Lethal, sublethal and transgenerational effects of insecticides labeled for cotton on immature Trichogramma pretiosum

The parasitoid Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is often released to manage lepidopteran pests in cotton crops. However, growers rely on multiple insecticide applications to manage cotton pests. A harmonious integration of control tactics is required for proper pest management, and the use of selective insecticides (i.e., those promoting effective pest control while causing little impact on natural enemies) fits within this scope. This study aimed to assess the lethal, sublethal and transgenerational effects of insecticides from varying chemical groups on T. pretiosum. The insecticides were sprayed on parasitized host [Ephestia kuehniella (Zeller), Lepidoptera: Pyralidae] eggs with developing T. pretiosum stages (egg-larva, prepupa, and pupa), and biological traits were assessed following adult emergence. Overall, pupae were more susceptible to insecticides. We found thiodicarb and chlorfenapyr to reduce F0 adult emergence in rates comparable to the positive control (methomyl). Adult F0 deformation was the highest on flupyradifurone-treated organisms, and both the F0 parasitism rate and female survival were reduced by the insecticides (except for teflubenzuron). The sex ratio (proportion female) was affected by thiodicarb and flupyradifurone. Transgenerational effects occurred on adult emergence, which was reduced on the offspring (F1) of thiodicarb, chlorfenapyr, and flupyradifurone treated T. pretiosum. In addition, thiodicarb decreased the F1 sex ratio (≤ 0.33) compared to the other compounds (sex ratio ≥ 0.45). These results indicate that teflubenzuron is the safest insecticide; the other insecticides are non-selective to T. pretiosum. Field and semifield studies are required to confirm the harmfulness of chlorfenapyr, flupyradifurone and thiodicarb toward T. pretiosum.


Introduction
Brazil is one of the top five cotton-producing countries . Cotton cultivated area surpassed 1.6 million hectares in 2019, resulting in 6.9 million tons of raw cotton (FAO 2021). Cotton wool is a commodity with multiple uses, and cotton cultivation provides substantial income and employment in Brazilian producing areas. The crop harbors dozens of yield-limiting arthropod species (Machado et al. 2019). These organisms are mainly managed by application of broad-spectrum insecticides in a calendar-based frequency, which can disrupt biological control exerted by beneficial arthropods (Moscardini et al. 2008).
The parasitoid wasp Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is often released in arable (e.g., cotton, corn, soybean, and vegetables) and wood crops for the management of lepidopteran pests (Smith 1996;Parra and Zucchi 2004;Laurentis et al. 2019). Under natural conditions, endemic populations of T. pretiosum parasitize up to 98% of eggs of the cotton leafworm Alabama argillacea (Hübner) (Lepidoptera: Noctuidae), and parasitism rates increase over the crop cycle (Fernandes et al. 1999). Inundative releases of this parasitoid have been reported to yield 76% egg parasitism of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) in cotton (Saavedra et al. 1997), but the establishment of augmented parasitoid populations is halted by the spraying of non-selective insecticides (Barros et al. 2018;Rakes et al. 2021;Milonas et al. 2021 Trichogramma pretiosum controls pests in the egg stage, which poses a major advantage since it can potentially prevents pests from causing economic damage (Figueiredo et al. 2015). However, due to its diurnal behavior, adult T. pretiosum may be exposed to direct insecticide spraying and fresh insecticide residue on foraging substrates (Pompanon et al. 1999;Reznik et al. 2009;Khan et al. 2015). In addition, immature parasitoid may come in contact with insecticide absorbed through the host egg chorion (Costa et al. 2014). Therefore, the use of selective insecticides is crucial for preserving T. pretiosum populations and achieving effective rates of parasitism in the field (Parra and Zucchi 2004;Costa et al. 2014). Conceptually, selective insecticides are those that effectively control pests while causing minimal toxicity on natural enemies and other beneficial organisms (Castle and Naranjo 2009;Bueno et al. 2017;Torres and Bueno 2018;Soares et al. 2019Soares et al. , 2020Carvalho et al. 2019).
Thus, studies of the insecticides' impacts on beneficial insects are central for integration of both biological and chemical control methods in pest management. The assessment of lethal and sublethal effects of insecticides is required to thoroughly examine their risk on non-target organisms (Desneux et al. 2007). Multiple studies have assessed the selectivity of insecticides to T. pretiosum (Moscardini et al. 2008;Souza et al. 2013;Khan et al. 2015;Paiva et al. 2018Paiva et al. , 2020Barros et al. 2018). However, most of these studies were designed with adults (Rakes et al. 2021), whereas the impact of insecticides on immature parasitoid stages (particularly egg and larva) has received less attention. Therefore, this study aimed to evaluate the lethal and transgenerational effects of insecticides used in cotton on immature T. pretiosum. The unveiling of insecticide toxicity to T. pretiosum will assist in selecting insecticides more compatible with releases of this parasitoid, allowing a more harmonious integration of chemical and biological control in cotton cropping systems.

