杀虫剂敌敌畏和除草剂丁草胺对斑腿树蛙蝌蚪的遗传毒性

敌敌畏和丁草胺是我国农田中使用最普遍的杀虫剂和除草剂,这些农田化学物质对当地的水生动物及种群造成很大威胁.本文以广泛分布于我国南方农田区域的斑腿树蛙蝌蚪为研究对象,用碱性单细胞电泳方法(或慧星试验)对暴露在不同浓度的敌敌畏(2.08,4.16,6.24,8.32,10.40 mg/L)和丁草胺(0.1025,0.205,0.410,0.820,1.230 mg/L)溶液中的蝌蚪进行了遗传毒性的检测.结果表明在实验室条件下,随着农药浓度的增加,蝌蚪的DNA损伤(DNA尾长与尾宽比)随之增加;敌敌畏浓度为2.08 mg/L和丁草胺浓度为0.41 mg/L时,对蝌蚪造成显著的损伤,而且农药的剂量与蝌蚪的DNA损伤(DNA尾长与尾宽比)呈显著的线性关系敌敌畏,y=1.136±0.0083x,r=0.957,P<0.01;丁草胺,y=0.968±0.0093x,r=0.964,P<0.01.本研究表明这两种农药对我国的两栖动物具有遗传毒性作用;同时也表明,在检测环境污染物对蝌蚪的基因毒性方面,碱性单细胞电泳分析是一种合适的方法[动物学报 51(3)447-454,2005].     

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro

Summary We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T₃) at a concentration higher than 10⁻⁹ M to the medium could induce cell death of larval epithelial cells, but T₃ alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 μg/ml) and cortisol (0.5 μg/ml) besides T₃ were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.     

Morphology and metamorphosis of Eupsophus calcaratus tadpoles (Anura: Leptodactylidae)

Eupsophus calcaratus, a leptodactyloid frog from the austral Andean forests of Argentina and Chile, has endotrophic, nidicolous tadpoles. We studied a metamorphic series from Stages 31 to 46 of Gosner's developmental table (1960). Other than the scarce pigmentation, proportionately large eyes, and massive developing hindlimbs, the remaining external characters are similar to those of generalized, exotrophic larvae. At the same time, internal morphology does not reveal any character state attributable to the endotrophic-nidicolous way of life; conversely, structures such as the hyobranchial skeleton and the mandibular cartilages are similar to those of exotrophic-macrophagous tadpoles. The metamorphic process is characterized by the delayed development of diverse structures (e.g., ethmoid region, palatoquadrate, and hyobranchial apparatus), and the retention of some larval characters (e.g., parietal fenestrae, overall absence of ossification) with the absence of development of some "juvenile" characters (e.g., adult otic process, several bones) in metamorphosed individuals. These heterochronic processes and truncation of larval development are related to a shorter larval life (when compared to other species of the austral Andean region) and to the small size at metamorphosis.     

Retardation of thyroxine-induced metamorphosis by Amphenone B in toad tadpoles

The effect of Amphenone B, an inhibitor of corticoid synthesis, on thyroxine (T4)-induced metamorphosis was studied in toad tadpoles kept in thiourea. Amphenone injections retarded T4-induced tail resorption markedly. The effect of Amphenone was nullified by aldosterone and corticosterone added to the water in which tadpoles were kept. Steroidogenic cells of adrenals in Amphenone-injected animals were enlarged markedly as compared with those in the saline-injected tadpoles or the Amphenone-injected tadpoles which were supplemented with corticoids. The results strongly suggest that endogenous corticoids act together with thyroid hormone to accelerate metamorphosis.     

Morphologic changes of the basal lamina in the small intestine of Xenopus laevis during metamorphosis

Further investigations of the epithelial and mesothelial basal lamina of the duodenum of Xenopus laevis during metamorphosis were performed by means of scanning electron microscopy (SEM) and histochemical techniques using polyethyleneimine (PEI) to demonstrate anionic sites as well as light- and transmission-electron-microscopic methods involving morphometric analysis. The basal lamina of the duodenal epithelial cells was smooth, and it was occasionally curved along the processes of the epithelial cells (stages 56-59). The basal lamina became thicker by folding, and the thickness of the folded basal lamina exceeded 1 micron (stages 60-62). Subsequently, the folded basal lamina disappeared gradually and became almost smooth again and consisted of only one layer (stages 63-66). After removing the epithelium by boric acid, SEM revealed that the small ridges of the basal lamina protruded like a mesh-work into the luminal side, and the luminal surface of the basal lamina became smooth at later stages of the metamorphic climax. The electron-dense granules of PEI-positive material were localized at both sides of the lamina densa at regular intervals (80-100 nm). The basal lamina of the mesothelial cells was almost smooth at stages 56-59 and started to show occasional slight folding. This folding became continuous and deeper (stages 60-62). The folded mesothelial basal lamina disappeared except for the cell-associated basal lamina and became smooth again at later stages of the metamorphic climax (stages 63-66). These morphologic changes of the basal lamina observed in the epithelium and mesothelium may be induced by common factors. We suggest that physical changes in the small intestine involving the shortening and narrowing should be a main factor to cause these changes in the basal lamina. Furthermore, morphometric analysis proposed that the basal lamina becomes more complex by adding newly synthesized basal lamina material, especially in the epithelium.     

