Review

Received: 13 October 2020 Revised: 2 January 2021 Accepted article published: 28 January 2021 Published online in Wiley Online Library: 15 February 2021

(wileyonlinelibrary.com) DOI 10.1002/ps.6306

Fitness studies of insecticide resistant strains:

lessons learned and future directions
Jamie C Freeman,a Letícia B Smith,a,b Juan J Silva,a Yinjun Fan,a
Haina Suna and Jeffrey G Scotta*

Abstract
The evolution of insecticide resistance is generally thought to be associated with a fitness cost in the absence of insecticide
exposure. However, it is not clear how these fitness costs manifest or how universal this phenomenon is. To investigate this,
we conducted a literature review of publications that studied fitness costs of insecticide resistance, selected papers that met
our criteria for scientific rigor, and analyzed each class of insecticides separately as well as in aggregate. The more than 170 pub-
lications on fitness costs of insecticide resistance show that in 60% of the experiments there is a cost to having resistance, par-
ticularly for measurements of reversion of resistance and reproduction. There were differences between classes of insecticides,
with fitness costs seen less commonly for organochlorines. There was considerable variation in the experiments performed. We
suggest that future papers will have maximum value to the community if they quantitatively determine resistance levels, iden-
tify the resistance mechanisms present (and the associated mutations), have replicated experiments, use related strains (opti-
mally congenic with the resistance mutation introgressed into different genetic backgrounds) and measure fitness by multiple
metrics. Studies on the fitness costs of insecticide resistance will continue to enlighten our understanding of the evolutionary
process and provide valuable information for resistance management.
© 2021 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: evolution of insecticide resistance; fitness costs; selection

1 INTRODUCTION resistance allele that results in decreased reproductive success

Insecticide resistance has been widely studied because it is a trac- by any mechanism, such as decreased fertility or shorter life span)
table evolutionary process (has a known selective pressure, quan- is based on multiple observations (see also Coustau et al. and
tifiable phenotype, etc.) and is important to pest management. Lenoramand et al.7, 8). First, resistance alleles are rare before selec-
Use of insecticides (or other pesticides) leads to the evolution of tion (excluding cases of cross-resistance). This observation is
based on direct measurements of resistance allele frequencies9
mutations that allow for survival. Mutations causing resistance
and by the uniform susceptibility of populations to novel insecti-
can reduce the fitness of individuals carrying them (relative to
cides (in the absence of cross-resistance). If selection is the driving
populations lacking the mutation) once insecticide use ceases.
force maintaining the genetic composition of a population, it fol-
This phenomenon has various names in different disciplines, such
lows that in the absence of insecticide, the common (susceptible)
as “cost” or “load.”1 Within the insecticide literature, it is most
allele is fitter than the rare (resistant) allele (whether due to the
commonly referred to a “fitness cost.” This can be quantified in
resistance alleles themselves or to linked loci). Second, resistance
terms of the rate of reversion to susceptibility or the fitness costs
often reverts in the absence of insecticide pressure. This was ini-
(in reproduction, growth, etc.) associated with resistance in the
tially proposed based on observations of reversion to susceptibil-
absence of insecticide pressure. It is generally assumed that in
ity in field populations,10 although in many cases immigration of
the absence of insecticide pressure (and without fixation of the
susceptible individuals could not be ruled out. Subsequently,
resistance allele), the resistant allele is less fit, and susceptibility observations on the instability of resistance in selected laboratory
will eventually dominate in the population. These ideas have populations maintained in the absence of insecticide were impor-
remained constant even though it is now widely understood that tant for developing the concept of a fitness cost of resistance.
populations contain multiple susceptible alleles and, in some
cases, multiple resistance alleles.2–5 An assumption of fitness costs
associated with insecticide resistance is the basis of many resis- * Correspondence to: JG Scott, Department of Entomology, Cornell University,
tance management strategies.6 Without these fitness costs, the Ithaca, NY 14853, USA. E-mail: jgs5@cornell.edu
rate at which insecticides become ineffective would be greatly
a Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY, USA
accelerated.
The assumption that insecticide resistance is associated with a
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b Laboratory of Malaria and Vector Research, National Institute of Allergy and

fitness cost in the absence of insecticide (herein defined as a Infectious Diseases, National Institutes of Health, Rockville, MD, USA

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 www.soci.org
Third, resistance mutations often result in altered gene function
for genes under strong selection, for example, the altered catalytic
activity of acetylcholinesterase (AChE) with resistance
mutations.11–13 Disruption of critical enzymes, receptors, and ion
channels is generally thought to be deleterious to fitness. We note
that there are exceptions to the observations described above
(e.g., Hartley et al.14). It is well understood that measurements of
fitness are only suitably carried out on strains that are highly
related (optimally congenic).15
Despite the assumption that insecticide resistance alleles have a
fitness cost (see above), research on this topic has not been com-
prehensively re-examined, except for a review of Bt resistance
published in 2009.16 Here, we assess the body of literature focus-
ing on the fitness costs of insecticide resistance. Although reviews
over the past decade have discussed papers of note or the theory
of the fitness costs of resistance more generally,17–19 none has
attempted to assess comprehensively whether, on the whole, fit-
ness costs of insecticide resistance are observed. Nor have any
previous reviews examined whether any specific parameters are
more commonly associated with a fitness cost across classes of
insecticides. In addition, we make recommendations for future
studies of the fitness costs associated with insecticide resistance.
