Does the dose make the poison? Neurotoxic insecticides impair predator orientation and reproduction even at low concentrations

Abstract

BACKGROUND

Pesticides can be noxious to non‐target beneficial arthropods and their negative effects have been recently recognized even at low doses. The predator Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) plays an important role in controlling insect pests in solanaceous crops, but its concurrent herbivory often poses relevant concerns for tomato production. Although insecticide side effects on N. tenuis have been previously studied, little is known on the potential implications of neurotoxic chemicals at low concentrations. We assessed the baseline toxicity of three neurotoxic insecticides (lambda‐cyhalothrin, spinosad and chlorpyrifos) on N. tenuis by topical contact exposure. The behavioral and reproduction capacity of the predator was then investigated upon exposure to three estimated low‐lethal concentrations (LC₁, LC₁₀ and LC₃₀).

RESULTS

Predator survival varied among insecticides and concentrations, with LC₃₀/label rate ratios ranging from 8.45% to 65.40% for spinosad and lambda‐cyhalothrin, respectively. All insecticides reduced the fertility of N. tenuis females at all estimated low‐lethal concentrations. Chlorpyrifos seriously compromised predator orientation towards a host plant even at LC₁, while the same effect was observed for lambda‐cyhalothrin and spinosad solely at LC₃₀. Lambda‐cyhalothrin (at all concentrations) and chlorpyrifos (at LC₁₀ and LC₃₀) also affected the time taken by N. tenuis females to make a choice.

CONCLUSION

The results indicate that all three insecticides can be detrimental to N. tenuis and should be avoided when presence of the predator is desirable. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Neurotoxic insecticides negatively affected the behavior and reproduction of the predator Nesidiocoris tenuis, even at low concentrations. Our study suggest that these compounds should be avoided when the presence of the predator is desirable.

1. INTRODUCTION

Pesticides have been incriminated for their negative consequences on biodiversity and its functioning, although their relevance in controlling plant pests effectively remains undeniable. ¹ , ² Pesticides can potentially affect non‐target organisms present in the agroecosystem, leading to disruption of the ecological services they provide, such as pollination, nutrient cycling and biological control. ³ , ⁴ For this reason, studies on the side effects of pesticides are encouraged to provide new insights to mitigate their negative impacts on non‐target beneficial arthropods. ⁵ , ⁶ , ⁷ , ⁸ , ⁹ This is especially relevant in integrated pest management (IPM) programs in which natural enemies are often deliberately released and/or conserved to reduce pest populations. ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴

Ecotoxicological screenings are usually based on guidelines developed by non‐governmental institutions, and in the European Union (EU) the ecotoxicological risk assessment of pesticides towards non‐target arthropods was developed in the Guidance Document on Terrestrial Ecotoxicology, ¹⁵ following the recommendation of the European standard characteristics of beneficials regulatory testing (ESCORT) of the Society of environmental toxicology and chemistry (SETAC) for non‐bee arthropods. ¹⁶ , ¹⁷ Most ecotoxicology studies consist of laboratory trials aimed at testing the highest pesticide dose recommended by manufacturers. However, pesticides are naturally degraded by biotic and abiotic factors, ¹⁸ , ¹⁹ and their drift may also occur in the field resulting in lower doses compared with their initial application. ²⁰ , ²¹ Therefore, non‐target organisms present in the agroecosystem can be exposed to chemical residues at low concentrations and, surviving individuals may experience related sublethal effects. ¹⁹ , ²² , ²³ , ²⁴ These effects may include lower fertility and a reduction in predation/parasitism ability, which can negatively affect the establishment of natural enemies in the field and bias their efficiency in reducing pest populations. ³ , ²⁵ , ²⁶

Hemipteran predators are of paramount importance for the biological control of insect pests in greenhouse crops because they are able to control populations of several arthropod pests. ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ Among mirid predators (Hemiptera: Miridae), the zoophytophagous Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is one of the most used species for biological control in the Palaearctic. Nesidiocoris tenuis has a multifaceted role for greenhouse pest control due to its high efficacy against a number of pests including aphids, whiteflies and lepidopterans, such as the South American tomato pinworm, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). ³² Moreover, the use of N. tenuis has been fostered because of its ability in priming induced plant defense mechanisms. ³³ , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ However, owing to its plant‐feeding activity when prey is scarce, N. tenuis can become a pest because this predator can cause plant damage at high population levels. ³⁹ , ⁴⁰ , ⁴¹ Despite this drawback, IPM programs still rely on the biological control provided by N. tenuis. Therefore, the predator can be often exposed to organic and/or synthetic insecticides routinely adopted in these programs. ⁴² , ⁴³ , ⁴⁴ , ⁴⁵

