Plant domestication is an evolutionary process whereby wild plants are modified to meet human needs (Meyer and Purugganan, 2013; Cornille et al., 2014). The suite of morphological and physiological traits that distinguish crops from their wild ancestors is known as the domestication syndrome (Harlan and De Wet, 1972; Koinange et al., 1996). Domestication traits are common in many crop species and generally include those that facilitate cultivation and harvesting, as well as other desirable traits, such as an increase in fruit size, yield, number of seeds, or plant growth (Wink, 1988; Evans, 1993; Meyer et al., 2012).

A study comparing 203 crops from 68 families showed that in 66% of these crops, the most common trait altered by domestication was the plant's secondary chemistry (loss or reduction of toxic or bitter compounds), followed by changes in plant morphology (e.g., aerial vegetative part or fruit size) (Chen et al., 2015). In recent years, there has been a surge in research examining how these domesticated traits have affected the interactions between crop plants and their associated herbivores. Overall, these studies reveal that often a decrease in chemical and physical plant defense results in increased insect performance (Idris and Grafius, 1996; Benrey et al., 1998; Gols et al., 2008). However, these patterns are not ubiquitous. For example, Turcotte et al. (2014) compared the performance of a leaf chewing herbivore (Spodoptera exigua) and a phloem-feeding aphid (Myzus persicae) on 29 pairs of crop plants and their wild relatives. They found that while domestication reduced the levels of morphological and chemical defenses in most of the examined crops, it did not always result in increased insect performance. In another study, Shlichta et al. (2018) found that in lima bean, Phaseolus lunatus, domestication has reduced the content of cyanogenic glycosides in seeds but not in leaves. Accordingly, the performance of the seed beetle Zabrotes subfasciatus, was better on seeds of domesticated varieties than on seeds of wild plants, whereas the performance of the generalist Spodoptera exigua, was not affected by the levels of cyanogenic glycosides (Shlichta et al., 2018). The authors argue that the trade-off between lower levels of defense and increased insect performance is supported for the seeds because this is the organ targeted by the domestication process.

Several other factors might be important to consider regarding the trade-off between decreased plant defense and increased insect performance. These include factors associated with the plant, the insect, or their interaction. For example, patterns of plant resistance in crops may vary across locations, degree of domestication and the organ or life stage attacked by the herbivore (Newell-McGloughlin, 2008; Chen et al., 2017). Crop domestication can also have a strong impact on the plant's nutritional content (Newell-McGloughlin, 2008; Delgado-Baquerizo et al., 2016; Gaillard et al., 2018) and insects may preferentially select and perform better on domesticated crops because of their higher nutritional quality, which may not always correlate with a decrease in defense traits (Tallamy et al., 1998). From the insect's side, feeding mode, host range, and/or level of specialization are important factors that can also influence their performance (Chen et al., 2015; Whitehead et al., 2017).

One factor that has been neglected in studies that examine the relationship between plant domestication and resistance to herbivores is the crop's purpose of domestication. This refers to the particular use for which a crop or a specific variety were domesticated, for example, for human or livestock consumption, fiber, as ornamental or medicinal use, and various other purposes depending on the type of crop. A large number of our food plants were originally selected for purposes other than food consumption (Meyer et al., 2012). For example, carrots were first domesticated for their seeds that were used as both spice and medicine (Simon, 2000) and saffron was used as a body paint, dye and perfume (Mousavi and Bathaie, 2011). Meyer et al. (2012) hypothesized that shifts in the primary used organs of a crop would cause changes in other organs, and further, that there would be a correlation between some organs used for food and non-food uses of other organs. In such cases, changes in chemical and physical defenses in a crop compared to its wild ancestor may be the result of the original desire for selection and not the current use of that crop.

