Males of many moths species display a stereotyped, very specific, and innate attraction response to the sex-pheromone released by conspecific females (Allison and Cardé, 2016a). They are also able to detect and avoid heterospecific sex-pheromones from related sympatric species, which favors reproductive isolation (Renou et al., 1996; Allison and Cardé, 2016b). These attraction/deterrence responses are crucial for moth reproduction. As these insects are exquisitely sensitive to their sex-pheromone, they have been studied a lot as models of odor specialist. Moreover, as many moth species are crop pests, behavioral manipulation by use of pheromones is an important tool for managing their populations (Cook et al., 2007; Witzgall et al., 2010; Cork, 2016; Evenden, 2016). Therefore, studying their responses to pheromones has also important applications. These features make moths important models to study how pheromone can trigger stereotyped behaviors.

While pheromones are classically described as elicitor of stereotyped behaviors, in bees and mammals non-sex-pheromones can modulate plasticity (Coureaud et al., 2006; Vergoz et al., 2007; Bredy and Barad, 2009; Jouhanneau et al., 2016). In particular, reports in bees point toward effects on gustatory learning and perception (Pankiw and Page, 2003; Urlacher et al., 2010; Baracchi et al., 2017). Many examples in moths show interactions between reproduction and gustatory perception and learning (Awmack and Leather, 2002; Geister et al., 2008; Shikano and Isman, 2009; Molleman, 2010; Minoli et al., 2012; Briscoe et al., 2013; Petit et al., 2015). Therefore, it is reasonable to hypothesize that sex-pheromones could also interact with the perception and learning of gustatory information in these insects.

Observing proboscis extension response (PER) is a convenient experimental procedure to investigate gustatory functions: when the insect antennae contact a sugar solution of sufficient concentration, a moth extends its proboscis (i.e., releases a PER). Thus, this reflex allows to assess sucrose-linked behaviors in restrained moths, which is relevant as sugars are their main food in nature (under the form of nectar). Using standardized dose-response curves, PER assays have been used to assess sucrose responsiveness in moths (Hostachy et al., 2019), as well as in bees (Scheiner et al., 2004a) and flies (Scheiner et al., 2004b). PER can also be used to train and study animals in a non-associative learning called gustatory habituation. In habituation, an animal decreases and stops its response to a stimulus, if this stimulus is ongoing or repeated and the animal does not undergo any consequence when stimulated; using a dishabituation test then confirms this response inhibition cannot be explained by fatigue or sensory adaptation (Rankin et al., 2009; Blumstein, 2016). Such dishabituation test consists in presenting of a more intense (or different) stimulus, and to observe whether the response to the original stimulus has been restored. It is easy to habituate the PER upon repeated presentations of a low-concentration sucrose solution on the antennae without feeding the animals: in that case, antennal stimulations cease eliciting the PER. Beyond moths, this protocol has been applied in bees and flies (Duerr and Quinn, 1982; Braun and Bicker, 1992; Dacher and Gauthier, 2008). Habituation can be modulated by non-sex-pheromones in bees (Baracchi et al., 2017). It is established that PER-based learning are relevant to natural conditions (Gerber et al., 1996; Sandoz et al., 2000; Chaffiol et al., 2005; Gil and De Marco, 2005; Riffell et al., 2013). Habituation leads to ignore irrelevant stimuli, which allows a reallocation of resources (Mennemeier et al., 1994; Dukas, 2002, 2004; Ramaswami, 2014; Turatto and Pascucci, 2016).

In this article, we took advantage of the PER to explore the links between reproduction and gustation in moths by assessing whether their sex-pheromone can modulate sucrose responsiveness and gustatory habituation. Male Agrotis ipsilon moths were thus pre-exposed to either their conspecific sex-pheromone or a heterospecific sex-pheromone compound of a related sympatric species (Agrotis segetum), which they can perceive and avoid (Renou et al., 1996). We then assessed whether this pre-exposure affects sucrose responsiveness or gustatory habituation either immediately or 24 h after exposition, as previous results suggest sex-pheromone elicits long-term effects (increase of the response to the sex-pheromone itself, Anderson et al., 2003, 2007). These pheromones were chosen with the idea that A. ipsilon sex-pheromone would be “positive” whereas A. segetum compound would be “negative” (as defined by Baracchi et al., 2017) as they are respectively attractive and deterrent for A. ipsilon males. Thus, we made the hypothesis that they would have opposite effects.

