Research Article

Received: 22 April 2020 Revised: 13 July 2020 Accepted article published: 6 August 2020 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/ps.6032

Synergism and unintended effects of the

association between imidacloprid and sodium
chloride (NaCl) on the management of
Euschistus heros
Hígor S Rodrigues,a Khalid Haddi,b Mateus O Campos,a
Noelio A Ferreira-Filho,a Raul Narciso C Guedes,a Philip L Newlandc and
Eugênio E Oliveiraa,d*

Abstract
BACKGROUND: The use of insecticidal solutions containing sodium chloride (NaCl) has been proposed as a more environmen-
tally friendly alternative to managing stink bug infestations of Neotropical soybean fields. The potential sublethal and unde-
sirable effects of this practice have, however, been overlooked, especially with novel insecticides. Here, we have evaluated
experimentally whether the addition of NaCl (0.5% w/v) to imidacloprid-containing solutions could alter insecticide toxicity
and modify the reproductive responses of the Neotropical brown stink bug Euschistus heros.
RESULTS: Adding NaCl to imidacloprid solutions significantly increased imidacloprid toxicity against E. heros. The exposure to E. heros
to sublethal concentrations of imidacloprid affected the insect's mating abilities in a concentration-dependent manner. The addition
of NaCl to solutions containing imidacloprid at concentrations as low as 0.126 ∼g a.i. cm−2 (i.e. the equivalent to 3% of field rate rec-
ommendation) also impacted the sexual behavior of E. heros, reducing mating duration. NaCl-exposed stink bugs, however, exhibited
higher fecundity and fertility rates than those insects that were unexposed to NaCl or those that were exposed to sublethal levels of
imidacloprid only.
CONCLUSIONS: The addition of low amounts of NaCl resulted in a higher toxicity of imidacloprid. This practice, however, can
also lead to undesirable effects as increasing reproductive output of E. heros that can potentially compromise the management
of these insect pests.
© 2020 Society of Chemical Industry

Keywords: pentatomid pest species; neonicotinoids; sexual behavior; reproductive fitness

1 INTRODUCTION
The gustatory system in insects is responsible for the detection
and processing of chemicals in the environment, and the avoid-
ance of insecticides and other toxic substances, through taste
organs (e.g. basiconic sensilla, gustatory bristles and pegs) distrib-
uted over the body and limbs.1–3 Advances in our understanding
of insect chemosensation have offered possibilities for the devel-
opment of novel insect pest control methods, such as the applica-
tion of tastants (e.g. salts and sugars) that may potentiate the
efficacy of existing insect control practices such as synthetic repel-
lents and synthetic insecticides.4–8
The utilization of sodium chloride (NaCl) at low concentrations
(0.5% w/v) in combination with synthetic insecticides is a com-
mon and empirically applied practice for controlling phytosucci-
vorous stink bugs in Neotropical soybean-growing fields.9,10
Such a control strategy was initially based on field observations
and other studies that suggested increases in the efficacy of insec-
ticides, allowing a reduction of up to 50% of the doses used.9,10
Most of these results, however, were obtained with insecticides
that were banned from commercial use in 2004, and despite a
recent study that has suggested that such practice does not
potentiate the unintended effects caused by modern insecti-
cides.11 There is, however, much that we do not yet know about
this control strategy, including the toxicity and synergism, espe-
cially at sublethal levels, when NaCl is applied with currently used
insecticides. For instance, the exposure of insect pests to low con-
centrations of NaCl can lead to an increase in their survival and
* Correspondence to: EE Oliveira, Departamento de Entomologia, Universidade
Federal de Viçosa, Viçosa, MG 36570-000, Brazil, E-mail: eugenio@ufv.br
a Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, Brazil
b Departamento de Entomologia, Universidade Federal de Lavras, Lavras, Brazil
c Biological Sciences, University of Southampton, Southampton, UK
d Departament of Entomology, Genetics and Neuroscience Programs, Michigan
State University, East Lansing, MI, USA
1

