archives@tulane.edu / The Eastern migratory monarch butterfly undertakes one of the longest annual migrations known among animal taxa, journeying from as far north as Canada down to central Mexico. A small subpopulation of monarchs has been found dropping out of migration in favor of breeding year-round along the U.S. Gulf Coast. The majority of these dropouts, known as winter-breeders, are feeding and breeding on a non-native milkweed species called tropical milkweed (Asclepias curassavica) that has greater concentrations of toxic cardenolides. The effects of tropical milkweed on monarchs are not yet fully understood, but it is correlated to breakage of reproductive diapause and migratory drop-out. The drop-out phenomenon is concerning due to the increased prevalence of infection by the monarch’s specialized protozoan parasite, Ophryocystis elektroscirrha (OE). OE is often physically damaging and can be lethal to monarchs. Here, we investigate the effects of a colder climate that winter-breeders experience on the Gulf Coast in the winter, exposure to OE, and a diet of exclusively tropical milkweed on larval development and adult wing morphology and pigmentation. Morphology and pigmentation are often functional traits that enhance fitness; thus, we use these measures as proxies for fitness components such as flight performance and immune function. In the first chapter, we found that monarchs reared on tropical milkweed developed faster and had larger wings and higher aspect ratios than monarchs reared on a low-cardenolide native milkweed species, swamp milkweed (Asclepias incarnata). In the second chapter, we found that monarchs reared in a suboptimal temperature developed slower and emerged with smaller, darker (redder and more melanized) wings than monarchs reared in a warmer or ‘normal’ temperature. Additionally, exposure to OE affected wing shape such that exposed monarchs had rounder wings with a slightly lower aspect ratio than unexposed monarchs. Lower temperature did not significantly affect melanism in monarch wings, but monarchs exposed to OE had less melanin deposited onto their wings than unexposed monarchs. Together, the findings from both chapters suggest that the conditions experienced by winter-breeders on the Gulf Coast are detrimental to monarch morphology. Smaller, rounder wings with lower aspect ratios are not conducive to migratory success, and paler color or less melanin are potential negative responses to exposure or infection by OE. While monarchs reared on tropical milkweed were larger and slightly redder which is a seemingly positive result, it may be limited to nonmigratory monarchs or winter breeders who may be adapted to tropical milkweed. Thus, it remains unclear how tropical milkweed is affecting the biology of migratory monarchs versus winter-breeders, but we suggest that the colder winter and increased risk of OE infection are negatively affecting winter-breeding populations and possibly migratory behavior. Migratory monarchs are already of conservation concern, and further monitoring and studying of the winter-breeding population and migrant populations are necessary to ensure the stability of monarch populations if and when they encounter tropical milkweed and whether the milkweed is driving population-level shifts in migratory behavior. / 1 / Caitlin Ducat

Given the rapid population decline and recent petition for listing of the monarch butterfly (Danaus plexippus L.) under the Endangered Species Act, an accurate estimate of the Eastern, migratory population size is needed. Because of difficulty in counting individual monarchs, the number of hectares occupied by monarchs in the overwintering area is commonly used as a proxy for population size, which is then multiplied by the density of individuals per hectare to estimate population size. There is, however, considerable variation in published estimates of overwintering density, ranging from 6.9-60.9 million ha(-1). We develop a probability distribution for overwinter density of monarch butterflies from six published density estimates. The mean density among the mixture of the six published estimates was similar to 27.9 million butterflies ha(-1) (95% CI [2.4-80.7] million ha(-1)); the mixture distribution is approximately log-normal, and as such is better represented by the median (21.1 million butterflies ha(-1)). Based upon assumptions regarding the number of milkweed needed to support monarchs, the amount of milkweed (Asciepias spp.) lost (0.86 billion stems) in the northern US plus the amount of milkweed remaining (1.34 billion stems), we estimate >1.8 billion stems is needed to return monarchs to an average population size of 6 ha. Considerable uncertainty exists in this required amount of milkweed because of the considerable uncertainty occurring in overwinter density estimates. Nevertheless, the estimate is on the same order as other published estimates, The studies included in our synthesis differ substantially by year, location, method, and measures of precision. A better understanding of the factors influencing overwintering density across space and time would be valuable for increasing the precision of conservation recommendations.