Earlier studies investigated the impact of insecticides on N. tenuis in terms of lethal and sublethal effects, by exposing N. tenuis adults to synthetic and organic neurotoxic compounds via different exposure routes (i.e., contaminated prey, direct spray and residual contact). ²⁷ , ²⁸ , ⁴⁶ , ⁴⁷ Nevertheless, most studies investigated only the maximum label rate of these compounds. To the best of our knowledge, there is no information regarding the effects of low insecticide concentrations on N. tenuis orientation capacity, which may ultimately affect the success of this predator as a biological control agent.

In this study, we hypothesized that low concentrations of neurotoxic insecticides might have detrimental effects on the physiology and behavior of N. tenuis. We tested this hypothesis through laboratory trials aiming to assess the fertility and olfactory response of N. tenuis adults topically exposed to three low‐lethal concentrations (LC₃₀, LC₁₀, LC₁) of insecticides, previously estimated for this mirid predator. Our findings may help in understanding the convolutions of pesticide side effects at low concentrations on natural enemies and provide new useful insights into the association between the predator N. tenuis and chemical insecticides in pest control.

2. MATERIALS AND METHODS

2.1. Biological materials

Nesidiocoris tenuis for laboratory rearing were obtained from periodic collections in organic open tomato greenhouses located in Fiumefreddo (Catania, Italy). Collected specimens were morphologically identified and reared in the laboratory as follows. Briefly, adults of N. tenuis (~150 individuals) were kept in entomological cages (32 × 40 × 70 cm) covered by fine net mesh and containing pesticide‐free sesame (Sesamum indicum L., variety T‐85 Humera) potted seedlings (~30 cm in height), as water and oviposition sources, according to the methodology described by Biondi et al. ⁴⁸ The commercial mixture of the alternative prey Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs and Artemia spp. cysts (i.e., Entofood® Koppert) was offered ad libitum to the predators as an additional food source. Nesidiocoris tenuis adults were kept on sesame plants for 3 days to allow mating and oviposition events; subsequently, N. tenuis adults were collected with a mechanical aspirator and transferred to new cages as described above. Sesame plants bearing N. tenuis eggs were isolated inside the cages for egg hatching and the development of newly hatched nymphs to adulthood. Half of the newly molted N. tenuis adults were collected with a mechanical aspirator and used for the bioassays, whereas the remainder were added to the rearing. New sesame plants and Entofood® were added to each cage twice a week. The rearing was maintained under laboratory conditions (25 ± 1°C, 55% ± 5% relative humidity, and a 14:10 h light/dark photoperiod) at the Department of Agriculture, Food and Environment of the University of Catania (Italy).

2.2. Insecticides

To assess the potential physiological and behavioral effects on N. tenuis, three neurotoxic insecticides were evaluated in this study. The insecticides, followed by their tradename, manufacturer, chemical group and mode of action, were: lambda‐cyhalothrin (Karate Zeon®, Syngenta Italia S.p.a.), a pyrethroid, Na⁺ channel modulator; spinosad (Laser®, Dow AgroSciences S.r.l.), a spinosyn, nicotinic acetylcholine receptor allosteric modulator; and chlorpyrifos (Dursban®, Dow AgroSciences S.r.l.), an organophosphate, acetylcholinesterase (AChE) inhibitor. Lambda‐cyhalothrin and chlorpyrifos are both synthetic insecticides used in conventional tomato crops in many countries, whereas spinosad is a naturally derived insecticide, therefore its use is allowed in both conventional and organic crops. These insecticides were selected due to their potential use in tomato crops to control hemipteran and lepidopteran pests (such as aphids, whiteflies and T. absoluta), which are also N. tenuis prey.

