非洲爪蟾肝细胞原代培养方法

非洲爪蟾是研究内分泌干扰物的良好模型动物,其体外肝细胞可用于类雌激素活性评价、污染物代谢等研究.论文探讨了非洲爪蟾肝细胞原代培养的方法,采用两步原位灌注法分离非洲爪蟾肝细胞,通过胶原酶的作用使细胞之间解离,后经过一系列转速的离心,获得纯化的肝实质细胞.研究结果表明,采用此法获得的细胞数量为2.5～5×106个,细胞成活率达95%以上,纯度在95%以上,细胞胞体透亮,折光性强,状态良好.培养24h后贴壁较好,每2d换液1次,可培养8～10d,细胞可满足多种后续实验的要求.     

Flash competition in male Photinus macdermotti fireflies

Summary The courtship of Photinus macdermotti fireflies involves a flash code in which the male emits a pair of flashes about two seconds apart and the female responds with a flash about 1.2 s after the second flash of the male pair. The male repeats his pair of courting flashes at varying intervals of four seconds or more. Lloyd (1979, 1981 a, b) observed that rival males in Florida sometimes interject flashes between the courtship flashes of the courting male. We have found that the competitive behavior of males of the same species on Long Island, New York, was somewhat different. Rival males timed their competing flashes from either the first or second of the original males's courting flashes. When a competing flash from the rival male was timed from the first courting flash it was synchronized with the original male's second courting flash. When a competing flash was timed from the second courting flash, a delayed flash resulted which appeared after the female's response. In either case the male's display period was preserved, insuring that the female would continue to respond. Either of these rival male flashes could sometimes induce the female to respond in his direction.     

除草剂乙草胺对非洲爪蟾性腺发育的影响

使用性腺发育对内分泌干扰作用敏感的两栖动物非洲爪蟾作为模型动物,通过考察性别比、性腺整体形态和组织学形态来揭示乙草胺对其性腺发育的影响,从而确定乙草胺是否具有内分泌干扰活性.46/47期蝌蚪经不同浓度(5μg·L-1、10μg·L-1、20μg·L-1)的乙草胺暴露处理至变态后1个月,在变态结束后3个月时,解剖性腺、鉴别雌雄并固定性腺做组织切片.实验中对照组所有性腺都为典型的卵巢或睾丸,雌性百分率为48.78%(20/41).而乙草胺暴露组却出现了明显的异常睾丸,其总体上是睾丸,但又带有部分卵巢的特征,在统计性别时将具有异常睾丸的爪蟾归为雄性.5μg·L-1、10μg·L-1、20μg·L-1暴露组的雌性百分率分别为:57.14%(24/42)、66.67%(22/36)、55.26%(21/38).虽经统计分析无显著性差异,但乙草胺暴露组中雌性多于雄性的趋势和出现带有雌性特征的雄性的现象,一定程度上却暗示了乙草胺可能对非洲爪蟾的性腺发育有雌性化作用.正常睾丸的组织学结构应有发育成熟的生精小管,其内有各个时期的精子囊和生精细胞及精子.但经乙草胺处理的爪蟾睾丸却表现出几种明显的异常:没有生精小管,相反却像发育早期的睾丸一样,充满大量的精原细胞,完全没有生精细胞和精子;有生精小管结构,但其内精子囊结构不明显,生精细胞少并且排列混乱,其间有很大的空隙;睾丸出现腔隙,并有大量体细胞存在,这种结构与57期的卵巢相似;睾丸中出现异常的生精细胞,与58期卵巢的卵细胞相似;睾丸组织中有明显的卵细胞散布.组织学研究发现的乙草胺导致的睾丸结构证明了乙草胺对非洲爪蟾的睾丸发育有抑制雄性化作用和雌性化作用,即乙草胺对非洲爪蟾的性腺发育有内分泌干扰作用.     

