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Article

Monitoring Resistance of Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) to Insecticides by Using Encapsulated Artificial Diet Bioassay

by
Cristiane Maria Tibola
1,2,*,
Leandro Silva
2,
Fernanda Sgubin
2 and
Celso Omoto
1
1
Department of Entomology and Acarology, Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba 13418-900, SP, Brazil
2
CL Empreendimentos Biológicos Ltd., Piracicaba 13424-586, SP, Brazil
*
Author to whom correspondence should be addressed.
Insects 2021, 12(7), 599; https://0-doi-org.brum.beds.ac.uk/10.3390/insects12070599
Submission received: 30 April 2021 / Revised: 11 June 2021 / Accepted: 26 June 2021 / Published: 1 July 2021
(This article belongs to the Section Insect Pest and Vector Management)
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Abstract

:

Simple Summary

The Neotropical brown stink bug Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) is currently one of the major soybean pests in Brazil, causing considerable and often irreversible damage to the crop. The main control strategy for regulating brown stink bug populations has been the use of insecticides, especially those belonging to the neonicotinoid class; however, the spraying of these insecticides does not achieve the desired control efficiency in some locations. Therefore, to improve the programs for detecting and monitoring E. heros resistance to insecticides, a new ingestion bioassay method by liquid diet encapsulation was evaluated in comparison with traditional bioassay methods. The new bioassay method of ingestion by encapsulation is very promising for detecting and monitoring resistance of E. heros populations to neonicotinoid insecticides.

Abstract

The novel ingestion bioassay method was developed for detecting and monitoring resistance of Euschistus heros by encapsulating an artificial liquid diet using Parafilm®. This methodology was compared with the tarsal contact (vial test) and topical application methods for thiamethoxam, imidacloprid, and lambda-cyhalothrin. The best bioassay method for the neonicotinoid insecticides thiamethoxam and imidacloprid was ingestion. For pyrethroid insecticide lambda-cyhalothrin, the best result was obtained by topical application. Using the best bioassay method for each insecticide, the susceptibility to these insecticides was monitored in 30 populations of E. heros collected from soybean crops in Brazil from 2018 to 2020. High variations in susceptibility to thiamethoxam (resistance ratios, 1.6–22 times), imidacloprid (resistance ratios, 1.6–22 times), and lambda-cyhalothrin (resistance ratios, 5–40 times) were detected among the evaluated E. heros populations. In order to monitor the susceptibility of E. heros to insecticides, diagnostic concentrations were defined based on the LC99 of the susceptible reference population: 5.65 µL of a.i./mL for thiamethoxam, 12.45 µL of a.i./mL for imidacloprid, and 0.20 µg of a.i./insect for lambda-cyhalothrin. Subsequently, we select an E. heros strain resistant to neonicotinoid insecticides and another to lambda-cyhalothrin. The resistance ratios obtained after seven selection cycles were 66, 41 and 44 times for thiamethoxam, imidacloprid and lambda-cyhalothrin, respectively.

1. Introduction

The Neotropical brown stink bug Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) is currently one of the major pests of soybeans (Glycine max (Merril)) in Brazil, causing significant and often irreversible damage to crops [1,2,3]. Brown stink bugs colonize soybean plants during the vegetative phase and are detrimental from pod formation until grain ripening. The damage caused by this species results from the insertion of the stylet in the soybean plants or pods, wherein they inject salivary secretions facilitating the feeding process. In addition, they can promote the development of fungal diseases and cause physiological disorders, such as leaf retention, compromising yields by up to 30% if they feed on pods throughout their reproductive stages [4,5,6,7,8,9]. Due to the severe damage caused, some authors acknowledge phytophagous brown stink bugs as the most important pest of soybean cultivation [10,11].
Overall pest control in soybean is practically based on the use of chemicals for insects and mites as well as for weeds and diseases affecting the crop [12,13]. It is estimated that four to eight insecticide applications are currently performed per soybean crop season [10,14,15]. Insecticide sprays for the control of phytophagous brown stink bugs have been frequently used, including insecticides belonging to three chemical groups (neonicotinoids, organophosphates, and pyrethroids), which can be used alone or in formulated mixtures [16]. The limited availability of insecticides and frequent use of the same mode of action may favor the selection of resistant populations, especially if insect resistance management strategies (IRM) are neglected [17,18,19]. The application of many of these insecticides has not achieved the desired control efficiency, and the high number of applications has not reduced the population increase of E. heros in different crops [10,11,14].
In Brazil, E. heros control measure failures have been reported for several insecticides, including beta-cyfluthrin, bifenthrin, lambda-cyhalothrin (pyrethroid), and imidacloprid (neonicotinoid) [19,20,21,22]. Until now, cases of E. heros resistance to endosulfan (cyclodiene), monocrotophos (organophosphate), methamidophos (organophosphate) [23,24], and imidacloprid [25] were detected using the tarsal contact method.
The methods currently used to detect and monitor brown stink bug resistance to insecticides have been based on topical and tarsal contact bioassays [23,24,26,27,28,29,30,31]. However, it is known that the detection of resistance can be affected by the chosen bioassay method [32,33,34,35], and topical and tarsal contact bioassays are based on direct contact of the insect with the insecticide, i.e., topical application to the insect integument or tarsal contact by vial test. Neonicotinoids, the main group of insecticides used to control sucking pests, have systemic properties, with physicochemical characteristics that allow their entry and translocation to all parts of plant tissues [36,37,38]. Further, neonicotinoids are rapidly degraded when the plant is exposed to UV radiation [39].
Developing resistance management strategies for E. heros requires a bioassay method for systemic insecticides, which can be reproduced easily for practical application, provides adequate dose−response data for statistical analysis, and enables detection of low resistance frequency levels [40]. Therefore, due to the inherent characteristics of insecticides belonging to the neonicotinoids class and increase of the E. heros population causing damage to soybean and other crops, the development of a new bioassay method that simulates the systemic action of insecticides and the form of suction feeding of brown stink bugs was needed. Therefore, the objective was to detect, monitor and select the resistance of E. heros to the insecticides thiamethoxam, imidacloprid and lambda-cyhalothrin using ingestion bioassay with encapsulated artificial diet compared to traditional methods of tarsal contact (vial test) and of topical application.

