Alina Avanesyan
Among the redwoods -- Muir Woods

Host-parasite interactions; cellular immune responses of pulmonate snails to infection by trematodes

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.

Location and morphology of the hematopoietic tissue

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.

Histological sections of the amoebocyte-producing organ (APO) in resistant Biomphalaria glabrata snails. (A) APO (black arrows) in non-infected snails; (mc) mantle cavity; (pc) pericardial cavity. (B) APO with divided cells (dc) (white arrows) after 3 days post infection (PI). (C) APO after 3 days PI. (D) APO after 5 days PI.

Hematopoietic activity in resistant and susceptible snails

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.

Encapsulation of Echinostoma caproni mother sporocysts developing in resistant Biomphalaria glabrata snails (from 3 to 7 days post infection). The sporocysts demonstrate different level of degradation, and sporocysts remains only are observed after 7 days post infection (the last picture).
Activity of the amoebocyte-producing organ (APO) during infection by Echinostoma caproni in resistant (A) and susceptible (B) Biomphalaria glabrata snails.

Germinal cell lineage in Echinostoma miracidia

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.

Longitudinal sections of miracidia of Echinostoma caproni (A) and Echinostoma paraensei (B,C) (e, embryo of mother redia; gc, germinal cells; n, neural mass; sc, secretory cells; uc, undifferentiated cells). Large arrow indicates miracidium without a redial embryo. Scale bar, 10 pm.

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.

Other projects

Genetic variation in littoral snail populations

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.

Peer-Reviewed Publications

Journal Articles

  1. Granovitch, А.I., Maximovich, A.N., Avanesyan, A.V., Starunova Z.I., and N.A. Mikhailova (2013) Micro-spatial distribution of two sibling periwinkle species across the intertidal indicates hybridization. Genetica 141 (7): 293-301. file_downloadfull text
  2. Ataev, G.L., Dobrovolskij, A.A., Avanessian, A.V., and E.S., Loker (2001) Germinal elements and their development in Echinostoma caproni and Echinostoma paraensei (Trematoda) miracidia. The Journal of Parasitology 87 (5): 1160-1164. file_downloadfull text
  3. Ataev, G.L., Avanessian, A.V., Loker, E.S., and A.A. Dobrovolskij (2001) The organization of germinal material and dynamics of mother sporocyst reproduction in the genus Echinostoma (Trematoda: Echinostomatidae). Parazitologia 35 (4): 307-319.(In Russian) exit_to_applink to publication file_downloadfull text

Published Abstracts

  1. Ataev, G.L., A.A. Dobrovolskij, A.V. Avanessian and C. Coustau (2000) Significance of amoebocyte-producing organ of Biomphalaria glabrata snails (strains selected for susceptibility/resistance) in cellular response to Echinostoma caproni mother sporocysts infection. Proceedings of the symposium on ecological parasitology at the turn of the millennium, St. Petersburg, Russia, 1-7 July, 2000. Bulletin of the Scandinavian Society for Parasitology 10 (2): 65. file_downloadfull text

Conference Presentations

  1. Avanesyan, A.V. and G.L. Ataev. (2001) The organization of amoebocyte-producing organ in different pulmonate snails. International Symposium: Animal Physiology, I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, St.-Petersburg, Russia. Poster presentation
  2. Ataev, G.L., A.A. Dobrovolskij, A.V. Avanessian and C. Coustau. (2000) Significance of amoebocyte-producing organ of Biomphalaria glabrata snails (strains selected for susceptibility/resistance) in cellular response to Echinostoma caproni mother sporocysts infection. The symposium on ecological parasitology at the turn of the millennium. St. Petersburg, Russia. Oral presentation

Non Peer-Reviewed Publications

Research Report

  1. Avanesyan, A. (2005) Structural changes in the amebocyte-producing organ of Biomphalaria pfeifferi snails during Echinostoma caproni infection. Functional Morphology, Ecology and Animal Life Cycles 5: 102-106. (In Russian)
  2. Avanesyan, A. and G.L. Ataev (2004) Hematopoiesis in gastropods. Functional Morphology, Ecology and Animal Life Cycles 4: 105-111. (In Russian)
  3. Ataev, G.L., and A. Avanesyan (2000) Snail defense responses to infection by trematodes. Functional Morphology, Ecology and Animal Life Cycles, pp. 118-122. (In Russian)