Insects
A colony of T. pretiosum was established in the Laboratory of Ecotoxicology at the Department of Entomology (Universidade Federal de Lavras, Brazil). The colony was kept at 25 ± 2 °C, 70 ± 10% RH and a 12:12 h L:D photoperiod and reared on UV-sterilized eggs of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) (Insecta Produtos Biológicos, Lavras, Minas Gerais, Brazil). Host eggs (≤ 24-h-old), glued with arabic gum (50%) onto blue paper cards (8 × 1 cm), were exposed to 24-h-old T. pretiosum kept in 1-L plastic containers sealed with plastic film. Then, the cards were transferred to new plastic containers and kept until adult parasitoid emergence. Newly emerged adult T. pretiosum were offered new host eggs and were fed honey droplets carefully placed on the container wall.

Bioassays
Twenty-five T. pretiosum females were individualized in glass vials (8.5 × 2.5 cm) sealed with plastic films and offered, for 24 h, UV-sterilized E. kuehniella eggs (n = 125) glued with arabic gum (50%) onto blue paper cards (5 × 0.5 cm). Then, females were removed and the vials were kept at rearing conditions for three pre-determined periods (1, 4 and 8 days) corresponding to the egg-larval, prepupal and pupal parasitoid duration, respectively (Cônsoli et al. 2001;Souza et al. 2014;Paiva et al. 2020). Thus, cards with host eggs containing T. pretiosum at the varying life stages were sprayed in groups of five under a Potter tower (Burkard, Uxbridge, UK) calibrated at 15 lb/pol 2 pressure to deposit 1.5 ± 0.5 µL/cm 2 , as recommended by the IOBC (Sterk et al. 1999). The sprayed cards were air-dried at room temperature and subsequently individualized in glass vials kept at rearing conditions until wasp emergence. Evaluations were performed with the aid of a stereoscopic microscope (40 × magnification) to determine egg parasitism (indicated by the blackening of the vitelline membrane of the host egg) and parasitoid emergence (presence of a wasp exit hole in the host egg) and, following adult emergence, the wasp sex (male or female) and deformation status (occurrence of antennaless and/or wingless adults). The bioassay was carried out in a completely randomized design (CRD) and a factorial layout (3 parasitoid stages × 6 insecticidal treatments), yielding 18 treatments with 5 replicates (a vial with 5 cards with parasitized host eggs). The assessed endpoints included the F0 emergence rate (emerged adults × 100 ÷ parasitized host eggs), F0 deformation rate (deformed wasp adults × 100 ÷ total wasp adults) and F0 sex ratio [proportion female = Σ♀ ÷ Σ(♀ + ♂)].
Transgenerational effects of the insecticides were also tested on the surviving F0 adults. Twenty adult females surviving from the insecticidal treatments were randomly collected and individualized in glass vials, as described previously. Each female was offered 125 UV-sterilized E. kuehniella eggs, free from insecticidal treatment, glued onto paper cards (5 × 0.5 cm) for 24 h. F0 adult female T. pretiosum were held in the vials, honey-fed, and assessed daily for survival until death, whereas the cards were transferred to new vials for further assessments. Evaluations were also done to determine the F0 parasitism (parasitized host eggs × 100 ÷ total host eggs) and, following the F1 cycle completion, the F1 emergence rate, F1 adult deformation rate and F1 sex ratio. The bioassay was held in a CRD and a factorial layout across a range of parasitoid stages (egglarva, prepupa and pupa) by insecticidal treatment (untreated control and five a.i.) combinations. Due to the low survival of F0 adults emerging from methomyl-treated T. pretiosum, transgenerational studies were not performed for this insecticide.

