Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Vasyl V. Tkach

Abstract

Digeneans are endoparasitic flatworms with complex life cycles that involve two or more different animals as definitive and intermediate hosts. Some digenean species harbor bacterial endosymbionts belonging to the genus Neorickettsia (Order: Rickettsiales, Family: Anaplasmataceae). Neorickettsia occur in all life cycle stages of digeneans and are maintained by vertical transmission. Far from benign however, Neorickettsia may also be transmitted horizontally by digenean parasites to their vertebrate definitive hosts. Once inside, Neorickettsia can infect macrophages and other cell types. For some vertebrate species (e.g. dogs, horses and humans), neorickettsial infections cause severe disease. With a few exceptions, studies of Neorickettsia have been traditionally carried out by bacteriologists, medical, and veterinary researchers, while helminthologists have rarely participated in these research endeavors. Despite the in-depth research published on different aspects of molecular biology, immunology, diagnostics and treatment of neorickettsiae and neorickettsial diseases, the quantitative aspects of transmission of these bacteria and their ecological and evolutionary interrelationships with their invertebrate and vertebrate hosts have received little attention. Recent progress in molecular techniques, particularly the polymerase chain reaction (PCR) and DNA sequencing has made possible the efficient and reliable detection of Neorickettsia at every step of their circulation, whether in the digenean host of the neorickettsiae or in the invertebrate and vertebrate hosts of the digenean. The same technology also allows for reliable identification of the digeneans. Taken from a mostly parasitological,

perspective, this study focused on the modes and quantitative aspects of Neorickettsia transmission and co-evolution with their digenean hosts through the use of molecular techniques.

To understand the biology and evolution of Neorickettsia within the digenean host we focused on four specific aims: 1) screen for Neorickettsia DNA a large collection(s) of diverse Digenea taxa from a wide variety of hosts and a broad geographic range using molecular methods and conduct molecular phylogenetic analyses of neorickettsiae in order to estimate interrelationships among all available genotypes; 2) develop and maintain a laboratory life cycle of a digenean, Plagiorchis elegans, harboring Neorickettsia sp.; 3) assess and quantify Neorickettsia vertical transmission efficiency through all stages of a digenean life cycle; 4) localize the bacterial endosymbiont within all stages of the digenean life cycle using immunofluorescent microscopy.

In this study (specific aim 1) we screened more than 3,000 digenean samples for Neorickettsia collected from various vertebrates and invertebrates in terrestrial, freshwater, brackish and marine habitats from multiple countries and continents. Neorickettsiae were detected using a real-time PCR protocol targeting the GroEL gene and verified with nested PCR and sequencing of a 1371 bp long region of 16S rRNA. Twenty isolates of Neorickettsia have been obtained. Bayesian phylogenetic analyses were conducted to estimate interrelationships among all known species/genotypes of Neorickettsia. We identified 14 new genotypes of Neorickettsia, more than doubling the number of known species level lineages. Additionally, we identified 14 new digenean species and 7 digenean families as hosts of Neorickettsia. Our findings suggest that further surveys from broader geographic regions and wider selection of digenean taxa are likely to reveal new Neorickettsia lineages as well as new digenean host associations and geographic records.

To accomplish specific aims 3 and 4 we for the first time, have maintained Neorickettsia sp. through multiple generations in the laboratory life cycle of a digenean, Plagiorchis elegans (aim 2). The laboratory life cycle of P. elegans consists of a snail first intermediate host, Lymnaea stagnalis, an aquatic arthropod second intermediate host, Culex pipiens (mosquito larva), and a vertebrate definitive host, Mesocricetus auratus (Syrian hamster). Using the newly developed laboratory life cycle we were able to quantify the number of bacteria within individual parasites at all stages of the digenean life cycle. To accomplish this we developed a quantitative real-time PCR assay targeting a 152 bp fragment of the heat shock protein coding gene, GroEL, using a g-block synthetic quantitative positive control. Furthermore, the laboratory life cycle has allowed us to localize the bacterial endosymbiont within eggs, sporocysts, cercariae, metacercariae, and adults of the digenean P. elegans, using immunoflourescent microscopy. Interestingly, unlike other genera of bacteria within the family Anaplasmataceae, Neorickettsia is not localized within the ovarian cells. The bacteria, is instead maintained from one generation of the digenean to another by infecting the vitellarium.

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