Study On Histopathology Of Pebrine And Preliminary Construction Of Digital Silkworm

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They can infect a wide variety of invertebrates and vertebrates, including human. Especially, microsporidia are important pathogens of the economic species, such as fish, silkworm and honey bee. Approximately 160 genera and over 1300 species have been reported to date. The first identified microsporidia species--Nosema bombycis can infect nearly all tissues of the silkworm, and cause destructive pebrine disease which lead to serious economic losses. Thus, N. bombycis is the only pathogen that should be quarantined in silkworm egg production. To investigate the pathological changes of infected silkworm is helpful to illustrate the pathogenesis. Although there were some reports on histopathology of pebrine, however, it is hard for us to obtain a comprehensive histopathology of pebrine. Here, we chose N. bombycis CQ1 isolate as the pathogen to challenge the silkworms, which is the subject of our genome sequencing and functional genomics research. So it is necessary for us to get the first hand results of pathology caused by CQ1 isolate.Tissue sections, scanning electron microscopy (SEM) and transmission electron microscopy(TEM) were employed to observe the histopathological changes of the main tissues of the infected silkworms. Meanwhile, the digital silkworm was constructed as software which integrated many pictures of the surface structure and histopathology. The main results were as following:1. Histopathology observation of the main tissues of silkworm infected by N. bombycisThe infected midgut, silk gland and fat body were collected for tissue sections stained by H&E stainning. Totally, about 600 images of tissue sections were obtained. In the infected midgut cells, the spore distribution were mainly in two forms, firstly, many spores clustered together inside the cell, the others were scattered in the cell, however, some distributed in the intercellular space. Notably, we found some spores seemed to be contained within a parasitophorous vacuole. In addition, N. bombycis did not infect all midgut cells, some cells seemed uninfected and no spores inside, their structures were unchanged compared to the normal midgut cells. For the infected silk gland, some milk-white spots in the silk gland were seen clearly, and the structure of gland cells was nearly destroyed, many spores were distributed in the cytoplasm. The fat body became swollen and the cell junctions were loosened in contrast with the normal fat body, a lot of spores were also present in cytoplasm, no structure of parasitophorous vacuole was observed in cells of infected silk gland and fat body.2. Ultrastructure observation of the main tissues of silkworm infected by N. bombycisTo see detail changes of tissues and the morphology of developmental spores, the infected tissues were embeded and cut into ultrathin slides for transmission electron microscopy. These results showed that the cell junctions of the midgut, silk gland, fat body infected by N. bombycis were obviously loosened, the cytoplasm were filled with parasites in different developmental stages of N. bombycis, including the schizont, sporont, sporoblast, and mature spore. The calathiform cells and cylindrical cells of midgut were filled with spores, the structure of mitochondria was intact and other organelles were hard to discern. Glandular cells of the infected silk gland were filled with different stages of spores, we found four schizonts were linked one by one inside the cell, all with two nuclei; plenty of spores proliferate in the cells of the infected fat body, the fat globules inside cells were reduced or even missed. We found the parasitophorous vacuole again in the cell of fat body. The membrane of the vacuole is intact, the shape of vacuole is oval with size about 28×16μm, more than 20 parasites in different stages are contained in the vacuole, more than half are nearly matured, and some are still in schizont stage.3. Preliminary construction of digital silkwormIn this part, we got more than 500 pictures of scanning electron microscopy and 147 pictures of transmission electron microscopy. We also obtained more than 600 pictures of tissue section. We integrated these images and constructed software DIGITAL SILKWORM 1.0. With this software, we can observe the structure of cell, tissue, and silkworm larva. The software provides many clear images which is convenient for the teaching and study of silkworm and N. bombycis.