2. Materials and Methods

2.1. E. heros Populations

To evaluate the traditional bioassay methods of tarsal contact and topical application, compared with the proposed new ingestion method, a susceptible reference strain of E. heros (SUS) was used. This strain was originated from insects collected in soybean fields at Areião Farm at Luiz de Queiroz College of Agriculture, University of São Paulo, Piracicaba, São Paulo, Brazil. The SUS strain has been maintained for more than 6 years in the laboratory of the company Pragas.com®, in the absence of selection pressure from insecticides (Table 1).
Studies of susceptibility to insecticides were performed in E. heros populations collected in the 2018/2019 and 2019/2020 seasons from soybean crops from different regions of Brazil (Table 1; Figure 1). After collection, brown stink bugs were brought to the laboratory and were maintained in aerated plastic containers (50 cm long × 30 cm wide × 15 cm high). The brown stink bugs were fed with green bean pods (Phaseolus vulgaris L.), soybean seeds, and peanuts (Arachis hypogaea L.). The rearing of E. heros and the bioassays were performed in an air-conditioned room at 25 °C ± 2 °C, 60% ± 10% RH, and 12:12-h photoperiod. The insects collected in the fields were kept under the above mentioned conditions and used the F1 generation of each field population for bioassays.

2.2. Insecticides

The technical insecticides used to evaluate the susceptibility of E. heros populations by traditional tarsal contact and topical application methods were as follows: lambda-cyhalothrin (≥95% a.i.; Sigma-Aldrich, Saint Louis, MO, USA), which is a modulator of sodium channels (IRAC MoA 3A pyrethroid group); thiamethoxam (≥98% a.i.; Sigma-Aldrich; Saint Louis, MO, USA) and imidacloprid (≥98% a.i.; Sigma-Aldrich, Saint Louis, MO, USA), which are competitive modulators of nicotinic acetylcholine receptors (IRAC MoA 4A neonicotinoid group).
Three commercial insecticides were used to evaluate the susceptibility of E. heros populations by the ingestion method: lambda-cyhalothrin (IRAC MoA 3A pyrethroid group; Karate Zeon 50 CS; 50 g a.i./L in 200 L/ha spray volume; Syngenta Protecção de Cultivos Ltd.a, São Paulo, Brazil), thiamethoxam (IRAC MoA 4A neonicotinoid group; Actara 250 WG; 250 g a.i./L in 200 L/ha spray volume; Syngenta Protecção de Cultivos Ltd.a, São Paulo, Brazil), and imidacloprid (IRAC MoA 4A neonicotinoid group; Evidence 700 WG; 700 g a.i./kg in 200 L/ha spray volume; Bayer, São Paulo, Brazil).

2.3. Bioassay Methods

2.3.1. Ingestion

The proposed new method of ingestion involved incorporation of the insecticide to be tested in an artificial diet. The artificial diet used was modified from Cerna-Mendoza et al. [41] (Table 2) to obtain a liquid diet with an even mixture of the insecticides. For diet preparation, the bean pods were washed under running water, cut, and frozen for 60 h to be lyophilized (Savant Freeze Dryer, model Novalyphe—NL150). Subsequently, the material was blended and stored in airtight containers. During diet preparation, peanuts were ground and later mixed with the other ingredients in the blender. The liquid diet was stored in a refrigerator for a maximum of 3 days.
The proposed bioassay ingestion method was developed by the encapsulation of a liquid artificial diet, thereby enabling even incorporation of the insecticides at different concentrations, which are ingested by the insect upon feeding. The encapsulation was adapted from Greany and Carpenter [42], using an acrylic mold (13 cm length × 8 cm wide) with a press attached to the vacuum pump (Tecnal, model TE-0582). The acrylic mold containing 24 wells was coated with sealing film (Parafilm M®, Bemis American, Neenah, WI, USA) for forming and shaping the wells with the aid of the vacuum pump for depositing 1.0 mL of the artificial liquid diet in each well. Then, a layer of the sealing film was placed over the diet-containing wells and pressed for sealing and closing the capsules. Each capsule was 1.2 cm in diameter and 0.5 cm in depth (Figure 2).
Five to nine concentrations prepared from commercially available products of each insecticide were diluted in water and added to the artificial liquid diet to be tested. Adult brown stink bugs were individualized in each cell of 24-well plates (Kasvi, model K12–024) containing one capsule of liquid artificial diet. Then, each plate was sealed with Parafilm M® to avoid that one insect moving to another cell. Feeding occurs by introducing the stylet into the capsule, perforating the Parafilm M® and sucking the liquid artificial diet treated with different concentration of insecticides (Figure 3).
For each concentration of the insecticides, four replicates were tested with 24 adult brown stink bugs per concentration of each insecticide (Figure 3). Assessments of insect mortality were performed 96 h after insect exposure. Adults of brown stink bugs that did not respond with vigorous movements when touched by a brush were considered dead.

2.3.2. Topical Application

For the topical application bioassay, five to nine concentrations of thiamethoxam, imidacloprid, and lambda-cyhalothrin (technical insecticides) diluted in acetone (99.5% purity; Sigma-Aldrich, São Paulo, Brazil) were used; only acetone was used for the control treatment. Using a single-channel pipette, 2 µL/insect of the products were applied to the pronotum of adult brown stink bugs. For each concentration of the insecticides, four replicates were tested with 25 adult brown stink bugs per concentration of each insecticide. The brown stink bugs were separated and distributed in 100-mL plastic containers, previously labeled according to the treatment. After the application, the brown stink bugs were kept in the same plastic containers with food (bean pods) and moistened filter paper. Mortality was evaluated 48 h after application, as indicated in the IRAC method 029 [24,30,43].