Monarch butterflies are famous for their annual long-distance migration. Decreasing temperatures and reduced daylight induce the migratory state in the autumn generation of monarch butterflies. Not only are they in a reproductive diapause, they also produce fat deposits to be prepared for the upcoming journey: Driven by their instinct to migrate, they depart from their eclosion grounds in the northern regions of the North American continent and start their southern journey to their hibernation spots in Central Mexico. The butterflies cover a distance of up to 4000 km across the United States. In the next spring, the same butterflies invert their preferred heading direction due to seasonal changes and start their northward spring migration. The spring migration is continued by three consecutive butterfly generations, until the animals repopulate the northern regions in North America as non-migratory monarch butterflies. The monarch butterflies’ migratory state is genetically and epigenetically regulated, including the directed flight behavior. Therefore, the insect’s internal compass system does not only have to encode the butterflies preferred, but also its current heading direction. However, the butterfly’s internal heading representation has to be matched to external cues, to avoid departing from its initial flight path and increasing its risk of missing its desired destination. During the migratory flight, visual cues provide the butterflies with reliable orientation information. The butterflies refer to the sun as their main orientation cue. In addition to the sun, the butterflies likely use the polarization pattern of the sky for orientation. The sky compass signals are processed within a region in the brain, termed the central complex (CX). Previous research on the CX neural circuitry of the monarch butterflies demonstrated that tangential central complex neurons (TL) carry the visual input information into the CX and respond to a simulated sun and polarized light. However, whether these cells process additional visual cues like the panoramic skyline is still unknown. Furthermore, little is known about how the migratory state affects visual cue processing. In addition to this, most experiments studying the monarch butterfly CX focused on how neurons process single visual cues. However, how combined visual stimuli are processed in the CX is still unknown. This thesis is investigating the following questions: 1) How does the migratory state affect visual cue processing in the TL cells within the monarch butterfly brain? 2) How are multiple visual cues integrated in the TL cells? 3) How is compass information modulated in the CX? To study these questions, TL neurons from both animal groups (migratory and non-migratory) were electrophysiologically characterized using intracellular recordings while presenting different simulated celestial cues and visual sceneries. I showed that the TL neurons of migratory butterflies are more narrowly tuned to the sun, possibly helping them in keeping a directed flight course during migration. Furthermore, I found that TL cells encode a panoramic skyline, suggesting that the CX network combines celestial and terrestrial information. Experiments with combined celestial stimuli revealed that the TL cells combine both cue information linearly. However, if exposing the animals to a simulated visual scenery containing a panoramic skyline and a simulated sun, the single visual cues are weighted differently. These results indicate that the CX’s input region can flexibly adapt to different visual cue conditions. Furthermore, I characterize a previously unknown neuron in the monarch butterfly CX which responds to celestial stimuli and connects the CX with other brain neuropiles. How this cell type affects heading direction encoding has yet to be determined. / Monarchfalter sind berühmt für ihre jährlichen Migrationsflüge. Sinkende