Squash, Cucurbita spp. represents a fascinating system to study the implications of domestication for ecological interactions (Lira et al., 2016). With 21 taxa, the genus has experienced independent domestication events in five species: C. pepo, C. ficifolia, C. moschata, C. maxima, and C. argyrosperma (Sanjur et al., 2002). Each domestication event occurred independently, sometimes on more than one occasion (Lira et al., 2016). Each variety was selected for specific traits, defined by the nutritional and cultural needs of early human populations in America (Zizumbo-Villarreal et al., 2010, 2012). Even though each species underwent a unique selection regime to meet local conditions and preferences, many domestication traits are common. One of the main traits altered with domestication is the content of a class of secondary metabolites called cucurbitacins. These molecules are oxygenated tetracyclic triterpenes, extremely bitter that render plants toxic or unpalatable to many invertebrates and vertebrate herbivores, including humans (Da Costa and Jones, 1971; Ferguson and Metcalf, 1985). However, cucurbitacins can also serve as feeding attractants for a number of specialized phytophagous beetles belonging to the Chrysomelidae family (tribe Luperini, Old World Aulacophora) (Chambliss and Jones, 1966; Castellanos-Morales et al., 2018). The second trait modified during domestication is the density of trichomes (Paris, 2016; Chomicki et al., 2020). Trichomes can develop on the surface of all plant organs and can be “glandular” or “non-glandular.” Non-glandular trichomes can serve as a physical defense against herbivores by interfering with their movement, causing physical entrapment or in some cases inflicting injury (Ashraf et al., 2018; Kaur and Kariyat, 2020). Whereas, glandular trichomes can synthesize and secrete toxic chemicals (Dimock et al., 1982; Wagner, 1991; Kang et al., 2010a; Mitchell et al., 2016). Indeed, high densities of foliar trichomes can increase plant resistance against herbivores (Tian et al., 2012; Vermeij, 2015).

Although detailed studies exist on the history of domestication of Cucurbita (Nee, 1990; Nigh and Diemont, 2013; Lira et al., 2016; Chomicki et al., 2020), the extent to which squash varieties selected for different purposes differ in physical and chemical traits associated with defense and affect the interaction with herbivores, is not yet known. In this study we examined how variation in defense traits in several squash varieties affects the interaction with two generalist herbivores, and whether plant resistance against these herbivores can be explained by the variety's purpose of domestication. To do this, we used several varieties of squash from the Cucurbita genus and investigated the relationship between levels of chemical (cucurbitacins) and physical defenses (trichomes), and the preference and performance of a leaf and a root herbivore. We addressed the following questions: (i) what is the level of variation in cucurbitacin content and leaf trichome density among varieties? (ii) does purpose of domestication (consumption vs. ornamental) explain differences in defense traits among varieties? and (iii) are herbivores feeding preferences and performance altered by defense traits of squash varieties? Therefore, we quantified the content of cucurbitacins in roots, cotyledons and leaves, and density of trichomes in leaves and cotyledons of several squash varieties. Then, we conducted performance bioassays with two generalist herbivores, a leaf-chewing caterpillar (Spodoptera latifascia) and a root-feeding beetle (Diabrotica balteata). As larvae of D. balteata can move freely in the soil where the adult female lays its eggs, we also conducted choice experiments to test whether beetle larvae showed differential preferences for squash varieties. Finally, we carried out a common garden experiment in Mexico, the place of origin of squash and quantified infestation and damage from native generalist and specialist herbivores.

We found great variation in cucurbitacin content and leaf trichome density across all squash varieties. Overall, we did not find support for our hypothesis on reduced defenses in consumption varieties. Our results also show that herbivore performance is not negatively affected by high cucurbitacin content nor by trichome density. Instead, larvae of the root herbivore, D. balteata preferred to feed on varieties with high levels of cucurbitacins.

The content of cucurbitacins in leaves for all varieties was very low, and relatively high in cotyledons and roots as has been shown in other studies (Metcalf et al., 1982; Hirsh and Barbercheck, 1996; Tallamy and Gorski, 1997; Brzozowski et al., 2019). The highest content was in cotyledons. Greater levels of chemical defense in young tissues are often found in plants (Zangerl and Rutledge, 1996; McCall and Fordyce, 2010). If cotyledons, the first photosynthetic organ, are damaged, the consequences for plant development will be more detrimental than if older organs are damaged (McCall and Fordyce, 2010; Barton and Hanley, 2013). For example, glucosinolates in Arabidopsis thaliana vary among plant tissues and the highest concentrations are found in seedlings (Hopkins et al., 2009). Similarly, in lima bean (Phaseolus lunatus), cyanogenic glycosides are found in higher quantity in cotyledons than in other plant vegetative tissues (Cuny et al., 2019).

