Selectivity of Insecticides to a Pupal Parasitoid, Trichospilus diatraeae (Hymenoptera: Eulophidae), of Soybean Caterpillars

Simple Summary Lepidoptera caterpillars are important pests around the world that decrease crop production, including that of soybeans. The use of insecticides and bioinsecticides is the main control strategy for these pests. Biological control with parasitoids, in combination with insecticides, is widely used as an alternative. However, the use of insecticides with parasitoids depends on tests to assess the survival/mortality of these natural enemies for each insecticide. Trichospilus diatraeae Cherian & Margabandhu, 1942 (Hymenoptera: Eulophidae) is a polyphagous pupal parasitoid that mainly comprises defoliating lepidopterans. This study evaluated the mortality of T. diatraeae from seven insecticides (acephate, azadirachtin, Bacillus thuringiensis (Bt), deltamethrin, lufenuron, teflubenzuron and thiamethoxam + lambda-cyhalothrin). The insecticides were sprayed on soybean leaves, which were left to dry and placed in cages with 10 T. diatraeae females. The insecticides azadirachtin, Bt, lufenuron and teflubenzuron did not affect T. diatraeae survival; deltamethrin and thiamethoxam + lambda-cyhalothrin presented low toxicity; and acephate was highly toxic, causing 100% mortality in this parasitoid. Azadirachtin, Bt, lufenuron and teflubenzuron are selective for T. diatraeae. Abstract Selectivity is an important aspect of modern insecticides to be able to target pests whilst maintaining beneficial entomofauna in the crop. The present objective was to assess the selectivity of different insecticides for the pupal parasitoid of soybean caterpillars, i.e., Trichospilus diatraeae Cherian & Margabandhu, 1942 (Hymenoptera: Eulophidae). Acephate, azadirachtin, Bacillus thuringiensis (Bt), deltamethrin, lufenuron, teflubenzuron and thiamethoxam + lambda-cyhalothrin at the highest recommended concentrations for the soybean looper Chrysodeixis includens (Walker, [1858]) (Lepidoptera: Noctuidae), as well as water in the control, were used against the pupal parasitoid T. diatraeae. The insecticides and the control were sprayed on the soybean leaves, which were left to dry naturally and placed in cages with T. diatraeae females in each one. Survival data were submitted to analysis of variance (ANOVA) and the means were compared using Tukey’s HSD test (α = 0.05). Survival curves were plotted according to the Kaplan–Meier method, and the pairs of curves were compared using the log-rank test at 5% probability. The insecticides azadirachtin, Bt, lufenuron and teflubenzuron did not affect T. diatraeae survival, while deltamethrin and thiamethoxam + lambda-cyhalothrin presented low toxicity and acephate was highly toxic, causing 100% mortality in the parasitoid. Azadirachtin, Bt, lufenuron and teflubenzuron are selective for T. diatraeae and could be used in IPM programs.


Introduction
Soybean monoculture (Glycine max (L) Merrill) favors the reproduction and development of insect pests, particularly lepidopteran caterpillars that feed on leaves, flowers and fruits, and that reduce crop productivity [1]. The use of insecticides is the main method to manage defoliating insects in soybeans, with lower initial costs, easy application and high efficiency [2]. However, integrated pest management (IPM) recommends the combined use of different control methods [3], including chemicals [4]. Selective products that cause high pest mortality and low impact on beneficial insects are preferable [5].
The insecticide acephate with the mechanism of inhibiting the action of acetylcholinesterase enzymes increases the nervous impulses, causing hyperexcitation and insect death. Deltamethrin is an insecticide from the group of sodium channel modulators. Its action mechanism causes the uncontrolled release of intracellular calcium, hyperexcitation, paralysis and insect death. Lufenuron and teflubenzuron are insecticides from the group of chitin biosynthesis inhibitors, preventing the growth and development of insects with a mechanism of action that mainly causes failure or accelerating ecdysis. Thiamethoxam + lambda-cyhalothrin is a mixture with the first as a neonicotinoid insecticide from the group of competitive modulators of nicotinic acetylcholine receptors and an acetylcholine-imitating mechanism, which stimulates the nerve cells and causes nervous hyperexcitation and insect death. The second, a pyrethroid from the group of sodium channel modulators, has an action mechanism that blocks sodium channels [6,7]. Azadirachtin, an insecticide obtained from the Azadirachta indica A. Juss plant, from the group of compounds with an unknown or uncertain mode of action is used as an alternative to synthetic insecticides. Bacillus thuringiensis, a biological insecticide from the group of microbial disruptors of the midgut membrane, is formed by bacteria that release crystals that dissolve into toxic proteins in the intestine following ingestion, destroying the midgut membrane and causing generalized infection (septicemia) and insect death. Bioinsecticides, such as azadirachtin and Bacillus thuringiensis (Bt), that present lower ecological harm and toxicity to non-target organisms, as well as faster breakdown, can complement the use of synthetic insecticides [8,9].
The high parasitism rate and low specificity yield mass rearing of T. diatraeae in pupae of different hosts [15]. Furthermore, this parasitoid may naturally be present in the field as an important agent in applied biological control programs [12].
The effects of insecticides on T. diatraeae are poorly understood, highlighting the need for further research for their use in conservative biological actions and/or inundative control to increase the success of IPM [16].
The objective of the study was to evaluate commercial insecticides in terms of their impact on the pupal parasitoid Trichospilus diatraeae Cherian & Margabandhu, 1942 (Hymenoptera: Eulophidae), a natural enemy of soybean caterpillars.

