Differential influence of temperature on the toxicity of three insecticides against the codling moth Cydia pomonella (L.) and two natural enemies

Abstract

Insecticide toxicity may strongly vary with temperature, and interspecifc diferences have been commonly reported for this relationship. A diferential infuence of temperature on insecticide toxicity between pests and their natural enemies may have important consequences on biological control in a global warming context. This study aimed to investigate cross efects between temperature and three insecticides—i.e., chlorantraniliprole, emamectin and spinosad—on the mortality of a major pest in orchards, Cydia pomonella L., and two of its natural enemies in southern France, the predatory earwig Forfcula auricularia L. and the introduced parasitoid Mastrus ridens Horstmann. We observed a decreased efciency of emamectin and spinosad with increasing temperature on mortality of codling moth, while no infuence of temperature on chlorantraniliprole efficacy was observed. Increasing temperatures increased the toxicity of spinosad and chlorantraniliprole against M. ridens and only for emamectin on F. auricularia. This study provides essential insight to make recommendations for using these insecticides in combination with two natural enemies to control the codling moth in prevision of global warming. Our results suggest that the use of spinosad may become sub-optimal under higher temperatures. In contrast, chlorantraniliprole should be suitable under warmer climatic conditions to control C. pomonella and the less detrimental against F. auricularia and M. ridens. To conserve the use of biological control strategies relying on F. auricularia, alternating use of emamectin during early spring, when its toxicity is the lowest on this natural enemy, chlorantraniliprole during summer could limit resistance risks in codling moth populations and reduce the insecticides’ impact on the populations of natural enemies.

Full text

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Differential inuence of temperature on the toxicity
of three insecticides against the codling moth Cydia
pomonella (L.) and two natural enemies
Marie Perrin (  marie.perrin@univ-avignon.fr )
Avignon University, Aix Marseille Univ, CNRS, IRD, IMBE, Pole Agrosciences
Nicolas Borowiec
INRAE, UMR INRAE-CNRS-Université Côte d’Azur “Institut Sophia Agrobiotech” (ISA)
Marcel Thaon
INRAE, UMR INRAE-CNRS-Université Côte d’Azur “Institut Sophia Agrobiotech” (ISA)
Myriam Siegwart
INRAE, Unité PSH, Equipe Controle Biologique par Conservation, Site Agroparc
Thomas Delattre
INRAE, Unité PSH, Equipe Controle Biologique par Conservation, Site Agroparc
Joffrey Moiroux
Avignon University, Aix Marseille Univ, CNRS, IRD, IMBE, Pole Agrosciences
Research Article
Keywords: Integrated pest management, climate change, biocontrol agents, chlorantraniliprole, spinosad,
emamectin
Posted Date: November 23rd, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2289037/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

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Abstract
Insecticide toxicity may strongly vary with temperature, and interspecic differences have been
commonly reported for this relationship. A differential inuence of temperature on insecticide toxicity
between pests and their natural enemies may have important consequences on biological control in a
global warming context. This study aimed to investigate cross effects between temperature and three
insecticides - i.e., chlorantraniliprole, emamectin and spinosad - on the mortality of a major pest in
orchards,
Cydia pomonella
L., and two of its natural enemies in southern France, the predatory earwig
Forcula auricularia
L. and the introduced parasitoid
Mastrus ridens
Horstmann. We observed a
decreased eciency of emamectin and spinosad with increasing temperature on mortality of codling
moth, while no inuence of temperature on chlorantraniliprole ecacy was observed. Increasing
temperatures increased the toxicity of all insecticides against
M. ridens
and only for emamectin on
F.
auricularia
. This study provides essential insight to make recommendations for using these insecticides
in combination with two natural enemies to control the codling moth in a warming world. Our results
suggest that the use of spinosad may become sub-optimal under higher temperatures. In contrast,
chlorantraniliprole should remain suitable under warmer climatic conditions to control
C. pomonella
,
conserve
F. auricularia
and facilitate the establishment of
M. ridens
. For conservation biological control
relying on
F. auricularia
, alternating use of emamectin during early spring, when its toxicity is the lowest
on this natural enemy, and chlorantraniliprole during summer could limit resistance risks in codling moth
populations and reduce the insecticides' impact on the populations of natural enemies.
Introduction
Since the second half of the 19th century, with the rst industrial revolution, climatic variations
accelerated considerably, increasing greenhouse gas emissions, which are responsible for the warming of
Earth's climate. All these climate changes induced by human activities are causing substantial changes in
the balance of ecosystems, including agroecosystems, that are of great concern as they are essential for
human existence.
Most studies agree that there will be an overall increase in crop losses with accelerated development of
resistance to certain control methods, such as the use of insecticides in some species and thus increased
damages. This possibility raises major questions about the sustainability of certain pest control
methods. Moreover, frequent applications of insecticides to control agricultural pests induce side effects
on human health, the environment, and benecial organisms (Pajač et al. 2011; Koureas et al. 2012;
Stehle and Schulz 2015). Due to climate change, their use is expected to increase signicantly in the
future, aggravating their impact and economic costs (Chen and McCarl 2001; Koleva and Schneider
2009). Several solutions have been developed to ensure a more sustainable agricultural transition. One of
them is Integrated Pest Management (IPM) strategy which is a science-based, decision-making process
that combines biological, cultural, physical, and chemical tools to identify, manage and reduce risk from
pests in a way that minimises economic, health and environmental risks (Stark et al. 2007; Dara 2019).