2.3.3. Tarsal Contact

The tarsal contact bioassay method was performed using the vial test. Five to nine concentrations of thiamethoxam, imidacloprid, and lambda-cyhalothrin (active ingredients) diluted in acetone (99.5% purity; Sigma-Aldrich, São Paulo, Brazil) were used. As indicated in IRAC method 030 [21,23,26,27,28,29,44], for 500 μL of each concentration, acetone was added into a glass bottle (20 mL) and distributed evenly throughout the internal surface of the bottle. The bottles were rotated at room temperature using a roller stirrer (Kasvi, model K45-8010) until the acetone had completely evaporated. Following this, two brown stink bugs were placed into each container and the container was closed using a veil tissue attached with an elastic band. For each concentration of the insecticides, four replicates were tested with 25 adult brown stink bugs per concentration of each insecticide. Mortality was evaluated 48 h after continuous exposition of stink bugs on treated surface using the same mortality criteria described before.

2.4. Monitoring Susceptibility of E. heros Populations to Insecticides

2.4.1. Estimated LC50/LD50 and LC99/LD99

The susceptibility of E. heros populations to insecticides was evaluated in populations collected during 2018/2019 and 2019/2020 seasons from soybean crops in different regions of Brazil (Table 1). For estimating LC50/LD50 and LC99/LD99, five to nine concentrations of lambda-cyhalothrin (active ingredient) were tested using the topical application method and commercially available thiamethoxam and imidacloprid were tested using the ingestion method, as described in Section 2.3.1 and Section 2.3.2.
The baseline susceptibility of E. heros to the insecticides was studied in the susceptible reference (SUS) strain to identify the diagnostic concentration, based on LC99/LD99, which could be used for monitoring populations of E. heros collected in different regions of Brazil, according to the criteria outlined by Ffrench-Constant and Roush [40] and Roush and Miller [45].
For the control treatment, only acetone or water was used according to the method tested. For each concentration of the insecticides, four replicates were tested with 24 adult brown stink bugs per concentration of each insecticide. Insects were kept on a natural diet for one generation before being used in the bioassays. The same mortality criteria described before was used to evaluate the bioassays.

2.4.2. Diagnostic Concentration

Based on the bioassay method defined for each insecticide and diagnostic concentration, based on LC99/LD99 was carried out the monitoring of brown stink bugs in populations collected during 2018/2019 and 2019/2020 seasons from soybean crops in different regions of Brazil (Table 1). For evaluating the bioassays, the ingestion bioassay method was used for thiamethoxam and imidacloprid, and the topical application method was used for lambda-cyhalothrin, as described in Section 2.3.1 and Section 2.3.2. The diagnostic concentrations used in monitoring were based on the LC99/LD99 previously identified using the baseline susceptibility of E. heros. Control treatment with the susceptible reference strain included six replicates of 25 brown stink bugs. The same mortality criteria described before was used to evaluate the bioassays.

2.5. Selection and Characterization of Resistance of E. heros to Insecticides

To obtain a population of E. heros resistant to the insecticides thiamethoxam, imidacloprid, and lambda-cyhalothrin, populations collected from Londrina−Paraná (PR) and Luís Eduardo Magalhães-Bahia (BA) in the 2018/2019 season were subjected to seven to eight cycles of selection in the presence of the insecticide under laboratory conditions using the technique of mass selection. These populations were selected because they showed lower mortality at diagnostic concentrations compared to other populations in susceptibility monitoring studies (item 2.4). At the beginning of the selection process, an intermediate concentration (between the LC50 and LC99) of the SUS was used. The surviving individuals from each selection cycle were retrieved and raised on a natural diet. The insecticide concentration used for the selection was increased in the fourth selection cycle to LC99 of the SUS: 0.20 µg of a.i./insect for lambda-cyhalothrin (topical application method) and 5.65 and 12.45 µg of a.i./mL of diet for thiamethoxam and imidacloprid, respectively (ingestion method).
Dose−response studies of E. heros subjected to five to eight selection cycles in the presence of thiamethoxam, imidacloprid, and lambda-cyhalothrin under laboratory conditions were performed using five to nine concentrations of each insecticide, according to the previously described experimental design and mortality criteria.

2.6. Statistical Analyses

Dose–response data were analyzed using Probit analysis on the Polo-PC statistical program [46] to estimate lethal concentrations, their respective confidence intervals (95% CI), and the slope. Mortality data were analyzed using the log−log complement model [47] to estimate the diagnostic concentration based on the LC99. The resistance ratio was estimated by dividing the LC50 of the field population by the LC50 of the SUS [48]. The survival percentage data of E. heros populations were transformed to arcsine (√X/100) and were subjected to analysis of variance at a significance level of α = 0.05 [49].