Temperaturen und die verkürzte Tageslichtbestrahlung induzieren die Migration in einer Herbstgeneration der Monarchfalter. Sie sind nicht nur in reproduktiver Diapause, sondern produzieren Fettreserven für die bevorstehende Reise: Getrieben von ihrem Migrationsinstinkt verlassen sie ihre Schlüpfstätten in den nördlichen Regionen des Nordamerikanischen Kontinents und starten ihre südliche Wanderung zu ihren Überwinterunsgstätten in Zentralmexiko. Dabei legen die Schmetterlinge Strecken von bis zu 4000 km durch die Vereinigten Staaten zurück. Im nächsten Frühling kehren die gleichen Schmetterlinge ihre Vorzugsrichtung durch die jahreszeitlich bedingten Veränderungen um und die Tiere bewegen sich nordwärts. Die Frühlingsgeneration wird insgesamt über drei Schmetterlingsgeneration durchgeführt, bis die Tiere die nördlichen Regionen in Nordamerika wieder als nicht-migrierende Monarchfalter besiedeln. Der Migrationsstatus der Monarchfalter ist genetisch und epigenetisch reguliert, was auch das gerichtete Flugverhalten einschließt. Demnach muss das interne Kompasssystem der Falter nicht nur die bevorzugte, sondern auch die aktuelle Flugrichtung prozessieren. Die interne Repräsentation der Flugrichtung des Falters muss jedoch mit der Umwelt abgeglichen werden, ansonsten droht das Tier von der ursprünglichen Flugrichtung abzuweichen und erhöht das Risiko den Wunschort nicht zu erreichen. Während des Migrationsfluges bieten visuelle Signale verlässliche Orientierungsinformationen. Dabei ist die Sonne ihre Hauptorientierungsreferenz. Zusätzlich zur Sonne nutzen die Schmetterlinge vermutlich noch das Polarisationsmuster des Himmels zur Orientierung. Diese Himmelskompasssignale werden im Gehirn in einer Gehirnregion, den Zentralkomplex, integriert. Vergangene Forschungsprojekte am Zentralkomplex haben gezeigt, dass tangentiale Zentralkomplex-Neurone (TL) die visuellen Signale in den Zentralkomplex leiten und auf eine simulierte Sonne und polarisiertes Licht sensitiv sind. Ob diese Zellen noch weitere visuelle Signale verarbeiten, wie zum Beispiel den Horizont eines Panoramas, ist nicht bekannt. Auch ist der Einfluss des Migrationsstatus auf die visuelle Signalverarbeitung im Zentralkomplex bisher unerforscht. Des Weiteren haben die meisten Experimente am Zentralkomplex des Monarchfalters den Fokus auf die Verarbeitung einzelner simulierter visueller Reize gelegt. Wie aber Kombinationen aus Stimuli im Zentralkomplex verarbeitet werden, ist nicht bekannt.   Diese Dissertation beschäftigt sich mit folgenden Fragen: 1) Wie beeinflusst der Migrationsstatus die visuelle Reizverarbeitung in TL-Zellen im Monarchfaltergehirn? 2) Wie werden mehrere visuelle Reize in TL-Zellen miteinander kombiniert? 3) Wie wird Kompassinformation im Zentralkomplex moduliert? In diesem Zusammenhang wurden TL-Neurone aus beiden Gruppen (migrierende und nichtmigrierende Monarchfalter) elektrophysiologisch mittels intrazellulärer Aufnahmen charakterisiert, während den Tieren unterschiedliche simulierte Himmelkompasssignale und visuelle Szenerien präsentiert wurden. Hierbei konnte ich zeigen dass die TL-Neuronen in migrierenden Tieren ein engeres Tuning zur Sonne aufwiesen, was den Tieren helfen könnte, eine gerichtete Flugrichtung zu halten. Außerdem antworten die TL-Neurone auf ein Panorama, womit der Zentralkomplex in der Lage wäre, Himmelskompasssignale mit terrestrischer Information zu kombinieren. In Experimenten mit zwei kombinierten simulierten Himmelskompasssignalen konnte ich zeigen, dass die TL-Zellen beide Signalinformationen linear miteinander verrechnen. Wenn die TL-Zellen jedoch mit einer visuellen Szenerie stimuliert werden, welche eine simulierte Sonne und ein Panorama beinhaltet, werden die einzelnen visuellen Signale unterschiedlich gewichtet. Die Ergebnisse sind ein Hinweis darauf, dass die Eingangsregion im Zentralkomplex sich flexibel an die visuellen Signalbedingungen anpassen können. Außerdem habe ich ein bis dahin unbekanntes Neuron während meiner Studien charakterisieren können, welches auf simulierte Himmelskompasssignale antwortet und den Zentralkomplex mit anderen Neuropilen im Gehirn verbindet. Wie dieser Neuronentyp Einfluss auf die Kodierung der Flugrichtung nimmt, muss in der Zukunft weiter erforscht werden.