非洲爪蟾胚胎用于发育神经毒性测试的方法

在美国材料与测试协会(ASTM)的非洲爪蟾胚胎致畸试验(FETAX)的基础上,以已知具有发育神经毒性的氯化甲基汞为模式化合物,探索一种以体征、运动神经元形态和运动行为参数为终点指标的研究发育神经毒性的方法.非洲爪蟾胚胎暴露氯化甲基汞3 d时,观察到暴露组胚胎的运动能力随暴露浓度(100—400 nmol·L-1)的增加而减弱.暴露4 d发现300 nmol·L-1和400 nmol·L-1暴露组胚胎体长和运动神经元明显短于对照组.暴露持续7 d,通过行为分析软件对蝌蚪运动行为定量,发现暴露处理的蝌蚪的游泳速率明显小于对照组.以上结果显示,非洲爪蟾胚胎可用来研究化学品的发育神经毒性,胚胎的体征、运动神经元形态和运动行为可以作为相对敏感的评价指标.     

非洲爪蟾蝌蚪脑神经细胞体外培养方法的建立(Establishment of an in Vitro Method for Culturing Brain Neurons from Xenopus laevis Tadpoles)

体外培养的神经细胞是神经毒性机制研究和神经毒物筛查的重要材料.目前鼠体外神经细胞的应用最为广泛.鉴于近年来发育生物学的模型动物非洲爪蟾越来越多地应用于毒理学研究,论文建立了一种体外培养非洲爪蟾脑神经细胞的方法.该方法取52～53阶段的非洲爪蟾蝌蚪的脑组织,在L-15培养液中直接吹打获得分散细胞,接种于包被多聚赖氨酸的培养板中,22℃培养.培养细胞状态良好,72小时后神经元细胞初步建立神经网络.用β2tubulin7B9可对脑神经元细胞进行荧光染色,鉴别神经突的生长和神经网络的形成.与鼠脑神经元细胞体外培养方法比较,该方法具有无需胰蛋白酶分离、无需胶质细胞共培养、可同时操作多只脑、能获得大量细胞等简单快捷的特点.因此,体外培养的非洲爪蟾蝌蚪脑神经细胞可作为目前体外神经毒理研究中鼠脑神经元的补充材料,用于神经毒性机制的研究和神经毒物的筛查.     

增味剂麦芽酚增加非洲爪蟾蝌蚪皮肤和神经黑色素团中色素的聚集

黑色素团是改变黑素体分布的色素细胞,使动物在伪装、体温调节和紫外线防护时显得更亮或更暗。一系列复杂的荷尔蒙和神经机制调节黑色素细胞的色素分布,因为它们可以迅速响应环境的变化,这些动态细胞可作为一种筛选毒物的有价值的工具。我们发现麦芽酚作为一种天然的增香剂和香料在非洲爪蟾蝌蚪体内以剂量-效应关系诱导黑色素聚集。为了确定麦芽酚是否影响伪装调整,我们将蝌蚪放在白色或黑色背景的麦芽酚中。结果显示,麦芽酚诱导的色素聚集具有相似的剂量依赖性,但与背景颜色无关。我们还比较了褪黑素处理组和麦芽酚处理组,发现麦芽酚诱导色素聚集的程度与褪黑素相似,但麦芽酚诱导色素聚集的时间要长得多。最后,麦芽酚对调节伪装相关色素聚集基因在大脑中的mRNA表达没有影响。我们的结果表明,麦芽酚并没有通过伪装调整机制或褪黑素相关机制发挥作用。这些结果首次证实了麦芽酚暴露在体内的假定毒理学效应,并排除了麦芽酚可能对其色素聚集起作用的几种机制。     

Sperm competition risk affects male mate choice copying

Mate choice copying was mostly described as a strategy employed by females to assess the quality of potential mates, but also males can copy other males’ mate choice. An open question in this context is whether and how copying males evaluate sperm competition risk, as mating with a female that has already copulated with another male obviously sets the stage for intense sperm competition (i.e., in species with internal fertilization). Using the livebearing Atlantic molly (Poecilia mexicana) as a model, we asked (a) whether males of that species indeed copy other males’ choices, and if they do so, (b) whether copying males strategically adjust their behavior to sperm competition risk. We used an approach where focal males could first choose to associate with a large or a small stimulus female. Mate choice tests were then repeated after an “observation phase” during which either no model male was present (treatment 1, control) or the previously non-preferred female could be seen associating (treatment 2) or physically interacting (treatment 3) with a model male. We found that, after the observation phase, males spent considerably more time with the previously non-preferred female in treatment (2), i.e., they copied the model male’s choice. This effect was much weaker during treatment (3) where sexual interactions between the model male and the formerly non-preferred female were allowed. Males, therefore, seem to adjust their copying behavior strategically to the perceived risk of sperm competition.     

