The aim of our study was to investigate the effect of capsaicin in domesticated chili peppers on a tritrophic interaction with the generalist herbivore Spodoptera latifascia and its ectoparasitoid Euplectrus platyhypenae. Overall, our results reveal that capsaicin had a negative effect on both insects, particularly at high concentrations. Indeed, we found that when larvae of S. latifascia were reared on pungent chili varieties, they had lower larval weight, reduced pupation and lower adult emergence than when caterpillars were reared on non-pungent varieties. Similar results were found when caterpillars where reared on spiked and control diets. The negative effects of capsaicin subsequently affected the third trophic level by reducing the parasitism of caterpillars when these were reared on fruits or diet.
To date, all published studies on the effects of capsaicin on insects have focused only on herbivores and most of these have used artificial diet or ground chili powder and not fresh fruits. These studies have mainly examined the benefits of capsaicin as a pesticide. For example, Cowles et al. (Cowles et al. 1989) found that oviposition of the onion fly (Delia antiqua) was reduced by 99.8 % and 95% respectively by applying both chili powder and synthetic capsaicin to the artificial diet. Interestingly, despite capasicinoids being naturally absent in leaves and vegetative organs of chili plants (Estrada et al. 2002), they are still effectively used as biopesticides against sucking insects attacking chili leaves, such as the green aphid Myzus persicae Sulz (Edelson et al. 2002; Koleva-Gudeva et al. 2013) or whiteflies, a major pest of pepper crops (Greer 2000). However, their mode of action remains unclear.
By using fresh chili fruits, we were able to assess the effect of natural capsaicin on the second and third trophic levels. Alternatively, the capsaicin-spiked diet allowed us to isolate the effect of capsaicin from other potential effects of the chili fruits on the insects. However, some differences were observed when using these two types of diets, particularly for the herbivore, where the negative effects of capsaicin were more evident when fed on fruits. Due to the high capsaicin content in the fruits, we could not mimic the exact levels because of the high toxicity we experienced while manipulating the pure synthetic capsaicin. Therefore, we would expect that the effect would be stronger if we increase the capsaicin content levels equivalent to those found in the fruits.
It is expected that the effects of capsaicin will be different on generalist and specialist herbivores. For instance, larval growth of the generalist herbivores Spodoptera frugiperda, Heliothis virescens and Helicoverpa zea was slower when fed with capsaicin-spiked diet, while the growth and survival of larvae of the tobacco budworm (Helicoverpa assulta), a specialist on Solanaceae, was not affected (Ahn et al. 2011a). The latter is considered to be one of the few insect herbivores capable of feeding on hot pepper fruits (Baek et al. 2009) and able to detoxify these secondary metabolites (Ahn et al. 2011b). Another species known to feed on chili pepper, is the pepper weevil, Anthonomus eugnii, a specialist primarily on fruits of Capsicum spp., but able to feed on other nightshade plants (eg. eggplants) (Rodriguez-Leyva 2006). This beetle is known to feed on highly pungent chili varieties such as Habanero and Scotch Bonnet (Seal and Martin 2016). Adults lay eggs on flower buds and complete their development inside the fruits (Riley and Sparks Jr 1995). Both larvae and adults were observed feeding on the fruit’s placenta (Chabaane, personal observation) where capsaicinoids are concentrated (Fujiwake et al. 1982). It is assumed that both larvae and adults of this species can handle the spiciness, but the mechanism remains unclear. In our study, even though S. latifascia is a generalist, caterpillars were able to tolerate diet and fruits with medium levels of capsaicin. We found that while feeding, caterpillars can sequester capsaicin in the haemolymph, but only when they feed on the highly pungent varieties (Fig. 4). However, when exposed to lower levels of capsaicin, sequestration did not occur, or the levels were under the detection limit. It is possible that caterpillars are able to detoxify or excrete this secondary metabolite when present at low levels as it has been shown for other herbivores exposed to nicotine, also an alkaloid (Barbosa et al. 1986).