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In IPM strategy, natural enemies, whether they are imported, conserved, or supplemented in
agroecosystems (Boivin 2001; Winkler et al. 2005), play a key role as biological control agents (Lacey and
Unruh 2005). Despite their prominent role, natural enemies commonly provide incomplete pest control,
and insecticides must be used sparingly as a complement (Stark et al. 2007). Nevertheless, most
insecticides cause lethal and sublethal effects on natural enemies, thus indirectly reducing pest control
(Desneux et al. 2007; Rajak et al. 2014). It is then important in IPM strategy to assess the impact of
insecticides on natural enemies and to select active substances with a low impact on biocontrol agents.
Such ecotoxicological studies have been commonly performed, but very few attempted to assess the
insecticides’ impact on natural enemies in a climate change context, even though temperature inuences
their toxicity on several pests (Glunt et al. 2013), predators (Mansoor et al. 2015) or parasitoid species
(Abbes et al. 2015). Contrasting results have been reported, depending on the insecticide class (Musser
and Shelton 2005; Boina et al. 2009), the active substance itself and the insect species. For example, the
toxicity of acetamiprid and chlorpyrifos increases with increasing temperature but decreases for λ-
cyhalothrin and spinosad (Mansoor et al. 2015) in the predator
Chrysoperla carnea
Stephens (Neuroptera,
Chrysopidae). Conversely, λ-cyhalothrin toxicity increases at high temperatures, while temperature does
not inuence spinosad toxicity in
Philodromus
spiders (Michalko and Košulič 2016). This species-
dependency of temperature-mediated toxicity of insecticides may be especially problematic with global
warming in IPM strategy if toxicity decreases with temperature for the pest but increases for natural
enemies. We may thus expect pest outbreaks because of both a reduced eciency of insecticides and a
drop in predator and parasitoids populations. To our knowledge, and despite the potential inuence of
global warming, no study investigated the impact of temperature and insecticides on a pest species and
its natural enemies.
The codling moth
Cydia pomonella
(L.) (Lepidoptera: Tortricidae) is one of the most important insect
pests in apple orchards worldwide, causing serious yield losses (Barnes 1991; Lacey and Unruh 2005).
Species from several insect orders have been reported to be its natural enemies, including predatory
Dermaptera, Neuroptera, Coleoptera and Heteroptera (Nikolic et al. 2018) and a diverse assemblage of
Hymenopteran parasitoids (Athanassov et al. 1997; Cross et al. 1999; Mills 2005). However, their
effectiveness in orchards is quite limited (Maalouy et al. 2013; Thorpe et al. 2016) and insecticides are
commonly used to manage
C. pomonella
and mating disruption (Witzgall et al. 2008; Grigg-McGun et
al. 2015; Ioriatti and Lucchi 2016). The intensive use of insecticides, associated with the multivoltinism
and high fertility of the codling moth, has, however, led to the development of resistance toward several
chemical groups of insecticides in wild populations (Sauphanor et al. 2000; Boivin et al. 2001; Bouvier et
al. 2001; Brun-Barale et al. 2005). There is thus a need to improve alternative or complementary methods
to chemical products for the management of this pest.
In this paper, we investigated the effect of temperature on the ecacy of three commonly used
insecticides against
C. pomonella
and two natural enemies of this pest occurring in southern France, the
predatory European earwig
Forcula auricularia
(L.) (Dermaptera: Forculidae) and the parasitoid
Mastrus ridens
Horstmann (Hymenoptera: Ichneumonidae).
Forcula auricularia
is an abundant native