Trichospilus diatraeae
Trichospilus diatraeae were obtained from a colony maintained in the "Laboratório de Controle Biológico de Insetos (LECOBIOL)" at the Universidade Federal da Grande Dourados (UFGD), Dourados, Brasil (22 • 11 56.7 S 54 • 56' 00.5 W). Adult insects were kept in glass tubes (length 8.5 cm; diameter: 2.5 cm), covered with cotton, and fed with a droplet of pure honey. One D. saccharalis pupa (natural host of the parasitoid) was exposed for 24 h for parasitism by five T. diatraeae females (48-72 h age) at 25 ± 2 • C, a relative humidity (RH) of 70 ± 10%, and for 14 h photophase (light hours) in a climate-controlled chamber. The emerged parasitoids were fed with honey droplets and allowed to mate for 48-72 h. After mating, females were isolated and used in the experiments [15].

Assessment of Pesticides Selectivity under Extended Laboratory Conditions (ASPECLE)
The ASPECLE System was adapted by Sanomia 2020 according to the International Organization for Biological Control (IOBC) and is a standard test used in experiments of insecticide selectivity to parasitoids [17].
Glass cylinders (Borosilicate, Laborglas™, São Paulo, São Paulo state, Brazil) that were 3.5 cm in diameter and 25 cm long were sealed with voile fabric and a perforated plastic cap was placed at both ends. A tube was inserted at one end for the ventilation system. The ventilation system consisted of a central tube connected to a compressor/vacuum cleaner (maximum vacuum 695 mmhg) (Dia-Pump Fanem™ vacuum pump, São Paulo, SP, Brazil) with 24 tubes (12 on the left and 12 on the right) where the glass cylinders were coupled. The ventilation system and the pump facilitated gas exchange, reducing the accumulation of toxic gases in the cylinders ( Figure 1) [18].
droplet of pure honey. One D. saccharalis pupa (natural host of the parasitoid for 24 h for parasitism by five T. diatraeae females (48-72 h age) at 25 ± 2 humidity (RH) of 70 ± 10%, and for 14 h photophase (light hours) in a clim chamber. The emerged parasitoids were fed with honey droplets and allow 48-72 h. After mating, females were isolated and used in the experiments [

Assessment of Pesticides Selectivity under Extended Laboratory Conditions (A
The ASPECLE System was adapted by Sanomia 2020 according to th Organization for Biological Control (IOBC) and is a standard test used in e insecticide selectivity to parasitoids [17].
Glass cylinders (Borosilicate, Laborglas™, São Paulo, São Paulo sta were 3.5 cm in diameter and 25 cm long were sealed with voile fabric an plastic cap was placed at both ends. A tube was inserted at one end for system. The ventilation system consisted of a central tube con compressor/vacuum cleaner (maximum vacuum 695 mmhg) (Dia-Pu vacuum pump, São Paulo, SP, Brazil) with 24 tubes (12 on the left and 1 where the glass cylinders were coupled. The ventilation system and the pu gas exchange, reducing the accumulation of toxic gases in the cylinders (Fi