The effect of capsaicin on S. latifascia was stronger than on its parasitoid E. platyhypenae. For the parasitoid, we found differences among treatments only for parasitism rate but not for clutch size. Yet, as the parasitoid larvae feed on the host’s haemolymph (Coudron et al. 1990; Nakamatsu and Tanaka 2003) where the capsaicin can be found, we could expect to find stronger effects of capsaicin on the parasitoid larval and adult stages. Thus, further studies should focus beyond the oviposition response of the parasitoid and examine the effects of capsaicin throughout parasitoid development, adult survival, size, and sex ratio. Moreover, parasitism rate was also reduced when Habanero fruits were replaced by no capsaicin diet during the exposure to wasps. This was probably caused by the capsaicinoids accumulated in the haemolymph. However, it remains to be investigated how long this accumulation will last once the exposure to capsaicin is stopped and the subsequent effects on the parasitoid. It would also be interesting to test the effects of capsaicin on other natural enemies with different life history strategies and feeding modes such as, endoparasitoids and predators.
Recently, a growing number of studies have examined the relationship between plant chemical defense as a result of domestication and insect performance (Chen et al. 2015; Whitehead et al. 2017). It is often found that lower chemical defense results in increased performance, but there are also many exceptions to this pattern (Shlichta et al. 2018; Turcotte et al. 2014). Chili pepper offers a unique model to examine this relationship, since we have varieties selected for lower capsaicinoid content than the wild, chiltepin, but also varieties that were selected for much higher pungency levels (Scoville 1912). In another study, we found that the capsaicinoid levels in chiltepin fruits collected from different populations along the Pacific coast of the state of Oaxaca, Mexico mainly ranged between the contents detected on Habanero and Cayenne varieties (Chabaane et al, 2020. unpublished data). Thus, we would expect that the performance of S. latifascia larvae on wild chili fruits would be intermediate relative to the highly pungent and mild varieties.
Here, we focused on the effects of capsaicin, the main plant trait targeted during varietal selection of chili pepper (Paran and van der Knaap 2007). Yet, the domestication syndrome of Capsicum species includes other traits such as, germination rate, fruit colour, position and size, foliar and phenological traits (Pickersgill 2016). It is likely that some or all of these traits will also affect insect choice and performance. Moreover, some of these traits might be correlated to the high pungency levels in fruits. For example, (Taiti et al. 2019) found that volatile organic compound (VOC) emissions from fresh chili fruits were correlated with their spiciness. It is known that parasitoids use plant volatiles to locate their hosts (Turlings et al. 1990; Vet and Dicke 1992). Therefore, future studies should investigate how multiple domesticated traits have influenced the tritrophic interactions in this important crop.
Our results also offer further insight into alternative strategies for pest management in chili pepper, one of the top ten vegetable crops in the world (FAOSTAT 2020). The use of mixed varieties in agriculture has been shown to reduce pest pressure by slowing the spread of insects with the presence of resistant varieties acting as barriers (Barot et al. 2017). For example, (Abdala-Roberts et al. 2015) showed that growing different genotypes of Capsicum chinense reduced attack by a leaf mining fly (Lyriomyza trifolii) by 25 % as compared to a monoculture setting. They suggest that plant genotypic diversity of their varieties (e.g. plant size, architecture, flowering phenology, and fruit size) played an important role in reducing insect attack. The importance of genotype diversity in chili peppers was also reported to decrease infestation by white fly (Bemisia tabaci) and yellow mite (Polyphagotarsnemus latus) (Datta and Chakraborty 2013), as well as aphid (Aphis craccivora) (Ofori et al. 2015) infestations on peppers. In this context, our results suggest that growing mixed chili varieties with different pungency levels might reduce pest pressure by generalist herbivores.
In conclusion, our study represents pioneering work regarding the effect of natural capsaicin on herbivores and the third trophic level. In the future, this knowledge could also be important for other crops as well, such as hot tomato produced by activating the inactive capsaicinoid biosynthesis pathway naturally present in this Solanaceae plant (Naves et al. 2019). Moreover, as chili peppers originate and were domesticated in Mesoamerica, by studying its interactions with native insects from this region, our results could provide insight into the selective pressures that have contributed to the crop’s phenotypic diversity and the relationship with its wild ancestor.