2 CRITERIA FOR INCLUSION AND
EVALUATION OF STUDIES
Literature searches were initially carried out using Web of Science
and Google Scholar. The former was found to be more optimal,
because it did not include papers in which the keywords were
found in the references. Thus, subsequent searches were done
using Web of Science. Initial searches were done without any
restriction on year, using the keywords fitness, insect, insecticide,
resistance, organophosphate (OP), carbamate, pyrethroid, neoni-
cotinoid, Bt, Bacillus thuringiensis, life table, reversion, and/or cost.
Approximately 250 papers were initially identified, and these
were manually scanned to identify additional references. This iter-
ative process continued until no new papers were identified. After
removal of duplicates, there were 597 references found. This pro-
cess ended on 30 June 2019 and subsequently published papers
were not included. Each of the five major classes of insecticides
(OP + carbamates, Bacillus thuringiensis (Bt), pyrethroids, neonico-
tinoids, and organochlorines) were examined separately. Studies
were more limited for other insecticides/acaricides and these
were grouped together (miscellaneous).
Papers identified from the literature searches were evaluated for
several criteria. We wanted to find papers in which the relative fit-
ness of related resistant and susceptible strains was studied
experimentally in the absence of pesticide exposure. First, papers
were screened to remove any that were off-topic (n = 194; e.g.,
organisms were not insects or mites, insecticide-resistant strains
were not used, neither fitness nor life history parameters were
studied). The remaining papers (n = 403) were then filtered using
quality criteria (most were rejected for a single reason, but some
met multiple criteria and so there is some overlap in the percent-
ages below). The compared resistant and susceptible strains had
to have a similar genetic background, which was achieved in most
studies either through the generation of congenic strains (with a
minimum of four backcrosses) or by making a collection that
was split and maintained as unselected and selected strains. Com-
parison of unrelated strains was the most common reason for
rejection (78% of those rejected). Papers using either technical
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grade or formulated insecticides were considered, unless the
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JC Freeman et al.
formulated materials contained more than one active ingredient
(12% of those rejected). Papers using more than one toxin were
excluded because the effects (resistance level or fitness) could
not be ascribed to a specific toxin. With one exception, all of
the papers employing a mixture of toxins were for Bt. Papers
in which the resistance ratio was less than five-fold were
rejected, accounting for 2.3% of the papers rejected. In some
cases, the levels of resistance were not reported, but the fre-
quency of known resistance alleles was, and these papers were
included. Four percent of papers were rejected for other rea-
sons (not in English and could not be translated, paper not avail-
able, link of measurement to fitness unclear, etc.). In cases in
which some strains or experiments within a paper fit our criteria,
but others did not (e.g., multiple resistant strains with only one
related to the susceptible), the appropriate experiments/strains
were included.
One fundamental observation that indicates resistance alleles
have fitness costs is reversion towards susceptibility in the
absence of insecticide treatment, as long as the strain was not
homozygous for resistance. In our efforts to summarize these
papers we had three fundamental challenges. Reversion experi-
ments were rejected if the data were not compelling and the
authors did not perform statistical analysis of their data (2.8% of
rejected papers). For laboratory studies, we included only experi-
ments about reversion of resistance if the paper also had some
compelling data to indicate that the strain was not homozygous
for resistance (commonly slope of the log dose/concentration
probit line) (3.3% excluded). Similarly, resistance can appear to
be lost from a field population if there is significant migration of
susceptible insects into an area. Thus, we included experiments
on the reversion of resistance under field conditions only if there
was some reasonable way to rule out migration of susceptible
insects as the cause (e.g., biology of the insect rules out migra-
tion). Following application of these filters, 183 papers were used
in our analysis. For the remainder of this review, we use “paper” to
refer to the publication and “experiment” to refer to a specific
study that was done. Thus, some papers contained multiple
experiments.
There was some variation in how authors defined the parame-
ters associated with reproduction. For standardization, we used
the following definitions: intrinsic rate of increase or r, calculated
as 1 = ∑lxmxe-rx (this is a slope and prediction of the rate of
increase, where lx is the proportion of females alive at day x and
mx is the number of daughters born to surviving females at age
x) or r = Nn + 1/Nn (this is a proportion, a rate of increase at a finite
time point, where Nn is the number of animals at time n), and “net
reproductive rate” or R0, where R0 = ∑lxmx = ∑lx{[(# eggs × egg
viability)/lx] × proportion female offspring}.20
Classes of insecticides were evaluated separately. Data were
extracted from each paper to compile a list of all the parameters
studied and to evaluate whether there was any fitness cost for
that parameter (see Supporting Information). These parameters
were grouped into six categories after reviewing the literature:
adult longevity, adult size, pre-adult development, reproduction,
reversion (of resistance or decrease in resistance allele frequency),
and sex ratio. If a parameter was studied that could not be
assigned a net benefit or cost (e.g., locomotion) it was not
included in the summary. Next, for each publication, all the
parameters within a specific category were evaluated to come
up with a final verdict for the summative effect on fitness associ-
ated with that category for that paper. In the Tables S1 to S6, we
term this the fitness consequence, to simplify any confusion that
Pest Manag Sci 2021; 77: 3847–3856
Fitness of insecticide resistant strains
could arise from a positive or negative “cost”. If a parameter had
experiments that gave both costs and benefits associated with
resistance a subjective decision was made for the score (+ or –
or +/− if the data were not compelling in one direction or the
other; +/− results were not tallied in the final tally of results). This
was mainly necessary in cases in which multiple similar parame-
ters were studied and the differences in effect sizes were small
(e.g., 10% less survival to pupation and 12% greater emergence
from pupation). For sex ratio, only a cost (deviation from expected
ratio) or no cost (expected ratio) was assigned. If the same resis-
tant strain was studied across multiple publications, all data for
the same strain were evaluated together. This led to construction
of Tables S1–S6. Herein, the classes of insecticides are ordered by
the number of experiments, from those having the greatest num-
ber to those having the least.