Pest Manag Sci 2020 www.soci.org © 2020 Society of Chemical Industry

 www.soci.org
reproductive performances, as has also been found in non-pest
species.12–15
Despite their potential environmental risks, neonicotinoid use
represents about 25% of the global insecticide market share.16,17
These systemic insecticides disrupt the transmission of nerve
impulses in insects by disturbing the function of certain types of
nicotinic acetylcholine receptors.18–20 The application of neonico-
tinoids, alone or mixed with other insecticides, is the most com-
mon practice used against the Neotropical brown stink bug
Euschistus heros.21–24 This species is the prevailing stink bug pest
species of South American soybean-growing fields.23,25,26 The fre-
quent overuse of neonicotinoid insecticides against this species
leads to unintended sublethal effects27,28 and control failures
due to insecticide resistance.21–23,29
In this study, we assessed the toxicity and evaluated whether
the sublethal exposure of E. heros to imidacloprid [at 0.126 μg
active ingredient (a.i.) cm−2 – the equivalent to 3% of the field rate
recommendation] alone, or mixed with low NaCl concentration
(0.5% w/v), could cause any unintended effects on the survival
and reproductive responses of these insect pests.
2 MATERIALS AND METHODS
2.1 Insect rearing
All insects used here originated from a mass-rearing colony kept
under controlled conditions [i.e. 27 ± 2 °C, 60 ± 20% of relative
humidity, and 14 h:10 h light/dark (L/D) photoperiod], as
described elsewhere.22,28,30
2.2 Concentration-mortality bioassays
Concentration-mortality bioassays were carried out using
methods that ensured insect exposure to dried imidacloprid resi-
due [isolated or with NaCl (0.5% w/v)]. The methods were adapted
from previous toxicological studies with the same
species.22,27,28,30–32 Briefly, a 2 mL aliquot of the imidacloprid-
containing solution was applied to coat the inner surface of a clear
250 mL glass vial. Insecticide solutions were made by diluting a
commercial formulation (water-dispersible granules at 700 g a.
i. L−1; Bayer CropScience, São Paulo, Brazil).
The potential synergism of the association of NaCl with imida-
cloprid was assessed by using four imidacloprid concentrations
(i.e. 0.042, 0.126, 0.42 and 1.26 μg a.i. cm−2 that were equivalent
to 1, 3, 10 and 30% of the recommended field rate) applied alone
or associated with NaCl (0.5% w/v). For control treatments, insects
were exposed either to distilled water alone or water containing
0.5% NaCl. We used newly emerged adults (< 24 h) and an expo-
sure time of 48 h. Ten replicates (i.e. a glass vial containing a
group of ten newly emerged adults) were used for each insecti-
cide concentration. The inner upper portion of each vial was lined
with Teflon PTFE (DuPont, Wilmington, DE, USA) and closed with a
piece of organza attached to the vial with a rubber band. Insects
were counted as dead when unable to walk the length of their
body when prodded with a fine hair brush.
2.3 Effects on mating behavior
Groups of newly emerged (< 24 h) male and female stink bugs
were individually exposed (48 h) to dry residues of imidacloprid,
initially applied at the concentration of 0.126 μg a.i. cm−2 that is
the equivalent to 3% of the recommended field rate, associated
or not with low concentration of NaCl (0.5% w/v). Other newly-
emerged stink bug couple groups were exposed to dried residues
of distilled water alone or mixed with 0.5% NaCl as control
2
wileyonlinelibrary.com/journal/ps © 2020 Society of Chemical Industry
HS Rodrigues et al.
treatments. Following exposure, groups of females or males were
kept apart in plastic containers for the next ten consecutive days
until reaching sexual maturity. During this time, all insects were
fed ad libitum with a mixture of fresh pods of green beans (Phaseolus
vulgaris L.), soybean seeds (Glycine max L.), raw peanuts (Arachis
hypogaea L.) and sunflower seeds (Helianthus annuus L.).33–35 Sup-
plies were replenished at 3-day intervals and the inner bottom of
the plastic containers were covered with paper towels to absorb
humidity.
Mating behaviors were digitally recorded for couples of sexually
mature virgin males and females that survived exposure to the
insecticide/NaCl combinations. Couples of insects were placed
inside a Petri dish (90 mm × 15 mm) and their mating activities
recorded for a 13 h period using a digital camera (HDR-XR520V,
Sony, Tokyo, Japan). Following the mating period, couples were
separated, and each individual maintained in a Petri dish. We used
the time to the first mating, the number of matings, the total and
averaged mating durations (in minutes) as the behavioral param-
eters. The total mating time encompasses the time between
mounting and dismounting. The average mating time was the
total mating time divided by the number of matings of each
couple.
2.4 Effects on survival, fecundity and fertility
All the procedures regarding insecticide exposure, mating and
post-mating periods were identical to those described earlier.
After the insecticide exposure and mating, we recorded the sur-
vival and reproductive output (i.e. fecundity and fertility) of at
least 20 couples per treatment. Insects were recognized as dead
when they were unable to walk after being prodded with a fine
hair brush. We used the daily and accumulated number of eggs
laid/female, the daily percentage of nymphs emerged/female,
and the accumulated number of emerged nymphs/females as
the reproductive parameters.
2.5 Statistical analyses
The concentration-mortality results were submitted to probit
analysis using the PROC PROBIT procedure.36 Synergism ratio
(SR50) was estimated by dividing the median lethal concentration
(LC50) value obtained for imidacloprid alone by the LC50 value
75
imidacloprid + NaCl
mortality (%)
LC50 = 0.33 (0.21 - 0.54) μg/cm2
(1,2) = 2.2; P = 0.33
2
50
25
imidacloprid
2
Lc50 = 1.23 (0.87 - 1.89) μg/cm
0
2
(1,2) = 3.0; P = 0.39
0 0.042 0.126 0.42 1.26
2
concentration ( μg a.i./cm )
Figure 1. Toxicity of dry residues of imidacloprid to adult Euschistus heros.
Lethal concentration (LC) values were estimated based on concentration-
mortality bioassays using probit analyses. Symbols represent the mean
mortality of ten replicates and the vertical bars represent the standard
error of the mean (± SEM). Blue triangles indicate the result obtained with
the application of imidacloprid alone. Red circles indicate the results
obtained with the application of imidacloprid with sodium chloride
(NaCl, 0.5% w/v).
Pest Manag Sci 2020
Effects of application of imidacloprid + NaCl against E. heros www.soci.org

