Causes of mortality of the Wyoming toad

Wyoming toads (Bufo baxteri) that died from January 1989 to June 1996 were submitted to the Wyoming State Veterinary Laboratory (Laramie, Wyoming, USA) for postmortem evaluation. These consisted of 108 free-ranging toads and 170 animals from six captive populations. Ninety-seven (90%) of 108 free-ranging toad carcasses were submitted during September and October. From 1989 to 1992, 27 (77%) of 35 mortalities in the captive populations occurred in October, November, and December. From 1993 to 1996, mortality in captive toads occurred without a seasonal pattern and coincided with changes in hibernation protocols that no longer mimicked natural cycles. Cause of mortality was determined in 147 (53%) of the 278 cases. Mycotic dermatitis with secondary bacterial septicemia was the most frequent diagnosis in 104 (71%) of 147 toads. Basidiobolus ranarum was found by microscopic examination of skin sections in 100 (96%) of 104 of these mortalities. This fungus was isolated from 30 (56%) of 54 free-ranging and 24 (48%) of 50 captive toads. This research documents the causes of mortality for both free-ranging and captive endangered Wyoming toads over a 7 yr period.     

人工诱导冬眠对中华蟾蜍血液和组织中宏量营养素的影响

为探寻冬眠期间两栖动物血液和组织中宏量营养素的适应性改变过程,经人工诱导冬眠,检测了中华蟾蜍(Bufo gargarizans)在冬眠第1、3、7、14、28、42、56天的体重和脏器指数,以及血液、心、肝和骨骼肌组织中宏量营养素的含量。结果显示:1)冬眠期间中华蟾蜍体重未出现显著性变化,无性别差异。雄性蟾蜍的心、肝和腓肠肌的脏器指数显著性大于雌性(P0.01),但同一性别的脏器指数在冬眠期间无显著性变化。2)血中葡萄糖浓度自冬眠第42天起显著下降(P0.01);总蛋白在冬眠后第56天显著降低(P0.05),总胆固醇变化不显著。血中宏量营养素无性别差异。3)肝糖原自冬眠第42天起显著下降(P0.01),肌糖原自冬眠第1天起显著性下降(P0.05),而骨骼肌和心肌组织蛋白含量无显著变化。组织中宏量营养素无性别差异。人工诱导冬眠条件下,中华蟾蜍血液和组织中的糖类含量先迅速下降,血液中的蛋白成分只在深眠时才显著减少,但血液和组织中的宏量营养素水平可在1个月内维持稳定,这可能是其适应冬眠的主要生理学机制之一。     

Dermatitis in captive Wyoming toads (Bufo baxteri) associated with Fusarium spp

From May 2007 to June 2008, 30 of 49 Wyoming toads (Bufo baxteri) kept at Omaha's Henry Doorly Zoo (Nebraska, USA) died showing clinical signs of ventral erythema, inappetance, lethargy, and delayed righting reflex. Treatment with antifungals and antibiotics was unsuccessful in all cases. Histopathologic analyses revealed dermatitis as the primary problem in 20 of 21 toads in which skin was examined. Fungal dermatitis was present in 17 toads, with hyphae approximately 1-3 μm in diameter, and parallel cell walls and frequent septations. In 14 animals, the fungal dermatitis was the main pathologic lesion. Several species of bacteria were associated with all cases. A few animals tested positive for Ranavirus using polymerase chain reaction. Fusarium sp. was consistently cultured from skin, feces, kidneys, and from powdered food provided to crickets. Four isolates were identified as Fusarium proliferatum, Fusarium oxysporum, Fusarium solani, and Fusarium verticillioides, which suggested a secondary role of fungi. A specific underlying cause of disease could not be found, although the roles of humidity and Ranavirus infection are discussed, along with the well-known susceptibility of bufonids to fungal dermatitis.     

Mortality of captive Canadian toads from Basidiobolus ranarum mycotic dermatitis

Twenty-six adult free-ranging Canadian toads (Bufo hemiophrys) were collected from northeastern North Dakota (USA) during the last week of August 1994 and placed in captivity. During late December and January 1995, 21 Canadian toads died. Clinical signs included increased time sitting in water bowls, darkened dorsal skin, constant arching of their backs, and hyperemia and sloughing of ventral epidermis. The condition progressively worsened until death occurred within 5 to 7 days after onset of clinical disease. Mycotic dermatitis due to Basidiobolus ranarum was diagnosed in all toads and the fungus was isolated from 11 (52%) of 21 toads. Histology of the ventral skin and digits revealed numerous fungal spherules and occasional hyphae without significant inflammatory reaction. This condition clinically resembled red leg associated with Aeromonas hydrophila and many other bacterial organisms, and the diseases could be confused without appropriate diagnostic tests. This also is the first report of B. ranarum causing clinical disease in a toad species.     