Intrasexual competition,territoriality and acoustic communication in male strawberry poison frogs (<Emphasis Type="Italic">Oophaga pumilio</Emphasis>)

In many species male reproductive success is limited by access to females. Territoriality is one behavioural strategy which helps to acquire females. In the present study, we investigated the correlation between territory size and (1) female availability and (2) rate of intrusion by conspecific males in strawberry poison frogs, Oophaga pumilio. Males defended smaller territories in areas with a high female density and high rate of intrusion by conspecific males. Only males with high body condition values were able to establish territories in areas of high female density probably due to better fighting abilities. Moreover, dominant calling frequency was lower during agonistic interactions. Because only males with high body condition values were able to produce very low dominant frequencies, the acoustic properties appear to be an honest signal containing information about fighting abilities. Thus, the negative correlation between dominant frequency and mating success of males found in previous studies seems to be in part the result of intrasexual competition between males.     

Size assessment in simulated territorial encounters between male green frogs (Rana clamitans)

We examined the ability of male green frogs to assess the size of an opponent based on the dominant frequency of their advertisement call, which is negatively correlated with size, using synthetic stimuli to simulate intruders of different sizes. In one field playback experiment, we broadcast a pair of stimuli representing a small and a large male; in a second experiment, we broadcast calls of a medium and a large male. In both experiments, males produced calls with significantly lower dominant frequencies in response to each stimulus. Contrast analyses revealed that males lowered the dominant frequency of their calls more in response to the large-male stimulus than in responses to the small- and medium-male stimuli. In the second experiment, males also responded to the large-male stimulus by calling at higher rates. There were no differences in mean note duration or the number of moves made toward or around the playback speaker in response to any stimulus. Thus, the frequency of an opponent's calls elicits a differential modification of calling behavior, primarily in the form of differential dominant frequency alteration, suggesting that males use dominant frequency to assess the size of opponents during aggressive encounters. Received: 17 April 1998 / Accepted after revision: 7 October 1998     

Growth rate and the cost of calling activity in male carpenter frogs,Rana virgatipes

Summary Energy stress during the breeding season and relationships between calling activity and growth were investigated in male carpenter frogs, Rana virgatipes. This species has a prolonged breeding season of up to three months in Southern New Jersey. Monthly collections made in 1985 revealed that both dry mass and percent body lipid decreased throughout the breeding season but sharply increased at the end of the breeding season. Observations of free-living males showed that small males were more likely to gain mass than large males during the breeding season. All males gained mass at higher rates after the breeding season. A simultaneous record of calling activity and mass change was obtained for 42 males. Males called on 95% of nights, indicating that they rarely ceased their reproductive activities. Small males tended to have low calling efforts and high growth rates. When the effect of initial mass was removed, growth rate was negatively correlated with calling effort among small males. This is the first demonstration of a direct tradeoff between a reproductive activity and growth in an anuran.     

