| Cadmium (Cd) is a highly toxic environmental pollutant that can affect adversely human health. In the agro-ecological environment, Cd could be transferred and accumulated in the food chains by uptake from the crops. Cd could affect the normal physiological and biochemical metabolism of the organisms, and also accelerate the deterioration of the agro-ecological environment, ultimately affect human health. Insects are the most widely distributed species in the nature, so they are vulnerable to metal stress and other environmental stress. Heavy metals in the environment can enter into the bodies of insect through breathing, integument, feeding, and so on. Excessive Cd in bodies can affect the growth, development and physiological function of insects, at the same time, insects have also developed a variety of defense mechanisms or detoxification mechanisms to reduce the hazards of excessive Cd and maintain the stability of the body environment.The rice grasshopper, Oxya chinensis (Thunberg)(Orthoptera: Acridoidea), is one of the important agricultural pests of rice. With the Cd entered into environment, its habits environment was affected, which resulted in Cd accumulation in different tissues of O. chinensis. In this research, we studied systemically the detoxification mechanism at the biochemical and molecular levels in O. chinensis. The study was divided into five parts:1. In order to study the accumulation pattern of heavy metal in O. chinensis, the grasshoppers were collected from Daixian County, Shanxi Province, China. The Cd and chromium Cr concentrations in their bodies were determined using atomic absorption spectrometry. The results showed that accumulation of Cd in O. chinensis were different in various parts of the insect. The order of Cd concentration in the female grasshoppers is midgut> hindgut> foregut> wings> head> ovary> legs>integument; for males, the order is midgut> foregut> hindgut> legs> head> integument and the accumulation of Cd in the wings and testis were not detected. The results indicated that the accumulation of Cd in the gut of O. chinensis was higher, and that that in the midgut was the highest and showed significant difference compared to those of the other parts (P<0.001). The Cd concentrations in the midgut were6.7984mg·kg-1and3.3477mg·kg-1for female and male, respectively. The order of Cr concentration in the grasshoppers is midgut> legs>integument>head>ovary> foregut> wings> hindgut for females and legs> integument> midgut> head> foregut> testis> wings> hindgut for males. The results of the tests of between-subjects effect showed that the Cd and Cr accumulation in the rice grasshopper O. chinensis was tissue-specific, and the accumulation characteristics between Cd and Cr were different.2. In order to study the accumulation and excretion of Cd in insects, Cd concentrations in the whole body, faeces, and exuviate of O. chinensis fed on diets contaminated Cd were detected using atomic absorption spectrometer. The results showed that high Cd significantly inhibited the growth of grasshoppers. At different developmental stages of the grasshopper, Cd accumulation in the bodies increased with the increase of Cd content in the treated diets (wheat seedlings) and showed significant difference among them. With the growth, Cd accumulation concentration increased initially and then declined slowly (from nymphs to adults). For faeces at controls, Cd accumulation in4th instar nymphs was the highest and exhibited significant difference compared with those in other stages. At the same developmental stage, Cd levels in faeces increased with the increase of Cd in diets, and there were significant differences in excretion capacity among developmental stages. For exuviates, in controls, Cd accumulation in the2nd instar nymphs was the highest and exhibited significant difference compared with those in other stages. Positive correlations were shown between Cd levels in diets and Cd accumulation in the bodies, faeces and exuviates of O. chinensis. The results might provide insight into the dynamic variation of Cd accumulation and excretion in the grasshopper O. chinensis.3. Glutathione S-transferases (GST) and carboxylesterases (CarE) play important roles in the detoxification of endogenous and exogenous compounds. In the present study, the biochemical effects of Cd on GST and CarE activities at different developmental stages of O. chinensis were studied. The results showed that GST and CarE activities exhibited significant difference among different developmental stages, which meant that GST and CarE played important roles in metabolism of O. chinensis. Cd concentration and developmental stage effects on GST activity were statistically significant. GST activity of nymphs increased in the highest Cd concentration, which meant that GST might involve in the detoxification of Cd. However, GST activity in adults was inhibited under Cd stress due to life-stage specific physiological characteristics. Substrate, stage, and Cd concentration have statistically significant effects on CarE activity. When a-NA used as substrate, Cd affected significantly the activities of CarE in the2nd-and5th-instar nymphs but had no effect on the other nymphs or adults. When β-NA used as substrate, Cd affected significantly CarE activity in the2nd-and3rd-instar nymphs but had no effect on the other nymphs or adults. Comparing the results of two substrates, the activity of CarE on the a-NA was greater than that on the (3-NA. The results suggested that the insect's life-stages and enzyme substrates should be considered when enzyme activity under Cd stress was studied.4. Metallothionein (MT) levels increased significantly to adapt or resist the hazards of heavy metals when the organism was under heavy metals stress. In this study, the relationship between Cd accumulation and MT levels were investigated in O. chinensis fed on wheat seedlings with Cd or injected with Cd solution. RT-PCR was used to clone MT gene fragment. The results showed that MT levels exhibited obvious tissue-specific and dose-dependent characteristics, and that the highest Cd and MT levels appeared in the midgut; MT expression levels in different developmental stages were significantly different, and the order of MT levels was:5th instar nymphs>3rd instar nymphs and adults>4th instar nymphs and2nd instar nymphs. At the same developmental stage, there was a significant positive correlation between the MT levels and Cd; Acute Cd injection failed to induce the expression of MT. 5. Insects synthesize heat shock proteins (Hsps) in response to the impacts of stress through molecular chaperone activity. As a molecular marker in ecological toxicology, Hsps is more sensitive than the traditional biological indicators. In this study, four full-length cDNA sequences of Hsps genes were acquired from the grasshopper O. chinensis. They were glucose-regulated protein (OcGrp78), heat shock protein70(Ochsp70), heat shock protein90(Ochsp90), and heat shock protein40(Ochsp40), respectively. Their transcriptional distribution in tissues and developmental stages of rice grasshopper were examined. The results showed that these four hsps were expressed with a tissues-and development stages-specific pattern under normal growth conditions in O. chinensis. In addition, we have investigated the expression profiles of these four Hsps genes in O. chinensis subject to Cd stress. The results showed that Cd acute injection could induce the expression of OcGrp78, Ochsp70, Ochsp90, and Ochsp40in O. chinensis and the expression pattern under Cd acute stress exhibited the same trend:the expression levels reached the maximum within a short time (6h), reduced significantly at12h, and dropped to the level of control or below the level at48h. For induction efficiency, Ochsp70was the most sensitive gene to Cd acute stress. The results of chronic Cd exposure showed that dietary Cd treatment induced up-regulation of OcGrp78, Ochsp90, and Ochsp40to some degree. But dietary Cd induced significant down-regulation of Ochsp70. Furthermore, we suggested that OcGrp78, Ochsp70, Ochsp90and Ochsp40could be used as molecular markers of acute pollution of Cd and that Ochsp70was the sensitive marker to Cd. Whether these hsps genes can be used as molecular markers of chronic Cd pollution remains to be elucidated.
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