Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)

Some chemicals have the potential to adversely affect sexual development through multiple endocrine actions. Prochloraz is an imidazole fungicide that displays diverse mechanisms of action, including inhibition of aromatase activity, inhibition of androgen synthesis, and antagonism of the androgen receptor. The objective of this study was to assess the effects of prochloraz on the sexual development of zebrafish (Danio rerio) in the Fish Sexual Development Test (FSDT) proposed as an OECD test guideline for detection of endocrine disruptors. Zebrafish were exposed to prochloraz (0, 16, 64 or 202 microg/L) for 60 days from 24 h post fertilization. Fish exposed to 202 microg/L prochloraz showed an increased proportion of males. Furthermore, the incidence of intersex and the stages of the gonads were altered in the treated fish compared to the control fish. A significant vitellogenin decrease was observed in both female and male zebrafish at an exposure concentration of 202 microg/L prochloraz. However, in the male fish, significantly increased vitellogenin concentrations were observed in the groups exposed to 16 or 64 microg/L prochloraz. This study serves as a part of the validation of the FSDT and indicates that the FSDT is suitable in detecting compounds with multiple endocrine actions. This is of importance in the assessment of the potential risk of existing and new chemicals.     

Effects of 17α-ethynylestradiol on sexual differentiation and development of the African clawed frog (Xenopus laevis)

Several studies have shown that exposure of amphibians, including the African clawed frog (Xenopus laevis), to potent estrogens at critical times during development results in feminization and/or demasculinization. However, genotyping of X. laevis has only recently become possible, so studies performed in the past were rarely able to make explicit linkages between genetic and phenotypic sex. Therefore, to further characterize this relationship, X. laevis tadpoles were exposed during development to 0.09, 0.84, or 8.81μg/L 17α-ethynylestradiol (EE2), which is the estrogen analog commonly used in oral contraceptives. Exposure to all concentrations of EE2 tested resulted in significant delays in time to metamorphosis. Genotyping showed that genetic sex ratios were similar among treatments. However, morphological evaluation revealed that a significant number of individuals with a male genotype displayed mixed sex and abnormal phenotypes. Additionally, both genetic males and females exposed to EE2 exhibited greater presence of vitellogenin protein relative to the respective controls. Since estrogens function downstream of the initial molecular signals of sexual differentiation, it is likely that genetic male animals received mixed endogenous male and exogenous female signals that caused disordered sexual development. The production of vitellogenin was probably temporally separated and independent from primary effects on sexual differentiation, and might have contributed to delays in metamorphosis observed in individuals exposed to EE2.     

Survival, development, and gonadal differentiation in Rana dalmatina chronically exposed to chlorpyrifos

Chlorpyrifos is an organophosphate pesticide among the most widely used in the world, which is suspected to be an endocrine-disrupting compound. To determine the capacity of chlorpyrifos to affect gonadal differentiation in Rana dalmatina, tadpoles were exposed to this pesticide during larval development until completion of metamorphosis at ecologically relevant concentrations (0.025 and 0.05 mg/L). No effects of chlorpyrifos exposure on survival, development, or metamorphosis were observed. After a 1 month metamorphosis, the gonadal phenotype was determined by gross morphology and histological examination. Morphological and histological analysis revealed normal ovaries or testes in froglets belonging to control group, whereas testes from several froglets exposed to chlorpyrifos were interspersed with testicular oocytes in histological sections. Chlorpyrifos exposure during the entire larval period did not affect sex ratio, but reduced the percentage of males with histologically normal testes. The findings suggest that chlorpyrifos exposure has significant effects on gonadal differentiation in some animals by inducing an intersex condition and alterations to testicular morphology, and that R. dalmatina is sensitive to endocrine disruption. Thereby, this study provides evidence that the ecologically relevant concentrations of chlorpyrifos, although not adversely affect the survival, development, or metamorphosis, may interfere with sex differentiation and reproductive development of R. dalmatina via endocrine-disrupting mechanisms.     

Induction of Vitellogenin Synthesis in Xenopus laevis Tadpoles

Oestradiol induces vitellogenin synthesis in vitro in liver taken from Xenopus laevis tadpoles that are in late metamorphosis. Inducibility first appears at the end of prometamorphosis, and the response to oestradiol increases during the completion of metamorphosis. Oestradiol continuously present during development does not influence the stage at which tadpole liver becomes inducible. It seems that the acquisition of inducibility is part of the normal development of the liver, and independent of both the supply of oestrogen and the sex of the tadpole.     

Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT)

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 μg/L and 101/293 μg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 μg/L and ~30/68 μg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.     

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10–500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.     

High temperatures influence sexual development differentially in male and female tadpoles of the Indian skipper frog, <Emphasis Type="Italic">Euphlyctis cyanophlyctis</Emphasis>

Although sex determination in amphibians is believed to be a genetic process, environmental factors such as temperature are known to influence the sex differentiation and development. Extremely low and high temperatures influence gonadal development and sex ratio in amphibians but the mechanism of action is not known. In the present study, effect of different temperatures on gonadal development, sex ratio and metamorphosis was studied in the Indian skipper frog, Euphlyctis cyanophlyctis. The embryos of Gosner stage 7 were exposed to 20, 22, 24, 26, 28, 30 and 32°C up to tadpole stage 42. The embryos (stage 7) were also exposed to 20 and 32°C up to tadpole stage 25 (non-feeding stages). Tadpoles of stage 25 were reared at 20 and 32°C up to stage 42 (feeding stages). The results show that exposure to higher temperatures (28, 30 and 32°C) during stages 7–42 produced male-biased sex ratio. Rearing of tadpoles at 32°C during stages 25–42 produced male-biased sex ratio, while exposure during stages 7–25 did not affect sex ratio. Embryos and tadpoles exposed to lower temperatures (20 and 22°C) died during the early stages. High temperatures stimulated testis development, and disturbed ovary development. Exposure to high temperatures resulted in the early metamorphosis of tadpoles with reduced body size. These results demonstrated that high temperatures influence gonadal development differently in male and female tadpoles, leading to male-biased sex ratio. These results suggest that high temperature probably acts through stress hormones and favours the small-sized sex.     

Tissue sensitivity to thyroid hormone in athyroid Xenopus laevis larvae

Tadpoles that spontaneously arrest development and remain as larvae occur occasionally in Xenopus laevis populations. These non-metamorphosing tadpoles continue to grow, and they develop into grossly deformed giant individuals which come as close as any anurans to being truly neotenic. Giant X. laevis tadpoles that fail to metamorphose lack thyroid glands. In this study, the hypothesis that the tissues of these tadpoles nevertheless remain thyroid hormone sensitive was tested, by exposing isolated tadpole tail tips to exogenous thyroid hormone in tissue culture. The tail tips from giant tadpoles significantly shrank in response to the thyroid hormone treatment, showing that their tissue was still capable of metamorphosis. However, the amount of shrinkage was less than that observed in tail tissue from normal tadpoles. It was hypothesized that complete induction of metamorphosis may not be possible in the giant tadpoles due to a disproportionate growth and development of tissues and organs.     

Malformations and mortality in the Asian Common Toad induced by exposure to pleurolophocercous cercariae (Trematoda: Cryptogonimidae)

Malformations and increased mortality due to infection by the digenetic trematode, Riberioa ondatrae have been reported for many species of amphibians. Severe malformations have also been reported in the Common Hourglass Tree Frog, Polypedates cruciger induced by pleurolophocercous cercariae in Sri Lanka in addition to the changes in the behaviour, development and survival of the host. We exposed pre-limb bud stage tadpoles (Gosner stages 25–26) of the Asian Common Toad, Duttaphrynus melanostictus to the same pleurolophocercous type cercariae under laboratory conditions. Molecular and morphological identification showed that these cercariae belonged Acanthostomum burminis infecting freshwater snakes as definitive hosts. These cercariae induced malformations (27.8%) and reduced survival to metamorphosis (53.8%). The magnitude of the effects increased with the dose of cercariae. Types of malformations were mainly axial, such as scoliosis and kyphosis. Severe limb malformations such as extra or missing limbs as reported for amphibians exposed to R. ondatrae were not observed in the D. melanostictus. Same authors reported a higher percentage of malformations previously when P. cruciger was exposed to the cercariae A. burminis compared to D. melanostictus. However, tadpoles of D. melanostictus, which are smaller compared to those of P. cruciger, experienced higher mortality than P. cruciger tadpoles. Trematode induced malformations and mortality in amphibians are highly variable and depend on multiple factors such as host species differences such as resistance to infection and tolerance, life-history characteristics such as size at metamorphosis and length of the metamorphosis period, and other factors such as size of the amphibian at the time of trematode exposure.     

