Abstract:

Objective To observe and analyze the ultrastructure of the metacercariae excystment of Clonorchis sinensis using scanning electron microscope and transmission electron microscope. Methods C. sinensis-positive fish pseudorasbora parva was subject to remove the head, scales and viscera, then weighed and minced. The minced fish was added with artificial digestive fluid (composed of 0.6 g of pepsin, 100 ml of normal saline, and 1 ml of concentrated hydrochloric acid) at a 1 g : 10 ml ratio and was digested overnight at 37 ℃, followed by repeated filtration. The mature metacercaria were separated under a stereomicroscope. Trypsin at a concentration of 0.025% (pH = 7.4) was added to the samples and incubated at 37 ℃ for approximately 3 min. The exysted cercaria, incompletely exysted metacercaria, metacercaria remain comparatively intact appearace, and existed empty sacs were collected separately and fixed with 3% glutaraldehyde supplemented and 1% osmium tetroxide. The fixed samples were dehydrated through a series of ethanol gradients (50%, 70%, 80%, 90%, and 100%, each for 10 min, with 100% ethanol repeated 3 times) and soaked in 100% hexamethyldisilazane 3 times (each for 10 min), dried, coated with gold, and observed using a scanning electron microscope. Gradient dehydration was performed using 50%, 70%, 90% ethanol, 1 : 1 mixture (90% ethanol with 90% acetone), 90% and 100% acetone (each stage was repeated 3 times for 10 min with 100% acetone dehydration). The samples were polymerized in the acetone with embedding reagent (epoxy resin 618) at 1 : 1 and 1 : 3 ratio or the whole embedding agent. The samples were trimmed, sliced and double-stained with uranyl citrate. The sample was observed by transmission electron microscope. Results The scanning electron microscopy revealed local swelling or shrinkage, folding and collapse, and separation of the inner from outer layers of the cyst wall; the linear crack formed during decapsulation was long and had smooth edges. The incompletely emerged metacestodes were wrapped in a soft collapsed cyst wall, with their epidermal spines piercing through the cyst wall; the decapsulated metacestodes were covered with body spines on the dorsal and ventral surfaces. The ventral sucker was significantly larger than the oral sucker, with different inner wall structures. The excretory bladder was filled with spherical excretory particles of various sizes. Transmission electron microscopy showed that the body wall of the decapsulated metacestodes was syncytial, with an electron-dense granular matrix underneath the cortical plasma membrane. The protrusions formed from the matrix towards the surface and numerous secretory granules and vesicles of various sizes within the matrix was seen. The spines originated from the basal membrane, penetrated through the matrix and emerged from the cortical surface. The basal layer had significant thickness variation and contained multiple high-electron-density secretory bodies; the outer circular muscle and inner longitudinal muscle were well-developed, extending into the cell layer with materials transported by cytoplasmic tubes forming vesicles at the distal end. The intercellular substance of the cortical cells was well-developed, filled with chaotically distributed tubular structures, vesicles of various sizes, and mitochondria. Conclusion The metacercariae exhibit vigorous movement via their well-developed muscle tissue, which plays an active role in exysting process. The dermal spines may assist the excysted metacercariae to rapidly escape from the softened wrapping cyst wall. The excysted metacercariae inside the cyst may stimulate the cyst wall via exercising to induce cortical sectetion, which could directly act on the cyst inwall, consequently, facilitate excystment by chemically softening the cyst inner wall.

Key words: Clonorchis sinensis, Excysted metacercaria, Ultrastructure, Scanning electron microscope, Transmission electron microscope