3. Results

3.1. Bioassay Methods

The best method for the systemic insecticides neonicotinoid thiamethoxam and imidacloprid was ingestion via encapsulated artificial diet. For the pyrethroid lambda-cyhalothrin, topical application showed better results (Table 3).
The ingestion bioassay of thiamethoxam incorporated in the artificial diet revealed a higher slope (4.99 ± 0.54) than that of topical application (3.98 ± 0.33) and tarsal contact (2.11 ± 0.13) bioassays of thiamethoxam. A high slope value provides a steeper dose−response curve, which facilitates the distinction between susceptible and resistant individuals in resistance monitoring programs. Other factors that contribute to the choice of the ingestion method for detecting and monitoring E. heros resistance to the systemic insecticide thiamethoxam were the small-width confidence intervals of the estimated LC50 and LC90, in addition to the adjustment of the data to the Probit model (Table 3).
A similar trend was observed for imidacloprid, i.e., the slope for the ingestion method (3.53 ± 0.26) was higher than those for the topical application (3.36 ± 0.27) and tarsal contact methods (2.29 ± 0.17). In addition, evaluation of imidacloprid using the ingestion method resulted in small-width confidence intervals of the estimated LC50 and LC90, an adequate χ2, adjusted more precisely to the Probit model (Table 3).
The topical application bioassay was more suitable for evaluating the susceptibility of E. heros to the insecticide lambda-cyhalothrin. The results of the probit analyses presented in Table 3 indicate that the slope of the dose–response curve was higher when the insects were subjected to the topical application method (3.62 ± 0.26) compared with those when the insects were subjected to the ingestion (2.56 ± 0.17) and tarsal contact methods (2.50 ± 0.17). The topical application method provided an adequate χ2 and adjusted more precisely to the Probit model (Table 3).

3.2. Definition of Diagnostic Concentrations

The LC/LD99 were estimated using the mortality data of the susceptible population of E. heros, which were considered as diagnostic concentrations for resistance monitoring programs. According to these analyses, LC/LD99 for each insecticide and corresponding methods, were as follows: thiamethoxam: 5.65 (CI 4.47–8.03) µL of a.i./mL of artificial diet, by ingestion method; imidacloprid: 12.45 (CI 10.03–16.47) µL of a.i./mL of artificial diet, by ingestion method; and lambda-cyhalothrin: 0.20 µg a.i./insect, by topical application method.

3.3. Monitoring the Susceptibility of E. heros to Insecticides

High variations in the LC50 of thiamethoxam, imidacloprid, and lambda-cyhalothrin were observed in the populations of E. heros collected from different regions of Brazil between 2018 and 2020 (Table 4 and Table 5; Figure 4).
The field populations of E. heros showed highly variable susceptibility to lambda-cyhalothrin using the topical application method. There was no mortality in the control treatment. The LC50 ranged from 0.026 (susceptible population) to 1.054 (Luís Eduardo Magalhães-BA population) µg a.i./insect, representing a resistance ratio of 40 times. Other populations also showed a high resistance ratio for lambda-cyhalothrin: 20.92 times (Não me Toque-RS), 27.73 times (Londrina-PR), and 19.74 times (Primavera do Leste-MT) (Table 4). Mortality using lambda-cyhalothrin at the diagnostic concentration (LC99) varied among the field populations, ranging from 52.0% and 100.0% for field populations in the 2018/2019 season and between 45.3% to 100.0% in the 2019/2020 season (Figure 4).
Using the ingestion bioassay method, the concentration responses to thiamethoxam were highly variable among the tested populations. There was no mortality in the control treatment. The LC50 ranged between 1.79 (SUS) and 39.52 (Luís Eduardo Magalhães-BA population) μg a.i./mL with an artificial diet. The resistance ratios ranged from 1.62 to 22.08 times for the populations of Uberlândia-MG and Luís Eduardo Magalhães-BA. respectively (Table 5). When the populations of E. heros were exposed to the insecticide thiamethoxam at the diagnostic concentration (LC99), the mortality varied among the populations. This ranged from 47.2% to 100.0% in the 2018/2019 season and 54.2% to 100.0% in the 2019/2020 season (Figure 4).
For the insecticide imidacloprid, there was also no mortality in the control treatment. LC50 ranged from 2.68 (susceptible population) to 60.73 (population of Luís Eduardo Magalhães-BA) μg a.i./mL of the artificial diet for imidacloprid using the ingestion bioassay method. The resistance ratio ranged from 1.64 to 21.92 times for the populations of Buri−SP and Luís Eduardo Magalhães-BA, respectively (Table 5). Mortality at the diagnostic concentration (LC99) ranged between 45.5% and 100.0% for field populations in the 2018/2019 season, and between 49.1% and 100.0% in the 2019/2020 season (Figure 4).

3.4. Selection and Characterization of E. heros Resistance to Insecticides

In the selection and characterization of the E. heros population resistant to thiamethoxam (THIAM−R), the estimated population LC50 after eight selection cycles was 118.66 μg thiamethoxam/mL. The slope (±standard error) was 2.03 (±0.14) and the χ2 value was 5.33 (six degrees of freedom), with a resistance ratio of 66.29 times (Figure 5). The test of parallelism and equality of concentration−response curves estimated by the probit analysis revealed that the slope of the susceptible strain (2.88 ± 0.17) was significantly higher than that of the thiamethoxam-resistant one (2.03 ± 0.14) (Figure 5). The highest slope of the susceptible population is possibly related to the greater homogeneity of that population. Some overlapping of the concentration−response curves of thiamethoxam (THIAM−R) was present. Thus, it was not possible to determine a discriminatory concentration, but it was possible to notice an increase in the resistance ratio between the fifth and eighth selection cycles. This may be indicative of the discriminatory concentration to be used in E. heros monitoring programs. The diagnostic concentration was maintained at 5.65 µg of a.i./mL; this concentration caused a mortality of approximately 99% in the susceptible strain.
In the selection and characterization of the E. heros population resistant to imidacloprid (IMIDA−R), the estimated LC50 for the population after seven selection cycles was 114.67 μg imidacloprid/mL. The slope (±standard error) was 2.12 (±0.15) and the χ2 value was 4.66 (6 degrees of freedom), with a resistance ratio of 41.40 times (Figure 5). The test of equality and parallelism of the concentration−response curves of the resistant and susceptible populations showed significant differences in response to imidacloprid. The estimated slopes for the SUS and IMIDA−R populations were significantly different. The highest slope estimated for the SUS population was possibly related to its greater homogeneity. The concentration−response curves of the SUS and IMIDA−R populations for imidacloprid overlapped. Therefore, it was not possible to establish discriminatory concentrations, but an increase in the resistance ratio between the fifth and seventh selection cycles suggested that there was scope for further refinement in future studies on monitoring the susceptibility of E. heros to imidacloprid using the discriminatory concentration.
The estimated LC50 for the LAMBDA−R population after seven selection cycles was 1.152 µg lambda-cyhalothrin/insect. The slope (±standard error) was 2.67 (±0.20) and the χ2 value was 9.07 (five degrees of freedom), with a resistance ratio of 44.31 times (Figure 5). The test of equality and parallelism of the dose−response curves of the resistant and susceptible populations showed significant differences in the response to lambda-cyhalothrin. The dose−response curves of the SUS and LAMBDA−R populations for lambda-cyhalothrin overlapped. Hence, it was not possible to establish discriminatory concentrations for monitoring the susceptibility of E. heros to lambda-cyhalothrin.

