Study On Molting And The Relevant Hormones Of The Freshwater Crab Sinopotamon Henanense

To investigate the regulatory mechanism of crustaceans molting, two eyestalks were ablated by burning and press process. First, the morphological changes in epidermic cells in carapace and setaes in the third maxilla of the freshwater crab Sinopotamon henanense were observed by conventional paraffin sections and entity microscopy to prepare for the calculation of molting cycle. Experiments lasted for 168 h were conducted in aquaria. Healthy crab with 3-5 cm head cuirass were selected and fed with specific amount of potato once a day. At the same time, the ecdysone level in haemolymph and chitinase level in epidermis were measured by ELISA, while N-acetyl-β-D-glocosaminidasev (β-NAGase) activity in the epidermis was measured by colormetry to provide theoretical basis for studying the regulatory mechanism of molting. Furthermore, we explored the toxic effects of cadmium (Cd2+) on the molting process by using in vitro cadmium chloride acute exposure, and measuring the ecdysone contents in haemolymph, as well as chitinase and β-NAG activity in epidermis. According to the LC50 (lethal concentration of 50%) of cadmium at 96 h,1/8、1/16> 1/32 of the LC50 were adopted to each group, Crabs were separated into one control group, one eyestalked ablation without cadmium group and three eyestalked ablation with cadmium groups which were exposed to Cd2+ concentrations of 0 (control group),7.25 mg·L-1,14.5 mg·L-1 and 29 mg·L-1 respectively.The results showed that:(1) Along with the duration, the heavy stained storage cells appeared in the carapace epidermal cells after eyestalks ablation for 72 h, red-stained appeared in the epidermis cells. To the 96 h, the epidermis cells with particles graduallymgrow into bubbles, and forned storage cells at 120 h. Furthermore the intercellular substances increased and epidermis cells decreased.(2) The epidermis in the third maxilla becomes thicken along with the gradually smearing and thinnering of the edge profile and finally formed a tubular-like structures. Meanwhile, the setal lumen became wider. At 168 h, the retraction of the epidermis tissue were showed and a transparent gap were formed bteween epidermis and the exoskeleton, suggesting the specific crab has entered the Do molting period.(3) The level of ecdysone in haemolymph was increased at first and then recovered, and reached the maximum value at 96 h. [(?):(23.25±4.56)ng/L;♀:(35.75±7.15)ng/L, P <0.01]. Similarly, the chitinase in epidermis also increased. After eyestalk ablation Reaching the higheast value at 48 h [(?):(16.29±3.91)ng/L;♀:(30.49±5.28)ng/L, P<0.01] and recovered subsequently at 120 h, the value in treated group is higher than that of control group. Activity of β-NAGase increased continuously by the eyestalk ablation and reached the maximum value at 144 h [(?):(400.44±21.00)U/g;♀: (216.94±23.97)U/g, P<0.05]. Then the activity of P-NAGase decreased gradually, but still higher than that of control group.(4) After Cd2+exposure, the eyestalk ablation group showed less ecdysone contents in haemolymph, as well as chitinase activity and P-NAGase activity in epidermis (compared to the eyestalked ablation group without Cadmium exposure, P<0.05) and displayed a concentration-dependent manner. When exposed to 29 mg·L-1 Cd2+ for 120 h, the level of ecdysone were decreased to that of the basal control. The chitinase activity was also reduced significantly in the eyestalked ablation group by 7.25 mg·L-1 Cd2+ for 72 h (P<0.01). In the 7.25 mg·L-1 Cd groups, the P-NAGase activity was slowly over time. However in the 29 mg·L-1 Cd2+ groups the P-NAGase activity dropped significantly and reacher the basal level after 144 h.Conclusions:(1) Along with the duration after eye-stalk ablation, the heavy stained storage cells appeared in the carapace epidermis, indicating that the synthesic activity of carbohydrate or lipid in the epidermis have been enhanced at that stage. The setaes in the third maxilla and its epidermis changed significantly and shortened the molting process. The intermolt was divided into:substage C1, substage C2 and substage C3.(2) Eyestalk ablation may affect the molting process by increasing the levels of ecdysone in the haemolymph, chitinase in the epidermis and the activity of β-NAGase. Molting hormone involved in the regulation of chitinase, but does not rule out the presence of other regulatory factors. Cadmium has inhibitive effect on molting process, probably by inhibiting the secretion of ecdysone, the restrain activity or inactivation of chitinase and β-NAGase.