Uncovering Skin Immune Proteins as Predictors of ResistanceAagainst WNS

Uncovering Skin Immune Proteins as Predictors of ResistanceAagainst WNS Uncovering skin immune proteins as predictors of resistance against WNS Uncovering skin immune proteins as predictors of resistance against WNS Marianne Moore, Liliana Dvalos, and Amy Russell Objectives: Our central aim is to test the hypothesis that the composition of bat skin immune proteins predicts resistance to white-nose syndrome (WNS) within and among species. Our goal is to discover the mechanisms underlying the survival of remnant populations in the WNS-affected area. To this end, we will characterize the diversity and relative abundance of skin immune proteins of five bat species that vary in observed rates of WNS-associated mortality, including the little brown myotis (Myotis lucifugus), big brown bat (Eptesicus fuscus), southeastern myotis (M. austroriparius), gray bat (M. grisescens), and Virginia (VA) big-eared bat (Corynorhinus townsendii virginianus). We will use high throughput protein sequencing (proteomics) to isolate, identify, and quantify skin immune proteins. Proteomic repertoires will then be compared across species to test the prediction that certain proteins related to anti-fungal responses are more prevalent in species that appear to suffer less from the effects of WNS, such as the gray bat and the VA big-eared bat. The little brown myotis and big brown bat will be more extensively sampled both within and outside of the WNS-affected area and their protein profiles will be compared across sites to test the prediction that proteins prevalent in survivors of more highly susceptible species are similar to those found in resistant species. Microsatellite genotyping will also be used to quantify levels of relatedness among sampled individuals, and this will allow for functional and adaptive similarity in immunological proteins to be differentiated from similarity due to common descent. We will focus on a set of proteins known as antimicrobial peptides (AMPs), which are known to kill or inhibit the growth of invading microorganisms such as fungi, but our analyses will also capture all other skin immune proteins. Our secondary aim is to investigate the potential for using host derived anti-fungal AMPs to mitigate WNS. To this end, we will select AMPs identified in resistant species, and in surviving populations of susceptible species, and will use growth-inhibition assays with cultured Pseudogymnoascus destructans (Pd) to test the ability of AMPs to kill the WNS etiologic agent. Strengths: We focus on the skin because Pd invades and destroys this organ. Effective immune responses against Pd must be initiated in this organ. AMPs have been selected as a primary focus because, unlike effectors of the adaptive immune system, they are constitutively expressed and likely to remain functional during hibernation. Many AMPs also have direct anti-fungal properties. These peptides are associated with resistance to chytridiomycosis in amphibians and have shown therapeutic potential against human fungal skin infections. Proteomics provides an efficient method to identify all expressed proteins in skin including those that are unique to a population and/or species. By genotyping all individuals, we will be able to account for genetic relatedness when modeling the effect of the proteomic composition of the skin on survivorship. We will use a relatively non-invasive method of tissue sample collection (i.e., small wing biopsies) that will allow us to study the endangered gray and VA big-eared bats, which may provide important clues to the nature of remnant populations, but cannot be studied using the invasive methods often required for immunological studies. Furthermore, biopsies will be collected during the active season when wound healing is maximal and can be collected by other researchers in combination with sampling for other studies. This approach will protect sensitive hibernating populations, significantly reduce the total number of bats handled, and make the proposed fieldwork more cost-efficient. Our team is particularly well equipped to conduct this study. Previous research by PI Moore demonstrated that the little brown myotis attempts immunological resistance against Pd and that big brown bats are relatively more resistant to infection by Pd compared to little brown myotis. Co-PI Russell has demonstrated that a small number of microsatellite markers can successfully characterize the variation of populations of the little brown myotis. Finally, co-PI Dvalos has demonstrated the use of proteomics with very small starting samples and its application to uncovering immune responses in mammals. Implications for Management: This study will directly relate to priorities identified in the WNS National Plan by (1) improving our understanding of why some species succumb to the disease while others appear completely resistant to the effects of WNS, and (2) by investigating anti-fungal skin proteins as a potential control method. Our approach will provide the perfect synergy between research and management since our findings will elucidate mechanisms underlying the differential impacts of WNS within and between species and help direct management efforts, while at the same time investigating a potential mitigation strategy to directly control the effects and spread of this devastating disease. Total Budget: The duration of this study will span three years with a budget of $159,560 for Year One and $158,895 for Year Two for a total cost of $318,455. Year Three is being requested as a no-cost extension.