Resistance of Spodoptera frugiperda (Lepidoptera:Noctuidae) to spinetoram:

Inheritance and cross-resistance to spinosad

Ewerton C. Lira, Anderson Bolzan, Antonio R.B. Nascimento, Fernando S.A. Amaral,
Accepted Article
Rubens H. Kanno, Ingrid S. Kaiser, Celso Omoto*

* Correspondence to Celso Omoto, Department of Entomology and Acarology, Escola Superior de

Agricultura “Luiz de Queiroz”, University of São Paulo (USP), Av. Pádua Dias 11, Piracicaba, São

Paulo 13418-900, Brasil, E-mail: celso.omoto@usp.br

Running title: Inheritance of spinetoram resistance in S. frugiperda

Abstract

BACKGROUND: The use of spinosyn insecticides is one of the major control

strategies of the fall armyworm, Spodoptera frugiperda (J. E. Smith) in Brazil. In this

study, we selected a spinetoram-resistant strain from a field-population of S.

frugiperda to characterize the inheritance of the resistance and cross-resistance

relationship between spinosyn insecticides.

RESULTS: The values of LC50 (95% CI) obtained from concentration-response

bioassays were 0.63 (0.55–0.73) µg spinetoram mL-1 for the susceptible strain

(SUS), and 1170.96 (1041.61–1323.89) µg spinetoram mL-1 for the strain resistant to

spinetoram (SPT-R). These values resulted in a resistance ratio of 1844-fold. The

SPT-R strain showed cross-resistance with spinosad (resistance ratio = 1196-fold).

The reciprocal crosses showed LC50 values of 3.91 (2.97–5.84) and 5.37 (4.52–6.52)
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 µg spinetoram mL-1 suggesting that the resistance of S. frugiperda to spinetoram is

autosomal and incompletely recessive. The backcrosses of the F1 progeny with the

SPT-R strain suggest a resistance with polygenic effect. Estimates of the effective

number of loci with equal contributions to the resistance effect were from 1.18 to
Accepted Article
1.76, suggesting that the resistance to spinetoram is associated with a few genes.

CONCLUSIONS: The inheritance pattern of resistance of S. frugiperda to spinetoram

was characterized as autosomal, incompletely recessive and polygenic. Cross

resistance between spinosyns was confirmed in S. frugiperda. The importance of this

information for implementing insect resistance management strategies is discussed

in this paper.

Keywords: Spodoptera frugiperda; fall armyworm; spinosyns; inheritance of

resistance; insect resistance management.

1 INTRODUCTION

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a polyphagous

pest causing severe damage in many crops.1,2 It is native to tropical and subtropical

regions of the Americas.3,4 However, recently it was reported as an invasive species

in Africa and Asia.5,6 In Brazil, fall armyworm is the main and most-destructive pest of

maize, with field attacks reported during the entire year in all regions of the country.7,8

The bioecological aspects of S. frugiperda, such as high polyphagia, high

reproductive capacity and adult dispersion associated with the intensive agricultural

system in Brazil favor the development of this pest.1,9,10

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 The use of insecticides is one of the main strategies for controlling fall

armyworm in Brazil. Spinosyns insecticides have been an important tool for the

management of this pest because of their unique mode of action (allosteric

modulators of nicotinic acetylcholine receptors) and low toxicity to beneficial

Accepted Article
insects.11 This chemical group is composed by two active ingredients. The first one is

spinosad, a naturally occurring mixture of spinosyns A and D, is the first spinosyn

insecticide introduced in the market. 12 The second and more recently insecticide of

this group is spinetoram, a semi-synthetic molecule with high efficacy and positive

toxicological attributes compared to spinosad.13

Due to the high efficiency against insect pests, there are several cases of

resistance to spinosad reported in the literature.11 Cases of resistance to spinetoram

have been reported only for some species, such as Drosophila melanogaster Meigen

(Diptera: Drosophilidae),14 Choristoneura rosaceana (Harris) (Lepidoptera:

Tortricidae),15 Diaphorina citri Kuwayama (Hemiptera: Liviidae),16 Plutella xylostella

(Linnaeus) (Lepidoptera: Plutellidae) and Frankliniella occidentalis (Pergande)

(Thysanoptera: Thripidae).17,18 So far there are only two records of S. frugiperda

resistance to spinosyns, one resulting from a laboratory-selected resistant strain to

spinosad in Brazil19 and the other from a field-evolved resistance to spinetoram in

Porto Rico,20 demonstrating the risk of resistance evolution of this pest to spinosyns

insecticides.