The counts table of results (supplementary table that contains
the counts (number of outcomes: cost, neutral, or benefitial) from
each accepted reference) was analyzed for deviation from a null
distribution where all three outcomes (cost, neutral, or benefit)
are equally likely using a chi-square goodness-of-fit test. To test
whether outcomes were different between insecticide classes or
fitness parameters, chi-square tests of independence were used,
and in the case that a significant association was found, post-
hoc chi-square tests with Bonferroni correction were used to com-
pare the outcomes in each class (or parameter) against the sum of
all others.
3 FITNESS COSTS ACROSS ALL
INSECTICIDE CLASSES: OVERALL TRENDS
Overall, 462 different measurements of fitness were evaluated
across all classes of insecticides and a fitness cost was the most
common result (61%), with neutral being the second most com-
mon (30%) (Figure 1). The number of experiments obtaining a
result of a fitness cost is overrepresented compared with a null
distribution using a chi-square goodness-of-fit test (χ 2 = 182.23,
2 df, P < 2.2 x 10-16). We were interested in whether there was
an association between the levels of resistance and the frequency
of finding a cost/benefit for resistance. Unfortunately, the level of
resistance was not defined (values not given or values not suffi-
ciently quantitative, e.g., >14-fold) in a sufficient number of
papers to have enough data to check this correlation.
FIGURE 1. Frequency at which a fitness cost was observed in resistant
strains across all insecticides, across six categories of fitness investigated.
n = number of experiments summarized.
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The number of experiments finding a fitness cost differed
between parameters (χ 2 = 99.1, 10 df, P < 2.2 × 10−16). The
parameter most frequently associated with fitness costs was
reversion, where 84% of experiments showed a cost and only
2% showed a benefit, and this greater frequency of finding a cost
represented a significant departure from what would be pre-
dicted based on the other parameters (χ 2 = 22.0, 2 df, adjusted
P = 9.78 × 10−5). This is perhaps logical because the reversion
phenotype is the summation of many other parameters. A num-
ber of papers cited previous observational data of reversion of
resistance over the course of normal rearing in the laboratory as
a rationale for testing for reversion,21 which may be a source of
bias in the data (strains that obviously revert are more likely to
be tested in the first place). For the other fitness categories mea-
sured, costs were most commonly observed for pre-adult devel-
opment and reproduction. Pre-adult development (n = 143) and
reproduction (n = 133) were also the most commonly investi-
gated fitness categories and fitness costs were observed in 58%
and 74% of these experiments (Figure 1). Costs were overrepre-
sented for reproduction relative to the other categories
(χ 2 = 12.4, 2 df, adjusted P = 0.012). For adult longevity (n = 45)
and size (n = 19), the differences between cost, neutral, and ben-
efit cases were not strong and were variable by insecticide class.
Based on all other parameters, costs were less likely for adult lon-
gevity (χ 2 = 12.7, 2 df, adjusted P = 0.010). Sex ratio (n = 37) is the
only category in which most experiments (84%) found a neutral
effect, making this category differ most significantly from all
others (χ 2 = 55.3, 2 df, adjusted P = 5.75 × 10−12).
Overall, there is great variation in results for the different cate-
gories across the insecticide classes. This suggests that a general-
ization about the fitness costs of insecticide resistance cannot be
made without information on the mechanisms of resistance,
although there were also considerable differences in the organ-
isms and parameters most commonly studied between insecti-
cide classes, which may also explain variations between classes.
A chi-square test of independence indicated a significant associa-
tion of insecticide class with outcome (χ 2 = 25.9, 10 df,
P = 0.00389). Post-hoc chi-square tests (with Bonferroni correc-
tion) indicate that only the organochlorines (χ 2 = 13.4, 2 df,
adjusted P = 0.00753) differ significantly from the overall trend,
which appears to be driven by the relatively high number of ben-
efit cases present for the organochlorines (Figure 1).
4 OP AND CARBAMATE RESISTANCE
4.1 Laboratory studies
The results from 47 publications that matched our criteria and
studied fitness of OP and carbamate resistance are summarized
in Table S1. Resistance was associated with a fitness cost in the
majority of experiments that measured reversion, pre-adult devel-
opment, or reproduction, being found in 74%, 73%, and 56% of
the experiments, respectively (Figure 2a). By contrast, experi-
ments of adult size most commonly found that the resistance cost
was neutral (40%) or had a benefit (40%). Resistance ratios varied
from 5- to 5300-fold. OP resistance studies were unique in that the
same resistant strains were often studied across multiple publica-
tions. Ten of the strains had known target-site resistance mecha-
nisms, and nine had known metabolic resistance mechanisms.
In five strains, three for Anopheles gambiae and two for Culex
pipiens, duplication of the OP target-site gene, acetylcholinesterase
(ace), was a mechanism of resistance. For the rest of the strains
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studied, nothing was known about the mechanisms involved in
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FIGURE 2. Frequency at which a fitness cost was observed in strains resistant to: (A) organophosphates and carbamates; (B) Bt; (C) pyrethroids; (D) neo-
nicotinoids; (E) organochlorines; and (F) miscellaneous insecticides, across six categories of fitness investigated. AL: adult longevity; AS: adult size; PD: pre-
adult development; Rep: reproduction; Rev: reversion; SR: sex ratio. n = number of experiments summarized.