Significance of spectral and temporal call parameters in the auditory communication of male grass frogs

Summary During the spawning period, male grass frogs (Rana temporaria L.) frequently produce short and long territorial calls in addition to mating calls. The calls differ in mean pulse number, duration, and pattern of amplitude modulation. Experiments in which recorded natural calls are played back reveal that male grass frogs are capable of discriminating the different conspecific calls. A male frog stimulated by mating calls always responds by producing mating calls in greater numbers (Fig. 3). Territorial calls presented at low intensity also cause an increase in the mating-call rate (Fig. 4), but at high intensity they clicit territorial calls and turning toward the loudspeaker. A combination of short and long territorial calls was especially effective in eliciting the phonotaxis response. As play-back experiments with simulated calls show, the carrier frequency and the pulse repetition rate are particularly important cues for recognition of conspecific calls (Fig. 5). A simulated call with a 400-Hz carrier frequency (the dominant frquency of the mating call) is just as effective as the natural call with the complete frequency spectrum (Fig. 3), whereas a 1100-Hz simulated call is ineffective (Fig. 5). The chief factors in discrimination among the conspecific calls are the call repetition rate and probably the amplitude and frequency modulation. Changes in the duration of the calls had little effect (Fig. 6). The available evidence suggests that the mating call has a reciprocally stimulating action on males in a chorus, whereas the territorial calls experess aggressiveness and give warning to other males.     

全氟辛磺酸（PFOS）对非洲爪蟾（Xenopus laevis）生长发育、甲状腺和性腺组织学的影响

为了揭示全氟辛磺酸(PFOS)的甲状腺激素和性激素干扰效应,并探索运用爪蟾动物模型同时检测多种环境内分泌干扰效应的可能性,将NF48阶段非洲爪蟾(Xenopus laevis)蝌蚪暴露于0.01、0.1和1mg·L-1PFOS中6个月,检测PFOS对爪蟾生长、变态、甲状腺和性腺的影响.结果表明,在各取样时间,PFOS组爪蟾体长、体重和蝌蚪尾长与对照组均无显著差异(p>0.05);2个月后,PFOS组比对照组平均慢1个发育阶段,4和6个月后,0.01mg·L-1PFOS组反而比对照组分别快1和2个发育阶段.6个月后,PFOS组甲状腺出现滤泡上皮细胞增生、胶质减少甚至空泡化等现象,且随着PFOS浓度的增加而加重;各组幼蛙性腺出现间性、睾丸珍珠状和先天性萎缩等畸形现象,外观为间性的性腺主要表现为睾丸组织中产生类似雌性个体的卵巢腔.对照组幼蛙的雌雄比为0.5:1,而暴露组分别为2.3:1(0.01mg·L-1组)、4.5:1(0.1mg·L-1组)和5:1(1mg·L-1组).上述结果表明,PFOS对爪蟾的变态过程具有小剂量刺激效应,能引起甲状腺组织结构的损伤,导致睾丸组织的雌性化和雌雄性比的异常升高,表现出明显的甲状腺激素和性激素干扰效应,可以认定为一种环境内分泌干扰物.实验同时表明非洲爪蟾可以用于多种环境内分泌干扰效应的同时检测.     

Apparent resource competition among embryos in the brood pouch of a male pipefish

Embryo success was studied in the paternally brooding pipefish Syngnathus typhle. During brooding, which lasts about a month, males provide embryos in their brood pouch with nutrients and oxygen via a placenta-like structure. Egg size depends on female size. In aquaria, males were mated with differently sized females to give the following treatments: M, mixed-egg-size broods of approximately half large and half small eggs; L, single-egg-size broods of large eggs; S, single-egg-size broods of small eggs; and F, field mated males. All males were kept in aquaria for a full brooding period. For each egg-size category, the number of newborn was compared with the number of eggs the male initially fertilized in his brood pouch. Within mixed-egg-size broods, a higher proportion of large eggs survived and large eggs resulted in heavier newborn than small eggs. Indeed, small eggs from a mixed-egg-size brood had significantly lower relative success (proportion of embryos surviving to birth) than those from a brood entirely composed of small eggs. The implication is that embryos compete for resources within the brood pouch, and that competitive success depends on egg size. Given that females produce eggs corresponding in size to their body size, and that females are known to compete indirectly for access to mates (i.e., the sex-roles are reversed), this intrabrood competition could be seen as an extension of female-female competition, but alternative explanations are discussed. Received: 28 April 1995/Accepted after revision: 28 October 1995     

Effects of sublethal concentrations of isoxaben on the development of the African clawed frog,Xenopus laevis

