Changes in the Oscillatory Electric Potential on the Olfactory Epithelium and in Reproductive Hormone Levels during the Breeding Season in the Toad (Bufo japonicus)

A slow transient electric potential change (electro-olfactogram, EOG) can be recorded through an Ag-AgCl electrode placed on the olfactory epithelium in response to stimulation with an air stream to the tissue in toads (Bufo japonicus). During the breeding season, oscillatory potential changes (OSC) superimpose on the EOG. In the present study the OSC amplitude was found to be highly correlated with the migratory behavior. Since toads track the route to and from the breeding pond using olfactory cues along the migration route, the enhanced OSC should be responsible for the breeding migration.A significant positive correlation was found between plasma gonadotropin levels and the OSC amplitude in males captured during the breeding migration. There was no significant relationship between plasma gonadotropin levels and the OSC amplitude in female toads during the breeding season, but there was a significant correlation between plasma progesterone levels and the OSC amplitude. In males, hypophysectomy just before the breeding season decreased the OSC amplitude. And testis weight was also positively correlated with the OSC amplitude in January. These results suggest that the appearance of the OSC is related to the timing of the activation of the reproductive system. However, treatment of toads with hCG (human chorionic gonadotropin), testosterone, estradiol or progesterone in the non-breeding season did not induce a significant change in the OSC amplitude. Other factor or factors may be required in activation of the olfactory system of the toad in a non-reproductive stage together with the hormones of the gonadal axis.     

Sexually dimorphic muscles in the forelimb of the Japanese toad,Bufo japonicus

During the breeding season, male anurans display clasping behavior by holding females with their forelimbs. This behavior is peculiar to males, and may require specializations in forelimb musculature. The present study revealed that five kinds of forelimb muscles were heavier in the male Japanese toad than in the female: the flexor carpi radialis (FCR), the flexor antibrachii medialis caput superius (FAMsup), the abductor indicis longus (AIL), the extensor carpi radialis caput superius (ECRsup), and the flexor antibrachii lateralis superficialis caput superius (FALSsup). In addition, one breast muscle, the coracoradialis (CR), was also heavier in males than in females. A quantitative analysis of muscle fibers processed for myosin ATPase activity showed that, in such “sexually dimorphic muscles” of the female, both fast (twitch) and slow (tonic) muscle fibers were of smaller diameter than in other forelimb muscles of both sexes (all male muscles plus “nondimorphic muscles” of the female). Moreover, both types of fibers were less numerous than in the corresponding muscles of the male. These results suggest that the “sexually dimorphic muscles” are used especially for clasping by the male and are degenerative or subnormal in the female. Slow muscle fibers were neither peculiar to, nor abundant in, these clasping muscles, although they may well be necessary for tonic and prolonged contractions of the forelimb muscles during clasping. The mechanism of sexual dimorphism may be a direct action of androgens on clasping muscles or an indirect action on clasping muscles via the innervating motoneurons.     

Local motion processing in the optic tectum of the Japanese toad,Bufo japonicus

Summary The results of previous behavioral studies can be so interpreted that the prey-catching behavior in the toad is elicited if there is a local motion restricted with-in a small part of the visual field, while it is suppressed if there is a global motion over a large part of the visual field. This has led us to design experiments to answer a specific question (yet a very essential one for understanding neural processes underlying this behavior): Are there local motion detectors in the toad's visual system that are not activated by global motion over a large part of the visual field but are activated by local motion confined within a smaller part of it? The present study showed that (1) the majority of the toad's tectal neurons exhibit properties of the local motion detectors as defined above, and (2) these properties can be explained from the receptive field structure revealed in the present experiments. Based on these results, we suggest that the tectal local motion detectors are essential for the detection and localization of small moving prey-objects in the natural environment while ignoring the large moving objects or the self-induced motion of the visual field.Abbreviations ERF excitatory receptive field - G1-5 group 1–5 neurons     

Ultrastructure and Atrial Natriuretic Peptide(ANP)-like Immunoreactivity of Cardiocytes in the Larval, Metamorphosing and Adult Specimens of the Japanese Toad, Bufo japonicus formosus