obtained for imidacloprid–NaCl association.37 The 95% confi- NaCl effects on mating performances
dence limits of these toxicity rate estimations were considered of E. heros
to be significantly different (P < 0.05) if they did not include the
value 1.37 The results of the survival bioassays were subjected to (A)
without NaCl with NaCl
survival analysis, which was performed using Kaplan–Meier esti-
mators (log-rank method) with SigmaPlot 12.0 (Systat Software, * control
*
San Jose, CA, USA). Overall similarity among the survival was
tested using the χ 2 log-rank test, and pairwise comparisons * imidacloprid *
(0.126 μg/cm2)
among the curves were carried out using the Holm–Sidak test
(P < 0.05). The second set of mating behavior bioassay was sub- 5 4 3 2 1 0 0 1 2 3 4 5
jected to factorial analysis considering an overall error level of
number of matings
P < 0.05 (PROC GLM),36 using imidacloprid (presence or absence)
and NaCl (presence or absence) as the factors. All multiple pair- (B)
wise comparisons, when necessary, were performed using the without NaCl with NaCl
Tukey's honestly significant difference (HSD) post hoc test
(P < 0.05) in SAS 9.0.36 For the reproductive bioassays, the results * control *
were subjected to regression analyses using the curve-fitting pro-
cedure of SigmaPlot 12.5. The regression model was chosen * imidacloprid *
(0.126 μg/cm2)
based on parsimony, lower standard errors, and steep increases
in R2 with model complexity. The regression models for each 100 80 60 40 20 0 0 20 40 60 80 100
treatment were considered different from each other if the confi- time elapsed up to the first mating (min)
dence limits of their parameters did not overlap.
(C)
without NaCl with NaCl
3 RESULTS control
3.1 Relative toxicity * *
The concentration-mortality results, estimated based on imidacloprid
* *
concentration-mortality bioassays, were satisfactory adjusted to (0.126 μg/cm2)
a probit model (P > 0.05) for both insecticidal solutions (imidaclo-
800 600 400 200 0 0 200 400 600 800
prid alone or in association with NaCl) (Fig. 1). Increases in the imi-
dacloprid concentration resulted in significantly higher mortality avegare mating duration (min)
levels for E. heros, which results in a LC50 of 1.23 (0.87–1.89) μg
a.i. cm−2 (χ 2(1,2) = 3.0, P = 0.39). The addition of NaCl also (D)
increased the toxicity for all imidacloprid concentrations, reduc- without NaCl with NaCl
ing the LC50 value to 0.33 (0.21–0.54) μg a.i. cm−2 (χ 2(1,2) = 2.2,
control
P = 0.33). Based on these LC50 estimations, the addition of low
concentration of NaCl association resulted in a SR50 of 4.09
imidacloprid
(2.06–8.10)-fold (Fig. 1). (0.126 μg/cm2)