2.3. Insecticides baseline toxicity toward Nesidiocoris tenuis

In this bioassay, we assessed the concentration–mortality response relationship of N. tenuis adult stage to lambda‐cyhalothrin, spinosad and chlorpyrifos by topical contact exposure. Newly emerged females (~2 days old) were exposed by topical spray to different concentrations of the insecticides. For each insecticide, six or seven concentrations, including of the highest label rate, were tested (see Table 1). Stock solutions were prepared with dilution of insecticidal formulations in distilled water, according to the manufacturer's recommendations. In addition, an untreated control with distilled water was included for all the insecticides (referred to as “zero concentration”). The insecticide dilutions were based on preliminary observations aimed at identifying the minimum dose needed to cause 100% mortality of N. tenuis females and the maximum dose that does not significantly affect the mortality of the treated insects in comparison with the untreated control.

TABLE 1.

Baseline toxicity of three insecticides toward Nesidiocoris tenuis females 48 h after topical contact exposure by spraying

Insecticide	Tradename	% a.i.	Label rate (ppm)	Slope ± SE	χ2 (df)	p‐value	Lethal concentration (ppm)	95% Confidence limits (ppm)	% LC/LR a
Spinosad	Laser®	44.20	0.3315	1.974 ± 0.260	33.355 (31)	0.353	LC1 = 3.37 × 10−3	1.35 × 10−3 to 5.88 × 10−3	1.08
							LC10 = 1.14 × 10−2	6.71 × 10−3 to 1.62 × 10−2	3.32
							LC30 = 2.75 × 10−2	2.00 × 10−2 to 3.60 × 10−2	8.44
Lambda‐cyhalothrin	Karate Zeon®	9.48	0.0236	1.301 ± 0.201	42.901 (36)	0.201	LC1 = 6.39 × 10−4	1.10 × 10−4 to 1.68 × 10−3	2.70
							LC10 = 4.06 × 10−3	1.49 × 10−3 to 7.33 × 10−3	17.13
							LC30 = 1.55 × 10−2	9.24 × 10−3 to 2.22 ∙ 10−2	65.40
Chlorpyrifos	Dursban®	44.53	0.3340	0.948 ± 0.202	35.563 (33)	0.349	LC1 = 8.87 × 10−4	2.30 × 10−4 to 4.09 × 10−4	0.27
							LC10 = 1.12 × 10−2	1.72 × 10−3 to 2.58 × 10−2	3.35
							LC30 = 7.05 × 10−2	3.34 × 10−2 to 1.14 × 10−1	21.11

^a % LC/LR is percentage of the estimated low‐lethal concentration in comparison with the highest label rate recommended in tomato crop.

An adapted methodology for insecticide topical contact application on N. tenuis adult stage was developed. Briefly, five N. tenuis females were isolated together in conical ventilated plastic tubes (Falcon®, 50 ml) and maintained at low temperature inside an insulated thermic box with ice packs for 3 h to reduce insect mobility. Thereafter, each group of N. tenuis females was placed in a plastic cup (100 ml) and topically sprayed with insecticide solutions using a hand‐sprayer (50 ml). The inside of the plastic cups was covered by absorbent paper to prevent the formation of insecticide droplets in the arena, preventing insect mortality via drowning. Clean and new absorbent paper was changed in each replicate for every insecticide–concentration combination. After spraying, each group of five N. tenuis females was transferred to an acrylic ventilated pot (5.5 cm in diameter × 3 cm height), along with a zucchini (Cucurbita pepo L.) leaf disc and Entofood®. Each pot containing five females was considered one replicate. Mortality caused by the insecticides on N. tenuis females was evaluated after 48 h. Eight replicates were performed for each insecticide–concentration combination.

2.4. Sublethal effects of insecticides on Nesidiocoris tenuis fertility

The aim of this bioassay was to evaluate whether low concentrations of lambda‐cyhalothrin, spinosad and chlorpyrifos could affect the fertility of the predator N. tenuis. Based on the results of the previous bioassay, newly molted N. tenuis males and females (2 days old) from the rearing were exposed to three low‐lethal concentrations (LC₁, LC₁₀ and LC₃₀) of the aforementioned insecticides. These concentrations were chosen to expose the predators to low concentrations that can occur under field conditions after environmental degradation of a full label spray, including a lethal range from very low mortality (LC₁) to moderate mortality (LC₃₀).