The distribution of an atrial natriuretic peptide (ANP)-like material in the cardiocytes of larval, metamorphosing, and adult specimens (both breeding and non-breeding) of the toad, Bufo japonicus formosus , was studied immunohistochemically, ultrastructurally and immunocytochemically. Histochemically, ANP-immunoreaction was positive in the atrium and ventricle in stage 33 larvae, while negative in the ventricle in stage 40 larvae. In adult toads, the reaction was stronger in the right than in the left atrium but quite weak in the ventricles, particularly those of non-breeding specimens. Electron microscopy indicated a very small number of secretory granules in the atrial and ventricular cardiocytes of embryos as early as the limb-bud stage (stage 28), and as development proceeded, the number of these granules increased rapidly in atrial but not in ventricular cardiocytes. In metamorphosing animals, a small population of larger granules (200–250 nm) was noted next to those of ordinary size (the median, 110 nm) in the same cell. In adult toads, granules of about 120 nm and 200 nm in median size were found in the same cell. Postembedding immunogold staining consistently indicated ANP-immunoreactivity in these granules in atrial and ventricular cardiocytes. The plasma content of immunoreactive ANP was considerably higher in breeding (20.5 ± 5.9 pg/ml) than in non-breeding toads (5.4 ± 1.7 pg/ml). These results are discussed in relation to presently available data on the physiological role of ANP.     

Genes induced during the early developmental stages of the Cane Toad, Bufo (Chaunus) marinus

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.     

黑眶蟾蜍卵母细胞体外成熟过程中生发泡迁移的研究(英文)

生发泡迁移（ＧＶＭ）是大多数两栖类动物中卵母细胞成熟之前都可以观察到的、涉及细胞核行为的现象。本实验在光镜水平上对激素诱导下的黑眶蟾蜍卵母细胞的ＧＶＭ现象、以及细胞骨架解聚剂类药物———秋水仙素、细胞松弛Ｂ（ＣＢ）对这种激素诱导作用的影响进行了研究。同时,采用ＡＺＡＮ染色法观察了ＧＶＭ过程中生发泡周边纤维骨架的结构变化。将取自刚脱离冬眠期雌体的卵母细胞按不同的培养液、分三个实验组,体外培养不同的时间后,固定、染色、观察。对照组培养液成分为Ｒｉｎｇｅｒ液中加入人绒毛膜促性腺激素和脑垂体;实验组分别增加秋水仙素或ＣＢ。Ｔａｂ．１和ＰｌａｔｅＩ１,４,５,６,７,８,９表明：经过体外培养４ｈ,各组生发泡均向动物极表面发生了迁移。但是,秋水仙素的作用在培养的前２ｈ,对ＧＶＭ表现为促进效应（ＰｌａｔｅＩ５）;而培养的后２ｈ,却表现为抑制（ＰｌａｔｅＩ８）;ＣＢ的作用始终是抑制（ＰｌａｔｅＩ６＆９）。６ｈ后,各组生发泡均告破裂（ＰｌａｔｅＩ１０,１１,１２）。正常情况下,生发泡周围被一环形纤维包围,其外侧有两个纤维化小体（ＰｌａｔｅＩ２）。发育较快者,纤维化小体消失,植物极附近纤维逐渐加厚（ＰｌａｔｅＩ２     

Breeding pond fidelity in the common toad, Bufo bufo

Two independent studies of adult common toad, Bufo bufo , movements between different spawning ponds/populations both within and between years were made in England and Sweden during the spring breeding periods of1987–1990. The results of the two studies were subsequently combined since they were found to be complementary. In addition, in England during 1984 and 1985, large numbers of 'toadlets' were marked so that they could be identified if recaptured as breeding adults.
The degree of relocation between ponds was negatively correlated to the distance between ponds. The proportion of both males and females that moved between ponds within a particular year was significantly less than the proportion relocating between years. In any year, between 79% and 96% of adults that survived to breed the following year, returned to the original pond. Similarly, of the toadlets marked during1984–85, 81% of the males that subsequently returned as breeding adults were captured in their pond of origin.
Isolation, in relation to both population dynamics and population genetics, is defined and its implications for the management of common toad populatins discussed.     

Electrical Response and Growth of Olfactory Cilia of the Olfactory Epithelium of the Newt in Water and on Land

A correlation between the length of the olfactory cilia and the electrical activity of the olfactory epithelium was studied in newts living in water and on land. The olfactory cilia grew when newts were transferred onto land. The cilia in the olfactory bud became longest in 108 hours after the transfer and then became shorter, while those in the interstitium only gradually elongated. Slow potentials were evoked in the epithelium by the application of odorous fluids but not by odorous vapors for 20 hours after the transfer. Thereafter, the slow potential began to appear in response to odorous vapors and reached maximal magnitude between 60 and 70 hours after the transfer, while it was not evoked by odorous fluids in this period. In the later stage, the slow potential to odorous vapors decreased in magnitude and disappeared 120 hours after the transfer, while it began to reappear in response to odorous fluids. When these changes in the slow potential were compared with those in the cilium, a discrepancy was found between the period of maximal potential magnitude and that of maximal cilium length.     