resistance. C. pipiens was the best examined organism, with 12 dis-
tinct resistant strains studied. Reversion was less commonly
observed for OPs and carbamates than for other conventional
insecticides. In total, for 35 tests in the laboratory, 74% found
reversion and 20% found no change in resistance.
4.2 Field studies
Five field-based studies were conducted on the fitness costs of OP
resistance. Four papers examined reversion of resistance in the
field. Two looked at reversion in the field over time, and both stud-
ies observed reversion. When insecticide use was halted on an iso-
lated farm for 7 years, decreases were observed in both the oral
and topical azamethiphos resistance of houseflies [with known
mechanisms target-site mutations, cytochrome P450 (CYP)- and
hydrolase-mediated metabolic resistance].22 After widespread
adoption of Bt cotton in northern China, collections of Helicoverpa
armigera were made over 8 years, and seven populations had a
measurable reduction in the quantitative level of resistance
against both endosulfan and phoxim [approximately fivefold
mean decrease in median lethal dose (LD50)].23 Two papers exam-
ined frequency of resistance genotypes/phenotypes over the
overwintering period for C. pipiens, where females overwinter as
adults and mortality is high.24,25 Both studies found a cost for
the ace-1 mutation G119S, but only one found a cost for overex-
pressed esterase. Only one paper examined life history traits in
the field,26 and found a cost of resistance for insensitive ace-1 as
well as two phenotypes of overexpressed esterases for adult size
in all conditions and for developmental time in most conditions.
4.3 Catalytic activity of AChE with resistance mutations
Alteration of the AChE protein as a resistance mechanism prompted
assays of the catalytic activity of the altered protein. The AChE pro-
3850
although in vitro studies of the protein cannot be directly related
to downstream effects on fitness in vivo, these studies do provide
potential explanations for the observed fitness costs. The effects
of four resistance mutations on the activity of the Drosophila mela-
nogaster protein were variable: from a 13% increase in activity for
the protein containing G265A to a 546% reduction in activity for
the protein containing G368A.11 The G119S mutation reduced activ-
ity by 77% in both the A. gambiae and C. pipiens proteins.12 Three
known resistance mutations in Tetranychus urticae (G228S, A391T,
and F439W) individually all reduced the activity of AChE and combi-
nations of mutations exacerbated this effect.13 The stability of the
altered D. melanogaster AChEs was also tested and all combinations
of mutations reduced stability of the protein.11
4.4 Fitness studies in which environmental stress was
applied
Several studies manipulated environmental conditions to find
how the fitness consequences of resistance changed, and the
most commonly altered variable was temperature. A
chlorpyrifos-resistant strain of Nilaparvata lugens was studied at
five temperatures.27 At 25°C and above (28 and 30°C) the resistant
strain copulated at a lower frequency than the susceptible strain,
but at 22°C there was no difference, and at 18°C the resistant
strain copulated at a higher frequency than the susceptible strain.
Fecundity was lower for the resistant strain in all treatments
except 18°C, and the resistant strain had a longer recovery time
from heat shock. Most measured life history traits of
chlorpyrifos-resistant Laodelphax striatella under two tempera-
ture conditions, 24 and 30°C, were not different between strains
(larval development time, larval survival, or adult longevity).28 At
24°C, the susceptible strain had higher fertility than the resistant
strain, but at 30°C the strains were not different. Multiple sub-

tein is essential for normal nervous system functioning, and strains of chlorpyrifos-resistant Plutella xylostella with the ace-1

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Fitness of insecticide resistant strains
mutations A201S+G227A were subjected to a series of tempera-
ture treatments.29 Overall, reversion of chlorpyrifos resistance
and resistance mutation frequency was observed in most cases
and the decrease appeared larger in the high temperature treat-
ments, although this was not statistically tested. Additionally,
fecundity and longevity were measured for the strains when
maintained at 25°C, 33.5°C, and under a temperature regime
mimicking a July day in Fuzhou, China (mean 31.9°C). At 25°C
no differences in longevity or fecundity were observed between
the strains, but at 33.5°C and under the variable temperature
regime the resistant strain had lower longevity and fecundity.
Exposure of adults to 40°C resulted in greater mortality in the
resistant strain after 48 and 72 h of exposure, although no differ-
ence was seen with 24 h of exposure. In a second paper, the
response of the ace-1 A201S+G227A resistant strain to six differ-
ent heat shock treatments during the pupal stage was tested.30
Pupal survival was less for the resistant strain in two treatments,
and adult emergence was lower for the resistant strain in all but
one treatment. A third paper31 examined heat damage to ovary
and sperm cells to explain the lower fecundity of the resistant
strain at higher temperatures, and found greater damage associ-
ated with heat treatment in the resistant strain. From these stud-
ies there is no clear indication that applying a stressor
(investigator defined) results in a larger fitness cost.
Larval density was used as a variable in two papers: one in the
field and two in the laboratory. In the field, increasing larval den-
sity of C. pipiens increased the difference in development time
between mosquitoes having at least one copy of ace-1 G119S
and the susceptible mosquitoes in 75% of the replicates.26 The
cost of ester1 and ester4 phenotypes on developmental time
had a more complicated relationship with density: between the
lowest and medium density treatments the cost increased, but
in the high-density treatments, there was little cost for ester1
and a slight benefit for ester4 compared with the susceptible
strain. For all three resistance phenotypes, there was no interac-
tion between resistance status, larval density, and adult female
wing size. The effect of increasing larval density on reversion of
temephos resistance in C. pipiens was also examined under labo-
ratory conditions, but there was an unclear relationship between
density and degree of reversion.32 Three larval rearing density
treatments were applied to Bactrocera dorsalis and there was no
effect of resistance status on development time, but susceptible
flies had a stronger negative response to density for pupal weight
and survival to emergence.33 Despite this, when both resistant
and susceptible flies were reared in the same fruit, the probability
of survival increased for susceptible flies and decreased for resis-
tant flies. This highlights the importance of competition experi-
ments in studying fitness costs.