Adult females (2 days old) were sprayed with the low‐lethal concentrations mentioned above and distilled water, as described in Section 2.3. Sprayed couples were kept in a ventilated plastic cup (400 ml) containing a green bean pod (Phaseolus vulgaris L., cv. ‘Garrafal enana’) as a water source and oviposition substrate, ²⁸ , ⁴⁹ and E. kuehniella eggs (1 g) as food supply in the arena. Each N. tenuis couple was kept in the aforementioned arena for 3 days to increase mating success and let the females oviposit into the bean. The experimental arenas containing green bean pods with N. tenuis eggs were maintained under laboratory conditions as described above, and the number of newly emerged nymphs was recorded daily under a stereomicroscope and removed with a soft paintbrush. The evaluation was conducted for 20 days until no new nymph emerged. For each pesticide–concentration combination and the control, the fertility of 25 N. tenuis couples (i.e., 25 replicates) was evaluated.

2.5. Sublethal effects of insecticides on Nesidiocoris tenuis orientation

The aim of this bioassay was to evaluate whether the orientation of the predator N. tenuis could be affected by the three low‐lethal concentrations (LC₁, LC₁₀ and LC₃₀) of lambda‐cyhalothrin, spinosad and chlorpyrifos. Adult females (2 days old) were sprayed with the low‐lethal concentrations mentioned above and distilled water, as described in Section 2.3. After being sprayed topically, N. tenuis females were starved for 24 h in transparent vials (1.5 cm in diameter × 6 cm height) with a wet cotton wad as the only water source. Thereafter, each N. tenuis female was transferred in a two‐way olfactometer (main arm and lateral arms 15 cm long and 4 cm internal diameter). The odor sources used were clean air and a sesame plant (~20 cm height). Sesame plant was chosen because previous studies demonstrated that this plant is highly attractive to N. tenuis. ⁴⁸ , ⁵⁰ A sesame plant was placed inside one of the cylindrical glass jars (5 L volume) connected to the lateral arms of the olfactometer. An air pump (Airfizz®, Ferplast) produced a unidirectional flow (150 ml min⁻¹) that passed through a water filter before entering the olfactometer system, conducting the air through the olfactometer lateral arms and reaching thus the main arm. The olfactometer was placed vertically on the bench surface and N. tenuis females were placed individually on the central arm. The bioassays were performed in a dark room, with controlled environmental conditions (25 ± 1°C, 60 ± 10% R. H.) and were conducted between 9:00 a.m. and 6:00 p.m. The olfactometer was illuminated by 22 W cool‐white fluorescent lamps, positioned 80 cm above the olfactometer arms, according to Naselli et al. ⁵⁰

The choice of each female was considered when it crossed half of the lateral arm. Each predator was observed for 5 min and, if no choice was made after that time, non‐responder N. tenuis females were discarded from the data set. After every two tested females, the olfactometer was inverted to reduce environmental interference in the insect response. For each insecticide–concentration combination, at least 30 replicates, each composed of an insect that have did a choice, were carried out. The time taken for N. tenuis females to make a choice (for insects that made a choice) was also recorded.

2.6. Statistical analyses

The baseline toxicity of lambda‐cyhalothrin, spinosad and chlorpyrifos on N. tenuis by topical contact exposure was carried out through a log‐probit regression model. ⁵¹ The preference data of N. tenuis towards sesame plants were analyzed using a chi‐squared goodness‐of‐fit to determine whether the female attraction to sesame plants was different from a 50:50 distribution.

Data regarding time taken by N. tenuis females to make a choice and fertility were tested for normality and homoscedasticity; ⁵² , ⁵³ however, these assumptions were not met. Therefore, these data were fitted to generalized linear models (GLMs), ⁵⁴ and potential interaction between factors (four treatments × three concentrations) was tested. The models were fitted using the Poisson family for fertility and negative binomial family for time taken by N. tenuis females to make a choice (Poisson and quasi‐Poisson families were first tested, but the negative binomial model presented a better fit). Means were separated by a post‐hoc Tukey's HSD test (p < 0.05). Probit analyses were performed in the statistical program SPSS v. 21.0 (IBM Corp.), whereas the analyses for the fertility and olfactory response bioassays were performed in R v. 3.6.0 (R Foundation for Statistical Computing), using the packages car and MASS for model fitting and the package multcomp to separate means. ⁵⁵ , ⁵⁶ , ⁵⁷

