As evolutionary biologists, we are often curious about the genomic origins of our favorite adaptations. Although some innovations certainly arose de novo, many more originated through the process of whole-gene or within-gene duplication. Following whole-gene duplication events, at least one gene copy is thought to be under relaxed selective constraints, meaning that mutations can accumulate within the gene and potentially give rise to novel adaptive traits. In this thesis, I aim to identify how gene duplication events have helped caterpillars cope with toxic host plants. Building upon the already-extensive literature on plant-insect coevolution, I highlight the complexity underlying detoxification phenotypes.

The research presented in Chapters II and III focuses on a family of genes coding for insect nitrile-specifier proteins (NSPs). These NSP-like genes are the canonical detoxification genes in Pierinae butterflies, allowing for the detoxification of the glucosinolates defenses present in their host plants. Importantly, the NSP-like gene family was formed through gene duplication events, with two key genes (NSP and MA) originating from the same ancestral gene. In Chapter II, Crispr-Cas9 methods were used to create lines of Pieris brassicae that lacked functional copies of NSP and/or MA. Through feeding assays on natural host plants, we showed that either NSP or MA are necessary for larval survival on plants containing aliphatic or benzyl glucosinolates – but not indole glucosinolates. Further, NSP seemed to be specialized for aliphatic glucosinolate detoxification, suggesting that some degree of subfunctionalisation occurred following gene duplication.  Expanding on these findings, we focused on the regulatory consequences of NSP-like family gene knockouts in Chapter III, looking specifically at the transcriptomic response to three host plants with vastly different glucosinolate profiles. We ultimately discovered that the response to host plant change was strongest when all NSP-like genes were functional, suggesting that the NSP-like gene family is part of a larger regulatory response to host plant defenses. 

While the above chapters center around gene duplicates that have already been associated with adaptations to host chemistry, there are likely more gene families out there that have been important for caterpillars overcoming ever-escalating plant defenses. In Chapters I and IV, I sought to identify some of these families using comparative genomic analyses. In Chapter I, I used genomes from across the Lepidoptera to see if diet breadth could be correlated with gene family sizes. I found that two serine protease families were larger in specialists and that a family of glutathione-S-transferases was larger in generalists. Due to the scope of the study, I was unable to associate gene duplication events with any particular host plant toxins. This knowledge gap ultimately led to the development of the work in Chapter IV, which centered specifically on instances of gene duplication and death in the Pieridae that occurred following major chemical changes in hosts. In addition to confirming that NSP-like genes are lost upon shifts away from Brassicales-feeding, we found that a subset of sulfotransferases existed in higher copy number in species that feed on glucosinolates.

Overall, this thesis shows that gene duplication may be important for insect dietary transitions, and that gene duplicates can become specialized to dynamically respond to host plant chemical profiles. It also provides a starting point for future studies, as open questions remain about the role of general detoxification mechanisms during initial transitions on to plants with novel chemical defenses. 

Comparative analyses of gene birth–death dynamics have the potential to reveal gene families that played an important role in the evolution of morphological, behavioral, or physiological variation. Here, we used whole genomes of 30 species of butterflies and moths to identify gene birth–death dynamics among the Lepidoptera that are associated with specialist or generalist feeding strategies. Our work advances this field using a uniform set of annotated proteins for all genomes, investigating associations while correcting for phylogeny, and assessing all gene families rather than a priori subsets. We discovered that the sizes of several important gene families (e.g. those associated with pesticide resistance, xenobiotic detoxification, and/or protein digestion) are significantly correlated with diet breadth. We also found 22 gene families showing significant shifts in gene birth–death dynamics at the butterfly (Papilionoidea) crown node, the most notable of which was a family of pheromone receptors that underwent a contraction potentially linked with a shift to visual-based mate recognition. Our findings highlight the importance of uniform annotations, phylogenetic corrections, and unbiased gene family analyses in generating a list of candidate genes that warrant further exploration.

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.

Coevolutionary innovations are thought to be a large driver of insect and plant biodiversity. Several such innovations have arisen from gene duplication events and subsequent divergence between gene copies, including many adaptations that allow insects to overcome defensive host plant chemistry. However, the role adaptive gene duplicates play in a wider transcriptional framework is still poorly understood. Here, we use short-term feeding assays and CRISPR-Cas9 modified lines of cabbage white (Pieris brassicae) caterpillars to explore how non-functionalization of different members of a family of specialized detoxification genes affects the larval transcriptome at large. We find that the transcriptional response to host plant changes is strongest when all genes in the detoxification family are functional, suggesting that the gene family is part of a larger regulatory response to host plant defences. Further, among individuals lacking specialized detoxification genes, we find that certain general detoxification genes are uniquely upregulated in response to stressful host plant switches. Our results shed light on the importance of transcriptional plasticity in plant-insect interactions and lead to new hypotheses about the initial colonization of mustards by early pierid butterflies.