Data analysis
All analyses were performed using R software (V. 3.5.3) and RStudio (V. 1.2.5001) (R Core Team 2019). Data on adult emergence and deformation (F0 and F1), parasitism (F0) and sex ratio (F0 and F1) were checked for normality (Shapiro-Wilk test) and homoscedasticity of residuals (Bartlett test). A two-way ANOVA (ExpDes package; Ferreira et al. 2018) was performed to verify the effect of parasitoid stage, insecticidal treatment and their interaction on the assessed biological traits. In case of significance of the main factors, the Scott-Knott cluster analysis was used to separate their means. Significant interactions were probed with additional ANOVA and Scott-Knott cluster analyses.
Data on F0 adult female survival were analyzed using Kaplan-Meier estimators to obtain survival curves and estimates of median lethal times (LT 50 ). The overall similarity of the survival curves was tested through the Log-Rank test, and pairwise comparisons among the curves were performed with the Holm-Sidak's test. These analyses were implemented with functions from survival and survminer packages (Therneau 2020;Kassambara et al. 2021).
Afterward, a hierarchical clustering analysis was performed to assist in interpreting of the bioassays' outcomes by applying functions (dist and hclust) from the stats package. Dissimilarity (Euclidean distance) between all insecticidal treatments was quantified using mean values of the assessed endpoints (emergence, deformation and sex ratio -for F0 and F1, and LT 50 and parasitism -for F0). The attributes (pooled mean values for each combination of insecticidal treatment with parasitoid stage) were standardized (mean = 0 and variance = 1) before the analysis (Wickham 2018). The dendrogram was constructed using the Ward's minimum variance method ("ward.D"), and the cut-off point was determined as per Mojena (Mojena 1977).
According to the IOBC classification, teflubenzuron was harmless (class 1) to T. pretiosum for all variables assessed (F0 emergence, F0 parasitism and F1 emergence), irrespective of the exposed parasitoid stage (Fig. 2). Thiodicarb was slightly harmful in most situations, whereas flupyradifurone and chlorfenapyr had mixed classes, ranging from harmless (class 1) to moderately harmful (class 3).
Based on the Ward.D' cluster dendrogram, the insecticidal treatments were categorized into two groups (safe and non-safe to T. pretiosum). Teflubenzuron was the only compound grouped with the untreated control (i.e., it was the safest insecticide to T. pretiosum) (Fig. 4).