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predator known to feed on codling moth eggs in apple orchards (Glen 1975; Unruh et al. 2016). It thus
plays a key role in conservation biological control (CBC).
Mastrus ridens
have been introduced in several
countries to control
C. pomonella
(Sandanayaka et al. 2018; Charles et al. 2019). It was introduced in
France between 2018 and 2021 in the frame of a classical biological control (ClBC) programme against
the codling moth aiming at the permanent establishment of this parasitoid to provide sustainable pest
control (Borowiec et al. 2020). To optimise eld practices, we tested insecticides that are commonly used
against codling moth and are promising for use in conjunction with biological control strategies: two
reduced-risk insecticides used in conventional apple orchards, emamectin and chlorantraniliprole
(European Food Safety Authority 2012; Redmond and Potter 2017), and one insecticide approved in
organic apple orchards, spinosad.
Our experiment was thus performed to investigate changes in insecticide toxicity on the codling moth and
its natural enemies as temperature increases and discuss the relevance of these substances in IPM
strategy relying on both methods to control
C pomonella
in a warming world.
Material And Methods
1. Insects
1.1 Cydia pomonella
L.
The laboratory strain of
C. pomonella
used for this study (later called
C. pomonella
strain NPP) originates
from northern France and has been mass-reared in the laboratory since 1995. It is used on an industrial
scale to produce Carpovirusine™ by Natural Plant Protection Firme, Pau, France. In our study,
C.
pomonella
strain NPP was reared on an articial diet (prepared according to Guennelon et al. 1981 recipe
without formaldehyde) at INRAE PSH Avignon (France). The rearing temperature was of 23°C with a
photoperiod of 16:8 h Light:Dark (L:D).
1.2 Mastrus ridens
Horstmann
Mastrus ridens
were reared at INRAE ISA (Sophia Antipolis, France) in insect-proof cages. The rearing
temperature was 23°C with a photoperiod of 16:8 h L:D. This strain results from a mix of several strains:
laboratory stock from New Zealand (2015) and Chile (2016) and individuals collected in Kazakhstan
(2018). Each cage contained 50 females and 35 males and was provided fresh honey and water daily.
For reproduction, 50 to 60 overwintering codling moth cocoons were placed in cages every 3–4 days.
1.3 Forcula auricularia
L.
Forcula auricularia
insects were sampled between March and June 2021 using traps cardboard sheets
in peach and apple experimental orchards of INRAE PSH Avignon, southern France, where very few
insecticides have been applied for the last 10 years. Fifty
F. auricularia
adults were placed in ventilated

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boxes with an articial diet (the same as
C. pomonella
) and cardboard sheets as shelters. The rearing
temperature was 25°C with a photoperiod of 16:8 h L:D until experiments.
2. Insecticides and bioassays
2.1 Insecticides
The commercial formulations of emamectin benzoate (Arm® 0.95%), spinosad (SUCCESS® 4 480 g/L)
and chlorantraniliprole (CORAGEN® 200 g/L) were purchased respectively from Syngenta (France), Dow
AgroSciences Distribution (France) and FMC Agricultural solutions (France). These products were used
diluted in water for all the bioassays by ingestion. The dilutions were made separately according to the
species (Tables1, 2 and 3). The pure active substances of insecticides (chlorantraniliprole PESTANAL®,
standard analytical purity ≥ 95.0%; emamectine benzoate PESTANAL®, standard analytical purity ≥
85.0%; spinosad PESTANAL®, standard analytical purity ≥ 95.0%) were purchased and used dissolved in
acetone to perform all the bioassay by contact. For these bioassays by contact, one unique dose
corresponding to the one used in the eld (X dose) was used for each insecticide: 2 mg/L for emamectin,
0.096 mg/L for spinosad and 0.035 mg/L for chlorantraniliprole.
2.2 Exposure methods
The application method differs for each species depending on the targeted stage in the eld. Codling
moths are targeted at the larval stage and are more likely to be exposed through ingestion. The two
natural enemy species used will be present in the eld at the end of their development as larvae and
adults for
F. auricularia
and only as adults for
M. ridens
. These two species can then be exposed
in
natura
by ingesting contaminated food (fruits, nectar, eggs, larvae and others) or by contact with the
surface of contaminated leaves and fruits.
2.3 Temperature ranges
The temperature range evaluated is based on (i) the IPCC warming forecasts projected for the 2005–
2010 average summer temperatures in Avignon and (ii) the thermal optima of the different species.
(i) According to the 5-year data (2005–2010) of the meteorological station of Avignon (Station
METAR/SYNOP 07563, 48 m.a.s.l., 43.95°N 4.82°E), the average maximum summer temperature was
30.13°C.
According to the IPCC 2021, climatologists predict an average warming of 1.8°C (best scenario) to 4.4°C
(worst case) by 2100 (IPCC 2021).
These combined data allowed us to set the maximum temperature tested in this study at 35°C, which is
consistent with the warming predictions in the Southern France region.
(ii) For natural enemies more sensitive to temperature changes, temperature selection was based on two
publications (Kharboutli & Mack, 1993; Devotto et al. 2010) and unpublished experimental data. For