Spinetoram has been frequently used to control fall armyworm in Brazil,

mainly because control failures and field-evolved resistance to several Bt maize

expressing a single or more insecticidal protein have been reported to S.

frugiperda.21–24 However, resistance to spinetoram in S. frugiperda has not been

reported yet in Brazil.

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 Understanding the genetic basis of insecticide resistance is important to

implement resistance management strategies. Information about the mode of

inheritance, degree of dominance and the number of genes involved determine the

rate of resistance evolution in the field. 25,26 Thus, the objectives of this study was to
Accepted Article
select a spinetoram-resistant strain from a field-population of S. frugiperda to

characterize the genetic basis of spinetoram resistance and evaluate the cross-

resistance to spinosad.

2 MATERIAL AND METHODS

2.1 Insects

The spinetoram-resistant strain (SPT-R) was selected from a field-collected

population in the second maize-growing season of 2016, in Barreiras, Bahia, Brazil

(11°45’42’’ S, 45°46’31’’ W). The susceptible strain (SUS) used as the reference has

been maintained at the Arthropod Resistance Laboratory (USP/ESALQ) for more

than 15 years, without selection pressure from insecticides. Larvae of both strains

were fed with artificial diet.27 The insects were maintained under controlled conditions

(25 ± 1 °C, 60 ± 10% RH and photophase of 14 h) in all development stages.

2.2 Bioassays

The concentration-response bioassays employed a diet overlay method,

conducted in 24-well acrylic plates (Costar®, Corning Inc., Corning, NY, USA), each

cell containing approximately 1.25 mL of artificial diet (1.9 cm2 area). For bioassays

using spinetoram, the commercial insecticide Exalt® (120 g.i.A.L-1; Dow AgroSciences

Industry Ltda., São Paulo, Brazil) was used; and for bioassays using spinosad, the

commercial insecticide Tracer® (480 g.i.A.L-1; Dow AgroSciences Industry Ltda.) was

used. The concentrations were prepared by diluting the commercial product in

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 distilled water, and the surfactant Triton™ X-100 (Labsynth, Diadema, SP, Brazil)

was added to each insecticide solution at 0.1% concentration. An aliquot of 30 µL of

the insecticide solution was applied per well. A solution containing only distilled water

and surfactant was used as the control treatment. The bioassays were conducted
Accepted Article
with 3rd instar larvae.

The bioassays were maintained in a climate-controlled chambers (25 ± 1 °C,

60 ± 10% RH and photophase of 14 h L). Mortality was assessed 48 h after

infestation, and larvae with no movement or uncoordinated movement when touched

were considered dead. Moribund larvae were considered dead because in

preliminary bioassays we confirmed that these larvae were dead after 96 h after

infestation.

2.3 Selection of spinetoram-resistant strain of S. frugiperda

The resistant strain (SPT-R) was selected using the F2 screening method.28

This method consists of the formation of isolines (pairs) with individuals from the

field-collected population, followed by endogamic crosses to obtain the F2 progeny

for each isoline. The bioassays were conducted using a diagnostic concentration of

5.6 µg spinetoram mL-1.29 After the evaluation (48 h), the surviving larvae from each

isoline were transferred to plastic containers (50 mL) containing artificial diet and

maintained until the adults emerged. A total of 78 isolines (Barreiras, BA) were

evaluated, using 120 larvae per isoline. Isolines that produced larvae that survived to

the adult phase were considered positive. These adults were grouped to form a strain

for selection. Selection initiated with the diagnostic concentration of 5.6 µg

spinetoram mL-1 and ended with the concentration of 3000 µg spinetoram mL-1 after

six generations of selection, obtaining the spinetoram-resistant strain (SPT-R).