External and internal control of patterns of feeding, growth and gonadal function in a temperate zone anuran, the toad Bufo bufo

Young toads were brought into the laboratory before hibernation in the autumn and exposed to alternating periods of low temperature and high temperature and feeding, thus simulating climatic cycles. In newly metamorphosed, non-hibernating toads, food intake was initially low and growth was stagnant, but subsequently food intake and lean growth increased strongly. Presumably, growth was initiated independently of feeding. In one-year-old, non-hibernating toads, food intake and growth declined with time, but high rates of feeding and growth were restored in the toads exposed to simulated hibernation. In both metamorphosed toads and one-year-old, hibernating toads, a period of predominant fat deposition succeeded predominant lean growth before food intake again declined to low levels. Initiation of sexual maturation in the female toads coincided with suspended growth, whereas sexual maturation in males was not correlated with changes in growth rate. In the female, but not the male toads, sexual maturation was greatly advanced under the experimental conditions, resulting in adult female sizes far below those typical of the wild population.     

Hormonal Control of Seasonal Growth in a Temperate Zone Toad Bufo bufo

Young toads were exposed to a light regimen of 12L/12D at 21°C and fed meal-worms before hibernation in the autumn, when the toads reduce feeding and discontinue growth, and after hibernation in the spring, when feeding and growth is resumed. In the autumn treatment of the toads with bovine growth hormone maintained feeding and growth; in the spring the treatment protracted the period of feeding and growth. Thyroxin had no obvious effects on growth. It is concluded that the annual growth pattern of temperate zone toads is controlled by an internal rhythm in the secretion of growth hormone.     

Experimental exposure of Canadian toads to Basidiobolus ranarum

Experimental transmission of the fungus Basidiobolus ranarum was induced in two treatment groups of Canadian toads (Bufo hemiophrys) and caused a fatal mycotic dermatitis. Seven of 10 (70%) toads that had their ventral skin mildly abraded and exposed to B. ranarum developed hyperemia, and sloughing of their ventral skin and died. Toads with abraded ventral skin or exposure to infected skin also were affected statistically at a higher rate than those with abraded skin and exposure to pure cultures of B. ranarum inoculated into their water source. Of toads showing clinical disease, B. ranarum was identified by both impression smears and histology in all cases, but not from toads that appeared clinically healthy. The organism was cultured from 5 of 7 (71%) toads with clinical disease but not from any toad that appeared clinically healthy (n = 28). This study documents methods of experimental transmission of B. ranarum, an organism responsible for causing a mycotic dermatitis that is fatal to toads.     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.     

Seasonal changes in the migratory behaviour of the toad Bufo bufo: direction and magnitude of movements

Summary The migratory behaviour of the toad Bufo bufo was studied from February 1985 to April 1986 in the submontane region of Bavaria, West Germany. Toads were fitted with a mechanical tracking device to record individual paths of migration. Three aspects of migratory behaviour were quantified: orientation in relation to the breeding site, straightness of path, and locomotory activity. The annual activity period began with migration from the hibernation sites to the breeding pond in April. The paths went straight towards the breeding pond independent of the distance (70–420 m). During the period of oviposition the preference for the breeding site direction vanished and toads moved away from the breeding pond, but in less straight paths than before. In summer migratory activity decreased considerably and was restricted to small areas, the home ranges, at distances of 55–1600 m from the natal breeding pond. The straightness of path was rather low, because toads often returned to their starting points. During rainy nights toads occasionally left their home ranges for extensive excursions. In autumn most toads again migrated towards the breeding pond, but paths were significantly less straight and direct than in spring. However, toads stopped before reaching the breeding pond and hibernated in holes or under the leaf layer. The mortality rate of tracked toads was about 45%. The relative influence of 17 environmental variables on locomotory activity was evaluated by principal component analysis and stepwise multiple regression. Temperature at night and rainfall variables accounted for significant amounts of variance, whereas temperature by day, air humidity, and atmospheric pressure showed no correlation. Activity decreased if temperature approached 0° C or after long periods without rainfall. Within a certain range of tolerance, however, the locomotory activity of the toads was widely independent of environmental factors, indicating that endogeneous factors are more important sources of variation in the migratory behaviour of these toads than commonly assumed.     

Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas)

The goals of this study were to test the effects of exogenous hormones and hibernation on breeding behavior and gamete release by boreal toads (Bufo boreas boreas). Each year, a subset of 77 toads was hibernated and then paired with hibernated or nonhibernated mates and treated with luteinizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotropin (hCG), or left untreated. Amplexus and egg and sperm production were recorded. At 1 yr of age, only 19% of pairs exhibited amplexus, and no sperm or eggs were produced. At 2 and 3 yr of age, most male toads treated with LHRHa exhibited amplexus (56.9% and 100%, respectively). Among 2-yr-old males, amplexus was more prevalent (P < 0.05) in those that were hibernated than in those that were nonhibernated (54.0% and 33.3%, respectively), but most males in each group (93.3% and 75%, respectively) produced sperm in response to LHRHa treatment. Only one 2-yr-old and two 3-yr-old females produced eggs. At 4 yr of age, eight females produced eggs, but two died from egg retention. More nonhibernated than hibernated females developed eggs (7 of 10 vs. 1 of 10, P < 0.05). Mean (±SD) weight of female toads producing eggs (58.9 ± 11.9 g) was greater (P < 0.05) than that of nonproducing females (43.6 ± 7.0 g). Similarly, four of seven nonhibernated females (58.8 ± 8.3 g) produced eggs at 5 yr of age. All eggs were produced by females treated once with LHRHa. Number of eggs per female varied (141 to 3307), and development to tadpoles was low (0 to 36.5%), although tadpoles did become toadlets. In conclusion, male and female boreal toads matured at 2 and 4 yr of age, respectively, and heavier females were more likely to produce eggs. To enhance breeding success, males should be hibernated and treated with LHRHa. In contrast, female productivity was enhanced by improving their body condition instead of subjecting them to hibernation prior to LHRHa treatment.     

The effects of density,rainfall and environmental temperature on body condition and fecundity in the common toad,Bufo bufo

The body length and body weight of all adult common toads (Bufo bufo) breeding at a pond in south Dorset were measured between 1983 and 1993. Each toad was placed into one of four categories depending on its sex and whether it was either a first time breeder or an animal that had previously bred. The body condition of each male and female toad for each year was compared with the average body condition of all the male and female toads captured over the 11 years of the study so that between-year differences in condition could be detected. Changes in body condition were compared with changes in body condition were compared with changes in toad density, rainfall and climatic temperature during the previous summer (March–September), during hibernation (October–February) and during the month preceding the start of spawning. During the study there was a decline in the body condition of all toad categories and these changes were significantly correlated with changes in toad density and climatic temperature. Toads were also more likely to enter hibernation in poor condition following a hot dry summer than after either cool wet or hot wet summers. Body condition explained 41% of the size-specific variation in fecundity.     

Dynamics of Oogenesis in a Lower Vertebrate,the Toad Bufo bufo

The patterns of production and growth of oocytes were studied in female toads brought into the laboratory after hibernation and kept at 20°C. The oogenic state of the ovaries was determined from the number and size frequency distribution of the oocytes, and change in oogenic state was followed by comparing ovarian biopsies with the ovaries at autopsy one month later. New oocytes grew within one month to sizes up to 0.16 mm or more in diameter, and were integrated in the existing pool of small oocytes. Production of new oocytes continued for more than one month, perhaps 2–3 months, to increase the pool of oocytes by about 50%. An episode of oogenesis sensu stricto is followed by a resting period, probably lasting for about 1–2 years. Oogenesis was not coordinated with ovulation or recruitment of oocytes to vitellogenic growth and it occurred also in the ovaries of sexually immature toads. Exogenous gonadotropin (hCG) and/or fasting for one month of toads in a good nutritional condition did not affect oogenesis, but no oogenic episodes were observed in the ovaries of starving toads that were depleted of energy reserves. It is concluded that oogenesis and growth of oocytes constitute a dynamic system that is only inadequately described by the widely accepted linear, sequential model of production of eggs in amphibians and teleosts.     

甘蔗糖蜜发酵生产多不饱和脂肪酸的菌种筛选

利用苏丹黑B染色和测定多不饱和脂肪酸碘值的方法,从土壤中筛选出一株适合用甘蔗糖蜜为原料生产多不饱和脂肪酸的霉菌LB1。研究表明,该菌株培养的最适糖蜜浓度为10°BX,通过单因素实验和正交实验设计,确定了优化培养条件:最适温度28℃、摇床转速为160r/min、pH值为6.0和培养天数为5d。在优化条件下,菌株的油脂含量为其生物量的57.08%,其中油脂中多不饱和脂肪酸的组成及含量为:油酸15.42%,亚油酸14.38%、γ-亚麻酸23.55%、α-亚麻酸3.06%、花生四烯酸9.87%、廿碳五烯酸8.14%、廿二碳六烯酸6.07%等。多不饱和脂肪酸的含量占总脂肪酸的80.49%。对LB1菌株进行形态特征、生理特征分析及5.8S rDNA基因两侧的内转录间隙进行序列分析推测该菌株为反屈毛霉。     

