANOVA
of cross type, but signiÞcant year and interaction ef-
fects occurred, suggesting any effect of resistance on
hatching success was lost.
Development to Hatch. Hours to hatch varied sig-
niÞcantly among resistant, susceptible, and hybrid
crosses in 1999 (ANOVA: F ? 5.082; df ? 2, 22; P ?
0.015) (Fig. 7). Resistant pairs laid clutches that took
9.7 h longer, on average, to hatch than susceptible
pairs, a signiÞcant difference using TukeyÕs HSD test,
and hybrids were intermediate in time to hatch.
beetles was almost one third less than that of suscep-
tible females. The hatching cost, when present, was
cundity in 1999 and 2004 in resistant and susceptible females
from Massachusetts. This Þgure combines means from three
analyses. The 1999 and 2004 Massachusetts means are from a
LS contrast from an ANOVA of fecundity on year, popula-
from an ANOVA comparing Massachusetts resistant, labora-
tory susceptible, and Maine resistant female fecundity in
Least-squares means ? SE of average daily fe-
resistant, laboratory susceptible, and hybrid crosses in 1999
Mean ? SE of hatching success in Massachusetts
tance on average fecundity and hatching success
Analysis of variance for the effect of year and resis-
Sourcedf SSF ratioProb ? F
Cross ? yr
Cross ? yr
1876JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 100, no. 6
?50%. The longer time to hatch for eggs of resistant
beetles will reduce population growth directly, but it
is likely to have a greater effect indirectly, through
increase in time to hatch (Hazzard et al. 1991). The
combination of reduced fecundity and slower devel-
opment will reduce intrinsic growth rates of resistant
genotypes. The slower sprint speeds observed in re-
sistant beetles also may act indirectly to select against
resistance, if it is indicative of reduced dispersal ca-
The fecundity cost is similar between the two geo-
graphically disjoint populations. One potential pitfall
of the present and several past studies of resistance
costs is that the resistant and susceptible populations
may differ in traits other than resistance, because the
sources may be from different geographical areas, or
they may have been held in the laboratory for differ-
ent numbers of generations. We tried to address this
by adding wild beetles to our susceptible colony each
summer to reduce inbreeding and adaptation to lab-
oratory conditions. One observation that lends conÞ-
dence that our results are not an artifact of selection
on a laboratory susceptible strain is that there was no
difference in fecundity between the laboratory sus-
entirely from the wild, and 2004, after several years in
Some costs, such as reduced fecundity and in-
creased developmental time, were highly conserved
over time. Loss of resistance costs has been ascribed
McKenzie and OÕFarrell 1993, Charpentier and
Fournier 2001), although in other well studied cases
cost reduction seems to have occurred through allele
replacement rather than addition of cost modiÞers
(Zhao et al. 2000b, Raymond et al. 2001). It is possible
that an allelic substitution occurred in Massachusetts
that expressed higher hatching success in 2004 than
1999, or that modiÞers arose to eliminate the differ-
ence in hatch rate. It is possible that the modiÞer or
variant was present in 1999, as different Þelds were
used as a source, and the closest Þeld from 2004 was
about 3 km from the 1999 source population.
The partial dominance in resistance observed here
contrasts slightly with the dominance of ?0.23 and
?0.11 measured at 3 and 7 d after imidacloprid treat-
ment in adult Colorado potato beetles from the Long
to determine whether the difference is signiÞcant, as
our conÞdence intervals overlap the measures of D
from Zhao et al. 2000a, and theirs did not include
in 1999 and 2004 all were slightly dominant, whereas
both of the earlier measures tended toward recessive
current study used larval assays. Dominance often
varies among life stages (McKenzie 2001), and it was
seen to decline in the Colorado potato beetle larvae
dose assay the dominance observed depends almost
wholly on the dose chosen (Roush and McKenzie
more comparable between studies.
Interestingly, both the current study and the study
by Zhao et al. (2000a) found evidence of polygenic
inheritance of resistance to imidacloprid in the Þeld.
Polygenic inheritance has been argued to occur as an
artifact of laboratory selection studies, because pop-
that can be applied, resulting in observation of only
the genes of small effect already present in the sus-
ceptible population (Roush and McKenzie 1987). Be-
cause most theoretical models advocating high-doseÐ
refuge approach assumed that resistance is controlled
by a single gene, a certain caution might need to be
exercised when applying their Þndings to managing
The recessive cost in fecundity observed in the
current study is probably not an artifact of heterosis,
because the populations were only in the laboratory
they were collected from Þelds a few kilometers from
each other in an area densely populated with potato
Þelds. Together with partially dominant of resistance,
this favors resistance genes when they are found in
Thus, there is likely to be a challenge to long-term
management using a refuge approach.
Certain caution needs to be exercised when using
our results to develop an applied resistance manage-
ment plan. First, systemic toxin imidacloprid is deliv-
ered by ingestion, which may constitute different nu-
the current study and by Zhao et al. (2000a). Second,
concentrations of toxin are likely to vary between
plant. Finally, there is likely to be genetic variation
among Colorado potato beetle populations from dif-
ferent geographic areas. Still, the signiÞcant costs
found do suggest that resistance management using
refuges, or temporal rotation of treatments with non-
neonicotinoids, can have an effect on prolonging ef-
Þciency of imidacloprid.
are not signiÞcantly different at ? ? 0.05 by using TukeyÕs
December 2007BAKER ET AL.: IMIDACLOPRID RESISTANCE COSTS
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Received 5 January 2007; accepted 6 August 2007.
December 2007BAKER ET AL.: IMIDACLOPRID RESISTANCE COSTS
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