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O lÃtex vegetal à produzido e estocado em sistemas de canais formados por cÃlulas altamente especializadas, os laticÃferos. Uma caracterÃstica marcante destes fluidos à a presenÃa de sistemas proteolÃticos complexos. Muitos estudos relatam que proteÃnas de defesa contra insetos e fungos sÃo encontradas em lÃtex. No entanto, alguns insetos sobrepÃem esta defesa e alimentam-se de plantas laticÃferas, como Pseudosphinx tetrio e Danaus plexippus, ambas da ordem Lepidoptera. As bases bioquÃmicas da resistÃncia de insetos Ãs proteÃnas defensivas do lÃtex ainda nÃo sÃo amplamente elucidadas. Esta problemÃtica foi abordada neste trabalho. Inicialmente a atividade proteolÃtica do extrato intestinal de P. tetrio foi caracterizada e avaliada sua capacidade de degradar as proteÃnas do lÃtex de sua planta hospedeira, Plumeria rubra, bem como de plantas laticÃferas nÃo hospedeiras (Calotropis procera e Cryptostegia grandiflora). AlÃm disso, foi avaliado se inibidores de proteases de fluidos laticÃferos (C. procera, P. rubra e Cr. grandiflora) inibem as proteases intestinais de P. tetrio e D. plexippus e vice-versa. Em adiÃÃo, foi analisado o efeito de enzimas laticÃferas sobre a membrana peritrÃfica (MP) de D. plexippus. As proteases intestinais de P. tetrio sÃo predominantemente do tipo serÃnica e suas atividades sÃo maiores em pHs bÃsicos. O extrato intestinal de P. tetrio rapidamente e completamente digeriu as proteÃnas do lÃtex de sua planta hospedeira e de C. procera, bem como digeriu parcialmente as proteÃnas do lÃtex de Cr. grandiflora. Larvas de D. plexippus se desenvolveram plenamente quando alimentadas com dieta artificial contendo 1% das fraÃÃes proteicas dos lÃtex de plantas nÃo hospedeiras. Ensaios in vitro indicaram que ambos, extratos intestinais e lÃtex das espÃcies em estudo, possuem inibidores de proteases serÃnicas e cisteÃnicas. Inibidores provenientes dos fluidos laticÃferos em estudo inibiram a atividade proteolÃtica dos extratos intestinais de ambas as larvas. Entretanto, anÃlise in vivo demonstrou que estes inibidores nÃo afetam o desenvolvimento de D. plexippus. Somente o extrato intestinal de D. plexippus apresentou atividade inibidora de proteases do lÃtex de sua planta hospedeira (Calotropis procera). Apenas discretas mudanÃas foram observadas no perfil proteico das MPs de D. plexippus submetidas Ãs fraÃÃes proteicas dos fluidos laticÃferos in vivo, enquanto que o tratamento in vitro resultou em danos mais acentuados. A partir dos resultados obtidos conclui-se que proteÃlise e a inibiÃÃo de proteÃlise fazem parte do sistema defensivo das larvas especialistas em plantas laticÃferas e de suas plantas hospedeiras. Embora inibidores de proteases de fluidos laticÃferos sejam capazes de inibir as proteases intestinais das larvas (in vitro), in vivo, a habilidade das proteases intestinais em prontamente digerir as proteÃnas do lÃtex parece ser crucial para a sobreposiÃÃo da defesa vegetal. / Plant latex is produced and stored in channels formed by highly specialized cells structures. A remarkable feature of these fluids is the presence of complex proteolytic systems. Many studies report that latex possesses a variety of defense proteins against insects and fungi. However, some insects overlap this defense and feed on latex-producing plants, for example Pseudosphinx tetrio and Danaus plexippus, both of the order Lepidoptera. The biochemical aspects of insect resistance to latex defense proteins are still not widely elucidated. This issue was addressed in this work. Initially, the