The differences in defense traits, cucurbitacins vs. trichomes, between tissues are in line with plant defense theories that predict a trade-off in resource investment for chemical and physical defenses (Freeman and Beattie, 2008; Moles et al., 2013). Similar trade-offs in plant tissues have been found in several wild populations of C. argyrosperma, in which roots and cotyledons have a high content of cucurbitacins, while leaves have high trichome densities and low levels of cucurbitacins (Jaccard et al., 2021). This suggests that although domestication reduced plant defense in squash (Metcalf et al., 1982; Nee, 1990; Brzozowski et al., 2019; Chomicki et al., 2020), it did not alter the trade-off between physical and chemical defense in different plant tissues. The different concentration of cucurbitacins and trichome density in cotyledons and leaves could indicate that plants switch defense strategies during their ontogeny toward the production of trichomes in leaves (Boege and Marquis, 2005; Barton and Koricheva, 2010). Brzozowski et al. (2020) found that, in one variety of Cucurbita pepo, there is a peak of cucurbitacin content in seedlings very early in the development, starting at the radical emergence, but cucurbitacins diminished once cotyledons were fully expanded. However, in a different variety of the same species containing less cucurbitacins, they did not detect changes in cucurbitacin levels throughout plant development. Thus, it would be interesting to further examine changes in defense traits throughout plant ontogeny for different species and cultivated varieties of squash.

Contrary to our expectations, we did not find lower levels of cucurbitacins or fewer trichomes in varieties domesticated for consumption compared to ornamental varieties. Several factors may explain these findings. First, other defense traits not considered in this study could have been reduced or selected during the domestication process. Alternatively, as fruits are the main edible part of the squash plant, it is possible that levels of cucurbitacins in leaves, cotyledons and roots do not reflect the direct target of domestication. In retrospective, it would have been informative to examine cucurbitacin levels in fruits and seeds. But this was not initially considered as we only examined the tissues eaten by the studied insects. However, because of the extreme bitterness of cucurbitacins, fruits of consumed cucurbit plants usually are void of these compounds. This is due to the original selection of non-bitter plants for food use by our ancestors and later by new breeders (Rymal et al., 1984; Gry, 2006). Yet, further studies should examine the levels of cucurbitacins present in the seeds and their effect on seed feeding insects.

Secondly, we found great variation in trichome density and cucurbitacin content among varieties domesticated for the same purpose, particularly among ornamental varieties and this variation may override differences resulting from the domestication purposes. Finally, the varieties used in our study belong to different Cucurbita species and it could be that the differences caused by speciation are higher than the putative effect of purpose of domestication. Ideally, to control for a phylogenetic signal, we should evaluate varieties of the same squash species. However, in this study we chose to use different species from the same clade that allowed us to test various uses of squash, which are not readily available within a single species. In the future, we expect to elucidate more questions regarding this aspect as we are currently exploring the relationship between purpose of domestication and plant resistance against herbivores with varieties of the species C. argyrosperma that have the same wild ancestor (Jaccard et al., 2021).

Changes in plant defense as a result of domestication are expected to alter the interaction with the plant's natural enemies (Levin, 1973; Chaudhary, 2013). Accordingly, we expected the performance of the two generalist herbivores to be lower on varieties with higher trichome densities and cucurbitacin levels. Cucurbitacins are among the bitterest plant secondary compounds found in plants (Rehm et al., 1957; Metcalf, 1986) and their roles are diverse (Hill et al., 1991), including protection against plant pathogens (Tallamy et al., 1998; Smyth et al., 2002) and deterrence of generalist insects and other arthropods (Tallamy et al., 1998). But they can also act as attractants and feeding stimulants for specialist insects, like Acalymma (Tallamy and Krischik, 1989; Tallamy and Gorski, 1997; Smyth et al., 2002). Interestingly, although S. latifascia and D. balteata are known to be very polyphagous insects (Howe et al., 1976; Habib et al., 1982), the content of cucurbitacins in cotyledons and roots did not affect larval performance. Moreover, choice experiments with D. balteata revealed that consistently, larvae preferred to feed on roots with high cucurbitacin content, which suggests that this species is adapted to these compounds. A likely explanation may lie in the common origin of genus Cucurbita and D. balteata, Mexico, where both plants and insects have been interacting for thousands of years probably well before the domestication of squash (Bach, 1980; Metcalf, 1986). Also in this region, squash is commonly grown in traditional mixed cropping with maize and beans (both crops readily consumed by D. balteata larvae). Thus, it is possible that during the generalist's long-time association with squash it has evolved to tolerate specific secondary defenses leading to an adaptation to the plant without being specialized on it. In addition, D. balteata belongs to the subtribe Diabroticina, which includes the specialist Acalymma, thus the preference toward cucurbitacins could be a shared trait among the Diabroticina. Because they can readily tolerate cucurbitacins the plants may provide a nutritional niche for the larvae, without much competition.

