# Introduction

Many plasticizers are considered endocrine disruptors, as they can disrupt the endocrine system of organisms and the biological functions controlled by hormones. Many plasticizers used in plastics are described as increasingly contaminating the environment. All environmental matrices (water, soil, air) seem to be affected. A new issue is emerging, more specifically for agro-ecosystems, where the literature is increasingly showing this type of contamination. Until now, the focus has been on aquatic environments and urban areas, but it is clear that these ecosystems are not spared. The sources are multiple: contaminated irrigation water, plastics from agricultural equipment, transfer of contaminants between environmental matrices. Among these substances, two in particular are being studied, as they are widely used in many everyday products and are increasingly found in agroecosystems: bis(2-ethylhexyl) (DEHP) and bisphenol A (BPA). They are found everywhere in the environment, particularly in sediments, soils and plants. They are recognized as endocrine disruptors in vertebrates. While invertebrates represent the major part of animal biodiversity and play critical roles in aquatic and terrestrial ecosystems, few studies have focused on the effects of these plasticizers on these species and particularly in terrestrial insects, despite the fact that the two lipid hormone families, ecdysteroids and juvenile hormones, are involved in the control of post-embryonic development and adult physiology in insects. In this context, we used Spodoptera littoralis, a polyphagous pest as a model to study the effects of larval exposure to DEHP and BPA on post-embryonic development, male sexual behavior and offspring development of treated males or females. We have shown that, as in vertebrates, these two compounds act as PEs in this species by affecting the ecdysteroid pathway, and modify post-embryonic development, male sexual behavior and the duration of life stages in the offspring.

# Material and methods

Larvae were reared under the laboratory conditions described by Aviles et al. [1] and Maria et al. [2] and exposed to DEHP or BPA via a semi-artificial diet from the third larval instar until pupation. Six environmentally relevant concentrations (100pg; 1ng; 10ng; 100ng; 1μg and 10μg per gram of feed) and three high concentrations (500μg; 5mg and 40mg per gram of feed) of DEHP were tested. For BPA, we tested environmental doses (e.g. 0.01, 0.1 and 1mg/g BPA) and high doses (e.g. 25mg/g). For the post-embryonic development study, the number and duration of larval and pupal stages, larval weight, food consumption and sex ratio were monitored. Emphasis was also placed on the last three days before pupation, i.e. days 5, 6 and 7 of the last larval stage (L7). Hemolymph samples were used to: 1) measure ecdysteroid titrations using enzyme-linked immunosorbent assays (EIA); and 2) perform metabolomic analyses using gas chromatography-mass spectrometry (GC-MS). RTqPCR analyses were also carried out on whole-body larvae to study the expression of ecdysteroid response genes. To verify actual DEHP exposure, DEHP concentrations were measured in feed using liquid chromatography-mass spectrometry (LC-MS). For the adult male experiments, males were collected three days after emergence during scotophase. Behavioral configurations were used to study male sexual behavior as well as to monitor mating and intergenerational effects. In addition, male antennae and hemolymph were collected to measure ecdysteroid response gene expression by RTqPCR and to measure ecdysteroid titration using EIA, respectively.

# Results and discussion

## Effects of DEHP and BPA on post-embryonic development

Only the two highest DEHP concentrations tested induced an increase in mortality, with 100% and 36% mortality respectively for treatments at 39.5 and 4.3mg/g DEHP. Exposure to the various DEHP doses used induced a general increase in the duration of larval and pupal stages. An increase in food consumption with no effect on larval growth curves was observed for low and intermediate DEHP concentrations (Fig. 1
                        ), suggesting an effect of DEHP on food absorption by the intestinal tract, or on energy metabolism, or an energetic cost of DEHP detoxification for the larvae. BPA had an impact on larval stage duration and larval growth (Fig. 1). Ecdysteroid concentrations and the expression of ecdysteroid response genes were disrupted in DEHP and BPA larvae, but in different ways, indicating a difference in the molecular effects of these two plasticizers.

## Effects of DEHP and BPA on male sexual behavior, mating and intergenerational effects

In adult males, DEHP was quantified even though the contamination was due to larval ingestion. We thus studied and observed a decrease in the expression of the ecdysteroid receptor (EcR) and its partner ultraspiracle (USP) in the antennae, except for treatments with 676ng and 1.1μg per gram of DEHP food (Fig. 2
                        ). In behavioral experiments, male sexual behavior was observed to be modified by DEHP, with changes depending on both the DEHP concentration considered and the concentration of female pheromone used. No effect of BPA on male sexual behavior was observed. Mating and intergenerational experiments showed that in DEHP-treated females mated with control males, the duration of mating was shortened and the duration of the larval and pupal stages of the offspring was altered.

# Conclusions

Overall, our results show that DEHP and BPA can affect the ecdysteroid pathway in S. littoralis, both in the larval and adult stages, and can therefore be considered as EPs in this species. In addition, altered post-embryonic development, male sexual behavior and mating, as well as intergenerational effects were observed following DEHP exposure, suggesting that this compound can potentially affect individual abilities and/or population dynamics in this species.

We are also starting to carry out studies on several trophic levels to try to understand the transfer from contaminated soils to plants, herbivores and vertebrates feeding on these organisms.