Four modes of lightening of coloration in desert populations of the Green Toad,Bufo viridis

Abstract

The Gréen Toad reaches its southern limit in the Israeli desert. Its coloration, green blotches on lighter background, may become pale in one or more of four ways: lighter hues, reduced number of blotches, reduced size of blotches, and reduced dark area of ring-shaped blotches.     

Plistophora myotrophica spec, nov., Causing High Mortality in the Common Toad Bufo bufo L., with Notes on the Maintenance of Bufo and Xenopus in the Laboratory

SYNOPSIS. Colonies of Bufo bufo kept for pregnancy diagnostic purposes were found infected with a new species of microsporidian, Plistophora myotrophica , causing atrophy of the striated muscles. Lysis of myofibrils by the parasite leads to the formation of fusiform spaces in the fibers packed with microsporidian spores. A cyst wall is not formed. Toads become emaciated and suffer heavy mortality.
In experimental infections the sporoplasm hatches in the gut. Fusiform granular bodies, not identifiable as blood cells, are found in the muscle capillaries at 18 days and possibly represent early stages of the parasite. Between 18 and 23 days division of the parasite occurs in the muscle fibers, first by binary fission, then multiple fission and finally by plasmotomy culminating in the formation of sporonts with 16 to 100 nuclei. Separation into sporoblasts precedes spore formation.
In old infections phagocytes, which are probably monocytes, infiltrate between the groups of spores and ingest them. Ingested spores which stain differently from their free counterparts, are dead. Muscle regeneration is initiated by the toad, with the formation of long chains of sarcoblasts, sometimes 30 or more in a chain, responsible for the regeneration and redifferentiation of myofibrils.
Tadpoles are refractory to infection. As toads normally lead a solitary life the high local incidence in the area from which the toads were collected is explained only if the toads acquire the infection from spore-contaminated ground in the breeding season.     

Effects of food availability and ambient temperature on hibernation in the Japanese dormouse,Glirulus japonicus

The effects of food availability and ambient temperature (Ta) on hibernation in the Japanese dormouse,Glirulus japonicus, were examined. When the dormice were deprived of food under natural environmental conditions in midwinter, they showed typical hibernation patterns; in contrast, the animals given ad lib food did not show continuous long-term torpor. Furthermore, exposure to constant low temperature did not affect the torpor patterns in the animals receiving adequate food. These results suggest that food availability plays a decisive role in the onset and maintenance of hibernation in this species, and that low Ta does not have a great influence on the torpor patterns in animals with access to food. This differs from the previous study that emphasized the importance of Ta in the occurrence of torpor and discounted the effects of food shortage.     

Influence of thyroid hormone on the development of preoptic-hypothalamic monoaminergic neurons in tadpoles of Bufo bufo japonicus

The effect of thyroid hormone on the development of diencephalic monoaminergic neurons was studied in tadpoles of Bufo bufo japonicus. Monoamine-containing neurons in the preoptic recess organ (PRO) appeared later than those in the paraventricular organ (PVO) and nucleus infundibularis dorsalis (NID). After deprivation of thyroidal primordium no fluorescent neurons developed in the PRO. Development of monoaminergic neurons in the PVO and NID was not affected by thyroidectomy. Thyroxine treatment brought about the fluorescent neurons in the PRO of the thyroidectomized tadpoles. Fluorescent terminals in the median eminence became conspicuous around the capillaries which penetrated to the median emience, when the tadpoles reached late prometamorphic stage. In the median eminence of thyroidectomized tadpoles, the monoaminergic axon terminals did not develop. Thyroxine induced both the fluorescent terminals and the capillary penetration in the median eminence of the thyroidectomized tadpoles. In the tadpoles hypophysectomized at tail-bud stage, thyroxine induced neither the fluorescent terminals nor the capillaries in the median eminence.     

DFP-sensitive multicatalytic protease complexes (proteasomes) involved in the control of oocyte maturation in the toad,Bufo japonicus

The inhibition of progesterone-induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0–0.5 GVBD₅₀ on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD₅₀) was set at 1.0], namely, before the beginning of GVBD. DFP-sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process. These findings raise the possibility that the activities of some endogenous DFP-sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo. © 1994 Wiley-Liss, Inc.     