Trichospilus diatraeae Exposure to Insecticides
Five chemical insecticides and two bioinsecticides, recommended for the management of defoliating caterpillars in soybean crop, were used at the maximum concentrations recommended by the manufacturer (Table 1). Table 1. Active ingredients (AI), trade names (TN), chemical group, commercial product (CP), active ingredient concentration (AI) and toxicological classification (TC) of the insecticides acephate, azadirachtin, Bacillus thuringiensis (Bt), deltamethrin, lufenuron, teflubenzuron (Teflub.) and thiamethoxam + lambda-cyhalothrin (T. + Lc), recommended for the management of defoliating caterpillars in soybean [6]. The insecticides/bioinsecticides were diluted in water at the maximum concentration indicated by the manufacturer and were sprayed onto soybean plants at the V4 phenological stage (three fully developed trefoils or four nodes), following the methodology of the IOBC [19], with a hand pressure sprayer (Brudden Practical 2000 ® , 1.5 L). Soybean seeds were sown in 7 L pots with 2/3 soil (distroferric red Latosol) and 1/3 organic matter (chicken manure) and were kept in a greenhouse until phenological stage V4.

AI
The products were applied on the plants until leaf runoff. The plants were then left to dry in the shade in an open environment. Soybean plants were collected and separated per treatment and taken to the laboratory with five leaves per plant per cage. Ten 48 h old T. diatraeae females were placed inside each cage with a piece of cotton moistened with water.
T. diatraeae forages in the field in search of hosts, flying over plants and walking on leaves [15]. At this moment, T. diatraeae makes contact with leaves sprayed with insecticides.

Trichospilus diatraeae Mortality
Groups of 10 T. diatraeae females that were 48 h old, mated and fed were placed in 12 cylinders, totaling 120 females per treatment. The number of dead individuals was counted 24 h after the experiment was completed.
The mortality data of the parasitoid T. diatraeae were submitted for analysis of variance (ANOVA) and the means were compared using Tukey's HSD test (α = 0.05) with the statistical program SASM-AGRI [21].

Trichospilus diatraeae Survival
A second group of 10 T. diatraeae females that were 48 h old, mated and fed were placed in 12 cages, totaling 120 females per treatment. This experiment lasted 120 h and the numbers of live individuals were counted daily. Parasitoids were considered dead if they remained immobile when touched with a brush.
Survival data were submitted to the SAS Proc LIFETEST [22] to estimate their means, which were compared in pairs using the log-rank test (α = 0.05). Data were used in the T. diatraeae survival curves with the Kaplan-Meier method using the Sigma Plot 10.0 program.

Discussion
Zero mortality of T. diatraeae female after 24 h classifies azadirachtin as non-toxic to this parasitoid [20]. This insecticide is a tetranortriterpenoid and, with its derivatives, is used to manage agricultural pests through repellence, antifeeding or interfering with the development of female sexual gonads, reducing or preventing oviposition [9,23]. In addition, the observed zero mortality of T. diatraeae may be related to contamination because azadirachtin mainly acts through ingestion [16] and the parasitoid only had contact with residues of this insecticide on soybean leaves. The similar zero mortality of T. diatraeae females when exposed to Bacillus thuringiensis kurstaki also classifies this insecticide as non-toxic [20]. This bacterium is specific to Lepidopteran caterpillars and, after ingestion, it releases its toxins, which bind to receptors in the gut wall, breaking it and causing septicemia [24]. Trichospilus diatraeae survival is due to the high specificity of

Discussion
Zero mortality of T. diatraeae female after 24 h classifies azadirachtin as non-toxic to this parasitoid [20]. This insecticide is a tetranortriterpenoid and, with its derivatives, is used to manage agricultural pests through repellence, antifeeding or interfering with the development of female sexual gonads, reducing or preventing oviposition [9,23]. In addition, the observed zero mortality of T. diatraeae may be related to contamination because azadirachtin mainly acts through ingestion [16] and the parasitoid only had contact with residues of this insecticide on soybean leaves. The similar zero mortality of T. diatraeae females when exposed to Bacillus thuringiensis kurstaki also classifies this insecticide as non-toxic [20]. This bacterium is specific to Lepidopteran caterpillars and, after ingestion, it releases its toxins, which bind to receptors in the gut wall, breaking it and causing septicemia [24]. Trichospilus diatraeae survival is due to the high specificity of B. thuringiensis var. kurstaki to Lepidopterans and because it is unlikely that this parasitoid will ingest dry residues of this bacteria sprayed on soybean leaves [8]. The zero mortality of T. diatraeae females after 24 h classifies the insecticides lufenuron and teflubenzuron, from the benzoylurea group, as non-toxic to this parasitoid [20]. These insecticides do not affect adult insects as they are growth regulators inhibiting chitin synthesis [25]. This is similar to that reported for the insecticides lufenuron and novaluron from the same IRACappointed insecticide action group, specific to insect larvae and nymphs with the parasitoid Diachasmimorpha longicaudata (Ashmead, 1905) (Hymenoptera: Braconidae) [16,26]. The 100% mortality of T. diatraeae females after 24 h exposure to acephate classifies this insecticide as toxic [20]. This insecticide, like other organophosphates, inhibits acetylcholinesterase (AChE) in the nervous system, causing paralysis and insect death [27], similar to the insecti-cide malathion for the parasitoid Palmistichus elaeisis Delvare & La Salle, 1993 (Hymenoptera: Eulophidae) [28]. Trichospilus diatraeae female mortality within 24 h from deltamethrin and thiamethoxam + lambda-cyhalothrin classifies these insecticides as mildly toxic to this natural enemy [20]. These insecticides, from the pyrethroid and neonicotinoid groups, act by keeping sodium channels open, causing hyperexcitation and blocking the insect's nervous system and or binding to acetylcholine (ACh), transforming it into nAChRs and causing diverse symptoms, including hyperexcitation, lethargy and paralysis [26]. Pyrethroids and neonicotinoids act on the nervous system and respiration at all stages of insect development [27,29].
After 120 h, with exposure to Bt, azadirachtin, lufenuron and teflubenzuron, may be due to the action mode of these insecticides and the behavior of this parasitoid [30]. Bt is more specific to a group of insects (lepidopteran caterpillars), and lufenuron and teflubenzuron only act during the early stages of insect development [9,24,25]. After 120 h, T. diatraeae female mortality in these treatments may be related to the natural mortality of this parasitoid, with an average longevity of 8.28 ± 1.01 days [15]. The death of all parasitoids with the insecticide acephate within 24 h confirms the high toxicity of this insecticide [16]. In addition, the lower survival with insecticides deltamethrin and thiamethoxam + lambda-cyhalothrin is due to the persistence of their dry residues on the leaves and the direct contact of the parasitoid, increasing its mortality over time [26,31].
The greater T. diatraeae female survival after 24 h of exposure confirms the safety of these insecticides for this parasitoid [32]. Botanical insecticides such as azadirachtin are considered safe for natural enemies, not causing significant mortality of the parasitoids Trichogramma nubilale Ertle & Davis, 1975 and Trichospilus pupivorus Ferrière, 1930 (Hymenoptera: Eulophidae) [33,34]. Even when Bt was ingested by chewing the chorion of the egg on which the insecticide was sprayed during parasitoid emergence, this did not cause mortality or reduce the survival of the parasitoid Trichogramma pretiosum Riley, 1879 (Hymenoptera: Trichogrammatidae) [8]. Lufenuron and teflubenzuron, from the benzoylurea group, are growth regulators in insects and do not affect the survival of adult natural enemies [32]. However, acephate is an organophosphate from a group of insecticides, generally with a broad spectrum and low selectivity, causing high mortality of parasitoids [35]. The lethal effects of the residues of deltamethrin [36] cause reduced survival of the parasitoid T. diatraeae after 120 h. This is similar to that reported for Trichopria anastrephae Lima, 1940 (Hymenoptera: Diapriidae), with a mean survival of 10% after 120 h of exposure to this insecticide, and Pachycrepoideus vindemmiae (Rondani, 1875) (Hymenoptera: Pteromalidae) with 100% mortality [27]. However, deltamethrin did not reduce P. elaeisis survival [36]. The reduced T. diatraeae survival rate with thiamethoxam + lambda-cyhalothrin is due to its toxicity as reported for the mortality of all Telenomus podisi Ashmead, 1893 (Hymenoptera: Platygastridae) individuals due to direct contact with insecticide residues [37].

Conclusions
Insecticides Bacillus thuringiensis, azadirachtin, lufenu ron and teflubenzuron were considered harmless, deltamethrin and thiamethoxam + lambda-cyhalothrin were considered low toxicity and acephate was considered toxic to females of parasitoid T. diatraeae. The latter should not be used with this parasitoid in pest management programs. Azadirachtin, Bt, lufenuron and teflubenzuron are selective to conserve T. diatraeae populations.
Because no sublethal impacts were observed, further testing with these chemicals is recommended to evaluate their effect on the longevity, fertility, mating and foraging behavior of this parasitoid prior to inclusion in IPM programs.  Data Availability Statement: All data sets presented in this study are included in the article and can be made available by the authors upon reasonable request.