The Effect Of Copper On The Growth And Development Of The Chinese Giant Salamander And The Antioxidant System And Endocrine System

Copper is an essential element crucial for many biochemical pathways, but it can be toxic at concentrations only slightly higher than the normal physiological range. It is a kind of metal that may be of particular significance to amphibian populations, and it is often present in aquatic habitats used by amphibian species. In recent years, concentrations of copper in aquatic systems have increased due to numerous anthropogenic activities including industrial discharges, urban or agricultural runoff, and mining operations; aquatic organisms can experience chronic exposure. In fact, copper concentrations ranging from 50 mg L^-1 to>560 mg L^-1 have been reported in polluted freshwater areas all over the world. This concentration is far higher than Freshwater-quality chronic copper criterion of the U.S. Environmental Protection Agency ?U.S. EPA? (9-13 ?g L^-1). Bufo gargarizans is a wide distributed amphibian species throughout the agricultural landscapes in China. This specie lives mainly in water during embryo and larvae development. Therefore, B. gargarizans larvae development becomes easier to be exposed to the copper.In the present study, to detect the effects of copper on growth and development of B. gargarizans tadpoles, each stochastic 30 larvae at Gs 26 were maintained in glass aquaria. Each treatment was conducted in quadruplicate. The nominal concentrations of copper used were 1,6.4,32 and 64 ?g L^-1 respectively. At Gs 42, we examined total length, weight, hind-limb length and tail length of B. gargarizans tadpoles; five tadpoles were randomly collected from both controls and copper treatments for thyroid histological examinations; total RNA was extracted from the whole body from three tadpoles for detecting the expression of Dios ?Dio2, Dio3? and TRs ?TRa, TR?? by real-time PCR; the expressions of Dio2, Dio3 TRa, TR?, HSP, SOD and GLU in tail, hind-limb and liver of B. gargarizans tadpoles were also examined. At Gs 46, total length and weight were measured; total RNA was extracted from the whole body from three tadpoles for detecting the expression of Dio2 and Dio3 and TRa and TR? by real-time PCR. Survival and metamorphosis were evaluated.Main results and conclusions are as follows:1. Exposure to copper for as long as 63 days affected the mortality of B. gargarizans. The highest copper concentration (64 ?g L^-1) significantly increased the mortality of B. gargarizans larvae ?16.36%? ?P?0.01?, while mortalities of groups exposed to the 1,6.4 and 32 ?g L^-1 copper groups was 4.72%,6.93%,8.43%, no significant difference was observed in 1,6.4 and 32 ?g L^-1 copper groups.2. The morphometric analysis showed that on day 16, significant decreases in the average total length were observed in 6.4,32 and 64 ?g L^-1 copper-treated groups ?P?0.01?. In addition, significant decreases in the average weight were also observed in 6.4,32 and 64 ?g L^-1 copper-treated groups ?P?0.01?. In contrast to the day 16 response, in 6.4,32 and 64 ?g L^-1 copper treatment, total length and weight did not show any significant effect on day 32. Moreover, total length and developmental stage could not show any significant effect on day The results demonstrated that the rapid growth reduction on day 16 reflected the initial shock phase of acclimation, whereas growth parameters returned to the reference values on day 32 and day 48, which represented a comparatively rapid recovery.3. Copper concentration significantly influenced experimental days to metamorphic climax ?Gs 42? and tail resorption time. Tadpoles in 6.4,32 and 64 ?g L^-1 copper treatment took significantly longer to reach Gs 42compared to the control ?P?0.01?; tail resorption time in 6.4,32 and 64 ?g L^-1 copper treatment was also significantly increased compared to the control ?P?0.01?. Our results demonstrated that copper delayed metamorphosis of B. gargarizans. As longer larval period is generally assumed to reduce fitness, the impacts of copper may cause a more dangerous threat to amphibian metamorphosis.4. At the climax of metamorphosis ?Gs 42?, significant decrease