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codling moths, unpublished experimental data on larval development at different temperatures allowed
the selection of the four most suitable temperatures.
2.4 Cydia pomonella
toxicity bioassay by ingestion
After a few days of mating, eggs were collected, washed with a water-based solution of dishwashing
liquid and bleach, and dried. Neonatal larvae were used for bioassays since it is the target of insecticide
treatments. We assessed the inuence of both temperature and insecticides on the mortality of
C.
pomonella
larvae.
To perform these tests, 96 microwell plates containing approximately 150 µL of articial diet (Stoney
diet Industries Ltd, Rochester, NY) per microwell were treated with different insecticide concentrations
diluted in osmosed water (Table1). Six µL of insecticide solution, or water for control, was deposited in
each microwell on the diet surface. After 20 minutes of drying, newborn larvae were individually
deposited, and microwells were closed with paralm strips. Plates were then placed in climatic chambers
at four different temperatures: 20°C, 25°C, 30°C and 35°C. The mortality rate was assessed at 105-degree-
days, i.e., 10 days at 20°C, 7 days at 25°C, 5 days at 30°C and 4 days at 35°C, to homogenise the larval
stages at the time of observation. A larva was considered dead when not responding to a probe with
dissecting forceps. Missing larvae were removed from the data (< 1%). The corrected mortality was
calculated using the Abbott formula (Abbott 1925). Each test consisted of 24 individuals per
concentration, insecticide, and temperature and was replicated three times. To ensure that potential
differences in toxicity with temperature were physiologically explained and not the consequences of
recording mortality at different times according to temperature (i.e., 105-degree-days), we also performed
three replicates with mortality observations at 5 days for all temperatures.
Table 1
Concentrations of active insecticide substance used for bioassays on
Cydia pomonella
neonate
larvae (for information, only doses in mg/L were converted in X dose corresponding to the eld
dose used in French orchards)
Insecticides Concentrations (mg/L)
Arm® (emamectin) 0.02
(X/100)
0.03
(X/67)
0.05
(X/40)
0.08
(X/25)
0.14
(X/14)
0.22
(X/9)
CORAGEN® (chlorantraniliprole) 0.3
(8.5X)
0.53
(15X)
0.95
(27X)
1.69
(48X)
3
(86X)

SUCCESS 4® (spinosad) 0.05
(X/2)
0.15
(1.5X)
0.45
(4.7X)
1.34
(14X)
4.01
(42X)
12
(125X)
Correctedmortality
= 100 × (1 − )
(
1 −
( ))
deadtreatedindividuals
totaltreatedindividuals
(
1 −
( ))
deadcontrolindividuals
totalcontrolindividuals

Page 7/22
2.5 Mastrus ridens
toxicity bioassay
2.5.1. By ingestion
In this experiment, we evaluated the inuence of both temperature and ingestion of insecticides on the
mortality of
M. ridens
. Since only adults are exposed to insecticides in orchards (larvae are
ectoparasitoids of cocoons), we used freshly (< 48 h old) emerged males of
M. ridens
for bioassays. To
perform this experiment, little pieces of paper (1 cm2) were soaked with 15 µL of a honey/water solution
(3/1) untreated for control or polluted with different insecticides concentration (Table 2). Soaked papers
were placed in 10 mL glass vial caps. After the preparation of control and treated vials, ve males were
placed per vial, and the caps were only partially screwed to allow the air to pass through. Bioassays vials
were placed in climatic chambers at 23°C, 28°C or 33°C. The mortality rate was assessed each day for 72
hours. Individuals were considered dead when not responding to a probe with dissecting forceps. Missing
individuals were removed from the dataset. The corrected mortality was calculated using the Abbott
formula (Abbott 1925), represented in section 2.4. Each test consisted of ve individuals per dose,
insecticide and temperature and was repeated four times.
Table 2
Concentrations of active insecticide substance used for ingestion bioassays on
Mastrus ridens
.
Concentrations are given in mg/L and converted in X dose corresponding to the eld dose used in
French orchards.
Insecticides Concentrations (mg/L and X dose)
Arm® (emamectin) 0.4
(X/5)
1
(X/2)
2
(X)
4
(2X)
10
(5X)
20
(10X)

CORAGEN® (chlorantraniliprole) 0.018
(X/2)
0.035
(X)
0.07
(2X)
0.175
(5X)
0.35
(10X)
1.75
(50X)
3.5
(100X)
SUCCESS 4® (spinosad) 0.02
(X/5)
0.05
(X/2)
0.096
(X)
0.2
(2X)
0.48
(5X)
0.96
(10X)

2.5.2. By contact
In this experiment, we assessed the inuence of temperature and insecticide exposure by contact on the
mortality of males. Little pieces of paper (1 cm2) were soaked with 15 µL of an untreated honey/water
solution (3/1) and placed in 10 mL glass vial caps. Glass vials were previously treated with 500 µL of
acetone for control or with 500 µL of insecticide-polluted acetone (see insecticides section). Control and
treated vials were gently laid down and rolled until complete evaporation of the solvent to homogenise
the distribution of the solution throughout the vial's surface. After 2 hours of drying, ve males were

Page 8/22
placed per vial. Caps were partially screwed on the vials, so air would always pass through. Bioassays
vials were placed in climatic chambers at 23°C, 28°C or 33°C. The mortality rate was assessed as in the
previous experiment. Each test consisted of ve individuals per dose, insecticide and temperature and
was repeated four times.
2.6 Forcula auricularia
toxicity bioassay
2.6.1. By ingestion
Since
F. auricularia
adults are more active than larvae and the most likely to be exposed to insecticides,
we tested the inuence of both temperature and ingestion of insecticides on their mortality. To perform
this experiment, individual ventilated Petri dishes were lled with humidied sand, and Eppendorf tube
caps were lled with the articial diet (Guennelon et al. 1981 without formaldehyde). For each cap, a 6 µL
volume of insecticide solution with different concentrations or no solution for control was deposited on
the diet surface (Table 3). After 20 minutes of drying, one cap was placed on a single petri dish, and one
individual of
F. auricularia
was introduced. Petri dishes were placed in climatic chambers at 23°C, 28°C or
33°C. The mortality was assessed each day at the same hour for ve days. Individuals were considered
dead when not responding to a probe with dissecting forceps. Missing individuals were removed from the
data. The corrected mortality was calculated using the Abbott formula (Abbott 1925), represented in
section 2.4. Each test consisted of one individual per dose, insecticide and temperature and was repeated
15 times per sex.
Table 3
Concentrations of active insecticide substance used for ingestion bioassays on
Forcula auricularia.
Concentrations are given in mg/L and converted in X dose
corresponding to the eld dose used in French orchards.
Insecticides Concentrations (mg/L and X dose)
Arm® (emamectin) 4
(2X)
10
(5X)
20
(10X)
40
(20X)
100
(50X)
CORAGEN® (chlorantraniliprole) 3.5
(100X)

SUCCESS 4® (spinosad) 0.096
(X)
0.2
(2X)
0.48
(5X)
0.96
(10X)
2
(20X)
2.6.2. By contact
We evaluated the inuence of temperature and insecticide exposure by contact on the mortality of
F.
auricularia
adults. Insecticides were used in the form of pure active substances dissolved in acetone.
Petri dishes were treated with 115 µL of acetone for control or with 115 µL of insecticide-polluted acetone

Page 9/22
(see insecticides section). Following application, the solvent was spread over the entire surface of the
Petri dish, including the sides. After 30 minutes, ve individuals of
F. auricularia
were placed in a single
Petri dish, and the articial diet (Guennelon et al. 1981 without formaldehyde) was provided. The Petri
dishes were placed in climatic chambers at 23°C, 28°C or 33°C. The mortality rate was assessed as in the
previous experiment. Each test consisted of ve individuals per dose, insecticide and temperature and
was repeated three times per sex.
3. Data analyses
Dose-response curves were tted with the package
drc
using R software (Ritz et al. 2015; R Core Team
2020 version 4.0.2) to calculate the different LD50 for each temperature and each insecticide. The
drm
function was used to calculate the different LD50, and the
EDcomp
function was used to compare each
concentration and temperature evaluated.
For contact bioassays, corrected mortality was calculated using the Abbott regression (Abbott 1925), and
differences in mortality between treatments, sexes (for
F. auricularia
) and temperatures were analysed
using a Chi-square test.
Results
Cydia pomonella
Based on observations at 105-degree-days, the temperature decreased the toxicity of emamectin by 1.5,
two and three times the dose of chemicals required to kill 50% of the larvae at 25°C (p = 0.01), 30°C (p =
0.008) and 35°C (p = 0.009) respectively, compared to 20°C (Fig.1A).Temperature also decreased the
toxicity of Spinosad with fourth the dose of chemicals required to kill 50% of the larvae at 25°C, 30°C and
35°C compared to 20°C (p = 0.04) (Fig.1B). However, no signicant difference was observed between the
three higher temperatures (p > 0.3) (Fig.1B). Temperature did not signicantly inuence the mortality of
codling moth larvae exposed to chlorantraniliprole (p > 0.6) (Fig.1C).
We observed similar results for mortality assessment at constant time, i.e., 5 days (Online Resource 1,
Table 1). Thus, our results are due to differences in the inuence of temperature on insecticides’ toxicity
and not by differences in reading time.
Mastrus ridens
Toxicity by ingestion
Spinosad toxicity increased with increasing temperatures when
M. ridens
ingested the insecticide. Four
times less product was needed to kill 50% of the individuals at 28°C (p = 0.008) and six times less at 33°C
(p = 0.009) compared to 23°C (Fig. 2B). Similarly, chlorantraniliprole toxicity increased with increasing
temperatures with eight and 15 times less product needed to kill 50% of the individuals at 28°C and 33°C
respectively compared to 23°C (p = 0.04) (Fig. 2C). For emamectin, a similar trend as for the other