4. Discussion

The new ingestion bioassay method using an encapsulated artificial diet was efficient in detecting and characterizing the resistance of E. heros to insecticides, as it enabled better discrimination between the susceptible and resistant populations. The proposed method of ingestion is a realistic method for systemic insecticides, especially for those belonging to the neonicotinoid class. The method is practical and can be easily reproduced. The important contributions of this method are the simulation of the systemic action of the insecticides while allowing feeding by brown stink bugs. The development of resistance management strategies requires the existence of a simple and reliable bioassay method that can provide adequate dose–response data for statistical analysis and enable the detection of low levels of resistance frequencies [50]. In addition, the capsules containing the artificial liquid diet used in the ingestion method have the thermoplastic characteristics of paraffin waxes and are flexible, odorless, moldable, malleable, translucent, colorless, easy to cut, non−toxic to insects, and easily pierced by the mouthparts of brown stink bugs.
The methods currently used to detect and monitor brown stink bug resistance are dipping bioassays of bean pods in a solution containing the insecticide, topical application bioassays, and tarsal contact bioassays using the vial test, which are based on direct contact of the insect with the product through application on the insect integument in the case of topical application or by tarsal contact of the bug in a container impregnated with the insecticide [23,24,26,27,28,29,30,31,43,44]. However, the methods of topical application and tarsal contact are not representative of the major group of insecticides used to control sucking insects. i.e., neonicotinoids. Besides, in the dipping bioassay method, which is dependent on the acquisition of bean pods, variations may arise depending on the variety, maturation stage, quality, and availability of bean pods. Therefore, realistic methods that are reproducible, regardless of the location, time of the year, and operator, are essential in integrated pest management (IPM) and IRM programs.
The resistance of E. heros to insecticides has not been detected using current bioassay methods, but control failures have been reported. In Brazil, failure to control E. heros populations has been reported for beta-cyfluthrin, bifenthrin, lambda-cyhalothrin (pyrethroid), and imidacloprid (neonicotinoid) [19,20,21,22]. Besides, there are numerous reports of farmers facing issues in controlling this species in soybean cultivation. To date, cases of E. heros resistance to the insecticides endosulfan (cyclodiene) and monocrotophos detected using the tarsal contact method and to the insecticide methamidophos (organophosphate) detected using the topical application method, have been reported [23,24].
One of the key objectives of resistance monitoring programs is to define a bioassay method that enables better discrimination of the susceptible and resistant pest populations [40,51]. A high slope enables maximizing the differences between the susceptible and resistant individuals; therefore, a high slope is one of the parameters for choosing the best bioassay method for evaluation [52]. In addition, a high slope allows for identifying resistance progression and genotypic variation in insecticide tolerance [53]. Therefore, high slopes obtained for thiamethoxam and imidacloprid using the ingestion method and for lambda-cyhalothrin using the topical application method indicate higher insecticidal activity and greater genotypic homogeneity of the tested populations.
The examination of the confidence intervals (95% CI) of LC50 for the selected bioassays, ingestion for insecticides with systemic action (thiamethoxam and imidacloprid), and topical application for insecticides with contact action (lambda-cyhalothrin) indicate that the selected bioassays are highly precise, since the 95% CI of the studied populations did not exceed twice the determined LC50 [54]. It was also observed that, compared with LC50, LC99 was able to better differentiate resistant individuals from susceptible individuals for the insecticides studied. Ffrench−Constant and Roush [40] reported that bioassays based on diagnostic concentrations were more efficient than those based on median lethal concentrations (LC50) for detecting low resistance frequencies and issues even at the initiation of resistance evolution.
Variation in susceptibility to insecticides among populations observed in distinct species of pests is not unusual [55]. From the perspective of resistance management, even a small susceptibility variation level is an indication of potential resistance selection [56]. The limited number of insecticides that can be used against brown stink bugs signifies that these insects are extensively exposed to the same active ingredients. This could result in future control failures, which can be attributed to resistance [20].
It is possible to verify an increase in the resistance ratio of some populations over the two seasons evaluated; this is likely due to the selection pressure imposed by the application of insecticides with the same active ingredient for controlling several species of insect pests throughout the crop cycle. The significant differences in the responses of the populations collected from Paraná and Bahia in the evaluated seasons indicate the possible existence of differences in the regime of insecticides used between these regions, which may be higher compared with those used in other regions of Brazil. However, the crop management and ecological situation of these locations are distinct. Londrina-PR is predominantly involved in the agriculture of soybean and maize, has a mild climate in the winter, and is warm in the summer. This causes brown stink bugs to enter dormancy in winter, which stay sheltered in straw for months and can feed on existing weeds or spontaneously grown soybean plants during off-season periods [3]. Conversely, Luís Eduardo Magalhães-BA is recognized as a hub of irrigated and high-performance agriculture of soybean and maize. Additionally, in the irrigated regions of western Bahia, brown stink bugs feed on alternative hosts since the agricultural areas succeed each other throughout the year. The number of applications of pest control chemicals is similar between the two regions, ranging from 8 to 12 applications per crop, only for agricultural pests [10,14,15]. In Londrina-PR, a commercially available mixture of lambda-cyhalothrin and thiamethoxam is most commonly used to control brown stink bugs in soybean; in contrast, in Luís Eduardo Magalhães-BA, imidacloprid, thiamethoxam, and a commercially available mixture of lambda-cyhalothrin and thiamethoxam is used to control brown stink bugs in soybean.
Our studies indicated high variation in susceptibility to insecticides in populations of E. heros in Brazil, as well as in responses to selection for resistance to thiamethoxam, imidacloprid, and lambda-cyhalothrin using a novel ingestion bioassay method. This reinforces that management practices are urgently needed to delay the evolution of insecticide resistance in E. heros populations in the field.

