Glyphosate, or N-(phosphonomethyl) glycine, is the active ingredient in Roundup, one of the world’s most widely used herbicides in small and large scale crop production (Benbrook, 2016). This molecule typically acts by inhibiting the 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), an enzyme that enables the biosynthesis of aromatic amino acids in plants, fungi and bacteria (Duke and Powles, 2008). Because this enzyme is not present in animals, it was long thought that exposure to glyphosate-based herbicide (GBH) would have no effect on mammals, birds and arthropods. However, a growing body of evidence indicates that this is not necessarily the case. In a taxonomically large number of species, exposure to GBH has been shown to alter fitness-related traits such as behaviour, development, and immunity (for review de Brito Rodrigues et al. 2017; Battisti et al. 2021; Talyn et al. 2023). For example, GBH exposure modifies locomotor activity in termites, cockroaches and in rats (Ekaye et al., 2022; Kanabar et al., 2021; Rocha et al., 2019), alters the moulting and metamorphosis process in juveniles of Spodoptera littoralis, Spodoptera frugiperda and honey bees (El-Sheikh et al., 2016; Vázquez et al., 2018), reduces immunocompetence and immunity in silkworms (Feng et al., 2023), as well as impairs reproduction in the mallard duck (Oliveira et al., 2007) and other birds population in wetlands (Linz et al., 1996; Santillo et al., 1989). The mechanisms driving these alterations remain unclear and are often attributed to the toxic impact of adjuvants present in GBHs (Defarge et al., 2018), GBH-induced changes in the host microbiota (Cullen et al., 2023; Vázquez et al., 2018), and/or endocrine disruptor properties of GBH (for reviews see Kalofiri et al., 2021; Levine et al., 2020; Muñoz et al., 2021). They may also involve other physiological parameters of the hosts, as suggested by the age-specific effects of GBH – stronger effects in juveniles than adults - reported in some mammals (Pope and Liu, 1997) and insects (Fogel et al., 2016; Stuijfzand et al., 2000).
In addition to the host traits detailed above, recent data indicate that GBHs may also alter the expression of social behaviours, such as maternal care, which have major implications for individual fitness and population dynamics (Cummings et al., 2010; Fong-McMaster et al., 2020). For instance, GBH exposure decreases the amount of time mothers spend caring for their offspring in rats (Dechartres et al., 2019) and reduces the expression of maternal defensive and grooming behaviours in rats and mice (Ait-Bali et al., 2020; Rocha et al., 2019). However, these data are mostly from studies in mammals, raising questions about the effect of GBH exposure on the expression of parental care in other animals, including insects. This lack of data on insects is surprising for three main reasons. First, parental care is a taxonomically widespread phenomenon in arthropods (Machado and Trumbo, 2018; Meunier et al., 2022), in which it is well documented that even subtle changes in the expression of this behaviour can dramatically affect reproduction and population dynamics, with major consequences for terrestrial ecosystems functioning, biodiversity maintenance, and crop protection (Schowalter et al., 2018). Second, studies suggest that exposure to various pesticides and chemical pollutants can alter insect parental care, opening the possibility of an effect of GBHs on this trait (de Oliveira et al., 2022; Gallegos et al., 2016). Finally, insects are frequently exposed to GBHs in crops and gardens (Mazzia et al., 2015). Studying their effects is, therefore, biologically relevant and timely, as it could enhance our understanding of the maintenance and population dynamics of many species that play crucial role in current agroecosystems.
The European earwig is an ideal species to study the effect of GBH on maternal care, as it is well known for the extensive care that mothers give to their eggs and newly hatched juveniles, and its ubiquity in multiple agricultural areas worldwide (Honorio et al., 2024; Meunier, 2024; Orpet et al., 2019). Earwig mothers typically care for their egg during 50 days, during which they regularly groom, clean and move their eggs during development (Koch and Meunier, 2014; Thesing et al., 2015). They then care for their juveniles (called nymphs) for two weeks, providing them with food, protecting them against pathogens and defending them against predators (Kölliker, 2007; Lamb, 1976). Unlike egg care, post-hatching care is facultative in this species, as newly hatched nymphs quickly become mobile and able to forage independently (Wong and Kölliker, 2012). Regardless of its maternal care, the European earwig is widespread in vineyards and orchards, where a wide variety of pesticides and herbicides are frequently used (Malagnoux et al., 2015b). This species is considered a crop auxiliary in pip-fruit orchards, where it feeds on aphids, moths and psyllids without eating the fruit, while it is considered a pest in stone fruit orchards, where it also feeds on leaves and fruit (Alins et al., 2023; Dib et al., 2011; Orpet et al., 2019). Previous studies have shown that exposure of earwig females to pesticides such as deltamethrin reduces the expression of maternal care and increases their investment in future reproduction (Mauduit et al., 2021a; Meunier et al., 2020). Similarly, a recent study showed that direct exposure of males to the GBH Roundup© altered their locomotor activity, but not their boldness, aggregation level or immunity (Pasquier et al., 2024). In contrast, female exposure to other chemicals such as cadmium, an heavy metal pollutant, and pyriproxyfen, another pesticide, does not alter the expression of maternal care (Honorio et al., 2023a, 2023b; Merleau et al., 2022).
In this study, we conducted two experiments in the European earwig to investigate the effects of (1) direct exposure of females to GBH on the expression of maternal care and the associated clutch fate, and (2) direct exposure of juveniles to GBH on their behaviour, pathogen resistance and expression of genes involved in the development. In the first experiment, we exposed 239 females to one of four solutions of a glyphosate formulation commonly used in domestic gardens (RoundUp GT Max®, Scotts France SAS) and then measured the expression of six classical forms of egg and nymph care, as well as the development time and hatching rate of their eggs, and the size and weight of newly hatched nymphs. In the second experiment, we exposed 282 nymphs to either a control solution or a solution containing the highest concentration of the same glyphosate formulation (detailed above) and then measured their general activity, the expression of genes involved in their development (JH pathway) and their survival after infection with the common entomopathogenic fungus Metarhizium brunneum. Overall, we predicted that female exposure to GBH induces changes in their expression of maternal care and the development/quality of their resulting nymphs, and that juvenile exposure to GBH alters their behaviour, gene expression of JH production and survival to pathogen infection.