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 2.4 Characterization of S. frugiperda resistance to spinetoram

2.4.1 Resistance ratio

Larvae from SPT-R and SUS strains were used in concentration-response

bioassays to estimate the LC50 (lethal concentration that kills 50% of the individuals)
Accepted Article
and the respective confidence intervals (95% CI). Both strains were exposed to

seven concentrations of the insecticide, spaced on a logarithmic scale, from 100 to

5600 µg spinetoram mL-1 for the SPT-R strain, and from 0.0 to 5.6 µg spinetoram mL-
1
for the SUS strain. The bioassays were conducted in completely randomized design

with three replicates, in total 72 individuals tested per concentration. The

concentration-mortality data for both strains were evaluated by Probit analysis,30

using the Polo Plus software.31 Parallelism and equality tests (P < 0.05) were

performed to test the hypotheses of equality and parallelism (slopes and intercepts

are not significantly different) between regression lines for each strain in the Probit

analysis.32 The resistance ratio of the SPT-R population was obtained by dividing the

LC50 of the SPT-R strain by the LC50 of the SUS strain (LC50 SPT-R/ LC50 SUS).

2.4.2 Cross-resistance with spinosad

Similarly to the method described in the previous topic (2.4.1), concentration-

response bioassays were conducted with strains SPT-R and SUS, using spinosad.

Strain SPT-R was exposed to seven concentrations spaced on a logarithmic scale,

from 100 to 3200 µg spinosad mL-1, while strain SUS was exposed to six

concentrations, from 0.1 to 5.6 µg spinosad mL-1. The bioassays were conducted in

completely randomized design with three replicates, in total 72 individuals tested per

concentration.

The concentration-mortality data for both strains were evaluated by Probit

analysis,30 using Polo Plus software.31 Parallelism and equality tests (P < 0.05) were

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 performed to compare the angular coefficients.32 The existence of cross-resistance

was determined by observing the resistance ratio of the SPT-R population obtained

for spinosad.

2.5 Inheritance of resistance

Accepted Article
2.5.1 Dominance

Then, reciprocal crosses were performed, using 20 pairs for each reciprocal

cross, kept in cylindrical PVC cages (10 x 20 cm) lined with paper (oviposition

substrate) for mating and oviposition. Each cage contained a container with a 10%

honey solution for feeding. Every two days, the eggs were removed and maintained

in a climate-controlled chamber (25 ± 1 °C and photophase of 14 h) until hatching (F1

progeny, heterozygotes H1 and H2). The larvae were maintained on an artificial diet

until reaching the 3rd instar. Concentration-response bioassays were conducted with

the F1 progeny. The larvae were exposed to seven concentrations of spinetoram

spaced on a logarithmic scale, between 1 and 32 µg spinetoram mL-1. The bioassays

were conducted in completely randomized design with three replicates, in total 72

individuals tested per concentration.

The concentration-response data for the SPT-R, SUS and heterozygote (F1)

strains were used to estimate the degree of dominance (D), based on the methods of

Stone33 to evaluated the dominance based on LC50 and Bourguet et al.34 to

evaluated the effective dominance considering the relationship between genotypes

and the insecticide concentration.

According to Stone33, the average degree of dominance is given by equation

[1]:

[1] ( – – ) ( – )

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 Where coefficients XF, XR and XS are logarithms of the LC50 estimated for the

heterozygote (F1), SPT-R and SUS strains, respectively. For the values of degree of

dominance (D) close to –1 (D ≅ –1), the resistance inheritance was considered

incompletely recessive, and for values close to 1 (D ≅ 1), the resistance was
Accepted Article

considered incompletely dominant.

The data were also analyzed using equation [2] proposed by Bourguet et

al.34

[2] ( – ) –

Where MRR, MSS and MRS represent the mortalities of the SPT-R, SUS and

heterozygote (F1) strains, respectively. For values of degree of dominance (D) close

to 0 (D ≅ 0), the inheritance was considered as incompletely recessive, and for

values close to 1 (D ≅ 1), it was considered incompletely dominant.