5. Conclusions

The new bioassay method of ingestion by encapsulation of an artificial liquid diet is very promising for detecting and monitoring resistance of E. heros populations to neonicotinoid insecticides. This data is essential for developing IPM and IRM programs that consider various brown stink bug control strategies and providing the most appropriate bioassay method and diagnostic concentrations for monitoring E. heros resistance to thiamethoxam, imidacloprid and lambda-cyhalothrin.

Author Contributions

Conceptualization. C.M.T. and C.O.; methodology C.M.T., C.O. and F.S.; software. C.M.T. and L.S.; validation. C.M.T., L.S. and C.O.; formal analysis. C.M.T. and L.S.; investigation. C.M.T. and F.S.; resources. C.M.T. and L.S.; data curation. C.M.T., L.S. and C.O.; writing—original draft preparation. C.M.T.; writing—review and editing. C.M.T. and C.O.; visualization. C.M.T. and C.O.; supervision. C.O.; project administration. C.M.T.; funding acquisition. C.M.T. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by CL Empreendimentos Biológicos Ltd.

Institutional Review Board Statement

Stink bug populations used in this study were registered in the Brazilian National System of Biodiversity Information (SISBIO license 63096-2).

Acknowledgments

This research was conducted in partial fulfillment by the senior author for the Doctoral Degree in Entomology at University of São Paulo. We acknowledge the assistance of team of employees of Pragas.com—www.pragas.com.vc (accessed on 10 January 2021) in the development of bioassays. We thank Crimson Interactive Pvt. Ltd. (Ulatus)—www.ulatus.com.br (accessed on 12 April 2021) for their assistance in manuscript translation and editing.

Conflicts of Interest

The authors declare no conflict of interest.