四川若尔盖湿地国家级自然保护区繁殖期黑颈鹤调查

于2012年5⁹月,对四川若尔盖湿地国家级自然保护区的黑颈鹤Grus nigricollis数量及分布进行了调查。共记录到287只黑颈鹤,其中5月70只,包括繁殖鹤群18对36只(占51.4%),3只以上的非繁殖鹤群3群27只(群体大小分别是4只、8只和15只),占38.6%,单只鹤7只(占10.0%)。7月份观察记录到黑颈鹤121只,繁殖鹤群有41对82只,占观察数量97只(除幼鹤外)的84.5%,单只鹤群3只,占3.1%,3只以上的非繁殖鹤群1群,12只,占12.4%,其余为幼鹤24只。9月份共观察记录96只(其中14只幼鹤),繁殖鹤群有38对,76只,占观察数量82只(除幼鹤外)的92.7%,单只鹤1只,占1.2%,3只以上的非繁殖鹤群1群,由5只个体组成,占6.1%。与之前调查结果相比,本次调查到的黑颈鹤分布区范围没有大的变化。整体上看,保护区内黑颈鹤的主要繁殖栖息地在尧拉乔、纳勒乔、纳洛乔一带和热尔大坝的哈丘湖、措拉坚、花湖等湖泊及湖滨沼泽。建议在这两个区域设置固定样地进行长期监测,并且在保护区内限制放牧强度,核心区内严禁放牧,调节好草场的季节性利用,使湿地生态系统得以合理的恢复使用,从而保护黑颈鹤及其栖息地。     

Immunolocalization of NGF and its Receptors in Ovarian Surface Epithelium of the Wild Ground Squirrel during the Breeding and Nonbreeding Seasons

The ovarian surface epithelium (OSE) plays an important role in normal ovarian physiology. During each reproductive cycle, the OSE takes part in the cyclical ovulatory ruptures and repair. The aim of this study was to investigate the immunolocalization of nerve growth factor (NGF) and its receptors, tyrosine kinase A (TrkA) and p75, in the OSE cells of the wild ground squirrels during the breeding and nonbreeding seasons. There were marked variations in ovarian weight and size between the breeding and the nonbreeding seasons. Histologically, cuboidal cells and squamous cells were identified in the OSE of both seasons. Yet, stronger immunostaining of NGF, TrkA and p75 were observed in cuboidal cells and squamous cells in the breeding season as compared to the nonbreeding season. In addition, plasma gonadotropin concentrations were higher in the breeding season than in the nonbreeding season, suggesting that the expression patterns of NGF, TrkA and p75 in the OSE were correlated with changes in plasma gonadotropins. These findings suggested that NGF and its receptor TrkA and p75 may be involved in the regulation of seasonal changes in the OSE of wild ground squirrel.     

Metallothionein present or induced in the three species of frogs Bombina orientalis, Bufo bufo japonicus and Hyla arborea japonica

A low molecular weight copper (Cu)- and/or zinc (Zn)-binding protein was present in the livers (and kidneys) of the three species of frogs Bombina orientalis, Bufo bufo japonicus and Hyla arborea japonica. Cadmium (Cd) accumulated in the livers (and kidneys) after repeated i.m. injections of Cd2+ was mostly bound to the same metal-binding protein as Cd, Zn- or Cd, Zn, Cu-thionein. Concentrations of Cd and several essential elements in the livers and kidneys of the three species of frogs were determined by inductively coupled plasma-atomic emission spectrophotometry. Metallothionein in the three species of frogs consists of a single isoform both in control and Cd-injected animals.     

Chemical characterization of sialyl oligosaccharides in milk of the Japanese black bear, Ursus thibetanus japonicus

Sialyl oligosaccharides were separated from two samples of Japanese black bear milk by extraction with chloroform/methanol, gel filtration on Bio Gel P-2, ion exchange chromatography on DEAE-Sephadex A-50 and high-performance liquid chromatography (HPLC) on a TSK gel Amido-80 column. They were characterized by ¹H-NMR spectroscopy. The structures of four sialyl oligosaccharides separated from the milk were the following:

Neu5Ac(α2-3)Gal(β1-4)Glc

Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3) Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6) Gal(β1-4)Glc

Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3) Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6) Gal(β1-4)Glc

Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc