The fundamental challenge to prevent species from going extinct is difficult but of grave importance. Halting species from going extinct minimizes the loss of biodiversity. One way of researching biodiversity is by studying species on a genetic level. This creates a dilemma as studying species genetically often requires using destructive sampling and is not desirable or even allowed when studying threatened species. Thus, there is a necessity for alternate sampling methods. In this study both non-lethal and lethal methods were used to gather tissues from the butterfly species Vanessa cardui. The DNA extractions turned out to give varying amounts of DNA, but it was successfully extracted from all the different tissue types. Amplifiable DNA was successfully gained using PCR and confirmed using gel electrophoresis. Existing and newly designed primers for multi-copy genes were used and several of them gave amplifiable DNA. Even if amplifiable DNA has been obtained in other studies, using various tissues, it turned out to only work with a live butterfly’s body in this study.

Migratory insects are vital components of global ecosystems and provide important ecosystem services, yet the migration phenomenon is understudied in insects compared to vertebrates. In this thesis, I aim to deepen our understanding of insect migration, using the monarch butterfly Danaus plexippus (L.) and the painted lady butterfly Vanessa cardui (L.) as model systems. Studying insect migration is notoriously difficult given the small size, high abundance, and short lifespans of insects. Isotope geolocation has shown promise for overcoming these obstacles. Here, I develop and apply metals and metal isotopes, specifically strontium isotope ratios (⁸⁷Sr/⁸⁶Sr), to increase the spatial precision of isotope geolocation and demonstrate how isotopic geolocation tools can advance our understanding of insect migration at the population level. In the first chapter, I test the validity of using ⁸⁷Sr/⁸⁶Sr, lead isotopes, and a suite of 23 metals and metalloids to estimate the natal origins of migratory insects, by investigating the pathways of metal incorporation into butterfly wing tissues. Using an 8-week diet-switching experiment, I show that the concentrations of many metals in insect wings can be altered through the adult diet or dust deposition, making them poor candidates for geolocation but potentially interesting tools to study insect physiology, diet, or toxicology. For example, lead was found to accumulate on butterfly wings from external sources, and lead isotopes could potentially be used to quantify the exposure of migratory insects to metal pollution. Some metals, including Ba, Cs, Mg, Na, Rb, Sr, Ti, Tl, and U, are good candidates for developing geolocation tools. I focused on ⁸⁷Sr/⁸⁶Sr and demonstrated that, despite some caveats, this tool is valid for isotope geolocation. In the second chapter, I outline the steps required to use ⁸⁷Sr/⁸⁶Sr for the geolocation of insects, including the calibration of a spatial model of isotopic variation (i.e., an isoscape) using random forest regression. I then combine hydrogen isotope values (δ²H) and ⁸⁷Sr/⁸⁶Sr into a dual assignment framework to estimate the natal origins of a single generation of monarch butterflies in eastern North America. I demonstrate that combining these two isotopes provides a more spatially constrained estimate of natal origin than using either isotope alone. In the third chapter, I apply this framework to characterize the migratory patterns and migratory connectivity of an insect species across a geographical barrier, the Sahara. Painted ladies journeying northwards across the Sahara appear to do so in a gradual progression, although spatiotemporal sampling limitations prevented a complete characterization of this movement. In contrast, painted ladies migrating southwards appear to journey in a broad front, parallel migration pattern with little longitudinal movement. Evidence for a leapfrog migration pattern was found in the western region, wherein butterflies of northernmost origin journey farther south than butterflies bred in more southerly regions. This leapfrog migration pattern suggests distinct migratory behaviours within painted lady butterflies wherein some individuals migrate longer distances than others. In the fourth chapter, I apply isotope geolocation to characterize the migration distances of multiple individuals and assess the potential genetic differentiation of butterflies migrating distinct distances. I use δ²H and ⁸⁷Sr/⁸⁶Sr-based geographic assignment to confirm that some painted ladies migrate up to 4,000 km from Europe to sub-Saharan Africa, while others migrate shorter distances from Europe to the circum-Mediterranean region. Despite these differences in migration distance, genome-wide analysis revealed a lack of adaptive variation between short- and long-distance migrants. Instead, variation in migration distance in painted lady butterflies is likely the result of a plastic response to environmental conditions. Overall, the methodological developments presented in this thesis are a step forward in studying insect migration. The development and application of metals and metal isotopes for insect geolocation opens new avenues to study the migration phenomenon at different scales with widespread relevance for conservation and pest management.