In Leptinotarsa decemlineata, altered acetylcholinesterase was
less sensitive to inhibition by ⊍-chaconine, a major steroidal glyco-
alkaloid in potato, so it was hypothesized that the resistant strain
would have an advantage on a high ⊍-chaconine potato culti-
var.34 The resistant strain had a shorter development time on a
high ⊍-chaconine diet, but no differences were observed on the
low ⊍-chaconine diet. In another paper with the same resistant
strain on a third diet, the resistant strain had a longer develop-
ment time than the susceptible strain.35
5 BT RESISTANCE
Forty-nine papers matching our criteria investigated the fitness
costs associated with Bt resistance (Table S2) and the most
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common results were a fitness cost (52%) or no difference (36%)
(Figure 2b). Pre-adult development (n = 35) and reproduction
(n = 28) were the categories most commonly studied. In both
cases the most common result was a fitness cost followed by no
difference. A fitness cost was observed in the single paper on
adult size. Reversion of resistance was seen in seven of ten exper-
iments. Levels of Bt resistance in the strains studied ranged from
5.7- to 4850-fold.
Resistance mechanisms were known for only seven papers and
in all seven (five using Pectinophora gossypiella and one using
H. armigera) these were cadherin mutations. For P. gossypiella, fit-
ness costs were associated with adult longevity, adult size, repro-
duction and pre-adult development, although not in every
experiment (Table S2). Cadherin mutations in Bt-resistant
H. armigera were associated with decreased reproduction
(Table S2).
The fitness costs of Bt resistance were also investigated under
different conditions. Gossypol, a cotton plant allelochemical,
was found to decrease larval weight in both susceptible and resis-
tant strains of P. gossypiella (resistance due to cadherin muta-
tions), but the effect was greater in the resistant strain.36 One
paper studied the fitness costs associated with Bt resistance due
to unknown mechanisms in Helicoverpa zea, under different
stressors. Using different protein/carbohydrate ratios in the diet
reduced pupal weight, but the effect was less in the resistant
strain than in the susceptible strain. Another experiment tested
found that a “low quality” plant diet resulted in a larger degree
of reversion of resistance in P. xylostella.37
The effect of Bt resistance on diapause in P. gossypiella was
investigated because proper timing and emergence from dia-
pause are critical for survival. By experimentally manipulating dia-
pause duration it was observed that Bt resistance was associated
with multiple changes.38 Resistant strains remaining in diapause
for a short period had reduced longevity compared with the sus-
ceptible strain, but resistant strains also had less weight loss and
better overwintering survival than the susceptible strain. These
results led the authors to conclude that “the evolution of resis-
tance to Bt cotton has pervasive effects on several traits associ-
ated with diapause.”38
A previous review of Bt resistance in 200916 found reversion of
resistance and reduced fitness in 62% and 34% of papers
reviewed, respectively. We found a higher percentage of experi-
ments with revision (70%) and fitness cost 48% (Figure 2b). Our
review is not exactly comparable with the previous one because
different criteria were used to decide which papers to include,
and an additional 22 papers on the fitness costs of Bt resistance
have been published since 2009.
6 PYRETHROID RESISTANCE
There were 30 published papers matching our criteria on the fit-
ness costs of pyrethroid resistance (Table S3). Most papers
reported on public health pests, especially mosquitoes (n = 14),
specifically Aedes aegypti (n = 6), C. pipiens (n = 5), A. gambiae
(n = 2) and Aedes albopictus (n = 1), followed by cockroaches (Blat-
tella germanica) (n = 2) and houseflies (Musca domestica) (n = 2).
The remainder of the papers reported on agricultural pests across
three orders, Lepidoptera (n = 6), Coleoptera (n = 3) and Hemi-
ptera (n = 2) plus the red spider mite (T. urticae; n = 1). Reported
resistance levels ranged from 19- to 1450-fold.
With the exception of papers on reversion of insecticide resis-
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tance, most measured multiple parameters to evaluate fitness
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costs. The types of parameters measured and the experimental
design for each parameter varied greatly between publications.
A fitness cost associated with resistance was observed in 57% of
the experiments, and 31% had neither a cost nor a benefit
(Figure 2c). All ten experiments on reversion of pyrethroid resis-
tance found a fitness cost. Apart from the reversion experiments,
the most common types of fitness categories measured were
reproduction (n = 20) and pre-adult development (n = 17), where
a fitness cost was reported in 70% and 59% of the experiments,
and a fitness benefit was reported in 10% and 6%, respectively.
Of the 13 experiments that measured adult longevity, six (46%)
found a cost and five (38%) found neither a cost nor benefit (both
cases were mosquitoes with Vssc mutations L1014F or F1534C,
herein we refer to Vssc mutations causing pyrethroid resistance
as knockdown resistance or kdr). Curiously, one of the two experi-
ments measuring reproduction that found a fitness benefit was
also a mosquito with kdr (F1534C mutation). For adult body size,
the results (n = 6) were equally divided among cost, neutral, and
benefit.