We also consider it possible that the larval feeding choices are the consequence of physiological adaptations in the adult Diabrotica beetles, which are known to be attracted to and feed on Cucurbita plants. Yet, it is not excluded that feeding on plants with cucurbitacins provides benefits that we have not yet uncovered.

Caterpillars of S. latifascia grew better on the consumption varieties, despite the higher density of trichomes. One reason may be the size and form of these trichomes, or the larval instar used in our study. Indeed, non-glandular trichomes only physically impede the movement of small arthropod herbivores on the plant surface (Dalin et al., 2008; Kang et al., 2010b). Moreover, like most caterpillars of the genus Spodoptera, S. latifascia have strong mandibles (Ma, 1976) which likely facilitated the chewing of the hairy leaves. Alternatively, selection on these varieties might have increased the nutritional quality of the leaves. This hypothesis remains to be tested.

Due to the low germination rate of the seeds in the field, the number of varieties and of plants of each variety that comprised the common garden experiment was small. Therefore, the conclusions that we can draw from this experiment are limited. Nevertheless, this experiment yielded interesting results. Overall, the ornamental varieties suffered the highest herbivore damage, particularly the variety Ott that did not grow well. However, we believe that these results do not necessarily reflect that insects preferred these plants. Rather, their poor growth might have compromised their ability to resist herbivore damage. In contrast, the Mexican hybrid varieties grew well and were less damaged by the natural herbivore populations, suggesting some degree of resistance, same for one variety selected for the fruit consumption (Fbg). One interesting and unexpected result was the low number of D. balteata adults that visited the plants, despite their high numbers in the field site. Instead, they preferred other crops in the site, such as beans, peanuts and maize. Individuals of Acalymma spp. on the other hand, were highly abundant in our experimental plots, but their abundance and amount of damage on the plants were not correlated with cucurbitacin levels or trichome density. This is not surprising as species in the genus Acalymma are known to be specialists on squash, adapted and attracted to cucurbitacins which they use as kairomones to locate their host plants and capable of sequestering them for their own defense (Bach, 1980; Batch, 1984; Benitez et al., 2014). Our results are in accordance with those from Theis et al. (2014). They found that the abundance of Acalymma vittatum in a squash field site in Massachusetts (USA), was not correlated with the cucurbitacin content of 20 cucurbit varieties, including cultivated and wild species. They suggest that the damage could be predicted by other plant traits and not by beetle presence and that floral sesquiterpenoids were driving these interactions. Similarly, in an ongoing study with wild and domesticated plants of C. argyrosperma, we found that larvae of the specialist A. vittatum were strongly attracted to cucurbitacins present in roots, but cucurbitacin content was not correlated with larval performance (Jaccard et al., 2021). Interestingly, these plants do not contain any cucurbitacins in the leaves which supports the hypothesis that other traits are likely involved in the attraction of adult beetles. Future work should explore the role of leaf and flower volatiles in this attraction.

In conclusion, our study suggests that varietal selection, but not the purpose of domestication in squash has altered cucurbitacin content and leaf trichome density, but the performance of the leaf and root herbivore was not affected by these defense traits. Further, the preference of D. balteata larvae to cucurbitacins may be likely due to the evolutionary history that this species has shared with wild and domesticated squash plants in their region of origin and domestication. Future efforts should be directed at elucidating the effects of domestication of this and other crops on their susceptibility to herbivorous pests. A better understanding of these effects could help in the development of crop varieties with enhanced insect and disease resistance.

The datasets generated for this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Materials.

CJ and BB originally formulated the idea. CJ, BB, and MC designed the experiments. CJ performed all the experiments and wrote the first version of the manuscript. GG developed the cucurbitacin extraction method. CJ, MC, and CB-S worked in the field in Mexico. CJ and CB-S did the DNA barcoding for field insect determination. LG performed the experiment on cotyledons. CJ, CB-S, and CA analyzed the data. All authors contributed to the elaboration of the design and to the writing of the last version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.