Effects of 17alpha-ethynylestradiol on sexual development of male western mosquitofish (Gambusia affinis)

Juvenile male western mosquitofish (Gambusia affinis) were exposed to different concentrations of 17alpha-ethynyl estradiol (EE2) in the diet during the period of sexual maturation. A clear inhibiting influence of EE2 on sexual development was apparent. The proportion of males in each treatment group that failed to complete gonopodial development during the 150-day observation period increased significantly with EE2 concentration. There were significant nonlinear trends toward shorter gonopodia in groups exposed to higher EE2 concentrations. Vitellogenin (VTG) was detectable in the blood of all fish exposed to 1.0 or more micro/g food and the concentration increased dramatically with increasing EE2 exposure. A significant negative association was seen between EE2 concentration and spermatophore counts. This study has demonstrated deleterious effects of EE2 exposure on sexual maturation and several indirect measures of reproductive fitness. It supports the biological relevance of vitellogenin in the blood and reduced gonopodium length as biomarkers for estrogen exposure and endocrine disruption in mosquitofish.     

Growth and development of larval green frogs (Rana clamitans) exposed to multiple doses of an insecticide

Our objective was to determine how green frogs (Rana clamitans) are affected by multiple exposures to a sublethal level of the carbamate insecticide, carbaryl, in outdoor ponds. Tadpoles were added to 1,000-l ponds at a low or high density which were exposed to carbaryl 0, 1, 2, or 3 times. Length of the larval period, mass, developmental stage, tadpole survival, and proportion metamorphosed were used to determine treatment effects. The frequency of dosing affected the proportion of green frogs that reached metamorphosis and the developmental stage of tadpoles. Generally, exposure to carbaryl increased rates of metamorphosis and development. The effect of the frequency of carbaryl exposure on development varied with the density treatment; the majority of metamorphs and the most developed tadpoles came from high-density ponds exposed to carbaryl 3 times. This interaction suggests that exposure to carbaryl later in the larval period stimulated metamorphosis, directly or indirectly, under high-density conditions. Our study indicates that exposure to a contaminant can lead to early initiation of metamorphosis and that natural biotic factors can mediate the effects of a contaminant in the environment.     

Mechanisms and Consequences of Developmental Acceleration in Tadpoles Responding to Pond Drying

Many amphibian species exploit temporary or even ephemeral aquatic habitats for reproduction by maximising larval growth under benign conditions but accelerating development to rapidly undergo metamorphosis when at risk of desiccation from pond drying. Here we determine mechanisms enabling developmental acceleration in response to decreased water levels in western spadefoot toad tadpoles (Pelobates cultripes), a species with long larval periods and large size at metamorphosis but with a high degree of developmental plasticity. We found that P. cultripes tadpoles can shorten their larval period by an average of 30% in response to reduced water levels. We show that such developmental acceleration was achieved via increased endogenous levels of corticosterone and thyroid hormone, which act synergistically to achieve metamorphosis, and also by increased expression of the thyroid hormone receptor TRΒ, which increases tissue sensitivity and responsivity to thyroid hormone. However, developmental acceleration had morphological and physiological consequences. In addition to resulting in smaller juveniles with proportionately shorter limbs, tadpoles exposed to decreased water levels incurred oxidative stress, indicated by increased activity of the antioxidant enzymes catalase, superoxide dismutase, and gluthatione peroxidase. Such increases were apparently sufficient to neutralise the oxidative damage caused by presumed increased metabolic activity. Thus, developmental acceleration allows spadefoot toad tadpoles to evade drying ponds, but it comes at the expense of reduced size at metamorphosis and increased oxidative stress.     

Gill alterations as biomarkers of chronic exposure to endosulfan in Bufo bufo tadpoles

Endosulfan sprayed on agricultural fields accumulates in temporary pools due to surface runoff or sediment transport and may result in high water concentrations in spring and summer, coinciding with breeding and crucial stages of amphibian larval development. In the present study, Bufo bufo tadpoles were exposed to three different concentrations of endosulfan (0.01, 0.05 and 0.1 mg/L) until they reached complete metamorphosis. The aim of the study was to investigate the effects of endosulfan, at environmentally relevant concentrations on gill morphology and ultrastructure. Modifications in ultrastructure and cell composition were observed at all concentrations after 96 h. The main gill effects recorded in treated animals were: mucous secretion, the appearance of tubular vesicles cells (TVC) and a degeneration phenomenon. Comparing these results with our previous findings in which we used growth, developmental rate and behaviour as endpoints, we also demonstrated that the first effect of endosulfan on Bufo bufo was gill alteration, thus supporting the role of a morphological approach in toxicological studies. This study provides additional information on the role of morphological studies in demonstrating the effects of exposure to environmental pollutants. In this context, the use of amphibian gills, as effective biomarkers, is a valuable approach in evaluating exposure to agrochemicals.     