For approximately half of the publications (16 of 30), the resis-
tance mechanism was unknown. For those publications that spec-
ified the resistance mechanisms involved, the most commonly
reported were kdr (n = 5), CYP-mediated detoxification (n = 4),
or both kdr and CYP (n = 5). Most publications, however, checked
for only one or two mechanisms, often genotyping for only a few
known Vssc mutations, a single CYP mutation or by using a CYP
inhibitor. The specific Vssc mutations varied between strains and
species. Publications using strains with kdr mutations had con-
trasting results. No fitness costs were associated with Vssc muta-
tion L1024V in the red mite, T. urticae.39 By contrast, in an
A. aegypti strain containing Vssc mutations V1016I + F1534C and
a slight increase in CYP activity, there was a fitness cost in three
of the four categories (including pre-adult development) mea-
sured.40 It is unclear whether the differences between these
results are due to the different Vssc mutations present, because
different species were used, or some other factor. The two
papers41,42 on CYP-mediated resistance used the same strain of
C. pipiens, one paper measured reversion and one measured other
fitness parameters, and they found a fitness cost for reversion, but
not for any of the specific parameters. Because of the small num-
ber of publications with known resistance mechanisms, it is not
possible to draw any conclusions on the fitness costs of the indi-
vidual or combined resistance mechanisms across populations
and/or species.
Several Xenopus oocyte experiments have demonstrated that
Vssc mutations alter channel gating in the absence of insecticides.
The effects of kdr mutations on channel function in oocytes are
visible as shifts in the voltage of activation/inactivation, size of
peak currents upon activation and/or changes in time of inactiva-
tion. For example, the L1014F and L1014H mutations shifted the
half-maximal voltage of both activation and inactivation in a
depolarizing direction, whereas L1014S produced a depolarized
shift in inactivation, but did not alter activation.43 The V410M
mutation was found to shift the voltage dependence of activation
in both oocytes (reviewed by Soderlund and Knipple)44 and in
neurons in the central nervous system.45 When peak current
amplitudes were examined, oocytes carrying the E435K or
C785R mutations both had reduced peak currents relative to
wild-type channels, but oocytes having both mutations were
not different from wild-type.46 The presence of M918T, T929I
and L1014F individual mutations reduced the decay time of inac-
3852
tivation relative to wild-type channels.44 Although these results
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JC Freeman et al.
suggest that some kdr mutations can alter the functionality of
the VSSC, how these changes might alter nervous system function
(and influence fitness in vivo) is not known.
7 NEONICOTINOID RESISTANCE
Fifteen papers matching our criteria examined the fitness costs of
resistance to neonicotinoid insecticides (Table S4). Across all cate-
gories, fitness costs were found in 72% of the experiments and no
differences were found in 25% of the cases. Experiments on rever-
sion of resistance (n = 17) and adult longevity (n = 3) found a fit-
ness cost in all cases (Figure 2d and Table S4). Of the
29 experiments that measured reproduction, 79% had a cost
and 21% were neutral. Experiments of pre-adult development
found a cost (52%) or were neutral (41%) in most cases. The levels
of resistance ranged from 16- to 140-fold. More than half of the
papers (n = 8) were done on hemipteran pests. In most cases
(21 of 25) the mechanism(s) of resistance were completely
unknown. Detoxification enzymes (identified by use of synergists
or gene expression) were the most common resistance mecha-
nisms (three cases). Another resistance mechanism, deletion of
nicotinic acetylcholine receptor ⊍1 by CRISPR/Cas9 in
D. melanogaster, resulted in 25-fold resistance to imidacloprid
and decreased courtship and longevity.47,48
8 ORGANOCHLORINE RESISTANCE
Of the 12 publications that matched our criteria on organochlo-
rine insecticides (Table S5), covering 16 resistant strains, there
was a fitness cost in 42% of cases and no cost in 33% of cases
(Figure 2e). Resistance ratios for the strains studied varied from
8- to 150-fold. The fitness categories of reproduction (12 of 12)
and pre-adult development (11 of 12) were measured in nearly
every experiment. Most experiments found a cost in reproduction
(67%) of organochlorine resistance, however, 25% found a repro-
ductive benefit for resistant strains. Curiously, all cases of a fitness
benefit associated with resistance were experiments with
D. melanogaster. Of the six experiments that measured adult lon-
gevity, none found a cost and half found a fitness benefit in resis-
tant strains. Eight of 12 experiments measured various fitness
parameters associated with dichlorodiphenyltrichloroethane
(DDT) resistance, mostly (n = 5) in D. melanogaster. Upregulation
of CYP6G1 was a major mechanism for DDT resistance in the
D. melanogaster resistant strains, associated with resistance levels
ranging from 8.2- to 14-fold. Experiments with these
D. melanogaster strains gave mixed results, with both costs and
benefits associated with reproduction and adult size. In
organochlorine-resistant mosquitoes, fitness costs were most
commonly found for reproduction (Table S5). DDT-resistant
Anopheles arabiensis were more able to survive as adults at 30°C
than their susceptible counterparts, although no difference was
observed at 25°C.49 No differences were observed in pre-adult
development at either temperature.