800 600 400 200 0 0 200 400 600 800

3.2 Mating behavior
The factorial analysis revealed that the exposure to NaCl, not the
imidacloprid, affected the mating behavior of E. heros (Table 1).
Couples of E. heros that were exposed to NaCl exhibited signifi-
cantly shorter averaged mating duration (F = 6.82; df = 1;
P = 0.01) and increase in the time elapsed to the first mating
attempt (F = 9.83; df = 1; P = 0.002) and the number of matings
(F = 5.47; df = 1; P = 0.02), regardless of imidacloprid exposure
total mating duration (min)
Figure 2. Mating behavior of Euschistus heros exposed to sodium chloride
(NaCl, 0.5% w/v) and imidacloprid. (A) The number of successful mating
attempts. (B) Time to first mating. (C) The averaged. (D) Total mating duration
(in minutes). Box plots indicate the median and range of dispersion (lower
and upper quartiles). Black points indicate 95% of data used to generate
box plots. Asterisks indicate statistical differences between groups in oppo-
site panels by Tukey's honestly significant difference (HSD) test (P < 0.05).

Table 1. Factor analysis of sexual behavior parameters (shown in Fig. 2)

Total mating duration Average mating duration Time elapsed up to the first mating Number of matings

Sources of variation df F P df F P df F P df F P

Model 3 0.21 0.89 3 2.89 0.04* 3 4.28 0.007* 3 1.86 0.14

Error 93 — — 83 — — 78 — — 93 — —
Insecticide (I) 1 0.44 0.51 1 1.84 0.18 1 2.05 0.16 1 0.08 0.78
Sodium chloride (SC) 1 0.06 0.80 1 6.82 0.01* 1 9.83 0.002* 1 5.47 0.02*
I × SC 1 0.14 0.71 1 0.00 0.95 1 0.96 0.33 1 0.02 0.88
*
Indicates significant difference at P < 0.05.
3

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 www.soci.org HS Rodrigues et al.

(A) survival of E. heros males (A) fecundity of E. heros

100 40
control control
imidacloprid imidacloprid
control + NaCl 30 control + NaCl
75 imidacloprid + NaCl imidacloprid + NaCl

eggs/female
survival (%)

20

50 a

10
ab

b ab
25
0
0 20 40 60 80
time (days)
0
0 20 40 60 80
(B) fertility of E. heros
time (days)
100 control
(B) survival of E. heros females imidacloprid
control + NaCl
100

(%)
80 imidacloprid + NaCl

eclosion/female
60
a
75
a
survival (%)

40
a
50 20

a
0
control
25 imidacloprid 0 20 40 60 80
control + NaCl
time (days)
imidacloprid + NaCl
Figure 4. Daily fecundity (A) and fertility (B) of Euschistus heros exposed
0 to sodium chloride (NaCl, 0.5% w/v) and imidacloprid. The lines represent
0 20 40 60 80 adjusted non-linear regressions. The symbols represent the average of the
time (days) observed data. The parameters of these equations are shown in Table 2.