Agricultural pesticide exposure is one factor implicated in the decline of amphibian abundance in aquatic ecosystems. Isoxaben is a cellulose biosynthesis inhibitor mainly used for roadside weed control, suggesting that isoxaben can run off during rain events and potentially affect amphibian development. This study followed the development of African clawed frog (Xenopus laevis) tadpoles exposed to different environmentally relevant concentrations of isoxaben. No significant adverse effects were observed on days to metamorphosis or size and weight at metamorphosis. This reinforces the conclusion that isoxaben herbicide application at low environmental concentrations is not a substantial risk to wildlife.     

The effect of mirrors on African clawed frog (Xenopus laevis) larval growth, development, and behavior

We examined the behavioral and developmental responses of Xenopus laevis larvae to their mirror images in three experiments. The mirrors allowed us to visually simulate increased density, without the tadpoles’ behavior being confounded by chemical cues from additional tadpoles. In the first experiment, we demonstrated that Xenopus tadpoles have a right eye preference for mirrors, contrary to the left eye preference of all other anuran species studied to date. This lateralized eye use disappeared, however, as tadpoles approached metamorphosis. Next, we examined how mirrored aquaria walls affected tadpole growth and development. We found that tadpoles raised in aquaria with partially mirrored walls showed depressed growth compared to tadpoles raised without mirrors, despite the fact that Xenopus larvae normally thrive when raised in visual contact with conspecifics. The tadpoles raised with mirrors had, though not significantly, proportionally larger bodies relative to their tail length (d = 0.51). This suggests that a phenotypically plastic response in body proportions was induced in these tadpoles solely by the sight of other tadpoles. The third experiment established that X. laevis tadpoles are more active in front of a mirror; i.e., they turn more often and spend more time in front of mirrored surfaces. We consider this increased activity to be an aberrant behavior of the tadpoles, which were attempting to school with their own images. We suggest that this extra activity reduced the amount of energy available for growth, accounting for the depressed growth seen in our second experiment.     

除草剂西玛津对非洲爪蟾生存和性腺发育的影响

为了研究三嗪类除草剂西玛津对两栖动物非洲爪蟾(Xenopus laevis)生存和性腺发育的毒性作用,并且与另一种三嗪类除草剂阿特拉津的毒性进行比较,将非洲爪蟾从46/47阶段开始暴露西玛津和阿特拉津到变态1个月后停止,再饲养2个月后将其解剖,取性腺做形态学和组织学观察.暴露期间,每天记录蝌蚪的生长发育情况和存活率.结果显示,在暴露的第1周内(蝌蚪处于46~50阶段),西玛津可导致蝌蚪死亡率明显升高,随后的时间内西玛津对非洲爪蟾的生存不再有明显影响,但却使发育阶段明显的不整齐.阿特拉津对非洲爪蟾的生存和发育则没有明显影响.西玛津和阿特拉津对非洲爪蟾性腺的总体形态和性别比没有明显影响,然而两种除草剂均在一定程度上导致了睾丸组织学的改变.西玛津可能与阿特拉津一样能够通过雌性化/去雄性化作用影响非洲爪蟾睾丸的发育.     

Scramble competition selects for greater tailfin size in male red-spotted newts (Amphibia: Salamandridae)

Darwin predicted that scramble competition for access to reproductive females would result in sexual dimorphism of locomotory structures, but direct evidence for this is extremely rare. I examined the relationship between variation in tailfin size and mating success in a field and laboratory study of red-spotted newts, Notophthalmus viridescens. Over three breeding seasons, male tailfin size was positively correlated with variation in male amplexus frequency, and indirectly correlated with male insemination frequency. In a laboratory study, I confirmed that males' ability to capture females is affected by variation in tailfin size. This is the first study to show that naturally occurring variation in male locomotory structures affects male mating success. It corroborates the prediction that scramble competition leads to sexual selection on locomotory structures and, potentially, to dimorphism in these structures. Received: 16 April 1999 / Received in revised form: 16 May 1999 / Accepted: 12 June 1999