Responsiveness to sucrose was not modulated by a pre-exposition to conspecific sex-pheromone in male A. ipsilon moths. By contrast, the major pheromone component of a sympatric species (Z5) increased sucrose responsiveness but only at the short-term exposure. Furthermore, habituation, a form of non-associative learning was hindered by both pheromones, but at different delays: immediately but not 24 h after exposure for Z5, and 24 h but not immediately after exposure for the conspecific sex-pheromone.

Experiments in bees and ants have shown that pheromone exposure modulates sucrose responsiveness and performance during learning, including gustatory habituation (Pankiw and Page, 2003; Vergoz et al., 2007; Urlacher et al., 2010; Minoli et al., 2012; Baracchi et al., 2017; Rossi et al., 2018). Overall, pre-exposure to pheromone sensitizes or desensitize insects (according to the “positive” or “negative” value of the pheromone, Baracchi et al., 2017), thereby affecting their sucrose responsiveness. In our experiments, exposition to the conspecific sex-pheromone did not affect sucrose responsiveness. Thus, an effect on habituation through a modulation of sucrose responsiveness is excluded. By contrast, the fact that Z5 increases sucrose responsiveness is consistent with its impairing habituation to sucrose (even though it did not initially decrease the PER rate in this experiment). A striking difference is the temporal difference of conspecific sex-pheromone and Z5: the later acts immediately but not after 1 day (except for a small effect on dishabituation rate), whereas sex-pheromone needs 1 day to have an effect and only affects habituation. This indicates that they act through different pathways.

The neurophysiological mechanisms underlying these effects of conspecific sex-pheromone and Z5 remain an open question. Such mechanisms could involve biogenic amines. Indeed, these neurotransmitters modulate responsiveness to sucrose and learning (Scheiner et al., 2006, 2014), and biogenic amines are involved in sex-pheromone’s actions (Duportets et al., 2010; Abrieux et al., 2014). A possible mechanism for pheromone effects would be that they could have a positive or negative valence for the animal: perceiving them as reward (conspecific sex-pheromone) or punishment (heterospecific sex-pheromone such as Z5) could explain their effect on learning (Coureaud et al., 2006; Vergoz et al., 2007; Jouhanneau et al., 2016; Baracchi et al., 2017). However, this is not what we observed; both conspecific sex-pheromone and Z5 had the same deterring effect on habituation rather than opposite ones.

Pheromonal modulation of gustatory habituation is an interaction between reproductive and feeding functions. Impairing gustatory habituation could mean moths are less prone to ignore food. In that frame, Z5 would do more than just prevent mating with incompatible females and would inform on the presence of competitors for food. In turn, habituation would be hindered (and sucrose sensitivity improved) upon detecting them, in order to promote eating in the context of competition for food. By contrast, this would not make sense anymore 24 h after. Similarly, conspecific sex-pheromone would indicate the presence of mates in the environment. While habituation should be maintained immediately after perceiving the pheromone, in the absence of mating 24 h after exposure food should not be ignored so as to gather resources to continue mate-searching. This is consistent with the observation that sucrose can sensitize the response to sex-pheromone (Minoli et al., 2012).

These hypotheses are arguably bold, but are testable as they make specific and strong predictions: presenting conspecific sex-pheromone both 24 h and immediately before mating would improve habituation, as the male would focus on mating rather than feeding.

To further investigate the functional relationships between feeding and reproduction, it would be interesting to assess the amount of sucrose consumed by A. ipsilon males having differing experiences with conspecific females (e.g., exposure, mating) and/or sex-pheromones (Sokolowski and Abramson, 2010). The same experiments could also be done with exposure to females of A. segetum and Z5. An interesting feature of males in some Lepidoptera species is puddling, i.e., drinking brackish water to get sodium needed for gamete formation (Smedley and Eisner, 1996; Boggs and Dau, 2004; Watanabe and Kamikubo, 2005; Molleman, 2010). Owing to our observations, it would also be relevant to assess whether puddling is modulated by exposure to sex-pheromones.

All datasets generated for this study are included in the article.

CH, ND, and MD designed the experiments. CH, PC, GP, MH-P, and MM performed the experiments. CH and MD prepared the figures and analyzed the data. MD wrote the first draft of the manuscript, and all authors contributed to manuscript revision, read and approved the submitted version.