Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

Summer 2020

Thesis Director

Jeffrey R. Laursen

Thesis Committee Member

Barbara S. Carlsward

Thesis Committee Member

Eloy Martinez

Abstract

Bluegills (Lepomis macrochirus) are common intermediate hosts for white grub (Posthodiplostomum minimum). They tolerate heavy infections with minimal effect on condition and continue to accumulate metacercariae as they age. This suggests that any immune response to this parasite might not be effective. This study was conducted to better understand the immune mechanisms underlying P. minimum infection in bluegills.

Infected organs (liver, kidney, and heart) were examined histologically, and serum from infected fish was tested for antibodies to white grub. Juvenile flukes were recovered from isolated metacercarial cysts. Polyclonal antibodies were produced in mice against white grub homogenate, bluegill serum, bluegill Immunoglobulins (IgM), green sunfish serum and bluegill mucus. These were used in serological assays (dot blots, Indirect ELISA, and Western Blots) to examine the immunological response of infected bluegills to the white grub antigen prep, as well as potential cross reactivity with other trematode antigens.

Histological assessment of infected tissues revealed P. minimum in double walled cysts surrounded by a clear zone and mild leucocytic infiltration. Melano-macrophage centers, characterized by brown pigmentation of cells, were observed in the surrounding tissues. In the heart, P. minimum metacercariae were confined to the epicardium.

There were shared epitopes between the white grub antigen prep and fish proteins, when tested using mouse antibodies against fish tissues. Although, mouse anti-white grub did not recognize fish proteins, it bound to shared epitopes in the white grub and yellow grub (Clinostomum sp.) preps. The ELISA assay proved unreliable. Absorbance values showed a general decrease to background levels across a serial dilution series. However, there was high variability between fish, and there was no difference in absorbance values between heavily versus lightly infected fish. I attempted to identify specific proteins that were recognized by serum from infected bluegills using western blot analysis. Rather than seeing additional bands when bluegill serum was added prior to mouse anti-bluegill serum, results revealed no bands. This implied that the ELISA absorbance was not due to fish antibodies binding to white grub antigens.

Thus, we could not detect specific systemic antibodies to P. minimum in infected fish, and this effective absence may have contributed to the persistent heavy infections commonly observed. Conversely, melanization an innate immune mechanism may be the prominent immunological reaction to white grub infection in bluegills. An examination of skin mucus is recommended, as this may provide insights into mucosal antibodies and their potential roles in P. minimum infection.

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