Figure 3. Survival curves of Euschistus heros males (A) and females

(B) exposed to sodium chloride (NaCl, 0.5% w/v) and imidacloprid. Curves
with different lowercase letters differ significantly from each other at
P < 0.001 (Holm–Sidak test).
(Fig. 2, Table 1). No significant differences were recorded for the
total mating duration (F = 0.06; df = 1; P = 0.80) (Fig. 2, Table 1).
3.3 Survival
Male survival differed significantly between treatments (log-rank
test: χ 2 = 9.63, df = 3, P = 0.022). Males of E. heros that were
exposed sublethally to imidacloprid + NaCl lived longer [esti-
mated median survival time (LT50) = 50.0 (42.7–57.3) days] than
males exposed to only imidacloprid [LT50 = 27.0 (26.0–28.0) days]
(Fig. 3(A)). Unexposed males [LT50 = 38.0 (31.1–48.0) days] and
males exposed to NaCl alone [LT50 = 42.0 (38.1–45.9) days] exhib-
ited intermediate results (Fig. 3(A)). There were no significant dif-
ferences between treatments for female survival (log-rank test:
χ 2 = 4.10, df = 3, P = 0.250), which exhibited a minimum LT50
value of 37.2 days and a maximum LT50 of 57.8 days (Fig. 3(B)).
3.4 Reproductive output
The daily fecundity results (i.e. daily number of laid eggs/females)
fitted well with a three-parameter Lorentzian model with a bell-
shaped curve skewed to the left and a long tail to the right
4
(Fig. 4(A), Table 2). NaCl-exposed couples exhibited a lower peak
of reproduction and slower reproductive decline, regardless of
imidacloprid exposure (Fig. 4(A)). Furthermore, unexposed and
imidacloprid-exposed couples exhibited a higher peak of daily
fertility, but with a quicker decline, than NaCl-exposed couples
(Fig. 5(B)). The longer reproductive period observed in the daily
fecundity and fertility curves were translated into higher levels
of reproductive output of NaCl-exposed couples, regardless of
imidacloprid exposure (Fig. 5(B), Table 3).
4 DISCUSSION
The use of salts and other substances that modify insect behaviors
have been used to enhance the efficacy of insecticides.6–8,10 Here,
we show that NaCl at 0.5% increases the lethal toxicity imidaclo-
prid to E. heros. NaCl, however, also increased the reproductive
performance of exposed insects, regardless of imidacloprid expo-
sure, which represents major negative consequence of such
NaCl use.
Previous investigations at glasshouse and field levels have pro-
posed that the addition of NaCl, at low concentrations, to certain
insecticide types can increase that insecticides efficacy
(e.g. organophosphates) against stink bugs.6,9,10,38 While not fully
understood as yet, the enhanced insecticidal activity achieved

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Table 2. Summary of non-linear regression analyses of daily fecundity and fertility (shown in Fig. 4)

Estimated parameters (± SE)

Variable Model Treatment a b x0 df F P R2

Eggs/ y = a/(1 + ((x Water 29.2 (26.1–32.3) a 20.0 (15.5–24.6) a 13.1 (9.9–16.3) a 23 108.5 <0.0001 0.89
female − x0)/b)2) Imidacloprid 27.9 (25.0–30.8) a 20.1 (16.0–25.4) a 12.45 (9.1–15.9) a 23 130.0 <0.0001 0.91
Water + NaCl 24.9 (21.6–28.2) a 31.8 (20.9–42.7) ab 15.7 (8.2–23.4) a 23 41.3 <0.0001 0.76
Imidacloprid 25.4 (22.2–28.5) a 48.7 (27.0–70.4) b 14.3 (−1.5–30.0) a 23 15.6 <0.0001 0.54
+ NaCl
Eclosion/ y = a/(1 + ((x Water 0.8 (0.7–0.9) a 22.5 (16.9–28.2) a 15.4 (11.7–19.2) a 23 95.3 <0.0001 0.88
female − x0)/b)2) Insecticide 0.9 (0.8–1.0) a 21.1 (16.5–25.7) a 16.4 (13.4–19.3) a 23 119.0 <0.0001 0.90
(%) Water + NaCl 0.7 (0.6–0.8) ab 38.2 (20.9–55.4) ab 16.3 (5.3–27.3) a 20 26.3 <0.0001 0.70
Imidacloprid 0.6 (0.5–0.6) b 37.8 (28.7–46.4) b 6.2 (−3.6–16.0) a 23 110.9 <0.0001 0.90
+ NaCl