Predator‐mediated phenotypic plasticity in tadpoles of the striped marsh frog,Limnodynastes peronii

Abstract We tested the phenotypic responses of larval striped marsh frogs (Limnodynastes peronii) to the odonate nymph predator, Aeshna brevistyla. When reared in the presence of dragonfly nymphs feeding upon conspecifics of L. peronii larvae the tadpoles showed a strong change in morphology. Morphological changes included an increase in total tail height, but also an unexpected marked change in head‐body shape. In addition, we examined how tadpole development, as well as mass and length at metamorphosis, was affected by exposure to dragonfly nymphs. Larval development of L. peronii was strongly influenced by exposure to the predatory behaviour of dragonfly nymphs. Predator‐induced tadpoles had significantly slower developmental rates than control larvae. Although metamorphs of non‐exposed L. peronii were approximately 33% lighter than predator‐exposed metamorphs and possessed lower jump distances, after adjusting for mass there was no difference in jump distance. The newly described morphological response may assist in more accurately relating morphological plasticity to fitness.     

Effects of developmental and growth history on metamorphosis in the gray treefrog, Hyla versicolor (Amphibia, Anura)

In ecological models, the timing of amphibian metamorphosis is dependent upon rate of larval growth, e.g., tadpoles that experience a decrease in growth rate can initiate metamorphosis early. Recent authors have suggested that this plasticity may be lost at some point during the larval period. We tested this hypothesis by exposing groups of tadpoles of the gray treefrog, Hyla versicolor, to different growth schedules. In endocrine models, metamorphosis is dependent on thyroxine levels and thyroxine is antagonized by prolactin (amphibian larval growth hormone), consistent with the idea that a rapidly growing tadpole can delay metamorphosis. Thus, we also manipulated the rate of development by supplementing or maintaining natural thyroxine levels for half of the tadpoles in each growth treatment. All tadpoles that received thyroxine supplements metamorphosed at the same time regardless of growth history. They also metamorphosed earlier than tadpoles not treated with thyroxine. Tadpoles not given thyroxine supplements metamorphosed at different times: those growing rapidly during day 15-34 metamorphosed earlier than tadpoles growing slowly. Growth rate before day 15 and after day 34 had no effect on metamorphic timing. The difference in larval period between these rapidly growing tadpoles and their sisters given thyroxine treatments was less than the same comparison for tadpoles that grew slowly during the same period. This apparent prolactin/thyroxine antagonism did not exist after day 34. These results are consistent with the hypothesis of a loss of plasticity in metamorphic timing.     

Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver.

Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained.     

Effects of the nematode Gyrinicola batrachiensis on development, gut morphology, and fermentation in bullfrog tadpoles (Rana catesbeiana): a novel mutualism

We describe a novel mutualism between bullfrog tadpoles (Rana catesbeiana) and a tadpole-specific gastrointestinal nematode (Gyrinicola batrachiensis). Groups of tadpoles were inoculated with viable or nonviable nematode eggs, and development, morphology, and gut fermentation activity were compared between nematode-infected and uninfected tadpoles. Nematode infection accelerated tadpole development; the mean time to metamorphosis was 16 d shorter and the range of times to metamorphosis was narrower in nematode-infected tadpoles than in uninfected tadpoles. At metamorphosis, infected and uninfected bullfrogs did not differ in body size or condition. Colon width, wet mass of colon contents, and concentrations of most fermentation byproducts (short-chain fatty acids: SCFAs) in the hindgut were greater in infected tadpoles. Furthermore, in vitro fermentation yields for all SCFAs combined were over twice as high in infected tadpoles than in uninfected tadpoles. One explanation for accelerated development in infected tadpoles is the altered hindgut fermentation associated with the nematodes. Energetic contributions of fermentation were estimated to be 20% and 9% of the total daily energy requirement for infected and uninfected tadpoles, respectively. Infection by G. batrachiensis nematodes potentially confers major ecological and evolutionary advantages to R. catesbeiana tadpoles. The mutualism between these species broadens our understanding of the taxonomic diversity and physiological contributions of fermentative gut symbionts and suggests that nematodes inhabiting the gut regions of other ectothermic herbivores might have beneficial effects in those hosts.     

