Most herbivorous insects are host-plant specialists that evolved detoxification mechanisms to overcome their host plant’s toxins. In the evolutionary arms-races between Pieridae butterflies and Brassicaceae plants, some plant species have evolved another defence against the pierids: egg-killing. Underneath the eggs, leaves develop a so-called hypersensitive response HR-like cell death. Whether some butterflies have evolved oviposition strategies to counter-adapt against egg-killing remains to be studied. In this study, we assessed the oviposition site location of pierid butterflies on their natural host plants. We described the plant tissue on which we located the eggs of the most common Pieridae in the Netherlands: Gonepteryx rhamni, Anthocharis cardamines, Pieris rapae, P. napi, P. brassicae, and P. mannii. Additionally, we assessed expression of HR-like cell death in response to the deposited butterfly eggs. We found that both A. cardamines and G. rhamni mainly oviposited on the floral stem and the branch, respectively, and oviposited on host plants from lineages not expected to express HR in response to pierid eggs. Accordingly, no HR responses were seen. All Pieris eggs found were located on leaves of their host, the only tissue found to express HR-like cell death. Furthermore, each Pieris species was found to at least occasionally oviposit on Brassica nigra. This was the only plant species in this survey that expressed HR-like cell death in response to the eggs of P. rapae, P. napi and P. brassicae. Our observations demonstrate that HR-like cell death remains an effective defence strategy against these Pieris species and as such did not find evidence for the hypothesised counterstrategies. Surveying certain key species and disentangling the micro-evolution of oviposition strategies within a species would allow us to further  investigate potential counter-adaptations that evolved against HR-like cell death. This study provides the basis for further investigation of potential counter-adaptations to egg-killing defences.

Fieldwork for this research was conducted in the provinces Gelderland and South Limburg in the Netherlands between April and July 2021. Eggs of G. rhamni were found at five locations in Renswoude, Wageningen and Wijchen. These locations were near roads and in forests. Eggs of A. cardamines were found at 12 locations in Wageningen, Wijchen, Arnhem and Vijlen (South Limburg). All locations were near streams or along paths. All Pieris eggs were found along paths, in fields, and gardens at 16 locations in Wageningen and Wijchen. All host plants were checked as they were found in the search locations. Therefore, some host plants were found more often than others. For all herbaceous plants, all parts of the plants were checked for eggs. For egg searching on Rhamnus frangula all leaves and twigs on branches within a height of ± 2 m to the ground were checked for eggs.The position of each egg was recorded by the following categories: branch, leaf, flower bud. Additionally, the presence of HR-like cell death was carefully observed with a magnifier or camera with a macro lens.