9 RESISTANCE TO MISCELLANEOUS
INSECTICIDES
Of the 35 experiments examining the fitness costs of resistance to
the miscellaneous group of insecticides (Table S6), the majority
found a fitness cost associated with resistance. All these experi-
ments were conducted under laboratory conditions and most
commonly measured fitness categories associated with pre-adult
Pest Manag Sci 2021; 77: 3847–3856
Fitness of insecticide resistant strains
development (n = 31) and reproduction (n = 28) (Figure 2f). Of the
various measurements of fitness (Table S6), a fitness cost was
found 69% of the time and a fitness advantage for the resistant
strain in only 3% of the experiments. Decreased reproduction
was commonly a resistance cost (found under at least one condi-
tion in 24 of 28 experiments). Reversion of resistance was found in
10 of 11 cases studied. Delayed pre-adult development was com-
monly a resistance cost (found under at least one condition in
23 of 31 experiments) as were adult longevity (three of four exper-
iments) and adult size (two of three experiments). There was no
difference in sex ratio associated with resistance (n = 10). Resis-
tance levels varied considerably for strains in the miscellaneous
category (from 5.9- to 11 500-fold) and in most cases the resis-
tance mechanism was unknown. The exception to this were the
elegant experiments on resistance to avermectins, bifenazate
and etoxazone (in each case resistance was due to mutations in
the target-site gene) in which three replicate near-isogenic resis-
tance lines for each resistance mutation were used.39 In these
cases, there was a fitness cost associated with each resistance,
but the categories showing the cost varied between the three dif-
ferent resistances (Table S6).
There were three experiments in which a stress treatment was
compared with normal rearing.50–52 In all cases, the stress
(increased temperature, nutrient poor diet or daily handling)
increased the fitness cost observed in the resistant strain. Unfortu-
nately, the mechanism of resistance was known in only one of
these papers.39
10 DISCUSSION
This review of the literature supports the idea that resistance
mutations often come with a fitness cost. Fitness costs were most
commonly observed as reversion of resistance and reduced
reproductive output, whereas sex ratio and adult longevity were
the two categories least commonly found to vary between resis-
tant and susceptible strains. For many of the other categories
there was considerable variation between and within insecticide
groups. For example, fitness costs were less commonly reported
for the organochlorines relative to other classes. Furthermore,
reversion of resistance was common for all classes of insecticides.
There was no apparent correlation between the levels of resis-
tance and the frequency of detecting a fitness cost, although
the data set was limited.
After reviewing the literature, we suggest that for maximal sci-
entific impact, several standards should be met by future studies
on fitness costs: quantitative determination of resistance levels,
identification of the resistance mechanisms present (and opti-
mally the associated mutations), fully replicated experiments,
use of related strains (optimally congenic with the resistance
mutation introgressed into different genetic backgrounds), and
fitness measured by multiple metrics. Each of these is discussed
below.
An intriguing aspect of the fitness costs associated with resis-
tance is whether there is any correlation between the strength
of the benefit (i.e., resistance) and the degree of the cost. This
can only be tested if resistance levels are quantitatively deter-
mined (i.e., obtain LC50 or LD50 values). Thus, the quantitation of
resistance in the strain(s) being evaluated for fitness costs should
be determined in future studies.
It can logically be assumed that not every resistance mutation
carries the same fitness cost, but would the same mutation in dif-
ferent species confer the same fitness cost? For example, the
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L1014F Vssc mutation is found in many insect species. It would
be helpful to know whether this generates the same fitness cost
across species or not. Additionally, many if not most field-
collected strains will contain multiple mechanisms of resistance
to a range of different insecticides, and without characterization,
comparison across studies is not possible. Where multiple mech-
anisms of resistance are present, characterization of the relative
fitness of different mechanisms can offer important useful infor-
mation on the evolutionary trajectory of populations.53
There was variation in what was considered a replication
between the reviewed papers. For some studies, individual
insects (commonly females) were used as a replicate, even if they
came from the same cohort of insects. However, in one paper in
which different cohorts were used as replicates, the variation
between cohorts was greater than the variation in insects within
a cohort.54 Thus, replications of fitness studies should optimally
use multiple cohorts. One of the most glaring problems associ-
ated with lack of replication was that most studies we reviewed
give a single R0 (or rm) value and thus no statistical analysis (owing
to no replicates). Another area in need of replication in the exper-
imental design is in reversion experiments, where multiple inde-
pendent replicate lines should always be used.
It is well established that related strains need to be used in fit-
ness studies.15 In the literature we reviewed, this was commonly
done in one of two ways. One approach was to generate congenic
strains, such that the resistance mutation was introgressed into
the background of a susceptible strain. This is the preferred
method, although introgressing the resistance mutation into mul-
tiple susceptible strain backgrounds is the most optimal because
differences can occur.55 The second method is to start with a field-
collected strain, split the population and select one line, leaving
the other unselected. Although this approach achieves the goal
of comparing related strains, the field-collected populations
already have resistance alleles (otherwise a resistant strain could
not be selected). Thus, the final strains are those in which the
selected population is likely homozygous for resistance, while
the unselected strain has some unknown frequency of the resis-
tance alleles, and comparisons between strains can be less clear.
Additionally, it is likely that field-collected strains will have multi-
ple mechanisms of resistance present, making it difficult to asso-
ciate the fitness cost with a single mechanism. Thus, the use of
congenic strains provides a better assessment of the fitness costs.
As CRISPR–Cas9 (Clustered Regularly Interspaced Short Palin-
dromic Repeats - CRISPR associated protein 9) editing to intro-
duce specific resistance mutations directly into pest species
becomes more feasible, single mutations can be assessed more
directly.