proteolytic activity of Pseudosphinx tetrio gut was characterized and evaluated in its ability to degrade latex proteins of its host plant (Plumeria rubra) and non-host plants (Calotropis procera e Cryptostegia grandiflora). Furthermore, we assessed whether protease inhibitors from latex fluids (C. procera, P. rubra and Cr. grandiflora) inhibit intestinal proteases from P. tetrio and D. plexippus and vice versa. The effect of latex enzymes on peritrophic membrane (PM) of D. plexippus was also assessed. Intestinal proteases from P. tetrio are predominantly of serine type and their activities are higher in basic pHs. P. tetrio gut proteases rapidly and completely digested latex proteins of its host plant and C. procera and partially digested proteins from Cr. grandiflora. D. plexippus larvae were not affected when fed on artificial diet containing latex proteins (1%) from non-host plants. In vitro assays detected serine and cysteine peptidase inhibitors in both gut homogenates and latex fluids. Protease inhibitors from latex inhibited the proteolytic activity of gut homogenates of both larvae. Nevertheless, in vivo analysis demonstrated that latex inhibitors do not affect the development of D. plexippus. Only the gut homogenate from D. plexippus showed inhibitory activity towards proteases latex from its host plant (Calotropis procera). Slight changes were observed in the protein profile of the PMs from D. plexippus subjected to latex protein fractions in vivo, whilst in vitro treatment resulted in more severe damage. This study concludes that proteolysis and inhibition of proteolysis are involved in the defensive systems of both caterpillars and their host plants. Even though latex peptidase inhibitors inhibit gut peptidases (in vitro), the ability of gut peptidases to promptly digest latex proteins (in vivo) regardless of their origin seems to be a pivotal event favoring caterpillars overcoming plant defense.

The eastern migratory population of monarch butterflies (Danaus plexippus plexippus) has declined by >80% within the last two decades. One possible cause of this decline is the loss of >= 1.3 billion stems of milkweed (Asclepias spp.), which monarchs require for reproduction. In an effort to restore monarchs to a population goal established by the US Fish and Wildlife Service and adopted by Mexico, Canada, and the US, we developed scenarios for amending the Midwestern US landscape with milkweed. Scenarios for milkweed restoration were developed for protected area grasslands, Conservation Reserve Program land, powerline, rail and roadside rights of way, urban/suburban lands, and land in agricultural production. Agricultural land was further divided into productive and marginal cropland. We elicited expert opinion as to the biological potential (in stems per acre) for lands in these individual sectors to support milkweed restoration and the likely adoption (probability) of management practices necessary for affecting restoration. Sixteen of 218 scenarios we developed for restoring milkweed to the Midwestern US were at levels (>1.3 billion new stems) necessary to reach the monarch population goal. One of these scenarios would convert all marginal agriculture to conserved status. The other 15 scenarios converted half of marginal agriculture (730 million stems), with remaining stems contributed by other societal sectors. Scenarios without substantive agricultural participation were insufficient for attaining the population goal. Agricultural lands are essential to reaching restoration targets because they occupy 77% of all potential monarch habitat. Barring fundamental changes to policy, innovative application of economic tools such as habitat exchanges may provide sufficient resources to tip the balance of the agro-ecological landscape toward a setting conducive to both robust agricultural production and reduced imperilment of the migratory monarch butterfly.