Discussion
Insecticides from varying chemical groups and modes of action were tested for lethal, sublethal and transgenerational effects on T. pretiosum. Overall, the compounds affected at least one of the assessed biological traits, and the degree of parasitoid response was mediated by the treated developmental stage.
Insecticides exhibited varying degrees of toxicity to T. pretiosum, likely resulting from the physicochemical properties (e.g., lipophilicity and molecular weight) of the tested products (Bacci et al. 2007). The reduced emergence of F0 individuals treated with chlorfenapyr can be attributed to the lipophilicity of this compound. Log K OW (a measure of compound miscibility in aqueous and organic phases) values range from -3 (very hydrophilic compounds) to + 10 (extremely hydrophobic compounds) (Cumming and Rücker 2017). Thus, chlorfenapyr (log Kow = 5.28) is highly lipophilic, making this compound easily absorbed through the host chorion and translocated up to its target site (Hoffmann et al. 2008). Although they did not disrupt T. pretiosum immature development, which was evidenced by the accumulation of urate granules and blackening of the host egg, methomyl and thiodicarb reduced adult emergence; in fact, we observed unemerged adults inside the host eggs. Such an effect results from the adult intoxication when opening the exit hole due to contact and/or ingestion of insecticide residues retained on the host egg surface (Cônsoli et al. 2001).
We found that all compounds, except for teflubenzuron, reduced T. pretiosum F0 parasitism rates. Teflubenzuron was the only insecticide that did not diminish F0 female survival, and it is reasonable to assume that, as the teflubenzurontreated females lasted longer, they could parasitize more eggs. Besides, the other compound might have impaired reproductive traits (e.g., sex discrimination, mate choice, and locomotor activity), leading to reduced parasitism by T. pretiosum (Dupont et al. 2010;Delpuech et al. 2012;Wang et al. 2018). The deleterious effects of thiodicarb and chlorfenapyr on T. pretiosum parasitism success have been previously reported, and they were both classified as slightly harmful (Moura et al. 2005;Bueno et al. 2008).
In flupyradifurone-treated immatures, F0 adult deformation might also have contributed to the lower parasitism rate. Adults had reduced mobility and did not fully distend their wings; some individuals even lacked these appendages. Another study reports higher deformation of adult T. pretiosum from neonicotinoid-treated host eggs (Moura et al. 2005), which corroborates our findings.
Some insecticides caused transgenerational effects on T. pretiosum emergence; thiodicarb, chlorfenapyr and flupyradifurone promoted lower F1 emergence, as opposed Fig. 1 Survival curves of Trichogramma pretiosum adult females emerging from immature stages [a) egg-larva, b) prepupa, and c) pupa] exposed to insecticidal treatments (control, teflubenzuron, chlorfenapyr, flupyradifurone, and thiodicarb). Curves with different lowercase letters differ significantly among themselves (P < 0.05, Holm-Sidak test) to the IGR teflubenzuron. In previous studies, triflumuron (another IGR) was found to not interfere with this biological trait of T. pretiosum (Carvalho et al. 2003;Vianna et al. 2008;Souza et al. 2013). The non-selectivity of flupyradifurone contrasts with a previous report of the harmlessness of neonicotinoids (acetamiprid and thiamethoxam) to the F1 emergence of T. pretiosum (Moura et al. 2005). Methomyl, thiodicarb and flupyradifurone reduced the F0 sex ratio, whereas only methomyl reduced the sex ratio of the offspring (F1). Like many egg parasitoids, T. pretiosum Fig. 2 Heatmap diagram of the classification (according to the International Organization for Biological Control-IOBC) of insecticides (teflubenzuron, chlorfenapyr, flupyradifurone, thiodicarb, and methomyl) based on their effects on Trichogramma pretiosum biological traits (F0 emergence, F0 parasitism and F1 emergence) when the parasitoid was treated at varying life stages (egg-larva, prepupa and pupa). Numbers 1 to 4 represent the IOBC categories (1 = harmless, 2 = slightly harmful, 3 = moderately harmful and 4 = harmful) Fig. 3 Sex ratio of the a F0 and b F1 generation of adult Trichogramma pretiosum emerging from immature stages [egg-larva, prepupa, and pupa] exposed to insecticidal treatments (control, teflubenzuron, chlorfenapyr, flupyradifurone, and thiodicarb). Within each T. pretiosum stage, bars (mean ± SEM) with different letters differ by the Scott-Knott test (P < 0.05) reproduces by arrhenotokous parthenogenesis. Thus, males originate from unfertilized eggs and females from fecundated ones. Insecticides impair sex ratios (i.e., the proportion of females) by causing selective male mortality (Matioli et al. 2019), male sterility (Umoru and Powell 2002), altered mating behavior (Wang et al. 2018;Kremer and King 2019), and reduced viability of stored sperm in females (Rosenheim and Hoy 1988). These effects, isolated or combined, might have reduced the number of fertilized eggs, ultimately leading to a male-biased sex ratio. Both teflubenzuron and chlorfenapyr did not affect the sex ratio of the F0 and F1 generations; these results are in line with earlier reports of innocuity of IGR (triflumuron) and chlorfenapyr to the sex ratio of T. pretiosum (Moura et al. 2005;Souza et al. 2013).
Trichogramma pretiosum is often released in cotton crops for the management of lepidopteran pests. Inundative releases of Trichogramma spp. contribute to increased crop yield and reduction of synthetic insecticide input (Figueiredo et al. 2015;Huang et al. 2020). Besides the direct effect on lepidopteran pests, recent works have shown that Trichogramma spp. are able to vector Beauveria bassiana (Balsamo) Vuillemin, an entomopathogenic fungus of multiple pests , which may provide a prospect for further use of T. pretiosum in cotton. Hence, chemical insecticides should be carefully combined with T. pretiosum releases, and the most selective compounds should be used rather than the toxic ones (Zang et al. 2021).
In this study, all tested insecticides, except for teflubenzuron, presented deleterious effects on T. pretiosum. Therefore, teflubenzuron should be prioritized in IPM programs of lepidopteran pests in cotton. Since environmental conditions (e.g., temperature, radiation and rainfall) mediate insecticide degradation and toxicity (Maia et al. 2016;Ricupero et al. 2020), further studies in semifield and field conditions are required to confirm the side effects of chlorfenapyr, flupyradifurone and thiodicarb on T. pretiosum.

Author contribution
GAC, MAC and EDA conceived and designed research. MAC and EDA conducted experiments. MAC and ESF analyzed data. MAC, GAC, ESF and VCC wrote the manuscript. All authors read and approved the manuscript. Fig. 4 Cluster dendrogram (built with Ward's method) of six insecticidal treatments (untreated control, teflubenzuron, flupyradifurone, thiodicarb, and chlorfenapyr) based on the dissimilarity (Euclidean distance) of their effects on biological traits of Trichogramma pretiosum. Two groups were formed, as indicated by the cut-off line