Detection of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in Amphibians Imported into the UK for the Pet Trade

There is increasing evidence that the global spread of the fungal pathogen Batrachochytrium dendrobatidis (Bd) has been facilitated by the international trade in amphibians. Bd was first detected in the UK in 2004, and has since been detected in multiple wild amphibian populations. Most amphibians imported into the UK for the pet trade from outside the European Union enter the country via Heathrow Animal Reception Centre (HARC), where Bd-positive animals have been previously detected. Data on the volume, diversity and origin of imported amphibians were collected for 59 consignments arriving at HARC between November 2009 and June 2012, along with a surveillance study to investigate the prevalence of Bd in these animals. Forty-three amphibian genera were recorded, originating from 12 countries. It was estimated that 5000–7000 amphibians are imported through HARC into the UK annually for the pet trade. Bd was detected in consignments from the USA and Tanzania, in six genera, resulting in an overall prevalence of 3.6%. This suggests that imported amphibians are a source of Bd within the international pet trade.     

<Emphasis Type="Italic">Batrachochytrium</Emphasis><Emphasis Type="Italic">dendrobatidis</Emphasis> and Chytridiomycosis in Anuran Amphibians of Colombia

In order to investigate the possible presence of the chytrid fungus Batrachochytrium dendrobatidis (Chytridiomycota: Chytridiales) in frogs (Amphibia: Anura) of Colombia, we made a retrospective examination of formalin-fixed specimens preserved in natural history collections. Using the staining technique of hematoxylin and eosin to identify B. dendrobatidis in histological slices, we found evidence of the fungus in 3 of the 53 frog species examined from a total of 672 specimens collected in 17 departments within Colombia between 1968 and 2006. The infected specimens were found dead or dying in recent years in high elevation sites, suggesting that chytridiomycosis (the disease caused by the fungus) may represent a significant threat to Colombian amphibians. We conclude that a more extensive search for B. dendrobatidis in museum specimens and wild-caught frogs should be undertaken as soon as possible, using both histological and molecular genetic techniques, in order to further characterize the geographic and taxonomic extent of infections of B. dendrobatidis.     

Invasion of the Fungal Pathogen <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> on California Islands

Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen, has infected >500 species and caused extinctions or declines in >200 species worldwide. Despite over a decade of research, little is known about its invasion biology. To better understand this, we conducted a museum specimen survey (1910–1997) of Bd in amphibians on 11 California islands and found a pattern consistent with the emergence of Bd epizootics on the mainland, suggesting that geographic isolation did not prevent Bd invasion. We propose that suitable habitat, host diversity, and human visitation overcome isolation from the mainland and play a role in Bd invasion.     

Early exposure to <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> causes profound immunosuppression in amphibians

Fungal pathogens have evolved a broad suite of strategies aiming at evading the host immune response. Amphibians are globally infected by the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd) and, while robust innate immune defences have been characterised, there is little evidence for the existence of effective adaptive immunity. We determine the immune response of the common midwife toad following challenge by Bd as larvae. Immune function was described for both the cell-mediated and antibody-mediated immune responses following infectious challenge as larval amphibians. While there were no significant differences in the ratio of neutrophils/lymphocytes between infected and uninfected individuals, early exposure of tadpoles to Bd significantly dampened the levels of circulating immunoglobulins (IgM and IgY) in the serum of juveniles after metamorphosis. Our results show that Bd immunosuppresses amphibians when infection occurs as larvae with potentially broad effects on the remodelling of immunity during metamorphosis.     

Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Infection in Water Frogs of the <Emphasis Type="Italic">Rana esculenta</Emphasis> Complex in Central Italy

Batrachochytrium dendrobatidis (phylum Chytridiomycota, order Chytridiales) is the causative organism of chytridiomycosis in amphibians, a disease associated with their population decline worldwide. In this work, we report a cutaneous infection in water frogs of the Rana esculenta complex in agricultural areas of Umbria, central Italy. Histological, immunohistochemical, ultrastructural, and molecular analyses demonstrated for the first time the presence of the Batrachochytrium dendrobatidis in this complex; to date, no association between the presence of chytrid fungal infection and mortality has been found, to our knowledge. However, the presence of Batrachochytrium dendrobatidis infection in the water frogs of the Rana esculenta complex is of concern because the frogs could act as a reservoir species and contribute to the decline of less resistant species.     

<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> and the Decline and Survival of the Relict Leopard Frog

Epizootic disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is a major driver of amphibian declines, yet many amphibians declined before the pathogen was described. The Relict Leopard Frog, Rana onca (=Lithobates onca), was nearly extinct, with the exception of populations within a few geothermal springs. Growth of Bd, however, is limited by high water temperature, and geothermal springs may have provided refuge during outbreaks of chytridiomycosis. We conducted field surveys and laboratory experiments to assess the susceptibility of R. onca to Bd. In the field, we found Bd at one of the two areas where remnant populations of R. onca still occur, but not in the other. In the laboratory, we infected juvenile frogs from these two areas with two hypervirulent Bd isolates associated with declines in other ranid species. In our experiments, these Bd isolates did not affect survivorship of R. onca and most infections (64%) were cleared by the end of the experiments. We propose that R. onca either has inherent resistance to Bd or has recently evolved such resistance. These results may be important for conservation efforts aimed at establishing new populations of R. onca across a landscape where Bd exists. Resistance, however, varies among life stages, and we also did not assess Bd from the local environment. We caution that the resistance we observed for young frogs under laboratory conditions may not translate to the situation for R. onca in the wild.     

