Abstract
Eggs of most species digenean flukes hatch in the external environment to liberate larvae that seek and penetrate a snail intermediate host. Those of the human liver flukes, Opisthorchis viverrini, hatch within the gastrointestinal canal of their snail hosts. While adult parasites are primarily responsible for the pathology in cases of human opisthorchiasis, their eggs also contribute by inducing granulomata and in serving as nidi for gallstone formation. In view of the peculiar biology of O. viverrini eggs and their contribution to pathology, we investigated embryogenesis in this species by light and transmission electron microscopy. Egg development was traced from earliest stages of coalescence in the ootype until full embryonation in the distal region of the uterus. Fully mature eggs were generally impermeable to resin and could not be examined by conventional electron microscopy methods. However, the use of high-pressure freezing and freeze-substitution fixation of previously fixed eggs enabled the internal structure of mature eggs, particularly the subshell envelopes, to be elucidated. Fertilization occurs in the ootype, and the large zygote is seen therein with a single spermatozoon wrapped around its plasma membrane. As the zygote begins to divide, the spent vitellocytes are pushed to the periphery of the eggs, where they progressively degrade. The early eggshell is formed in the ootype by coalescing eggshell precursor material released by approximately six vitelline cells. The early eggs have a thinner eggshell and are larger than, but lack the characteristic shape of, mature eggs. Characteristic shell ornamentation, the “muskmelon” appearance of eggs, appears after eggshell polymerization in the ootype. Pores are not present in the shell of O. viverrini eggs. The inner and outer envelopes are poorly formed in this species, with the outer envelope evident beneath the eggshell at the opercular pole of the mature egg. The miracidium has a conical anterior end that lacks the distinctive lamellar appearance of the terebratorium of other digeneans, such as the schistosomes. The miracidium is richly glandular, containing an apical gland in the anterior end, large cephalic gland, and posterior secretory glands. Each gland contains a secretory product with different structure. The paucity of vitelline cells associating with eggs, the reduced size of eggs, and reduced complexity of the extraembryonic envelopes are interpreted as adaptations to the peculiar hatching biology of the miracidia.
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Acknowledgment
We are grateful for the financial support of this work from the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant number PHD/0005/2550) to student Miss Panita Khampoosa and advisor Assoc. Prof. Dr. Smarn Tesana. We also acknowledge the support of the Higher Education Research Promotion and National Research University Project of Thailand, through the Health Cluster (SHeP-GMS), Office of the Higher Education Commission, Khon Kaen University. Malcolm Jones acknowledges the support from the Australian Research Council and National Health and Medical Research Council of Australia. We thank Dr. Kathryn Green for the assistance and advice with electron microscopy.
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Video 1
Movement biology of O. viverrini miracidium. The video commences in mature egg incubated in DW for 20 days to prove maturation of miracidium within the eggshell (MPG 3,111 kb)
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Khampoosa, P., Jones, M.K., Lovas, E.M. et al. Light and electron microscopy observations of embryogenesis and egg development in the human liver fluke, Opisthorchis viverrini (Platyhelminthes, Digenea). Parasitol Res 110, 799–808 (2012). https://doi.org/10.1007/s00436-011-2557-3
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DOI: https://doi.org/10.1007/s00436-011-2557-3