Managing Monarch butterfly overwintering groves: making room among the eucalyptus Proper management and conservation of the coastal California overwintering sites used by western Monarch butterflies (Danaus plexippus L.) is critical for continued use of these sites by monarchs. Many management efforts are currently concentrating on eucalyptus-only sites because of the prevailing notion that monarchs prefer eucalyptus over native tree species. Yet, whether a preference exists or not has never been tested. Herein, we test the “eucalyptus preference” hypothesis with data from five overwintering sites comprised of blue gum eucalyptus (Eucalyptus globulus) and at least one other native tree species from fall 2009 to spring 2012. We found that when monarchs clustered disproportionately on a tree species relative to its availability, they clustered significantly more than expected on native trees and significantly less than expected on eucalyptus. Also, in years when the overwintering population was highest, monarchs clustered disproportionately on native conifers, and they often switched from clustering on eucalyptus in the early winter to native conifers in the middle or late winter. Our results suggest that overwintering groves should be managed to include a mixture of tree species. We cannot recommend simply planting more eucalyptus. At overwintering sites in central coastal California, native conifers such as Monterey cypress (Hesperocyparis macrocarpa) and pitch canker-resistant Monterey pine (Pinus radiata) should be planted as replacements for blue gum eucalyptus in areas where trees are likely to fall, and around the perimeter of groves. Testing the Monarch butterfly eucalyptus preference hypothesis at California overwintering sites Western Monarch butterflies (Danaus plexippus L.) overwinter in groves of native and non-native trees along the California coast. Eucalyptus is abundant in coastal counties, and overwintering monarchs utilize this type of tree more than any other. This has led to the belief that monarchs prefer eucalyptus. Yet whether a preference exists has never been tested. We tested the “eucalyptus preference” hypothesis at five California overwintering sites with canopies comprised of eucalyptus and at least one native conifer species. We found that at no time over the course of three years did monarchs cluster on trees in proportion to their availability in the canopy. Overall, they did not cluster on one tree species significantly more frequently than another, indicating that monarchs do not prefer eucalyptus—or any tree species—all of the time. However, more often than not monarchs clustered significantly more than expected on native trees, particularly at midseason when the weather was most inclement. They also clustered disproportionately on native conifers when the overwintering population size was highest. At most sites monarchs exhibited tree switching, shifting from eucalyptus to native conifers in the middle or late winter. Based on these results, we reject the “eucalyptus preference” hypothesis. In its place, we propose the “conditional preference hypothesis”, wherein monarchs are predicted to prefer cluster trees according to microclimate conditions and prefer alternate trees within a site as climatic conditions change. Rejection of the eucalyptus preference hypothesis suggests that sites comprised exclusively of eucalyptus may not offer monarchs a suitable range of microhabitats, and further suggests we should rethink “eucalyptus-centric” management. Monarch butterflies overwintering in coastal California: low site fidelity and high intersite movement Western monarch butterflies (Danaus plexippus L.) overwinter in large aggregations at hundreds of sites along the California coast. Management plans and census methods are both founded on the assumption that individual monarchs arrive at an overwintering site in the fall and stay at that site for the winter. Though populations potentially coalesce en masse from autumnal sites onto climax overwintering sites, very little individual movement between sites is inferred. Monarch movement is therefore thought to be primarily into sites (as opposed to out of or among them). We refer to this assumption and inference as the accrual hypothesis. In light of previous studies that provide evidence for movement among sites, we propose that overwintering monarchs may belong to a superpopulation. The existence of a superpopulation comprised of individuals moving in and out of sites would force us to rethink our ideas of landscape-level resource use by monarchs, our site-centric (rather than landscape-level) management strategies, and our abundance estimation techniques, which employ closed population models. We tested the closed population model, the accrual hypothesis, and the superpopulation model at three California overwintering sites using a mark-resight study design. We found that a large proportion of the monarchs at a site moved among (into and out of) monitored sites, both while the population size increased in October and November, and while the population appeared to exhibit an equilibrium winter maximum. The pattern of abundance of both tagged and untagged monarchs at monitored sites leads us to reject the closed population model and the accrual hypothesis. We found that monarchs at all three study sites are part of a larger superpopulation, though the sites do not contribute to the superpopulation equally. We determined that mark-resight is a viable alternative to existing population estimation techniques, though mark-resight methods would need to be explored further before being applied routinely. Our results suggest we need to move away from site-based management and manage instead for landscape-level overwintering (superpopulation) dynamics.

Butterfly weed (Asclepias tuberosa) is a primary larval food source for the monarch butterfly (Danaus plexippus). Planting more butterfly weed may stimulate declining monarch populations. To that end, a habitat suitability map was created for Mississippi in GIS using soil pH, soil texture, and land cover. Herbarium data were derived from the Southeast Regional Network of Expertise and Collections (SERNEC) database. Environmental data were from the USDA National Resource Conservation Service geospatial data gateway. Frequency analysis was used to assign scores to environmental variables of SERNEC occurrences using a suitability index. Global positioning systems (GPS) locations of butterfly weed were collected to validate the model. The most suitable model with 78.9% of GPS points in medium to high suitability was a weighted sum overlay with land cover 50%, soil pH 25%, and soil texture 25%. The suitability map may enable conservationists to identify suitable sites for butterfly weed in Mississippi.

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.