2.5.2 Number of genes

To estimate the number of genes associated with resistance, backcrosses

were conducted between the F1 progeny (heterozygotes H1 and H2) and the SPT-R

strain (parental strain that was phenotypically more distinct from F 1), as proposed by

Tsukamoto35 and Roush & Daly36 (Fig. 1). Four backcrosses were formed, each with

20 pairs, and kept in cages and reared as described in the previous topic. The

concentration-response bioassays were conducted with the F2 progeny (R1, R2, R3

and R4), using four concentrations between 3.2 and 18 µg of spinetoram mL-1. The

bioassays were conducted in completely randomized design with four replicates, in

total 96 individuals tested per concentration. The mortality data were used to

evaluate the hypothesis of monogenic inheritance by the chi-square test, given by

equation [3].37

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 [3] –

Where Ni represents the mortality observed in concentration i p is the

expected mortality, calculated based on the Mendelian model [4] 38; ni is the number

of individuals tested; and q = 1 – p.

Accepted Article

[4]

The monogenic inheritance hypothesis was rejected when the calculated chi-

square was higher than the table value (χ2 calculated ≥ χ2 table) at one degree of

freedom (P < 0.05), accepting the hypothesis of polygenic inheritance.

To estimate the number of loci related to the resistance of S. frugiperda to

spinetoram, equation [5] was used, which estimates the minimum number of genetic

factors (nE) that contribute quantitatively to differentiating two populations for any

type of selection.39

( – )
[5]
( )

Where µp2 and µp1 correspond to log10 of LC50 of strains SPT-R and SUS,

respectively, and corresponds to the phenotypic variations estimated by the

inverse value of the angular coefficient squared, using equation [6] proposed by

Lande39 to estimate the minimum number of genes contributing to the resistance

effect.

[6] –* +

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 Where , , , and correspond to the phenotypic variations of

the backcrosses R1 and R2/R3 and R4 and the heterozygote cross (F1), for the SPT-

R and SUS strains, respectively.

3. RESULTS
Accepted Article

3.1 Selection of spinetoram-resistant strain of S. frugiperda

Using the F2 screening method, 78 isolines of Barreiras, BA were evaluated

at the diagnostic concentration of 5.6 µg spinetoram mL-1 and 53 isolines were

positive.

3.2 Characterization of spinetoram-resistant strain of S. frugiperda

The LC50 values (95% CI) were 0.63 (0.55–0.73) µg spinetoram mL-1 for

strain SUS and 1170.96 (1041.61–1323.89) µg spinetoram mL-1 for strain SPT-R.

The resistance ratio obtained for strain SPT-R was 1844-fold, with rejection of the

hypothesis of equality (χ2 = 868, df = 2, P < 0.05) and parallelism (χ2 = 20.25, df = 1,

P < 0.05) between slopes and intercepts of strains (Table 1).

For spinosad, strain SPT-R showed an LC50 (95% CI) of 1949.74 (1601.24–

2470.51) µg spinosad mL-1, while the value for strain SUS was 1.63 (1.05–2.51) µg

spinosad mL-1. Thus, the resistance ratio obtained was 1196-fold, with rejection of

the hypothesis of equality (χ2 = 524, df = 2, P < 0.05) and parallelism (χ2 = 16.06, df =

1, P < 0.05) between strains (Table 1). Because of the high resistance of strain SPT-

R to spinosad, we accepted the existence of cross-resistance between spinetoram

and spinosad in S. frugiperda.

3.3 Resistance inheritance pattern

The LC50 (95% CI) values for both heterozygous strains were calculated. For

H1 (♀ SUS × ♂ SPT-R) and H2 (♂ SUS × ♀ SPT-R) the values were 3.91 (2.97–5.84)

µg spinetoram mL-1 and 5.37 (4.52–6.52) µg spinetoram mL-1, respectively. This

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 overlapping of the confidence interval of the heterozygotes indicates that the genes

related to S. frugiperda resistance to spinetoram are located in autosomal

chromosomes (Table 2).

Although the heterozygotes showed a susceptibility response closer to strain

Accepted Article
SUS, the resistance ratio ranged from 6.15 to 8.45-fold, differing from strain SUS by

rejection of the hypothesis of equality (χ2 = 550, df = 4, P < 0.05) and parallelism (χ2

= 57.64, df = 2, P < 0.05) between slopes and intercepts of heterozygotes and

susceptible strain (Fig. 2; Table 1). Similarly, the heterozygous differed from the

resistant strains, by rejection of the equality hypothesis (χ2 = 660, df = 4, P < 0.05).