3. RESULTS

3.1. Insecticides baseline toxicity toward Nesidiocoris tenuis

The probit models were fitted to observed data for all the treatments (i.e., there were no significant differences between the observed and the expected data), validating the low‐lethal concentrations for all the tested neurotoxic insecticides (Table 1). All insects treated with distilled water only (“zero concentration”) survived throughout the evaluation period. Lambda‐cyhalothrin was the insecticide with the lowest values of LC₁, LC₁₀ and LC₃₀, being the most lethal active ingredient for N. tenuis females. Spinosad and chlorpyrifos also presented high toxicity to the predator as highlighted by the low LC₃₀ values estimated for these compounds. Nevertheless, despite lambda‐cyhalothrin being the most toxic active ingredient, it was observed that the proportion values between the estimated LC₁₀ and LC₃₀ and the maximum label rate were higher for this insecticide (17.13% and 65.40%) in comparison with those observed for spinosad (3.32% and 8.45%) and chlorpyrifos (3.35% and 21.11%) (Table 1).

3.2. Sublethal effects of insecticides on Nesidiocoris tenuis fertility

Although the GLM revealed no significant insecticide × concentration interaction (χ² = 12.023, df = 6, p = 0.061), all the tested insecticides significantly reduced the fertility of N. tenuis females at all the evaluated concentrations (LC₁: χ² = 64.642, df = 3, p < 0.001; LC₁₀: χ² = 73.707, df = 3, p < 0.001; LC₃₀: χ² = 118.560, df = 3, p < 0.001). The reduction in fertility was higher for chlorpyrifos at LC₃₀ (χ² = 9.939, df = 2, p = 0.007), whereas no differences were observed among the concentrations for lambda‐cyhalothrin (χ² = 2.659, df = 2, p = 0.265), spinosad (χ² = 1.008, df = 2, p = 0.604) and the control (χ² = 0.427, df = 2, p = 0.808) (Figure 1).

FIGURE 1.

Mean (±) fertility values for Nesidiocoris tenuis females after topical contact exposure to three insecticides at three low‐lethal concentrations and distilled water (untreated control). Different upper case letters indicate significant differences among treatments in a concentration, whereas different lower case letters indicate significant differences in the concentrations for a treatment (GLM – Poisson distribution, Tukey's HSD test, p < 0.05).

3.3. Sublethal effects of insecticides on Nesidiocoris tenuis orientation

A significant attraction towards sesame plants was expected for insects that did not experience any insecticide exposure, ⁵⁰ and it was confirmed for all control treatments. Therefore, this was taken as a reference for the percentage of insects orienting toward sesame compared with clean air for the treatments with insecticides. The preference of N. tenuis females for sesame plants instead of clean air was not affected by lambda‐cyhalothrin or spinosad at LC₁ and LC₁₀. However, the choices of insects treated with chlorpyrifos did not differ between sesame and air for these two low‐lethal concentrations. At LC₃₀, all insecticides affected N. tenuis orientation, resulting in no difference between the proportion of choices for sesame and clean air (Figure 2).

FIGURE 2.

Response of Nesidiocoris tenuis females topically exposed to three insecticides at three low‐lethal concentrations (LC₁, LC₁₀ and LC₃₀) and distilled water (untreated control) towards the volatiles produced by a Sesamum indicum plant. The percentages indicate the proportion of choices for sesame and clean air. Asterisks indicate differences in the attraction to S. indicum and clean air according to the likelihood chi‐squared (p < 0.05).

Differences in the time taken by N. tenuis females to make a choice were observed in all low‐lethal concentrations (LC₁: χ² = 9.358, df = 3, p = 0.024; LC₁₀: χ² = 22.566, df = 3, p < 0.001; LC₃₀: χ² = 33.291, df = 3, p < 0.001) (Figure 3). Insects treated with all the tested concentrations of lambda‐cyhalothrin took longer to make a choice in comparison with the control treatment. The same was observed for insects treated with chlorpyrifos at LC₁₀ and LC₃₀. For females treated with all concentrations of spinosad time taken to make a choice was not affected in comparison with the control treatment. No differences were observed among concentrations for any of the treatments (control: χ² = 0.508, df = 2, p = 0.777; lambda‐cyhalothrin: χ² = 0.634, df = 2, p = 0.729; chlorpyrifos: χ² = 4.981, df = 2, p = 0.083; spinosad: χ² = 3.589, p = 0.166) (Figure 3). There was no interaction between treatments and concentrations for the time taken by N. tenuis females to make a choice (χ² = 9.066, df = 6, p = 0.170), therefore the data were evaluated separately.

FIGURE 3.