Mapping the Relative Probability of Common Toad Occurrence in Terrestrial Lowland Farm Habitat in the United Kingdom

Introduction

The common toad (Bufo bufo) is of increasing conservation concern in the United Kingdom (UK) due to dramatic population declines occurring in the past century. Many of these population declines coincided with reductions in both terrestrial and aquatic habitat availability and quality and have been primarily attributed to the effect of agricultural land conversion (of natural and semi-natural habitats to arable and pasture fields) and pond drainage. However, there is little evidence available to link habitat availability with common toad population declines, especially when examined at a broad landscape scale. Assessing such patterns of population declines at the landscape scale, for instance, require an understanding of how this species uses terrestrial habitat.

Methods

We intensively studied the terrestrial resource selection of a large population of common toads in Oxfordshire, England, UK. Adult common toads were fitted with passive integrated transponder (PIT) tags to allow detection in the terrestrial environment using a portable PIT antenna once toads left the pond and before going into hibernation (April/May-October 2012 and 2013). We developed a population-level resource selection function (RSF) to assess the relative probability of toad occurrence in the terrestrial environment by collecting location data for 90 recaptured toads.

Results

The predicted relative probability of toad occurrence for this population was greatest in wooded habitat near to water bodies; relative probability of occurrence declined dramatically > 50 m from these habitats. Toads also tended to select habitat near to their breeding pond and toad occurrence was negatively related to urban environments.     

产二十二碳六烯酸等多不饱和脂肪酸真菌的筛选*

从土壤中筛选出一株产二十二碳六烯酸（DHA）的丝状真菌,菌丝含油21.23％,DHA占总脂肪酸2.51％;同时含二十碳五烯酸（EPA）,占总脂肪酸的0．41％;不饱和脂肪酸占总脂肪酸的80％。经鉴定为头孢霉属（Caphalosporiumsp．）真菌。同时发现两株菌含EPA,经鉴定为小克银汉霉（Cunninghamellasp.）和毛霉（Mucorsp．）。在这几个属中发现DHA和EPA尚属首次。头孢霉菌DHA产量及百分含量和斜面菌种在不同温度下储藏有关。菌种在20℃储藏10天,在液体PDA培养基上发酵,DHA可占总脂肪酸11．27％,产量达63．35mg／L。     

达乌尔黄鼠实验室饲养、繁殖及其冬眠阵

为探索实验室条件下达乌尔黄鼠饲养与繁殖的方法及冬眠阵的发生规律,参照野生黄鼠冬眠洞穴的主要生态环境参数,建立人工冬眠屋,采用传统锯末技术记录冬眠阵。结果显示: (1) 处于春季繁殖期的黄鼠应以大鼠饲料为主,辅以少量黄瓜等,夏季活跃期交叉饲喂大鼠饲料与兔饲料,辅以多水的瓜果蔬菜,秋季育肥期以大鼠饲料为主,辅以高脂肪高蛋白的花生、豆类等。(2)雌鼠怀孕期为28 d 左右,哺乳期约一个月,雌鼠每窝产仔4 ～ 8 只,平均5.52 只;初生幼鼠两周内忌换垫料,并避免将异味带入鼠房。(3)黄鼠冬眠期从当年11月下旬至次年3 月上旬,平均93.95 d;冬眠阵睡眠时长平均7. 44 d,阵间激醒时长平均1.36 d,睡眠天数占整个冬眠期的89.9% ;整个冬眠期,黄鼠冬眠阵平均7. 55 个。(4)2009 年秋至2011 年春季,自野外共捕回黄鼠185 只, 存活146 只,存活率78. 9% 。在2006、2009 和2011 年的黄鼠繁殖期,共配对25 对,产仔138 只,成活92 只,成活率为66.7% 。结果表明,野生达乌尔黄鼠可在人工饲养条件下实现繁殖,并可在人工冬眠屋成功冬眠。     

Glycosphingolipids as a possible signature of microbial communities in activated sludge and the potential contribution of fungi to wastewater treatment under cold conditions