3.3.1 Dominance

The degree of dominance obtained by the method proposed by Stone 33 was

–0.52 and –0.43 for reciprocal crosses H1 and H2, respectively, determining that the

resistance of S. frugiperda to spinetoram is incompletely recessive. However, by the

method of Bourguet et al.34, the degree of dominance varied with the concentration,

and was considered incompletely dominant in lower concentrations (1 to 3.2 µg

spinetoram mL-1), incompletely recessive in concentrations intermediaries close to

the concentration recommended for S. frugiperda control in the field (≈ 7.6 µg

spinetoram mL-1), changing to completely recessive with increasing concentrations

(>56 µg spinetoram mL-1) (Fig. 3).

3.3.1 Number of genes

The observed and expected mortalities of backcrosses R1, R2, R3 and R4 were

compared, using the chi-square test. Significant chi-square values (P < 0.05) were

found for most concentrations tested, rejecting the monogenic hypothesis (Table 3).

These results may indicate that the resistance of S. frugiperda to spinetoram has a

polygenic effect. However, according to the equation proposed by Lande,39 the

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 effective number of loci with equal contributions to the resistance was 1.18 to 1.76,

suggesting that the resistance of S. frugiperda to spinetoram is associated with a few

genes.
Accepted Article
4 DISCUSSION

In this study, we selected and characterized the resistance of S. frugiperda to

spinetoram from the field-collected population from Barreiras, Bahia. High level of

resistance to spinetoram was observed in S. frugiperda strain selected (SPT-R)

(1844-fold). However, control failures of fall armyworm to spinetoram has not been

reported yet in Brazil. Although, this indicates that the resistance alleles to spinosyns

are present in the field populations of S. frugiperda in Brazil. It is an alert to the risk of

resistance evolution under selection pressure. Furthermore, the SPT-R strain was

obtained in the same state where Okuma et al. 19 selected a spinosad-resistant strain

of S. frugiperda. This situation may indicate the intense use of spinosyns to control

fall armyworm in western of Bahia state, since we observed in this study that there is

cross resistance between spinosyns in S. frugiperda.

High cross-resistance observed between spinetoram and spinosad in S.

frugiperda favors the evolution of spinosyns resistance, due to pressure from both

insecticides selection under the same population. In addition, the occurrence of

cross-resistance between these insecticides makes the joint use of these compounds

inefficient for resistance management of S. frugiperda. Cross-resistance between

spinosyns has been reported for other species. 11 However, cross-resistance with

other insecticide chemical groups is not found in most cases, and when it occurs, the

resistance levels are low (3–20-fold).11. This fact increases the potential to use

insecticides with different mode of action (MoA) to management spinosyn resistance

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 in fall armyworm. An effective strategy to insecticide resistance management

recommended by Insecticide Resistance Action Committee - IRAC (https://www.irac-

online.org/).

When evaluating the response of heterozygotes exposed to spinetoram, the

Accepted Article
results indicate that the genes related to S. frugiperda resistance to spinetoram are

located in autosomal chromosomes, therefore shared by males and females.

Autosomal inheritance is commonly associated with resistance of fall armyworm to

19,40–46
insecticides, including spinosad. Our results about degree of dominance

suggests as incompletely recessive spinetoram resistance in S. frugiperda. However,

dominance levels varied depending upon the concentration. Resistance was

characterized as dominant at the lowest concentration (1 µg AI/mL), incompletely

recessive when exposed to concentrations close to the field-recommended rate (≈

7.6 µg AI/mL) and completely recessive at the highest concentration tested (56 µg

AI/mL). Effective dominance of resistance is the relationship between phenotypes

and three different genotypes (resistant homozygotes, susceptible homozygotes, and

heterozygotes) that can vary according to environmental conditions and

concentrations used.34,36. Thus, it is possible that heterozygotes can survive under

field conditions mainly when the pesticide residue decays.