Note: The parameters characterize different attributes of the curves, where a is the location of the peak response value on the time axis, b is the max-
imum value of the dependent variable, and x0 is the skewness (or rate of change) of the response as a function of time. Parameter values followed by
different letters in the columns were significantly different (based on non-overlapping of confidence limits).

with NaCl on stink bugs has been attributed to either an increased caused by NaCl to imidacloprid's effect recorded here, however,
attractiveness to NaCl-impregnated surfaces or to a prolonged goes beyond that of a phagostimulant or phagoarrestant. Probing
probing behavior initiated by the salt.10 The enhanced toxicity behavior was prevented in our study, which forced the stink bugs
to detect NaCl and imidacloprid residues through chemorecep-
tors located on their legs and body parts other than their mouth-
parts. This is commonplace in many insect species, where
(A) cumulated fecundity of E. heros chemoreceptors located on the insect's tarsi are involved in the
initial stages of food selection2,39 and plant-derived insecticide
500
compounds (e.g. decaleside) act via tarsal gustatory sites.5
Sodium ions (Na+) play relevant roles in osmoregulation and
neurophysiological functions.40,41 Furthermore, higher internal
number of eggs

375
concentration and availability of Na+ can cause hyperexcitation
of the insect nervous system, which may enhance imidacloprid-
250
mediated mortality.42–45 Imidacloprid molecules may also
control
imidacloprid undergo modifications in the presence of NaCl [e.g. insertion of
125 control + NaCl the chloride ions (Cl−)] increasing its affinity to its binding sites
imidacloprid + NaCl at nicotinic acetylcholine receptors (nAChRs), thereby resulting
in a higher insecticide efficacy.46,47
0
By contrast to increasing insecticide toxicity, NaCl also
0 20 40 60 80
enhanced the sexual and reproductive performance of E. heros.
time (days) The potential contributions of NaCl-containing salts on neuro-
physiological functions that control mating behaviors and repro-
(B) cumulated fertility of E. heros ductive performance was initially presumed by ancient
600
civilizations.48,49 Nowadays, it is well known that females undergo
major changes in their nutritional demands to invest in progeny
number of nimphs

450
production and elevated internal Na+ contents maximize repro-
ductive output in several species.13,50–53 Other studies have also
300 demonstrated the involvement of Na+ in several processes such
as sperm motility,54 and amino acid uptake by the digestive
control tract,55 making these ions more readily available for growth and
150 imidacloprid reproduction.
control + NaCl Other studies indicate a higher appetite for Na+-containing salts
imidacloprid + NaCl
0 during the reproductive period of several species.13,56,57 In insects,
0 15 30 45 60 females can eat more amino acids to obtain the raw materials
required to produce eggs,56 and males of several Lepidoptera
time (days)
species that frequent mud puddles (activity also termed as pud-
Figure 5. Cumulative egg production (A) and emerged nymphs (B) of dling) to imbibe Na+ increased their mating success by increasing
Euschistus heros to sodium chloride (NaCl, 0.5% w/v) and imidacloprid their neuromuscular activity.12 These puddling males can transfer
(0.126 μg a.i. cm−2 – the equivalent to 3% of field rate recommendation).
The lines represent adjusted non-linear regressions. The symbols repre-
the gained Na+ ions to females during mating, which may end up
sent the average of the observed data. The parameters of these equations in their progeny.58 Curiously, the sublethal exposure to imidaclo-
are shown in Table 3. prid has been shown to induce differential reproductive
5

