Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb.

Coevolutionary interactions are responsible for much of the Earth's biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes' importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

Composition of Strawberry Floral Volatiles and their Effects on Behavior of Strawberry Blossom Weevil, Anthonomus rubi.

The strawberry blossom weevil (SBW), Anthonomus rubi, is a major pest in strawberry fields throughout Europe. Traps baited with aggregation pheromone are used for pest monitoring. However, a more effective lure is needed. For a number of pests, it has been shown that the attractiveness of a pheromone can be enhanced by host plant volatiles. The goal of this study was to explore floral volatile blends of different strawberry species (Fragaria x ananassa and Fragaria vesca) to identify compounds that might be used to improve the attractiveness of existing lures for SBW. Floral emissions of F. x a. varieties Sonata, Beltran, Korona, and of F. vesca, were collected by both solid-phase microextraction (SPME) and dynamic headspace sampling on Tenax. Analysis by gas chromatography/mass spectrometry showed the floral volatiles of F. x ananassa. and F. vesca were dominated by aromatic compounds and terpenoids, with 4-methoxybenzaldehyde (p-anisaldehyde) and α-muurolene the major compounds produced by the two species, respectively. Multi-dimensional scaling analyses separated the blends of the two species and explained differences between F. vesca genotypes and, to some degree, variation between F. x ananassa varieties In two-choice behavioral tests, SBW preferred odors of flowering strawberry plants to those of non-flowering plants, but weevils did not discriminate between odors from F. x ananassa and F. vesca flowering plants. Adding blends of six synthetic flower volatiles to non-flowering plants of both species increased the preference of SBW for these over the plants alone. When added individually to non-flowering plants, none of the components increased the preference of SBW, indicating a synergistic effect. However, SBW responded to 1,4-dimethoxybenzene, a major component of volatiles from F. viridis, previously found to synergize the attractiveness of the SBW aggregation pheromone in field studies.