The evaluation of backcrosses showed that the resistance of S. frugiperda to

spinetoram is associated with multiple genes, polygenic effect. A change in the

target-site, especially in the α6 subunit of nicotinic acetylcholine receptor is the main

mechanism of resistance reported to spinosyn. 11. Although, some studies have

reported spinosyns resistance associated with metabolic mechanisms, by the action

of detoxification enzymes15,47–51. Furthermore, studies are necessary for the better

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 understanding of the genes related to resistance and the molecular mechanisms of

spinetoram resistance in S. frugiperda.

The inheritance pattern of resistance to spinetoram in S. frugiperda was

characterized as autosomal, incompletely recessive, and polygenic. The same

Accepted Article
inheritance pattern was obtained by Okuma et. al.19 when characterized the

inheritance of S. frugiperda to spinosad. Given this, we may assume that the

resistance inheritance pattern of S. frugiperda is the same for both spinosyns. This

inheritance means that the heterozygotes presented phenotype closer to susceptible,

facilitating their control. This is favorable for resistance management, since

heterozygote individuals are the main carriers of the resistance alleles and are

present in higher proportion in the field than are resistant homozygote individuals. 36

When the resistance shows a polygenic effect, its evolution in the field is slower,

because polygenic character depend on the combination of more than one gene and

additive effects.52 Thus, the higher the number of genes involved in resistance,

slower may be the evolution. Other reason is due the resistance alleles are diluted by

the influx of susceptible alleles when an individual showing polygenic resistance

migrates to a susceptible population,25 especially in the case of recessive

inheritance.

In Brazil, spinetoram is an important management tool for S. frugiperda,

since it is one of the most effective insecticides against this pest in Bt and non-Bt

maize.21 For this reason, in recent years the use of spinetoram in the field has

increased due to control failures of S. frugiperda in Bt crops already reported in

Brazil,21–24 increasing the risk of resistance evolution to spinosyns due to increased

selection pressure, considering that both spinosyns are recommended against the

same species in maize, soybean, cotton, rice and sorghum. Analysis of the

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 inheritance pattern of resistance to spinetoram associated with inheritance for

spinosad19 provides useful information for implementing management tactics to

prolonging the useful life and efficacy of spinosyns group for fall armyworm control.

For this, it is necessary to monitoring the frequency of resistance in the field and
Accepted Article
implement proactive strategies, such as rotation of MoA that can delay the evolution

of resistance as well as restore susceptibility, since Okuma et al. 19 have proven that

there is fitness cost to spinosad in S. frugiperda. The success of rotation requires that

there is fitness cost of resistant individuals and that there is no cross-resistance

between MoA.53

In conclusion, we characterized S. frugiperda resistance to spinetoram as

autosomal, incompletely recessive and polygenic, and confirmed cross resistance

between spinosyns for fall armyworm. We showed that the risk of resistance

evolution is high in Brazil and provided results to understand de genetics bases of

this resistance to implement proactive strategies to delay the evolution of S.

frugiperda resistance to spinosyns.

ACKNOWLEDGMENTS

We thank the National Council for Technological and Scientific Development

(CNPq) for granting the scholarship to ECL (Process # 132014/2016-5) and research

fellowship to CO (Process # 312086/2013-0). We also thank the Brazilian Insecticide

Resistance Action Committee (IRAC-BR) for providing partial financial support for

this study.

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Accepted Article

Table 1. Concentration-response of susceptible (SUS) and resistant (SPT-R) strains

of S. frugiperda to spinetoram and spinosad.

Strain Insecticide n Slope (±SE) LC 50 (95% CI) a χ2 df b RR d

SUS 641 5.40 (±0.52) 0.63 (0.55-0.73) 6.75 5 -
Spinetoram
SPT-R 575 3.04 (±0.24) 1170.96 (1041.61-1323.89) 2.30 5 1844
SUS 645 2.12 (±0.19) 1.63 (1,05-2,51) 11.97 4 -
Spinosad
SPT-R 611 3.69 (±0.37) 1949.74 (1601.23-2470.5) 9.18 5 1196
a -1
LC50 (µg mL ) and confidence interval.
b
df = degrees of freedom.
c
Resistance ratio (RR) = LC50 of resistant strain/ LC50 of susceptible strain.