Red light accelerates and melatonin retards metamorphosis of frog tadpoles

Background  

Earlier studies from this laboratory reported that light and its spectra influence reproduction in the Indian skipper frog Rana cyanophlyctis through both ocular and extra ocular photoreception. During the course of our ongoing studies on chromotactic behaviour of the tadpoles, we noticed that tadpoles held in red light metamorphosed earlier than those held in white or other colours of light. The focus of the present study therefore was to examine the effect of red light on metamorphosis of the tadpoles.     

Connective tissue is involved in adult epithelial development of the small intestine during anuran metamorphosis in vitro

Summary The role of connective tissue in metamorphic changes of the small intestinal epithelium inXenopus laevis tadpoles was investigated by using organ culture techniques and electron microscopy. Tissue fragments isolated from various parts of the small intestine at stage 57 were cultivated. Larval cell death of the epithelium was induced by thyroid hormone in all fragments, whereas adult epithelial development was observed only in fragments isolated from the anterior intestinal region containing the typhlosole where most of the larval connective tissue was localized. The epithelium was then cultivated in recombination with homologous or heterologous non-epithelial components. The adult epithelium developed only in recombinants containing a thick connective tissue layer from the typhlosole. There was no regional difference in the developmental potency of the epithelium itself. In all explants where adult epithelium developed, the connective tissue increased in cell density just beneath the epithelium, which was rapidly proliferating and forming typical islets. At the same time, fibroblasts possessing well-developed rough endoplasmic reticulum differentiated close to epithelial cells and often made contact with them. These results indicate that the connective tissue originating from the typhlosole plays an important role in adult epithelial development of the anuran small intestine, probably via direct cell-to-cell contacts or some factor(s) synthesized by the fibroblasts.     

Inhibition of thyroxine-induced metamorphosis by prolactin in tadpoles, Rana catesbeiana.

Thyroxine (T4)-prolactin interactions on hepatic arginase and ornithine transcarbamylase (OTC) as well as hind legs, tail, digestive tract and median eminence were investigated in tadpoles, Rana catesbeiana. Prolactin completely blocked T4-induced tail resorption, but failed to suppress hind-leg growth, shortening of digestive tract and promotion by T4 of the median eminence development. Prolactin blocked T4-induced increase in hepatic arginase activity but not in hepatic OTC activity. A possibility that T4 and prolactin are regulating the hepatic arginase indirectly is discussed.     

Effect of gutimine derivatives on the metamorphosis of Rana temporaria tadpoles

A one-time addition of gutimine (0.5 g/l), amtizol (0.2 g/l), and unithiol (0.25 g/l) to the medium at early stages of tadpole development was shown to inhibit metamorphosis only upon addition of unithiol. Repeated addition of gutimine, amtizol, and unithiol to the medium leads to reliable delay in metamorphosis. The delay is more pronounced at addition of the compounds to the medium at late stages of tadpole development (beginning at 40 stage). This suggests that the studied compounds may have anti-thyroid activity. All drugs under study induce the skin clearing.     

Electron microscope studies of phosphatases in the small intestine of Rana temporaria during larval development and metamorphosis

Summary Changes in the ultrastructure, and distribution of phosphatases in the intestinal epithelium of Rana temporaria during development were consistent with other developmental changes. Alkaline phosphatase AMP-ase and ATP-ase were always associated with sites of absorption of foodstuffs into the cell. Initially, these were only the yolk platelets but at the onset of feeding the brush border lateral wall, membranes and associated absorption vesicles all became sites of activity. At metamorphosis when the larvae cease feeding, the enzyme activities decreased and became difficult to detect.In the early larval stages, acid phosphatase activity was confined principally to the lateral cell-wall membranes. This soon disappeared but was followed at metamorphosis by a dramatic increase in both the number of sites and their activity. In general, acid phosphatase appeared to be associated with areas of degeneration. The new epithelial cells which developed during metamorphosis appeared under the old epithelium. The cell debris from the larval epithelium was then expelled into the lumen of the intestine. The new epithelium contained sites of enzyme activity similar to those of the adult. Acid phosphatase was now present only in lysosome-like bodies and very sparsely on the brush border.These results are discussed in relation to dietary and structural changes. It is suggested that the presence of the enzymes at any site can be related to and anticipate these changes, possibly under hormonal control.