Page 10/22
insecticides was observed. However, differences between temperatures were not signicant (p > 0.8),
probably because of a high variability occurring at 23°C (Fig. 2A).
Toxicity by contact
Table 4
Effects of temperature and insecticides at eld dose (X dose) on the mortality
of
Mastrus ridens
adults. Different letters indicate signicant differences
between treatments. (Chi-square χ² test).
Insecticide
(X dose)
Corrected mortality (%)
23°C 28°C 33°C
emamectin 64.74a (
χ²
= 0) 100b (
χ²
= 8.17) 100b (
χ²
= 8.17)
spinosad 68.27a (
χ²
= 0) 100b (
χ²
= 6.97) 100b (
χ²
= 6.97)
chlorantraniliprole 100 (
χ²
= 0.40) 100 (
χ²
= 0.15) 100 (
χ²
= 0.15)
Toxicity bycontact of emamectin and spinosad increased signicantly with temperature as it rose from
less than 70% at 23°C to 100% mortality at 28°C and 33°C for both insecticides. The mortality reached
100% when chlorantraniliprole was applied, regardless of the temperature.
Forcula auricularia
Toxicity by ingestion
Emamectin toxicity increased at 28°C (p = 0.008) and 33°C (p = 0.009) but did not at 23°C (Fig. 3A) when
earwigs ingested the insecticide. A similar trend was observed for spinosad, but differences between
temperatures were not signicant (p > 0.8), probably because of a high variability occurring at 23°C (Fig.
3B). For chlorantraniliprole, no mortality occurred at the single dose tested (3.5 mg/L, corresponding to
100× the approved eld rate), regardless of temperature.
Toxicity by contact
We tested a single dose corresponding to the approved eld dose of each insecticide substance. No
mortality was observed for
F. auricularia
adults regardless of the insecticide or the temperature evaluated.
Discussion
Climate change may differentially affect the eciency of insecticide treatments against pests and
benecial insects through a wide range of factors in the eld. This study rst wants to assess the effect
of temperature on three insecticide toxicity in the controlled condition in the laboratory to avoid the
complex interactions between factors, including the behaviour or ecology of arthropods
in natura
. It
provides a rst step of whether the ecacy of these insecticides may change with the climate using the