Mean (±SE) time taken (s) by Nesidiocoris tenuis females to make a choice between the volatiles emitted by a Sesamum indicum plant or clean air after topical contact exposure to three insecticides at three low‐lethal concentrations and distilled water (untreated control). Different upper case letters indicate differences among treatments in a concentration, whereas different lower case letters indicate differences in the concentrations for a treatment (GLM – Negative Binomial distribution, Tukey's HSD test, p < 0.05).

4. DISCUSSION

In many systems, broad‐spectrum insecticides, such as neurotoxic insecticides, are the most used compounds for pest control because of their effectiveness in controlling pests. However, a vast literature has documented concerning detrimental effects on beneficial organisms caused by these effective tools. ³ , ⁵⁸ , ⁵⁹ To preserve the ecological functions of beneficial organisms in the agroecosystem (including biological control) selective insecticides should be preferred in pest management. ⁵ , ²² , ²⁶ , ⁶⁰ , ⁶¹ , ⁶² Additionally, insecticides can cause sublethal effects that can bias the biological control provided by predators and parasitoids. ³ , ²⁴ These alterations can be observed in individuals that survived both full label rates of selective compounds and lower concentrations of broad‐spectrum insecticides, which can occur after natural degradation under field conditions. ²² , ⁶³ , ⁶⁴ , ⁶⁵

Probit models are often used to estimate concentration–mortality of pesticides to pests and natural enemies, in order to select efficient and safe compounds, respectively. ⁴⁹ , ⁶⁵ , ⁶⁶ , ⁶⁷ , ⁶⁸ In our observations, lambda‐cyhalothrin was the most toxic compound for the predator, because lower values were estimated for all lethal concentrations. Spinosad was the least toxic insecticide at LC₁ towards N. tenuis; however, at LC₃₀ this insecticide was more toxic than chlorpyrifos. The highest slope estimated for spinosad treatment indicates that a slight increase in insecticide concentration may lead to high predator mortality. ⁶⁹ Moreover, due to the lower active ingredient concentration in the lambda‐cyhalothrin based insecticide, the difference between the maximum label rate and the estimated LC₁₀ and LC₃₀ are lower for lambda‐cyhalothrin than for the spinosad and chlorpyrifos commercial products. Therefore, despite the higher LC₁₀ and LC₃₀ values observed for spinosad and chlorpyrifos, these insecticides might be even more toxic than lambda‐cyhalothrin under field conditions.

Besides mortality, all insecticides at the three evaluated concentrations reduced the fertility of the predator N. tenuis. Reproductive parameters are among the most sensitive biological characteristics to insecticides and the most important in terms of population dynamics. ⁷⁰ Similar to our results, a reduction in N. tenuis progeny was also observed for the pyrethroids cypermethrin and deltamethrin. ²⁸ , ⁴⁶ Additionally, pyrethroids can be used at sublethal concentrations to contaminate insect‐proof nets, and Biondi et al. found that the continuous exposure of T. absoluta adults to such nets can cause a variety of chronic sublethal effects rather than acute toxicity. ⁷¹

Lower concentrations of the organophosphate chlorpyrifos were also frequently reported as causing negative effects on the reproduction of natural enemies. At LC₃₀ several sublethal effects were observed on the hemipteran Andrallus spinidens Fabricius (Hemiptera: Pentatomidae), such as reduction in fertility and enzyme activity, and alterations in life table parameters. ⁷² Fernandes et al. observed negative effects on reproduction after chlorpyrifos exposition at LC₂₀ for the predator Orius insidiosus (Say) (Hemiptera: Anthocoridae). ⁶⁷ Moreover, spinosad reduced the offspring of the predatory bugs, such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae), ²² Macrolophus pygmaeus (Rambur) (Hemiptera: Miridae) ²⁷ , ⁷³ and Deraeocoris brevis (Uhler) (Hemiptera: Miridae). ⁷⁴

Negative effects were also observed in the behavioral traits of N. tenuis. In the insecticide treatments, spinosad and lambda‐cyhalothrin at LC₃₀ and chlorpyrifos at all concentrations affected the orientation ability of the predator. Moreover, the two synthetic insecticides also increased the time taken by N. tenuis females to make a choice. Because of their neurotoxic action, all three insecticides can affect the capacity of the nervous system to react to external stimuli. ⁵⁹ , ⁶³ , ⁷² , ⁷⁵