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Insects 12 00599 g001 550
Figure 1. Collection sites of the populations of E. heros used in the bioassay for selection and characterization of susceptibility to insecticides.
Figure 1. Collection sites of the populations of E. heros used in the bioassay for selection and characterization of susceptibility to insecticides.
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Figure 2. (A) Ingredients of the artificial diet; (B) mixing in a blender; (C) preparation of the capsules with the mold and the press coupled to the vacuum pump; (D) capsules containing artificial diet already sealed; (E) capsules containing the artificial diet; (F) E. heros feeding on the artificial diet capsule.
Figure 2. (A) Ingredients of the artificial diet; (B) mixing in a blender; (C) preparation of the capsules with the mold and the press coupled to the vacuum pump; (D) capsules containing artificial diet already sealed; (E) capsules containing the artificial diet; (F) E. heros feeding on the artificial diet capsule.
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Figure 3. E. heros feeding on the artificial diet capsules containing the insecticides concentrations in 24-well plate sealed with Parafilm M®.
Figure 3. E. heros feeding on the artificial diet capsules containing the insecticides concentrations in 24-well plate sealed with Parafilm M®.
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Figure 4. Percent mortality of populations of E. heros collected in crop seasons 2018/2019 and 2019/2020 exposed to a diagnostic dose of (A) thiamethoxam (5.65 µg of a.i./mL) in ingestion bioassays. (B) Imidacloprid (12.45 µg of a.i./mL) in ingestion bioassays and (C) lambda-cyhalothrin (0.20 µg a.i./insect) in bioassays by topical application.
Figure 4. Percent mortality of populations of E. heros collected in crop seasons 2018/2019 and 2019/2020 exposed to a diagnostic dose of (A) thiamethoxam (5.65 µg of a.i./mL) in ingestion bioassays. (B) Imidacloprid (12.45 µg of a.i./mL) in ingestion bioassays and (C) lambda-cyhalothrin (0.20 µg a.i./insect) in bioassays by topical application.
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Figure 5. Concentration/dose-mortality responses of E. heros populations to the insecticides (A) thiamethoxam (µg of a.i./mL), (B) imidacloprid (µg of a.i./mL) and (C) lambda-cyhalothrin (µg a.i./stink bug), submitted the seven cycles of selection with insecticide in laboratory conditions, by the technique of mass selection.
Figure 5. Concentration/dose-mortality responses of E. heros populations to the insecticides (A) thiamethoxam (µg of a.i./mL), (B) imidacloprid (µg of a.i./mL) and (C) lambda-cyhalothrin (µg a.i./stink bug), submitted the seven cycles of selection with insecticide in laboratory conditions, by the technique of mass selection.
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Table
Table 1. Populations of E. heros used in the evaluation of bioassay methods for selection and characterization of susceptibility to insecticides.
Table 1. Populations of E. heros used in the evaluation of bioassay methods for selection and characterization of susceptibility to insecticides.
Crop SeasonsPopulationsCollection SiteLatitudeLongitudeCollection Date
SUSPiracicaba—SP−22.413.920−47.382.900January 2013
2018/20191Cesário Lange—SP−23.221.722−47.879.901January 2019
2Santo Ângelo—RS−28.268.440−54.222.377February 2019
3Buri—SP−23.797.690−48.555.636February 2019
4Uberlândia 2—MG−19.039.666−48.214.689January 2019
5Londrina—PR−23.270.385−51.096.414January 2019
6Belmonte—SC−26.864.231−53.579.014February 2019
7Rio Verde—GO−17.800.507−50.888.559January 2019
8Canarana—MT−13.545.447−52.326.032February 2019
9Santa Bárbara do Oeste—SP−22.826.796−47.469.803January 2019
10Santa Cruz das Palmeiras—SP−21.846.312−47.248.144January 2019
11Luís Eduardo Magalhães—BA−12.158.124−45.781.697January 2019
12Sete Lagoas—MG−19.447.287−44.174.600January 2019
2019/202013Cesário Lange—SP−23.221.823−47.882.271December 2019
14Santo Ângelo—RS−28.271.968−54.229.174February 2020
15Buri—SP−23.797.690−48.555.636February 2020
16Uberlândia 2—MG−18.931.495−48.165.026January 2020
17Londrina—PR−23.246.961−51.119.236January 2020
18Não me Toque—RS−28.450.782−52.844.472January 2020
19Rio Verde—GO−17.854.921−50.947.760January 2020
20Santa Bárbara do Oeste—SP−22.825.832−47.470.452December 2019
21Santa Cruz das Palmeiras—SP−22.185.233−47.242.628December 2019
22Chapadão do Sul—MS−18.757.929−52.557.225December 2019
23Luís Eduardo Magalhães—BA−12.137.713−45.808.886January 2020
24Sete Lagoas—MG−19.456.500−44.173.400December 2019
25Lucas do Rio Verde—MT−13.035.200−55.574.500January 2020
26Primavera do Leste—MT−15.505.000−54.296.000January 2020
27Sorriso—MT−12.355.670−55.446.880January 2020
28Cristalina—GO−16.934.871−47.678.666January 2020
29São Desidério—BA−12.343.002−44.996.204January 2020
30Rolândia—PR−23.161.728−51.284.844January 2020
Table
Table 2. Composition of the artificial diet of E. heros for the ingestion bioassay.
Table 2. Composition of the artificial diet of E. heros for the ingestion bioassay.
IngredientsArtificial Diet bModified Artificial Diet c
Lyophilized and ground green beans35.00g100.00g
Crushed peanuts35.00g35.00g
Sucrose5.00g10.00g
Tetracycline0.01g0.01g
Sorbic acid0.10g0.15g
Ascorbic acid- 0.30g
Nipagin1.00g1.00g
Fatty acid- 1.00mL
Vitamin solution a- 5.00mL
Water30.00mL1000.00mL
a Composition of the vitamin solution: Niacinamide 1 g, Calcium Pantothenate 1 g, Thiamine 0.25 g, Riboflavin 0.5 g, Pyridoxine 0.25 g, Folic Acid 0.25 g, Biotin 0.02 mL, Vitamin B12 1 g, added to 1000 mL of distilled water. b Artificial diet of Cerna−Mendoza el al. [41]. c Artificial diet modified from Cerna−Mendoza el al. [41].
Table