Page 11/22
example of a pest and two of its natural enemies used in IPM programmes. Indeed, the consequences of
climate change on biological control caused by a differential inuence of temperature on insecticide
toxicity between pests and their natural enemies have never been considered, although such an inuence
is known to be species-specic. This topic is especially relevant for organic farming and IPM strategy,
which rely on both natural enemies and insecticides. The worst scenario is that insecticide toxicity
decreases with global warming for a pest but increases for its natural enemies.
In this study, we observed that higher temperature (i) decreased the toxicity of emamectin and spinosad
but did not inuence the toxicity of chlorantraniliprole against the pest
C. pomonella
, while it (ii) increased
the toxicity of these three insecticides on the parasitoid,
M. ridens
, and (iii) increased the toxicity of
emamectin on the predator,
F. auricularia
. Our results suggest a possible problem in future
C. pomonella
population control in the eld because of a decreased eciency of insecticides on the pest coupled with
the opposite effect on natural enemies’ populations.
Our results on
C. pomonella
agree with those reported for spinosad on another pest species, the cotton
mealybug
Phenacoccus solenopsis
Tinsley (Mansoor et al. 2014). However, our results on
C. pomonella
contrast with those obtained on
P. solenopsis
(Mansoor et al. 2014) and
Plutella xylostella
L., on which
toxicity of emamectin increased with temperature as for chlorantraniliprole on
P. xylostella
(Li et al. 2004;
Teja et al. 2018). Indeed, interspecic differences in temperature-mediated insecticides’ toxicity between
pests have been frequently reported (Boina et al. 2009), although underlying mechanisms still have to be
investigated.
Compared to pests, few studies have been conducted on natural enemies. Still, increased toxicity at high
temperatures was reported for chlorantraniliprole on the parasitoid
Bracon nigricans
Szépligeti (Abbes et
al. 2015), as we observed for
M. ridens
when the substance was ingested. However, in our study,
spinosad toxicity increased with temperature for
M. ridens
. At the same time, it was not inuenced by
temperature in
B. nigricans
(Abbes et al. 2015) and decreased with increasing temperature in the
lacewing
Chrysoperla carnea
Stephens (Mansoor et al. 2015). The same interspecic differences for
pests were also observed for natural enemies.
The positive relationship between toxicity and temperature observed for the three substances in
M. ridens
and emamectin ingested by
F. auricularia
may result from several mechanisms that should be
investigated in further studies. First, high temperature may inuence detoxication enzymes (Yan et al.
2013; Zhang et al. 2015; Liu et al. 2017) or Heat Shock Protein (Ge et al. 2013; Su et al. 2018) activities
and expression. Second, metabolic rate increases with temperature (Brown et al. 2004), consequently
increasing food consumption, insect locomotion (Gillooly 2001; Medrzycki et al. 2010), and substance
penetration in the insect's body (Boina et al. 2009).
This study was conducted in a simplied system where insects are "forced" into contamination.
In natura
,
other complex behavioural or ecological parameters might limit contact between insects and insecticide.
For example,
F. XXXuricularia
is a nocturnal species that hunt at dusk, which could allow them to avoid

Page 12/22
diurnal substances spraying (Vancassel 1973). Moreover, some parasitoid species also avoid hosts
resistant to some insecticides to minimise contamination (Alfaro-Tapia et al. 2021).
Whether the two natural enemies ingested the insecticides or were exposed by contact,
M. ridens
was
more sensitive to substances than
F. auricularia
. The three insecticides caused 100% mortality in
M.
ridens
above 28°C at the authorised eld dose, while no mortality was detected in
F. auricularia
. This
interspecic difference may be due to differences in the composition or thickness of their cuticle
(Fernandes et al. 2010) and/or insecticide penetration, depending on the anity between the cuticle and
the substance (Leite et al. 1998). The smaller size of
M. ridens
compared to
F. auricularia
may also
account for this difference, as the specic target area of the insecticides decreases with increasing body
size, resulting in reduced insecticide exposure (Picanco et al. 1997; Bacci et al. 2007).
Such differences may also explain that toxicity was mediated by temperature for the three insecticides
for
M. ridens
, while it was only true for emamectin in
F. auricularia
. These interspecic differences in the
relationship between temperature and insecticides’ toxicity underline the need to study specically each
pest-natural enemy’s system. This approach would allow for selecting insecticides that will be the most
reliable with global warming. Ideally, insecticides used in IPM programmes and organic agriculture should
be less toxic to natural enemies than to target insect pests (Zhao et al. 2012) and remain harmless as
temperature increases.
From our results, spinosad was as toxic to the codling moth as it was to the European earwig and was
even more toxic to
M. ridens
, regardless of the temperature. It was also more toxic to natural enemies
than emamectin and chlorantraniliprole, considering the tiny dose needed to kill 50% of parasitoids
compared to the two other insecticides. Lethal and sublethal effects of spinosad and other spinosyns on
benecial arthropods have been previously reported in several studies (Biondi et al. 2012; Abbes et al.
2015), and yet, this product is authorised and widely used in organic farming while emamectin and
chlorantraniliprole are not (Biondi et al. 2012). Moreover, our results indicate that higher temperatures
emphasise differences in spinosad toxicity between the two trophic levels. Among the insecticides
evaluated, it is likely to cause problems managing codling moth populations under high-temperature
conditions. The combination of spinosad with strategies of regulation based on natural enemies should
be avoided. Its approval for organic production may raise questions in the future considering its
increased toxicity on some benecial organisms as temperature increases.
Surprisingly, chlorantraniliprole may be the most promising insecticide in a warming world for strategies
relying partly on biological control by conservation. The toxicity of this substance indeed remained very
effective at high temperatures against
C. pomonella
while it did not cause any mortality on
F. auricularia
,
regardless of temperature or exposure method. Moreover, it was relatively safe against
M. ridens
when
parasitoids ingested the substance. Despite the product's increased toxicity at high temperatures, the
lethal doses for 50% of individuals remained high (equivalent to 10–50× the recommended eld dose).
However, the substance caused 100% mortality during contact for
M. ridens
. Thus, this substance may be