Pest Manag Sci 2020 © 2020 Society of Chemical Industry wileyonlinelibrary.com/journal/ps

 www.soci.org HS Rodrigues et al.

responses in males and females of E. heros27,28,30–32 and cause

Note: The parameters characterize different attributes of the curves, where a is the location of the peak response value on the time axis, b is the maximum value of the dependent variable, and x0 is the
0.98
0.99
0.98
0.98
0.98
0.99
0.98
0.99

skewness (or rate of change) of the response as a function of time. Parameter values followed by different letters in the columns were significantly different (based on non-overlapping of confidence limits).
R2
stimulatory reproductive responses in other insect
species.27,28,59–62 Some studies suggest potential sex-biased pat-
terns for insecticide detection that may reflect differences in the

<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
distribution and morphology of their sensilla.27,63,64
Collectively, the findings described here have revealed that
P
adding NaCl to imidacloprid-containing solutions can increase
the efficacy of an insecticide against E. heros, but NaCl also
enhances reproductive performance, which may represent a neg-
406.4
521.2
486.6
317.9
329.0
416.4
399.4
515.2
F

ative consequence for the integrated management of these

insect pests.
13
12
12
13
9
9
10
10
df

ACKNOWLEDGEMENTS
29.2 (25.8–35.6) ab The provision of stock colonies of E. heros by Dr R. Laumann
27.4 (24.4–30.5) b
29.4 (25.5–33.4) b

33.8 (27.5–40.1) b
20.6 (18.6–22.7) a
19.9 (18.1–21.6) a

19.6 (17.2–22.0) a
23.2 (20.5–25.8) a

(EMBRAPA Genetic Resources and Biotechnology, Brasília, DF, Bra-

zil) was greatly appreciated. This work was supported by the
x0

CAPES Foundation (Finance code 001), the National Council of Sci-

entific and Technological Development (CNPq), and the Minas
Gerais State Foundation for Research Aid (FAPEMIG).
Estimated parameters (± SE)

CONFLICT OF INTEREST
14.6 (11.1–18.2) b
9.6 (7.5–11.7) ab

11.8 (9.4–14.1) ab
13.0 (10.5–15.5) a
13.6 (10.4–16.9) a
9.6 (7.7–11.6) a
9.0 (7.4–10.6) a

8.6 (6.5–10.6) a

The authors declare that they have no conflict of interest.

b

ETHICAL APPROVAL
All applicable international, national, and institutional guidelines
Table 3. Summary of non-linear regression analyses of cumulative egg and nymph production (shown in Fig. 5)

for the care and use of animals were considered in the present
investigation.
438.3 (407.0–469.6) b
486.1 (443.6–528.6) b

572.0 (477.4–666.7) b
544.5 (492.3–596.8) b
347.9 (332.1–363.8) a
327.6 (314.1–341.1) a

356.0 (330.5–381.4) a
383.0 (351.7–414.3) a

INFORMED CONSENT
a

All the authors of this manuscript accepted that the article is sub-
mitted for publication in the Pest Management Science, and report
that this article has not been published or accepted for publica-
tion in another journal, and it is not under consideration at
another journal.
Imidacloprid + NaCl

Imidacloprid + NaCl
Treatment

AUTHORS' CONTRIBUTIONS
Water + NaCl

Water + NaCl
Imidacloprid

Imidacloprid

HSR: conceptualization, methodology, investigation, formal analy-

sis, visualization, writing – original draft. KH: conceptualization,
Water

Water

methodology, formal analysis, writing – review and editing.

MOC: investigation. NAF-F: investigation. RNCG: writing – review
and editing, resource, funding acquisition. PLN: writing – review
y = a/(1 + exp(−(x − x0)/b))

y = a/(1 + exp(−(x − x0)/b))

and editing, funding acquisition. EEO: conceptualization, method-

ology, formal analysis, writing – review and editing, resource,
funding acquisition. All authors read, corrected and approved
Model

the manuscript.

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