Endocrine mechanisms underlying plasticity in metamorphic timing in spadefoot toads

Amphibian larvae respond to heterogeneous environments by varyingtheir rates of growth and development. Several amphibian speciesare known to accelerate metamorphosis in response to pond dryingor resource restriction. Some of the most extensive studiesto date on developmental responses to pond drying have beenconducted on species of spadefoot toads (family Pelobatidae).We have found that tadpoles of two species of spadefoot toadaccelerate metamorphosis when exposed to water volume reductionin the laboratory (to simulate a drying pond). Furthermore,Western spadefoot toad (Spea hammondii) tadpoles acceleratedmetamorphosis in response to food restriction, which was intendedto simulate a decline in resource availability in the larvalhabitat. Metamorphic acceleration was accompanied by increasedwhole body 3,5,3'-triiodothyronine and hindbrain corticotropin-releasinghormone content by 24 hr after transfer of tadpoles from highto low water. Food restriction for 4 day accelerated metamorphosisand elevated whole body thyroid hormone content. Although tadpolesaccelerated metamorphosis and activated their thyroid axis inresponse to the two environmental manipulations, the kineticsof the responses were greater for water volume reduction thanfor resource restriction. The modulation of hormone secretionand action by environmental factors provides a mechanistic basisfor plasticity in the timing of amphibian metamorphosis, andthe neuroendocrine stress axis may play a central role in developmentalplasticity.     

Effects of thyroxine on the ontogeny of the vitellogenic response in Pleurodeles waltli of both sexes

Summary In the newt, Pleurodeles waltli, vitellogenin synthesis can be induced by oestrogen prior to metamorphosis and well before vitellogenin synthesis is normally observed in females. Vitellogenin indicubility has been shown to occur during normal development at stage 53 in both sexes. Immersion of young larvae in thyroxine at concentrations that did not result in early morphological metamorphosis led to a precocious acquisition of liver responsiveness to oestrogen. Treatment of larvae with thiourea greatly diminished the ability of oestradiol to induce vitellogenin synthesis, although it did not abolish the hormonal response.     

葡萄糖和维生素C对镇海林蛙蝌蚪生长 及其三种酶活性的影响

营养源对动物生长发育具有重要作用。本文采用静水实验浸泡法探究葡萄糖和维生素C对镇海林蛙(Rana zhenhaiensis)蝌蚪生长及其苹果酸脱氢酶、乳酸脱氢酶及淀粉酶活性的影响。在葡萄糖实验组共分为0.5、1.0和2.0 g/L三个浓度组,维生素C实验组共分为10.0、20.0和30.0 mg/L浓度组,另设1组加曝气水作为对照组。在葡萄糖实验组中,蝌蚪存活率在不同实验组间的差异不显著(P0.05)。变态时间在0.5 g/L和1.0 g/L实验组最短,分别为(43.0±4.0)d和(43.0±3.4)d,2.0 g/L实验组最长,为(46.2±5.4)d,且实验组间差异显著(P 0.05)。变态时,0.5 g/L实验组的体重和体全长最大,且各实验组间的体重差异显著(P 0.01),但体全长差异不显著(P 0.05)。增重率在0.5 g/L实验组最高,为(9.67±1.71)mg/d,对照组最低,为(7.54±1.22)mg/d,且各实验组之间差异显著(P0.05)。维生素C实验组中,存活率在各实验组间差异不显著(P 0.05)。所有实验组蝌蚪的发育历期在实验第7、14、21和28天时均大于对照组。变态时间在所有的实验组相似,为43 d左右(P 0.05)。变态时,蝌蚪的体重(P 0.05)和体全长(P 0.05)在20.0 mg/L实验组和30.0 mg/L实验组最大,对照组最小,但各实验组间差异不显著。蝌蚪的增重率在20.0 mg/L和30.0 mg/L实验组最高,10.0 mg/L实验组最低,但不同实验组间差异不显著(P 0.05)。生化酶活性检测中,苹果酸脱氢酶(MDH)活性在葡萄糖实验组和维生素C实验组中均随实验浓度的增加而增加。乳酸脱氢酶(LDH)和淀粉酶(AMS)活性分别在葡萄糖的1.0 g/L实验组和维生素C的10.0 mg/L实验组达到最高。本研究结果表明,10.0mg/L维生素C或1.0g/L葡萄糖为镇海林蛙蝌蚪的最适外源物添加浓度,能促进蝌蚪的生长和体内酶活性。该研究结果将为镇海林蛙的养殖提供一定的理论参考数据。