| Digestion is important physiological function of organisms. It controls the nutrient absorption and there after affects the growth of animals. Cadmium (Cd) is a heavy metal which is considered as one of the most toxic pollutants in aquatic ecosystems, which causes a series of toxic effects in aquatic organism. The present research investigated the structures of various organs of digestive system in freshwater crab Sinopotamon henanense used a stereoscopic microscopeand optical microscope, and studied systematically its morphological structure and histological characteristics of the digestive system at first. To explore the effects of Cd on the digestive system of aquatic animals, we applied acute toxicity experiment (Cd concentrations being 7.25, 14.50,29.00 mg/L; exposure time being 4 d) and subchronic toxicity experiment (Cd concentrations being 0.725,1.450,2.900 mg/L; exposure time being 21 d), and examined the effects of Cd onthe activities the 7 of digestive enzyme (sucrase, lactase and maltase, amylase, lipase and trypsin and pepsin) in 3 digestive organs (hepatopancreas, intestine and stomach) in S. henanense. To further explore the mechanism of the changes in digestive enzyme activities, the levels of Cd accumulation in hepatopancreas, intestine and stomach after the two Cd exposure modes were assessed using the flame atomic absorption method. The changes in microscopic and submicroscopic structures of esophagus, midgut, hindgut and hepatopancreas in S. henanense in 29.00 and 2.900 mg/L Cd treatment groups were documented. Finally, colorimetric method was used to quantify the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and malondialdehyde (MDA) level of two main digestive organs (hepatopancreas and intestine) after the two different modes of Cd exposure. Moreover, the real-time fluorescent quantitative PCR technology was used to detect the mRNA of metallothionein (MT) gene expression in the same two tissues, to explore the mechanism of digestive system responses to Cd toxicity.The results showed:1. Morphological observation and histological studies on the digestive system of S. henanense:The digestive system comprises of digestive tract and digestive glands. The digestive tract is composed of esophagus, stomach, midgut and hindgut. The digestive gland is hepatopancreas. The stomach of the crab is divided into cardiac stomach and pyloric stomach. There is a gastric mill with calcified teeth, ridges, spines and setae in the cardiac stomach, which is used to grind food. There are several special structures such as combs in the pyloric stomach, which are for food filtration. The results of light microscopy showed that the wall of esophagus, midgut and hindgut is surrounded by mucosal, muscular and connective tissue layers. The ridges in the surface of epithelium mucosa of each segment of the digestive tract wall take the shape of multigrade wrinkles. The muscular layers consist of longitudinal muscle, striated muscle and radiant muscle. In the midgut there are typical microvilli. The epithelium of the digestive tract is covered by cuticle except the midgut. There are more developed cilia on the surface of epithelial mucosa of esophagus and hindgut. The hepatopancreas is composed of several ramose hepatic ducts. Each hepatic duct consists of four types of cells:resorptive cells (R cell), blister-like cells (B cell), fibrillar cells (F cell) and embryonic cells (E cell).2. Effects of Cd on activities of digestive enzyme in S. henanense:Digestive enzyme and their activities were different in different tissues: sucrase and maltase activities in intestine were higher than those in other two tissues; the activities of lactose, amylase, trypsin and lipase were higher in hepatopancreas than in other tissues; pepsin activity was only detected in stomach; and lipase activity was not detected in intestine. The digestive enzyme activities in S. henanense were affected significantly by Cd, and the impacts were different between the two exposure modes. In the acute Cd treatment groups, the lipase activity in both hepatopancreas and stomach were up-regulated, trypsin activity was increased and then decreased with the increasing concentrations of Cd. The activities of the rest 5 digestive enzyme were inhibited. After subchronic Cd stress, the digestive enzyme activities of all treatment groups were decreased to various degrees, except that the lipase activity was significantly higher than that of the control group when Cd was used at 0.725 mg/L.3. Cd caused damages to digestive system of S. henanense:The Cd accumulation was higher in hepatopancreas than intestine and stomch, and the bioaccumulation in two treatment modes showed obvious concentration-dependent manner. The level of Cd in the three tissues reached its peak after 29.00 mg/L Cd exposure for 4 d. The microscopic results showed that damages in acute 29.00 mg/L Cd treatment group to the digestive system were more severe than those in the subchronic group: swelling and vacuolization were observed in the epithelial cells; edema and hyaline degeneration appeared in muscle fibers of esophageal and hindgut; and large areas of epithelial cells were necrotic in hepatopancreas and midgut, which detached from the basal lamina. The damage degree of 2.900 mg/L Cd was less severe. There was no significant change in esophageal morphology, while moderated damages were noted in hepatopancreas and midgut. The transmission electron microscopy (TEM) results were basically in agreement with those of light microscopy, the effects of 29.00 mg/L Cd treatment group had a greater influence on the cellular structures of four tissues than 2.900 mg/L group. In all of the tissues examined, the two sets of Cd treatment both resulted in cytoplasmic hypervacuolization, nuclei deformation, condensation and margination of nuclear chromatin, swelling and lysis of mitochondrial membrane, and collapse of the mitochondria cristae. In addition, the structure of microvilli, rough endoplasmic reticulum, lysosome as well as Golgi complex in epithelial cells of hepatopancreas and midgut were altered. Edema and loose muscle fiber bundle appeared in esophagus and hindgut.4. Biochemical changes in the digestive system of S. henanense in responsive to Cd stress:The antioxidant enzymes activies, MDA content, and MT mRNA expression were higher in hepatopancreas than those in the intestine in the two modes of Cd treatment. In acute toxic treatment, the SOD and CAT activities in intestine were increased in the 7.25 mg/L group, and the activities of 3 antioxidant enzymes were inhibited in both tissues after 29.00 mg/L Cd exposure. In subchronic group, the antoxidant enzymes activities in both two tissues were decreased in various degrees. MDA contents were increased with the increasing of Cd concentration in the two treatment modes; the MDA contents were slightly higher in subchronic group than in the acute group. The MT mRNA expression was increased in hepatopancreas and intestine to Cd stress; it was higher in the subchronic group than that in the acute group.The results indicated that:1. The digestive enzyme activities of S. henanense were reduced after Cd exposure, which seriously affected the digestive ability of crabs. The sensitivity of digestive enzyme activities to Cd stress can reflect the toxicity level on the digestive system of crustaceans, and thus can be used as biological indicators of water ecological environmental pollution caused by Cd;2. Cd accumulation and damage degree of digestive system in S. henanense were higher in acute Cd treatment group than subchronic group. The pathological changes of hepatopancreas and intestine were most serious, and the large area epithelial necrosis was even observed, which showing that the two tissues are target organs of Cd toxicity. These injuries may influence the synthesis and secretion of digestive enzyme, leading to a loss of its normal digestive function;3. The changes of SOD, CAT, GPx activities and MDA contents indicated that, in the two mode of Cd treatment, the antioxidant defense system of S. henanense was damaged. The degree of Cd toxicity to aquatic animals is related to oxidative stress and lipid peroxidation;4. In the subchronic low Cd concentration, MT mRNA expression in the hepatopancreas and intestine were higher than acute groups, which indicate that acute Cd toxicity impairs the defense system. The ability to detoxify of MT may provide main self-protection mechanism for digestive system of S. henanense after chronic Cd stress. |
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