Page 13/22
appropriate for classical biological control relying on the introduction of parasitic wasps, although eld
experiments should conrm our laboratory observations.
For both natural enemies, the LD50 remained very high at low temperatures for emamectin, indicating that
a high quantity of this insecticide should be applied to kill 50% of the individuals. These results are
consistent with the literature on other natural enemies for this substance (Argentine et al. 2002; Khan et
al. 2018). Emamectin has been recommended in IPM strategies for a long time because of its low toxicity
on benecial organisms, high selectivity for pests and rapid environmental degradation (Argentine et al.
2002; López et al. 2010). However, we observed that its toxicity increased rapidly with temperature for
F.
auricularia
and
M. ridens
and decreased for the codling moth. In this sense, its ecacy may decrease
with global warming despite being the most effective insecticide against
C. pomonella
among the three
tested in terms of effective doses at high temperatures.
Contrary to chlorantraniliprole, emamectin toxicity on
F. auricularia
increased strongly with temperature.
Alternating the use of these insecticides in conservation biological control, with preferential use of
emamectin during the colder periods (e.g., early spring) and the use of chlorantraniliprole (e.g., during the
summer) could limit the risks of resistance appearance in pest’s populations while limiting the impact of
chemical substances on the European earwig. However, its use in CBC programmes involving the
introduction of
M. ridens
should be avoided considering its toxicity at high temperatures.
The present study highlights opposite cross effects of temperature and insecticides (spinosad,
emamectin, chlorantraniliprole) on
C. pomonella
and two of its natural enemies, one exotic (
M. ridens
)
and one native (
F. auricularia
) in southern France. According to the future climate change predictions, we
recommend using these insecticides to preserve natural enemies associated with
C. pomonella
. The use
of spinosad should be avoided, while emamectin can be used in conjunction with the conservation of
F.
auricularia
. Only chlorantraniliprole appears suitable for controlling
C. pomonella
, conserving
F.
auricularia
and establishing
M. ridens
. The present study remains a simplied but essential tool to
understand the cross effects of temperature and three insecticides on the target pest and two of its
natural enemies, allowing us to propose an adaptation of strategies relying on insecticides in apple
orchards in a changing climate. We focused on the mortality of natural enemies, but the effects of
sublethal exposures, which affect insects’ behaviour and life history traits, should also be investigated
since they may be critical to the long-term conservation and/or establishment of natural enemies (Saber
2011; Poorjavad et al. 2014).
Declarations
Ethical Approval
Not applicable.
Competing interests

Page 14/22
The authors have no relevant nancial or non-nancial interests to disclose.
Authors’ Contributions
Perrin M participated in the design of the study, performed the bioassays, performed the statistical
analysis and drafted the manuscript. Borowiec N participated in the design and coordination of the study
and helped to draft the manuscript. Moiroux J and Siegwart M conceived of the study, and participated in
its design and coordination and helped to daft the manuscript. Delattre T helped to draft the manuscript.
Thaon M helped to performed the bioassays. All authors read and approved the nal manuscript.
Funding
This work was supported by FranceAgriMer (‘BIOCCYD-Mastrus’ 2019-2022), the PACA region, France,
and the experimental station “la Pugère”. The author N. Borowiec has received research support from
FranceAgriMer (‘BIOCCYD-Mastrus’ 2019-2022) and the author M. Perrin has received research support
from the PACA region, France, and the experimental station “la Pugère” for doctoral fellowship.
Availability of data and materials
The datasets generated during and/or analysed during the current study are available from the
corresponding author on reasonable request.
Acknowledgements
This study was supported by FranceAgriMer (‘BIOCCYD-Mastrus’ 2019-2022). We are grateful to the PACA
region, France, and the experimental station "la Pugère" for the nancial support of M. Perrin doctoral
fellowship.
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Figures
Figure 1
Effects of temperature on doses of emamectin (A), spinosad (B) and chlorantraniliprole (C) needed to kill
50% (LD50) of codling moth neonatal larvae. Different letters indicate signicant differences between
temperatures. (EDcomp, α = 0.05).

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Figure 2
Effects of temperature on doses of emamectin (A), spinosad (B) and chlorantraniliprole (C) needed to kill
50% (LD50) of Mastrus ridens adults. Different letters indicate signicant differences between treatments.
(EDcomp, α = 0.05).
Figure 3

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Effects of temperature on doses of emamectin (A) and spinosad (B) needed to kill 50% (LD50) of
Forcula auricularia
adults. Results for males and females were pooled since there were no signicant
differences between the sexes. Different letters indicate signicant differences between treatments.
(EDcomp, a = 0.05).
Supplementary Files
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