Seven strains of fungi were isolated from activated sludge and identified as Mucor sp., Geotrichum sp., Trichosporon sp., Candida sp., and Trichoderma sp. by 28S rDNA D2 region sequences analysis. The structures of the main ceramide monosaccharides (CMSs) from these fungi were identified as glucosylceramide (GlcCer) consisting of ceramide moieties of 9-methyl-octadeca-sphingadienine (9-Me d18:2), with 2-hydroxyhexadecanoate (h16:0) (Mucor sp. and Geotrichum sp.), 2-hydroxyoctadecanoate (h18:0) (Trichosporon sp. and Candida sp.), and 2-hydroxyoctadecenoate (h18:1) (Trichoderma sp.). Seasonal changes in glycosphingolipids in activated sludge suggest the possibility that microbial flora in activated sludge changes with the seasons, and that fungi adaptable to low temperatures dominate in the cold period, resulting in the maintenance of stable effluent quality. Mucor sp., Geotrichum sp., and Candida sp. satisfactorily reduced the BOD of synthetic sewage at 10 degrees C. These results indicate that fungi in activated sludge can contribute to wastewater treatment in cold conditions.     

Isolation, identification and antifungal susceptibility of lemon pathogenic and non pathogenic fungi

Numerous species of filamentous fungi were isolated from lemon on different plantations in the province of Tucuman, Argentina. The techniques suggested by the Subcommittee of Antifungal Susceptibility of the National Committee for Clinical Laboratory Standards, (USA) were adapted. The effect of three different concentrations of the fungicides imazalil, guazatine, SOPP and thiabendazole on the fungi Fusarium oxysporum, Fusarium moniliforme, Aspergillus niger, Aspergillus flavus, Aspergillus clavatus, Geotrichum candidum, Rhizopus sp, Penicillium sp, Penicillium digitatum and Mucor sp were studied. All the tested strains were resistant to thiabendazole. We assayed a mixture of SOPP (5%), guazatine (350 ppm) and imazalil (100 ppm), which showed a synergic effect on Rhizopus sp. Mucor sp was the only fungus resistant to the four fungicides tested as well as to the above mentioned mixture.     

The Influence of Parasite Infection on Mating Success in Spadefoot Toads, Scaphiopus couchii

The desert toads, Scaphiopus couchii, have an annual activityseason of less than 8 weeks and experience only one significantparasite infection: the monogenean Pseudodiplorchis americanusis transmitted during host spawning and provides a natural systemfor testing the influence of parasite burden on host matingsuccess. The 10 month hibernation involves total starvationduring which the blood-feeding parasites reduce fat reservesand haematocrit. The toads emerge and spawn on the first nightafter rainfall, before they replenish depleted reserves. Malechorusing is energetically very demanding and mate selection,limited to a 7 hr nocturnal assembly, is determined principallyby female choice. Parasite transmission triggered by host sexualactivity results in 100% prevalence and high intensities ofinfection amongst males. Around 50% of toads lose their burdensentirely but the rest carry chronic infections throughout hosthibernation. Field data show a consistent reduction in intensityeach year strongly suggesting resistance. Parasite infectionis pathogenic and creates extra stress during hibernation; therefore,to the extent that elimination of infection is heritable, toadsentering spawning assemblies with heavy burdens should makepoor mates. However, extensive field studies show no correlationbetween mate success and parasite burden. Although infectioncan prejudice survival, it is only one of several inter-relatedfactors (including feeding success, tolerance of hibernation).The condition of successful and unsuccessful males in spawningassemblies indicates that all exceed a threshold at which parasite-inducedpathology is significant. Males which are debilitated by infection—orother factors—are selected against before mate choicebegins     

An autoradiographic study of the uptake of tritiated proline by osteoblasts during hibernation

Twenty-four LSH and LVG strain golden hamsters, Mesocricetus auratus, were used. Experimental animals were maintained at 5 C and allowed to hibernate. Control animals were kept at 27 C. Six animals (3 experimental, 3 control) were injected subcutaneously with 1 microCi of 3H-proline/gm body wt. (Spec. act. 3 Ci/mM) after hibernation lasting 12 hours, 1 day, 3 days, or 7 days. Animals were killed 1 hour after injection and autoradiographs were prepared from 5 microns thick decalcified sections of femurs. A greater number of endosteal cells were labeled than periosteal cells and also exhibited a greater magnitude of labeling throughout the study. Differences between endosteal and periosteal cells both in percentage of cells labeled and magnitude of labeling were maximum in control animals and progressively decreased with increasing periods of hibernation. A reduction in synthesis of matrix proteins during the early period of hibernation was seen and was attributed to a significant reduction both in average cell activity and in the number of active cells during hibernation. The latter phenomenon apparently made a large contribution to the reduced matrical synthesis. 3H-proline uptake by osteoblasts probably reflects the reduced requirements of matrical synthesis during hibernation.