James Graves, Jr.
The University of Georgia
College of Veterinary Medicine
Class of 2019
Characterization of Dirofilaria immitis amphids by fluorescent staining
James L. Graves Jr., Christopher C. Evans, Bobby E. Storey, Andrew R. Moorhead, and Adrian J. Wolstenholme
Department of Infectious Diseases (Graves Jr., Evans, Storey, Moorhead, Wolstenholme), Center for Tropical and Emerging Global Diseases (Wolstenholme), University of Georgia, Athens, GA
Macrocyclic lactone (ML) resistance to canine heartworm (Dirofilaria immitis) has been documented. Heartworms arrive in the heart and pulmonary artery after a lengthy migration through host tissues, and while the mechanism of parasite killing by MLs is still poorly understood, it is possible the parasite’s ability to migrate normally is interrupted in this process. Amphids are sensory neurons in nematodes used to navigate the environment and respond to stimuli. The purpose of this study is to test the hypothesis that D. immitis amphid structures are altered in ML-resistant parasites, as previously observed in other species. To test this hypothesis, D. immitis third-stage larvae were stained with DiO and DiI. Then, amphid staining was assessed visually using fluorescence microscopy. Caenorhabditis elegans served as a positive control, as its amphids were successfully stained using standard procedures. While established methods were unsuccessful in staining D. immitis, the procedures were modified in attempt to obtain clear amphid staining. Collagenase treatment, molting to fourth-stage larvae, and incubation in different media with EDTA did not improve stain uptake. However, the most successful procedure involved staining D. immitis during overnight incubation with DiO at 378C, which yielded fluorescent staining localized at the amphids. Further development of an amphid staining procedure in D. immitis will allow for better characterization of amphid structure between susceptible and resistant isolates. This information could aid in determining the role of amphids and migration in the ML mode of action.
Research Grant: Filariasis Research Reagent Resource Center (FR3)
Student Support: NIH Office of Research Infrastructure Programs, Grant Number 2T35OD010433-11