in average total length and weight were found in 6.4,32 and 64 ?g L^-1 copper group ?P?0.01?, which indicated copper inhibited body size of tadpoles at metamorphic climax.5. Significant increase in the average hind-limb length and tail length were observed in 64 ?g L^-1 copper group ?P?0.01?. We inferred that longer larval period of B. gargarizans could be attributed to the increase in hind-limb length and tail length. At the completion of metamorphosis ?Gs 46?, average total length was significantly decreased in 6.4 ?g L^-1 copper group, no significant differences were found between the control group and all other treatments, which indicated copper may not have obvious inhibition effects on body size at this stage.6. The histological analysis of the thyroid glands showed that at Gs 42, regular shaped follicles were lined by a single layer of tightly arranged follicular epithelial cells in control group, while follicular cell hyperplasia in thyroid gland can be observed in 64 ?g L^-1 copper group. Follicular cell hyperplasia is characterized by multiple layers of loosely-arranged irregular follicular epithelial cells. Histological examination of thyroid glands in both control and copper treatments did not reveal other noted differences in the cellular architecture.7. Quantiative real time PCR analysis of deiodinases ?Dio2 and Dio3? showed that at Gs 42, the mRNA expression of Dio2 was not affected by the treatment. Exposure to 32 and 64 ?g L^-1 copper significantly upregulated mRNA expression of Dio3 ?P<0.05?. At Gs 46, compared with the control, a significant decrease in Dio2 mRNA levels was observed in 64 ?g L^-1 copper. Exposure to 32 and 64 ?g L^-1 copper significantly upregulated the mRNA expression of Dio3 ?P<0.01?. Increasing levels of Dio3 expression and decreasing levels of Dio2 expression would cause decreased TH concentrations in target tissues.8. Quantiative real time PCR analysis of TRa showed that in 32 and 64 ?g L^-1 copper, mRNA expression of TRa was significantly downregulated ?P<0.01?. TRa mediates the early events of metamorphosis that are mainly growth programs. Downregulation of TRa inhibited the growth of tadpoles.9. Quantiative real time PCR analysis of TR? showed that at Gs 42 and Gs 46, in 32 and 64 ?g L^-1 copper, mRNA expression of TR? was also significantly downregulated ?P?0.01?.TR? expression peaked at the metamorphic climax, and then reduced to normal level rapidly by the end of metamorphosis, which indicated TR? expression is believed to be correlated to TH levels. A decrease of TR? in the normal tadpole is associated with delay of metamorphosis. Thus we hypothesized that copper delayed metamorphosis by inhibiting TR? expression, and copper might have the endocrine-disrupting effect.10. At the end of the experiment ?on day 63?, the mean metamorphosis percent of larvae was 50% in control groups, while in 6.4,32 ?g L^-1 and 64 ?g L^-1 copper, the mean metamorphosis percent of larvae were 39.30% and 35.67%,28.77%, respectively. Exposure to 6.4,32 and 64 ?g L^-1 copper significantly decreased the metamorphosis percent of B. gargarizans larvae ?P<0.01?.11. Quantiative real time PCR analysis showed that in all tissues examined, at Gs 42, exposure to 32 and 64 ?g L^-1 copper significantly downregulated mRNA expression of HSP and SOD in hind-limb, tail and liver ?P<0.01?. In 32 and 64 ?g L^-1 copper groups, GLU mRNA levels were significantly decreased in hind-limb and tail ?P<0.01?. Our results indicated that high concentrations of copper might cause increased oxidative damage and disrupt the antioxidant system in B. gargarizans tadpoles.12. In all tissues examined, exposure to 32 and 64 ?g L^-1 copper significantly downregulated mRNA expression of Dio2, TRa and TR?. Exposure to 64 ?g L^-1 copper significantly also downregulated mRNA expression of Dio3 in hind-limb and liver ?P<0.01?. Copper might induce a considerable reduction of TH levels through downregulation of Dio2 and Dio3 mRNA levels in peripheral tissues. Decreased TH levels may thus decrease the expressions of TRa and TR?. Moreover, Decreased TH levels also determine a reduction in percent metamorphosis and extension of time to metamorphic climax.