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Accepted Article

Table 2. Concentration-response to spinetoram of susceptible (SUS) and resistant

(SPT-R) strains of S. frugiperda, and heterozygotes (H1 = ♀ SUS × ♂ SPT-R and H2

= ♂ SUS × ♀ SPT-R) from reciprocal crosses (F1 progeny).

Strain n Slope (±SE) LC 50 (95% CI) a * χ2 df b RR c

SPT-R 575 3.04 (±0.24) 1170.96 (1041.61-1323.89) a 2.30 5 1844.03

SUS 641 5.40 (±0.52) 0.63 (0.55-0.73) b 6.75 5 -
H1 835 2.81 (±0.29) 3.91 (2.97-5.84) c 13.42 6 6.15
H2 770 2.05 (±0.16) 5.37 (4.52-6.52) c 6.02 8 8.45
a -1
LC50 (µg mL ) and confidence interval.

* LC50 values followed by the same letter did not differ significantly between the confidence intervals

(95%).
b
df = degrees of freedom.
c
Resistance ratio (RR) = LD50 of resistant strain/LD50 of susceptible strain.

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 Table 3. Chi-square analysis ( 2) of the mortality data from the backcrosses between
Accepted Article

spinetoram-resistant (SPT-R) and F1 progeny of the reciprocal crosses (H1 = ♀ SUS

× ♂ SPT-R and H2 = ♂ SUS × ♀ SPT-R) exposed to different concentrations of

spinetoram.

Concentration Backcrosses mortality 2

n χ ρ
(µg.mL) Obs. a Exp. b
R1 = ♂ SPT-R x ♀ (♀SUS x ♂SPT-R)
3.2 96 9 (9.38) 17.36 (18.09) 4.27 * 0.039
5.6 95 18 (18.95) 33.65 (35.42) 9.19 * 0.002
10 96 25 (26.04) 46.67 (48.61) 16.41 * 0.0001
18 91 40 (43.96) 45.50 (50) 0.09 0.767
R2 = ♀ SPT-R x ♂ (♀SUS x ♂SPT-R)
3.2 72 6 (8.33) 13 (18.09) 4.62 * 0.032
5.6 96 17 (17.71) 34 (35.42) 13.16 * 0.0003
10 72 18 (25) 35 (48.61) 16.07 * 0.0001
18 72 31 (43.06) 36 (50) 1.39 0.239
R3 = ♂ SPT-R x ♀ (♂SUS x ♀SPT-R)
3.2 96 5 (5.21) 16.27 (16.95) 9.4 * 0.002
5.6 96 12 (12.50) 26.55 (27.66) 11.03 * 0.001
10 96 20 (20.83) 33.70 (35.11) 8.58 * 0.003
18 96 31 (32.29) 42 (43.75) 5.12 * 0.024
R4 = ♀ SPT-R x ♂ (♂SUS x ♀SPT-R)
3.2 96 10 (10.4) 16.3 (16.95) 2.91 0.088
5.6 91 17 (18.7) 25.2 (27.66) 3.67 0.056
10 94 26 (27.7) 33 (35.11) 2.29 0.130
18 93 29 (31.2) 40.7 (43.75) 5.97 * 0.015
a
Observed mortality.
b
Expected mortality, based on Mendelian inheritance.

* Significant difference (P < 0.05, degree of freedom = 1) between observed and expected mortalities.

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Accepted Article

Figure legends

Figure 1. Reciprocal crosses and backcrosses of F1 progeny and the spinetoram-

resistant strain SPT-R of S. frugiperda.

Figure 2. Concentration-response to spinetoram on logarithmic scale of susceptible

(SUS) and spinetoram-resistant (SPT-R) strains, and heterozygotes (H1 = ♀ SUS ×
♂ SPT-R and H2 = ♂ SUS × ♀ SPT-R).

Figure 3. Effect of the dominance of resistance to spinetoram in S. frugiperda as a

function of spinetoram concentration. (H1 = ♀ SUS × ♂ SPT-R; H2 = ♂ SUS × ♀
SPT-R).

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Accepted Article

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Accepted Article

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Accepted Article

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