Dissertation. Candidate of Science: The effect of defense responses of snails on development of trematode partenitae (with a focus on the family Echinostomatidae) (Alina Avanesyan, 2002; Herzen State University, St. Petersburg, Russia)
Advisor: Dr. Gennady Ataev, Professor, Department Chair, Department of Zoology, Herzen State University, St. Petersburg, Russia
The focus of my Candidate of Science dissertation was cellular immune response of Biomphalaria snails to infection by Echinostoma trematodes. Biomphalaria snails are freshwater pulmonate snails, native to Caribbean and South America. In my research, I used two species, B. glabrata and B. pfeifferi. Biomphalaria glabrata has been a primary model species for investigating snail defense mechanisms because it is an intermediate host for the human blood fluke, Schistosoma mansoni, a dangerous parasite that infects millions of people worldwide and causes the disease that is called schistosomiasis. For my research, I used other, less dangerous, but still wide-spread trematodes – Echinostoma caproni and E. paraensei, which are intestinal parasites of mammals and birds and which also undergo their larvae development in Biomphalaria snails. In previous studies on B. glabrata, two strains of this species have been identified: a strain which exhibits resistance to infection by E. caproni (my primary study species) and a strain which is susceptible to infection by this trematode. In my research, I was interested in exploring the role of hemocytes in snail defense mechanisms and how a hemocytic response might differ between resistant and susceptible snails.
I worked under the direction of Dr. Gennady Ataev in the Laboratory of Experimental Zoology at the Department of Zoology at Herzen State University. Snail defense mechanisms to infection by trematodes was the main focus of the lab research. For my dissertation project, my primary objectives were (1) to identify and characterize the hematopoietic tissue in B. glabrata snails, (2) to compare the structure, mitotic activity of the hematopoietic tissue, as well as encapsulation of parasites by hemocytes in resistant and susceptible strains (both qualitatively and quantitatively), and (3) to perform morphological analysis of Echinostoma larvae (sporocyst) development in B. glabrata snails.
By the time I started this project, the location of the hematopoietic tissue in invertebrates, and particularly in snails, was poorly understood. We found that the hematopoietic tissue in Biomphalaria snails (so called amebocyte-producing organ, or APO) is located between the pericardium and the mantle epithelium. APO is composed of numerous cellular aggregations; they are located near the external surface of the pericardium and lacunas of the blood system. The number of cell aggregations In non-infected snails did not exceed 5; the cross-sectional area of each aggregation ranged from 25-50 µm. The aggregations varied from round, oval, and elongated to amoeboid shape, and comprised of clustered cells with the basophilic cytoplasm and oval-shaped nuclei.
To further explore defense mechanisms B. glabrata snails, we performed a comparative histological analysis of the structure and mitotic activity of the hematopoietic tissue, as well as migration of hemocytes from the APO to the location of E. caproni sporocyst in resistant and susceptible snails. Snails were dissected in certain time intervals (from 1h to 30d post infection); tissues were then fixed and embedded in paraffin. Sections (5 µm thick) were prepared using a microtome, stained (using Erlich’s hematoxylin-eosin), and screened under the microscope. We found that there was a greater cellular immune response in resistant snails compared to that in susceptible snails – i.e. increased cell proliferation in the APO, increased migration of hemocytes to the location of the sporocyst, and successful encapsulation of the sporocyst. In susceptible snails, however, even though we also observed increased cell proliferation in the APO and aggregations of hemocytes near the sporocyst, this cellular response had never resulted in the encapsulation of the parasite, and the sporocyst continued its development and migration to the snail heart area.
As part of my dissertation, I explored development of Echinostoma trematodes in Biomphalaria snails. I focused on the germinal elements of Echinostoma miracidia and performed comparative morphological analysis of germinal cells development in E. caproni and E. paraensei. Germinal material in Echinostoma miracidia is represented by germinal cells (primary and/or secondary) and undifferentiated cells. The germinal cells are quite large (cross-sectional area is 27.0 ± 0.8 µm2), with a large bubble-shaped nucleus and a basophilic cytoplasm. We found that miracidia of E. paraensei already contained embryos, and their sporocysts released mother rediae a few days earlier than sporocysts of E. caproni. Overall, our analysis of germinal cells development provided additional support to previous findings that species E. caproni and E. paraensei distinct species.
Additionally, I explored the cellular composition and activity of the hematopoietic tissue in B. pfeifferi, as well as several local gastropods, such as Planorbis, Planorabarius, and Succinea snails, and collected data on the temperature effect on the trematode development in their intermediate hosts.
I worked on this project while I was a part-time researcher at the Institute of Cytology, RAS in St. Petersburg, Russia. I worked at the Laboratory of Cell Biology in Culture under the direction of Dr. Natalia Mikhailova. The project was on molecular phylogeny and ecological adaptations of the Cerastoderma and the Littorina snails collected in the intertidal zone of North-European region. We have recently published our paper on the evidence of hybridization between two species of littoral snails in the intertidal sites of the Barents Sea, using RAPD nuclear marker.