LINKING SPECIES, SPACE, AND SURVEILLANCE: ADVANCING TOOLS FOR WILDLIFE DISEASE ECOLOGY

Abstract

Zoonotic spillover events are facilitated and influenced by numerous ecological, environmental, biological, and behavioral factors. Because spillover events occur in a complex and multifactorial system, it is essential to gather field-based data to inform ecological models of disease dynamics. In Chapter 1, I review two processes that modulate zoonotic disease, biodiversity and vector movement, and provide a review of current tools used to track bats and mosquitoes. Horseshoe bats are known reservoirs of zoonotic viruses, yet cryptic species complexes within the genus hinder accurate host identification during virus surveillance. In Uganda, two sympatric morphotypes of bats in the Rhinolophus fumigatus/eloquens species complex, one small and one large biometric profile, were observed co-roosting in caves. In Chapter 2, I integrate biometric, genetic, and acoustic analyses to investigate their evolutionary relationship with other Afrotropical horseshoe bats. Maximum-likelihood phylogenies of mitochondrial cytb revealed paraphyly, with the small morphotype closely related to the large morphotype within the Rhinolophus fumigatus/eloquens species complex. Additional phylogenies inferred from nuclear introns revealed mito-nuclear discordance: morphotypes shared similar mitochondrial cytb sequences but dissimilar nuclear intron sequences. These findings are consistent with evolutionary processes such as introgression or incomplete lineage sorting. Biometrics and acoustic data congruent with nuclear intron phylogenies support two distinct lineages, including a previously undescribed small profile lineage. Viral screening and sequencing identified coronaviruses, including a sarbecovirus (Coronaviridae, Betacoronavirus, Sarbecovirus) in both lineages. These data suggest cross-species viral circulation among co-roosting horseshoe bat populations. Our findings highlight the importance of integrative taxonomy for accurate host identification and provide new insights into viral ecology of bat-associated coronaviruses in cave roosting horseshoe bats. Very little is known regarding Afrotropical horseshoe bat movement patterns, in part due to their small size limiting tracking methods. Because bat foraging behavior, movement relative to landscape structures, and the impact of land-use change on flight paths has implications for both conservation policies and zoonotic disease prevention, it is important to gather data on how horseshoe bats move during short-term foraging bouts and long-term seasonal dispersal. In Chapter 3, I evaluate the utility of miniature store-on-board GPS data loggers to collect information on nightly foraging behavior and long-term trends in foraging locations. A total of 36 GPS units were attached to horseshoe bats from two caves in eastern Uganda, 8 of which were recovered and yielded information on foraging trends in dry and wet seasons. Units were recovered from recaptured bats several days after deployment and from the cave floor, presumably after it was naturally discarded. No long-term data were collected as units were shed sooner than had been reported for insectivorous bats in temperate climates. We found no difference in recapture rates between GPS-tagged and untagged bats. This data represents the first of its kind in tracking horseshoe bats in the region and offers insights into foraging and flight patterns for horseshoe bats bordering a protected national park. Mosquito Mark-Release-Recapture studies are essential for gathering information on population size, dispersal, survivorship, and assessing mosquito control measures. Current marking techniques are limited by labor or materials costs, longevity, impact on mosquito survival and behavior, or limited diversity of markers. In Chapter 4, I conduct a pilot field trial and supplementary laboratory trials to evaluate the effectiveness of DNA-loaded microcrystals as a novel marking strategy for larval Culex spp. vectors in Fort Collins, CO, USA. Larval marking of mosquito vectors in field conditions was stymied by contamination events and limited mark-success (3.3%); however, wild Culex spp. mosquito vectors successfully colonized marking bins during both field trials (7/12 and 7/14 in 2021 and 2022, respectively). Laboratory trials identified potential external marking of adult mosquitoes exposed to crystal-marked water, artificially inflating mark-success in laboratory conditions. Crystal imaging identified large (up to 380um) microcrystal clusters and the presence of unstructured protein agglomerate competing for guest DNA, which may contribute to low-mark-success in larvae. Study results suggest additional optimization and batch control is required, though the technology may be useful for passive marking of adult mosquitoes. In Chapter 5, I offer concluding remarks on insights gleaned from these investigations within the broader context of pathogen surveillance, disease ecology, and wildlife conservation. Collectively, these chapters represent the application of innovative tools and approaches to surveil and track vertebrate and invertebrate vectors of viral pathogens. The information derived from these studies has resulted in higher taxonomic resolution for a cryptic horseshoe bat species complex, a deeper understanding of Afrotropical horseshoe bat foraging patterns, and revealed challenges in the development of novel mosquito marking technologies. The work provides valuable insights and data to inform predictive models and develop better tracking methods for vectors.