The behavioral results in this study are consistent with neurotoxicity associated with lambda‐cyhalothrin. The deleterious effects caused by pyrethroids result from a blockage in electrical stimulus conduction as a consequence of the permanent opening of sodium channels while the insecticide is acting, leading to behavioral and physiological impacts. ⁵⁹ , ⁷⁶ Desneux et al. also observed that sublethal doses of lambda‐cyhalothrin (LD_0.1) affected the orientation behavior of the parasitoid Aphidius ervi Haliday (Hymenoptera: Braconidae). ⁶³ The parasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae) exhibited a reduction in parasitism and longevity after treatment with sublethal concentrations of lambda‐cyhalothrin. ⁷⁷ Soares et al. also observed alterations in N. tenuis behavior caused by lambda‐cyhalothrin. ²⁶

Similarly, a lack of coordination is associated with chlorpyrifos intoxication. ⁷² The biased orientation of N. tenuis females treated with chlorpyrifos at LC₁, LC₁₀ and LC₃₀ indicates that this insecticide can affect the predator behavior even at very low concentrations. The time taken by N. tenuis females to make a choice also increased after treatment with LC₁₀ and LC₃₀. Fernandes et al. observed negative effects on predation rate after chlorpyrifos exposure at LC₂₀ for the predator O. insidiosus. ⁶⁷ The predator M. pygmaeus also showed behavioral alterations (reduced attack rate and increased handling time) after treatment with chlorpyrifos at LC₃₀. ⁷⁸

By contrast, studies regarding the side effects of spinosad on the behavior of beneficial insects are scarce. For example, Barbosa et al. observed alterations in walking activity in the stingless bee Melpona quadrifasciata Depeleiter (Hymenoptera: Apidae) after exposure to spinosad. ⁷⁹ Nevertheless, owing to the toxic effect on the nervous system, insects intoxicated by spinosad may present symptoms like a lack of coordination, trembling of appendages and a compromised perception of external stimuli, which can ultimately result in reduced predatory capacity for a natural enemy. ⁷⁵ , ⁸⁰ , ⁸¹ , ⁸²

Plants from different botanical families, such as Asteraceae, Solanaceae and Pedaliaceae, are suitable for N. tenuis biological development. These plants could serve as water and oviposition sources, and are also plants where N. tenuis prey can be found in the field. ³⁹ , ⁴⁸ For this reason, disrupting the predators’ capacity to locate host plants directly influences their survival and success as biological control agents. The misorientation caused by lower concentrations of insecticides could also compromise N. tenuis capacity to locate plants infested with herbivorous prey, as observed for the predator Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae) after exposure to the pyrethroid deltamethrin. ⁸³ Further study is needed on this point of the system.

In summary, we observed that spinosad, chlorpyrifos and lambda‐cyhalothrin can be toxic to the predator N. tenuis, even at low concentrations, with effects on fertility and orientation. In addition to the laboratory results, field trials should be performed to confirm the toxicity of the compounds exploring different exposure routes (i.e., residual contact and ingestion of contaminated prey) and/or by testing the potential side effects toward insect parasitoids exploited in tomato crops. ¹³ , ⁸⁴ , ⁸⁵

5. CONCLUSIONS

The baseline toxicity showed that the insecticides were toxic to N. tenuis females. The sublethal effects caused by the tested concentrations of the three insecticides were also relevant. Even at LC₁ and LC₁₀ the fertility of N. tenuis females was compromised by all the insecticides. In addition, sublethal effects on predator orientation were observed. We concluded that the three insecticides were noxious to N. tenuis and should be avoided when the presence of the predator is desirable. Nevertheless, field trials must be carried out to confirm their sublethal toxicity and overall risk (interaction of exposure, hazard and other factors).

Additionally, the negative effects on N. tenuis orientation observed in the current study provide a basis for further research aiming to elucidate how neurotoxic insecticides impair N. tenuis capacity to locate host plants or herbivorous prey, by investigating alterations in the gene expression of odorant‐binding and chemosensory proteins that might be involved in plant volatile reception by employing electro‐antennography and quantitative real‐time polymerase chain reaction bioassays. ⁸⁶ , ⁸⁷ , ⁸⁸ Moreover, the results highlight the importance of investigating other insecticides that might have a narrow spectrum and that would be more compatible with sustainable IPM.

CONFLICTS OF INTEREST

The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.