| The “muscle whitish disease” occurred in a marine polyculture of ridgetail whiteprawn, Exopalaemon carinicaudand swimming crab, Portunus trituberculatusin Qidong,Jiangsu province, China. Morphological and structural analysis of pathogens confirmedthat they were two different microsporidian by using three microscopic techniquesinitially. The universal microsporidia primers were used to amplify the SSU rRNA geneand themolecular identifications were conducted further. The pathological analysis ofinfected E. carinicaud and P. trituberculatus were also performed. The results were asfollows:The microsporidan with sporophorous vesicle structure was observed in the prawnE. carinicaud. One sporophorous vesicle containing eight oval-shaped uninucleatespores was measured5.4±0.55(4.4-6.6μm), mature and fresh spore was approximately2.3±0.25(1.7-2.8μm)×1.5±0.19(1.2-1.9μm) with9-10polar filament coils whichaligned irregularly in two ranks and it was2.0±0.16（1.7-2.3μm）×1.3±0.15（0.96-1.6μm）after fixing. The Morphological characteristics shared similarity with the genusThelohania.A cloned ribosomal SSU-ITS-LSU (partial) gene fragment of1873bp wasobtained from the microsporidian. Phylogenetic analysis suggested a closest affinitywith P. morhaphis. Their sequences identity was91%but significant differences werefound on morphological characteristics. Due to the contradiction between traditionalmorphological and molecular taxonomic data, this microspridian was placed into theunclassified members as Microsporidium sp. Pathological analysis showed that theparasite invaded the gill, stomach, gut and muscle tissues of host and caused differentpathological changes, especially worst in the skeletal muscle. The muscle of abdomen,cephalothorax, pereiopoda, tail fan showed white appearances and opacity. Musclefibres of the abdomen were largely replaced by spherical sporophorous vesicles.Myofibres were ruptured apparently. The number of vacuoles increased and themitochondria disappeared. The hypertrophy and vacuolization, part of gill membranesnecrosis, disappearing of nucleoplasm, cytoplasm and organelles were observed in gill cells. The electron densities of cytoplasmic matrix in the stomach cells increased andthe spores lived around the lipid droplets in connective tissue. The nucleoplasm of thegut epithelial cell condensed and some muscularis disappeared. Parasites locatedbetween the submucosa and muscle layer in gut.The microsporidan found in P. trituberculatus did not possessed the sporophorousvesicle structure. The special microvillus projections which presented on the surface ofspore exosporium shaped radial. Mature uninucleate spores were almost oval-shaped,approximately1.8±0.11(1.6-2.1μm)×1.4±0.08(1.2-1.6μm), with9coils of polarfilament which aligned regularly in single rank. The morphological features of the crabparasite were consistent with members of the genus Ameson. The amplified fragment ofmicrosporidian ribosomal SSU-ITS-LSU (partial) gene was about1684bp. Phylogeneticanalysis of the SSU rRNA gene sequences revealed that the crab microsporidian had aclose relationship with the genus Ameson. The results of morphological classificationwere consistent with that of the molecular taxonomy, hence the crab microsporidian wasidentified as Ameson sp. Pathological analysis showed that the parasite invaded the gill,stomach, gut and muscle tissues of host and it caused different pathological changes,especially in the skeletal muscle. The muscle of cheliped,ambulatory legs andswimming legs showed distinctive white and opaque appearances. Large number ofspores parasitized in muscle cells. The normal fibrillar structure was indistinct and mostdisappeared apparently. The nucleus deformation, heterochromatin concentration anddisappearing of mitochondrial cristae were also observed in muscle cells. Cell necrosis,mitochondrial edema and cristae disappearance were observed, the nucleoplasmmarginalized, nuclear disintegrated and disappeared, the cytoplasm and organellesdissolved. The phenomenon was found in the gill epithelial cells which had lost thetypical cell structure. Microsporidian parasites in stomach caused the cell membrane todisappear, mature spore, the majority of organelles and cytoplasm were released into theextracellular milieu, nuclear chromatin condensed on the inner edge of nuclearmembrane.The structure of intestinal cell membrane was destroyed by microsporidian,the number of lysosomes around the nucleus increased, nucleus enriched, broken oreven disappeared, organelles disappeared, and the cell became vacuolization. |
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