Fitness studies most commonly investigate parameters associ-
ated with growth, development, reproduction, and reversion of
resistance, including allele competition/population cage
experiments,2,4 but a more reliable measurement of fitness comes
from multiple measurements across a diverse array of assays, par-
ticularly when these assays measure different aspects of the biol-
ogy. For example, one could find no apparent differences
between resistant and susceptible strains in a life table analysis,
but a fitness cost could be evident in an allele competition study
(where factors in addition to those measured in a life table analy-
sis determine the outcome).16,54 There is a potential danger of
multiple-testing in measuring parameters until a cost is discov-
ered. One potential design may be to start with a competition
experiment, and only if a difference in reproductive output is vis-
3853
ible, follow up with a study of life history parameters.
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 www.soci.org
In terms of understanding fitness costs, field studies and labora-
tory studies are both valuable, but present unique challenges and
limitations. Field studies have the advantage of multiple simulta-
neous factors (biotic and abiotic) being applied as a test of the fit-
ness cost and are also most useful for practical determinations of
fitness costs that could potentially be used for pest control.8 How-
ever, because biotic and abiotic conditions vary geographically,
seasonally, and by year, these studies have limited value in differ-
ent locations and under different conditions. Greater numbers of
animals and replicates are needed to account for the variability in
environmental conditions. Laboratory studies have the advantage
of being able to control the variables that are used in the evalua-
tion of fitness costs, which allows researchers to understand the
physiological and mechanistic cause and effects of different resis-
tance mutations. The limitation with laboratory studies is that
conditions are artificially simplified and limited by the variables
that can be manipulated, and so may not accurately estimate
the real costs seen in the field, making them potentially less rele-
vant for pest control. It should be pointed out that when a given
experiment finds no cost (or even a benefit) associated with resis-
tance, that does not mean that there is no cost under field condi-
tions where the total fitness costs (from diseases, predators,
environmental conditions, fecundity, fertility, etc.) determine the
degree of fitness impairment in the absence of insecticide. Opti-
mally both field and laboratory studies would be done to examine
fitness costs.
Assessing fitness costs across multiple environmental condi-
tions or challenges can give greater understanding of the rele-
vance of the observed fitness costs to field conditions. Overall,
there was no clear trend in the relative performance of resistant
strains under laboratory-applied stress conditions when com-
pared with no-stress conditions. This may simply reflect the myr-
iad of different resistance mutations and variable stressors
between the reviewed papers. In our review of the literature,
one aspect of fitness, namely response to biotic challenges, was
almost entirely missing with only five publications examining this
phenomenon.56–61 Although manipulation of predators, para-
sites, or pathogens can be experimentally challenging, how resis-
tance mutations alter an organism's ability to run/hide or
otherwise defend itself could likely play a role in fitness costs in
the field. Thus, exploration of the fitness costs associated with
biotic challenges is encouraged.
Under field conditions it is common for both homozygous and
heterozygous resistant individuals to be present.2,4,5,24,25,32 Thus,
an understanding of the fitness costs for both homozygous and
heterozygous insects is desirable. Although a few studies have
conducted these types of analyses, there were not enough studies
to extrapolate any type of pattern, and additional work along
these lines would be very valuable.
It is well known in the antibiotic resistance field that although
resistance mutations are costly, they are commonly followed by
the evolution of compensatory mutations that help to minimize
the fitness cost.62 Thus, it could be expected that this is happen-
ing in cases of insecticide resistance as well. The first and cleanest
demonstration of a fitness modifier of insecticide resistance was
seen in 1987 in the Australian blowfly, Lucilia cuprina.63 In this
case, the modifier (which rescued delayed development associ-
ated with resistance) was identified because it was on a different
linkage group,63 and because a difference in the fitness of field-
collected resistant strains was noticed over a period of approxi-
mately 10 years. It was subsequently reported that resistance to
3854
diazinon was conferred by mutations of the carboxylesterase
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JC Freeman et al.
E3,64 and proposed that Scalloped wings was the fitness modi-
fier.64 It has been proposed that duplication of ace-1 in C. pipiens
and A. gambiae is another example of a modifier locus (keeping
together one copy of the susceptible allele with a copy of the
resistant allele to compensate for the poor enzyme activity of
the resistant copy against acetylcholine),65,66 but separating the
resistance allele and the modifier for experimental tests of fitness
is in that case much more difficult.53 Overall, the concept of com-
pensatory mutations or fitness modifiers draws attention to the
fact that mutations cannot be classified as advantageous or dele-
terious on their own: the context of their genetic background can
be critical. Characterization of compensatory mutations can help
us better understand both the physiological consequences of
resistance mutations and the dynamics that drive their evolution-
ary trajectory.
Insecticide resistance is both an intriguing evolutionary puzzle
and an area of tremendous practical importance because resistant
populations make control of agricultural, urban, and medically
important pests much more difficult and expensive. In this regard,
the fitness costs of resistance in the absence of insecticide use are
a powerful ally in our efforts to control pests. In fact, fitness costs
can greatly extend the lifetime of a given class of insecticide
against a pest, in at least one case.67 However, sustainable control
of pest insects cannot rely on fitness costs alone and resistance
management strategies68,69 must be a central tenant of sustain-
able pest control.
In conclusion, numerous studies on the fitness costs of insecti-
cide resistance show that there is commonly a cost to having
resistance. We suggest that future papers will have maximum
value to the scientific community if they quantitatively determine
resistance levels, identify the resistance mechanisms present (and
the associated mutations), have fully replicated experiments, use
related strains (optimally congenic with the resistance mutation
introgressed into different genetic backgrounds) and measure fit-
ness with multiple metrics. Studies on the fitness costs of insecti-
cide resistance will continue to enlighten our understanding of
the evolutionary process and provide valuable information for
resistance management.
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
article.
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