Microspores are intraspore parasites with tiny spores. Microspores parasitize many crustaceans, insects, flukes, and several species of which usually parasitize shrimp and cause infectious diseases.
First, an overview of microspores
Structural characteristics of microsporidium: The body structure of microspores is very simple, with a membrane formed by the base butine material on the outside, a spore-like cyst, and a pole filament inside.
Figure 1: Microspore structure published by researchers in 1998.
Figure: Microspore development cycle
Second, diseases caused by microsporidium in shrimp
1. The epidemic history of microspores
In Thailand in 2000-2002 (according to Kanokporn Chayaburakul, 2004), severe slow-growing disease was detected in farmed P. monodon, caused by microspores that parasitize the pancreatic liver. In 2002, the disease caused nearly $300 million in damage.
In 2009, Somjintana Tourtip and CTV reported that the pathogenic microspores in shrimp pancreatic liver are parasitic endospores (Enterocytozoon hepatopenaei).
Enterocytozoon hepatopenaei (EHP) microspores were identified as parasitic on shrimp liver. The stage from asexual spore synphysis to adult spores is parasitic in the cells of tubular pancreatic hepatocytes (host). There are many asexual spore nuclei that adhere directly to the cellular material and contain a large number of small particles (Bleb) on the surface of shrimp cells.
Microspore mitotic reproduction occurs in the early stages of parasite development, and a large number of sporoblasts are formed within the parasite cells. Dense discs and filamentous precursors grow in the cytoplasm of the early parasite, forming prospores from the surface of the fertilized egg. The spores are oval, 0.7x1.1 μm in size, mononucleated, with 5-6 annular filamentous bodies in front of the cyst and vacuoles in the back, and the tip of the cyst has a disc-like attachment to the filamentous body, and the cell wall is thin and dense.
The cell wall consists of a plasma membrane, an inner layer of light-emitting cells about 10 nm thick, and an extracellular dense layer about 2 nm thick. Primers designed to amplify DNA fragments of the parasite SSU rRNA genome have a size of 848 bp on genbank (GenBank FJ496356). The nucleotide sequence showed 84% homology, and the amplified fragment sequence was similar to that of Enterocytozoon bieneusi.
According to the characteristics of the results, the parasite belongs to the family Intestinal Cell Worms. The cytoplasm and SSU rRNA sequences of the worm are different from those of E. Bienseusi is 16%, so the parasite is considered a new species in the genus Enterocytozoon.
2. Microspores pathological signs
Status: Sick shrimp move slowly, swim to the edge of the pond, and die (Figure 2). The mortality rate of shrimp increases rapidly, 60-70% of shrimp deaths within 1-2 weeks, and can be as high as 100% if the shrimp is seriously ill.
External signs: Diseased shrimp have no typical signs of disease, and usually shrimp have slow growth and flocks (Figure 3).
Internal signs: when the disease is slightly infected, there are no obvious symptoms, in severe cases, shrimp pancreatic liver necrosis is serious, when the constitution of shrimp is weak, then liver and pancreatic rot is very fast.
Signs of pathological tissue: The pancreatic hepatic duct model is mostly necrotic (hollow) and contains a large number of fat droplets (Figure 4). Some cells on the large tube model are filled with small pellets (Figures 5, 6, 7, 8).
Figure 2: Shrimp swim to shore and die (August 2010 sample)
Figure 3: Slow and large shrimp in groups (August 2010)
Figure 4: In shrimp pancreatic hepatocytes, mature spores are filled with small granules.
Figure 5: Adult spores in shrimp pancreatic liver.
Figure 6: Hepatopancreatic necrosis.
Figure 7: Pancreatic hepatotube cells open and filled with small granules (H&E staining, August 2010)
Figure 8: Pancreatic hepatogeniocytes are open and filled with small granules.
3. Microspore pathogens
Initial investigations of the pathogens in some shrimp farms determined that they resemble small spores of the family Enterocella family, which are intracellular parasites.
Gram staining of mature spores appears purple in the cytoplasm of black tiger shrimp hepatopancreatic cells (Figure 9, 10).
Adult spore electron microscope cut vertically into ovals measuring 0.60 x 1.86 μm with a cystic pole (PP) in front, an adhesion disk (ad) at the anterior end, a vacuole (PV) behind, a nucleus (N) in the middle, and filaments (pf) on the upper sides (Figures 10-11). The spore stage develops in the host cytoplasm (hepatopancreatic cells), which contain many small particles (vesicles) on the surface.
Figure 9: Adult spores in shrimp pancreatic liver
Figure 10: Adult spores in shrimp pancreatic liver
Figure 11: The spores are cut oval in shape with a cystic pole (PP) on the front, an attachment disc at the anterior end (ad), a vacuole (PV) at the back, a nucleus (N) in the middle, and filaments (pf) on both sides of the rod.
Figure 12: Mature cells in shrimp pancreatic hepatic duct epidermal cells
4. Distribution of microsporidium disease
Microspores parasitize many crustaceans, and more than 50 species have been described: Octospores, Pirizoma, Monopolar worm, Enterocytozoon hepatopenaei and so on, which are found in 30 species of marine shrimp (including: Penaeus vannamei, Penaeus monodon, etc.), freshwater shrimp, crab and lobster. In addition, microspores parasitize insects, flukes.
According to (Somjintana Tourtip et al., 2009), microsporidium was found in Thailand to parasitize the hepatopancreas of Penaeus monodon; （M. Toubiana and CTV, 2004) showed that microsporidium was found in three species of shrimp in Madagascar (Penus hindui, Penaeus monodon and P. brachygo; (GD Stentiford et al., 2007) showed that microsporidiosis (Enterospora canceri) was found in crabs.
Preliminary investigations indicate that microsporidium can infect shrimp fry, and disease can occur between 20 and 120 days after seedling. This disease often occurs when the weather fluctuates (e.g., rainfall, temperature changes, water pollution).
3. Diagnosis and prevention methods of microspore disease
1. Diagnostic methods for microspore diseases
- Pathological histological methods: Gram stain, Giemsa stain, H&E stain
- Electron microscopy.
- Molecular Biology Methods: DNA Amplification Techniques for SSU rARN Genomes
2. Prevention methods of microspore disease
Microspores belong to the group of spore parasites, they have a fairly stable crust, so it is difficult to use therapeutic drugs, so the main measure is the prevention of diseases in farmed shrimp, based on the following principles:
- Killing pathogens from the environment: using TCCA to kill pathogens at the bottom of ponds and in the water environment; Use microbial agents to clean the environment, reduce water change, prevent pathogens from entering the external environment, and if water change must be taken from a disinfected sedimentation tank.
- Increase the disease resistance of shrimp: Feeding shrimp with some microbial preparations (β-glucan, vitamins, etc.) increases the disease resistance of farmed shrimp.
- Repair shrimp liver and pancreatic necrosis: take drugs such as multi-amino acids, enzymes, multivitamins, and various trace elements to quickly restore the liver and pancreas of shrimp.