Table 3. E. heros concentration/dose−mortality responses to the insecticides thiamethoxam, imidacloprid and lambda-cyhalothrin by ingestion methods (µg of a.i./mL), topical application (µg a.i./insect) and tarsal contact (µg a.i./cm2).
Table 3. E. heros concentration/dose−mortality responses to the insecticides thiamethoxam, imidacloprid and lambda-cyhalothrin by ingestion methods (µg of a.i./mL), topical application (µg a.i./insect) and tarsal contact (µg a.i./cm2).
InsecticideBioassayn aSlope (±SE)LC50/LD50 (95% CI) bLC90/LD90
(95% CI) b		χ2(d.f.) cp
thiamethoxamIngestion6724.99 (±0.54)1.93 (1.76–2.16)3.49 (2.98–4.39)3.19(4)0.5271
Topical application7003.98 (±0.33)0.027 (0.02–0.03)0.058 (0.052–0.068)2.62(4)0.0203
tarsal contact8002.11 (±0.13)0.196 (0.164–0.235)0.795 (0.614–1.114)5.05(5)0.0004
imidaclopridIngestion6723.53 (±0.26)2.73 (2.28–3.26)6.30 (5.04–8.59)4.89(4)0.2982
Topical application7003.36 (±0.27)0.028 (0.023–0.036)0.068 (0.050–0.115)8.04(4)0.0032
tarsal contact8002.29 (±0.15)0.315 (0.264–0.377)1.138 (0.882–1.591)5.26(5)0.0001
lambda-cyhalothrinIngestion7682.56 (±0.17)17.85 (14.87–21.49)56.53 (43.92–79.33)6.19(5)0.0005
Topical application8003.62 (±0.26)0.046 (0.042–0.050)0.104 (0.091–0.122)2.42(5)0.7878
tarsal contact8002.50 (±0.17)0.169 (0.143–0.199)0.549 (0.436–0.745)5.25(5)0.0043
a Number of tested. b LC50/LD50 e LC90/LD90: concentration/dose of insecticide required to kill 50% and 90% of stink bugs, respectively. c Degrees of freedom.
Table
Table 4. Dose−mortality responses (LD) of lambda-cyhalothrin (0.20 µg a.i./insect) in bioassays by topical application.
Table 4. Dose−mortality responses (LD) of lambda-cyhalothrin (0.20 µg a.i./insect) in bioassays by topical application.
PopulationCrop SeasonsLD50 Estimated
n aSlope (±SE)LD50 (95% CI) bχ2d.f. cRR50 d
Lambda-Cyhalothrin
Susceptible reference2019/20208003.23 (±0.20)0.026 (0.023–0.029)3.565-
Buri—SP7002.78 (±0.17)0.135 (0.116–0.158)6.4655.12
Não me Toque—RS8003.90 (±0.24)0.551 (0.488–0.620)8.24620.92
Londrina—PR5603.20 (±0.24)0.731 (0.621–0.773)2.99527.73
Cristalina—GO4903.22 (±0.25)0.291 (0.259–0.326)4.83511.02
Primavera do Leste—MT5602.89 (±0.21)0.521 (0.445–0.611)5.57519.74
Uberlândia—MG5602.95 (±0.21)0.132 (0.118–0.147)3.7655.00
Luís Eduardo Magalhães—BA4322.31 (±0.19)1.054 (0.831–1.366)4.44439.98
a Number of tested. b LD50: dose of insecticide required to kill 50% of stink bugs. c Degrees of freedom. d Resistance Ratio.
Table
Table 5. Concentration−mortality responses (LC) of thiamethoxam (5.65 µg of a.i./mL) and imidacloprid (12.45 µg of a.i./mL) in ingestion bioassays.
Table 5. Concentration−mortality responses (LC) of thiamethoxam (5.65 µg of a.i./mL) and imidacloprid (12.45 µg of a.i./mL) in ingestion bioassays.
PopulationCrop SeasonsLC50 Estimated
n aSlope (±SE)LC50 (95% CI) bχ2d.f. cRR50 d
Thiamethoxam
Susceptible reference2018/20198642.88 (±0.17)1.79 (1.63–1.98)4.376-
Cesário Lange—SP4322.49 (±0.23)4.53 (3.99–5.12)0.4342.53
Buri—SP6482.39 (±0.16)5.27 (4.32–6.47)8.9962.94
Santo Ângelo—RS3362.75 (±0.27)4.36 (3.35–5.58)4.0142.44
Londrina—PR7681.68 (±0.13)14.27 (11.51–18.57)5.1357.97
Rio Verde—GO5042.87 (±0.23)3.09 (2.68–3.56)3.2041.73
Canarana—MT5042.69 (±0.22)2.80 (2.40–3.24)3.5141.56
Santa Bárbara do Oeste—SP6712.43 (±0.17)3.51 (3.06–4.00)2.7541.96
Luís Eduardo Magalhães—BA3842.03 (±0.25)19.20 (15.51–25.37)3.46410.73
Buri—SP2019/20204322.75 (±0.22)4.67 (4.04–5.38)1.8842.61
Não me Toque—RS2882.55 (±0.25)4.15 (3.44–4.97)1.0142.32
Londrina—PR5041.96 (±0.14)25.15 (18.94–33.58)8.18514.05
Cristalina—GO4202.69 (±0.21)3.17 (2.72–3.67)1.8441.77
Uberlândia—MG4322.45 (±0.20)2.90 (2.46–3.38)2.6941.62
Lucas do Rio Verde—MT5762.21 (±0.15)3.79 (3.28–4.35)1.8042.12
Luís Eduardo Magalhães—BA5042.23 (±0.16)39.52 (33.84–46.15)3.49522.08
Imidacloprid
Susceptible reference2018/20195763.27 (±0.22)2.77 (2.37–3.20)4.924-
Cesário Lange—SP3603.40 (±0.34)9.67 (5.96–13.65)7.1243.49
Buri—SP5043.31 (±0.27)4.59 (3.64–5.69)5.5441.66
Santo Ângelo—RS3362.59 (±0.25)4.80 (3.99–5.73)1.3041.73
Londrina—PR5042.41 (±0.19)15.36 (12.00–19.90)7.8055.55
Rio Verde—GO5042.78 (±0.23)5.53 (4.79–6.34)2.6742.00
Canarana—MT5042.84 (±0.23)5.46 (4.74–6.26)2.8841.97
Santa Bárbara do Oeste—SP5043.44 (±0.34)9.54 (7.47–12.22)7.3043.44
Uberlândia—MG6482.49 (±0.18)15.19 (12.66–18.58)8.2065.48
Buri—SP2019/20204203.10 (±0.25)4.55 (3.97–5.19)1.9941.64
Não me Toque—RS2882.62 (±0.25)4.73 (3.94–5.64)1.3541.71
Londrina—PR5042.32 (±0.17)28.97 (22.51–37.09)7.51510.46
Cristalina—GO4322.69 (±0.22)5.43 (4.69–6.25)0.9941.96
Lucas do Rio Verde—MT4202.66 (±0.21)5.27 (4.56–6.08)0.8441.90
Primavera do Leste—MT5043.05 (±0.25)20.41 (16.36–25.80)7.9457.37
Uberlândia—MG6002.68 (±0.19)17.62 (13.67− 23.13)8.7846.36
Luís Eduardo Magalhães—BA4322.49 (±0.20)60.73 (52.27–70.39)2.35421.92
a Number of tested. b LC50: concentration of insecticide required to kill 50% of stink bugs. c Degrees of freedom. d Resistance Ratio.
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Tibola, C.M.; Silva, L.; Sgubin, F.; Omoto, C. Monitoring Resistance of Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) to Insecticides by Using Encapsulated Artificial Diet Bioassay. Insects 2021, 12, 599. https://0-doi-org.brum.beds.ac.uk/10.3390/insects12070599

AMA Style

Tibola CM, Silva L, Sgubin F, Omoto C. Monitoring Resistance of Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) to Insecticides by Using Encapsulated Artificial Diet Bioassay. Insects. 2021; 12(7):599. https://0-doi-org.brum.beds.ac.uk/10.3390/insects12070599

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Tibola, Cristiane Maria, Leandro Silva, Fernanda Sgubin, and Celso Omoto. 2021. "Monitoring Resistance of Euschistus heros (Fabricius) (Hemiptera: Pentatomidae) to Insecticides by Using Encapsulated Artificial Diet Bioassay" Insects 12, no. 7: 599. https://0-doi-org.brum.beds.ac.uk/10.3390/insects12070599

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