Immmunological Clearance of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Infection at a Pathogen-optimal Temperature in the Hylid Frog <Emphasis Type="Italic">Hypsiboas crepitans</Emphasis>

Amphibian declines worldwide have been linked to the fungal disease chytridiomycosis. Its causative agent (Batrachochytrium dendrobatidis, hereafter Bd), however, also infects many nondeclining species. Experimental infections have shown species-specific and temperature-dependent frog responses to Bd infection. Although Bd infection may be eliminated by housing amphibians at temperatures above those tolerated by the fungus, the question of whether frogs can eliminate infection under more favorable conditions remains unanswered. Repeated diagnostics using real-time polymerase chain reaction (rt–PCR) assays of postmetamorphic individuals at 28, 38, 45, 53, and 62 days after exposure demonstrated that Hypsiboas crepitans is able to clear infection within a few weeks at 23°C. Thus, we demonstrate a temperature-independent and likely immunological mechanism for the clearance of Bd in a resistant amphibian species. Future studies are needed to determine the generality of this mechanism among amphibians and to describe the immune factors affecting different outcomes of Bd exposure including resistance to infection, tolerance of infection, and clearance of infection.     

Potential distribution of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in Argentina: implications in amphibian conservation

Within the last two decades Batrachochytrium dendrobatidis (B.d.), the causative agent of chytridiomycosis, seems to have become a pandemic parasite, and is proposed as one of the more important causes of amphibian declines worldwide. In Argentina, the first report was in 2002 in Leptodactylus ocellatus. Since then, the fungus has expanded through different environments and species. In this study, we predict B.d. distribution in Argentina applying niche modelling based on reports of infected amphibians and environmental variables. The distribution hypothesis showed regions with highest suitability for B.d. including habitat types with (1) the most diverse amphibian fauna in Argentina such as the Paraná River Basin, (2) endangered species, such as north Patagonia and northwest Andean highlands, and (3) wide range of optimum precipitation and temperatures allowing development of B.d.     

Prevalence and Seasonality of the Amphibian Chytrid Fungus <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Along Widely Separated Longitudes Across the United States

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines on almost all continents. We report on prevalence and intensity of Bd in the United States amphibian populations across three longitudinally separated north-to-south transects conducted at 15 Department of Defense installations during two sampling periods (late-spring/early summer and mid to late summer). Such a standardized approach minimizes the effects of sampling and analytical bias, as well as human disturbance (by sampling restricted military bases), and therefore permits a cleaner interpretation of environmental variables known to affect chytrid dynamics such as season, temperature, rainfall, latitude, and longitude. Our prevalence of positive samples was 20.4% (137/670), and our mean intensity was 3.21 zoospore equivalents (SE = 1.03; range 0.001–103.59). Of the 28 amphibian species sampled, 15 tested positive. Three sites had no evidence of Bd infection; across the remaining 12 Bd-positive sites, neither infection prevalence nor intensity varied systematically. We found a more complicated pattern of Bd prevalence than anticipated. Early season samples showed no trend associated with increasing temperature and precipitation and decreasing (more southerly) latitudes; while in late season samples, the proportion of infected individuals decreased with increasing temperature and precipitation and decreasing latitudes. A similar pattern held for the east–west gradient, with the highest prevalence associated with more easterly/recently warmer sites in the early season then shifting to more westerly/recently cooler sites in the later season. Bd intensity across bases and sampling periods was comparatively low. Some of the trends in our data have been seen in previous studies, and our results offer further continental-level Bd sampling over which more concentrated local sampling efforts can be overlaid.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Presence and Diversity of <Emphasis Type="Italic">Streptomyces</Emphasis> in <Emphasis Type="Italic">Dendroctonus</Emphasis> and Sympatric Bark Beetle Galleries Across North America

Recent studies have revealed several examples of intimate associations between insects and Actinobacteria, including the Southern Pine Beetle Dendroctonus frontalis and the Spruce Beetle Dendroctonus rufipennis. Here, we surveyed Streptomyces Actinobacteria co-occurring with 10 species of Dendroctonus bark beetles across the United States, using both phylogenetic and community ecology approaches. From these 10 species, and 19 other scolytine beetles that occur in the same trees, we obtained 154 Streptomyces-like isolates and generated 16S sequences from 134 of those. Confirmed 16S sequences of Streptomyces were binned into 36 distinct strains using a threshold of 0.2% sequence divergence. The 16S rDNA phylogeny of all isolates does not correlate with the distribution of strains among beetle species, localities, or parts of the beetles or their galleries. However, we identified three Streptomyces strains occurring repeatedly on Dendroctonus beetles and in their galleries. Identity of these isolates was corroborated using a house-keeping gene sequence (efTu). These strains are not confined to a certain species of beetle, locality, or part of the beetle or their galleries. However, their role as residents in the woodboring insect niche is supported by the repeated association of their 16S and efTu from across the continent, and also having been reported in studies of other subcortical insects.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Survey of Pathogenic Chytrid Fungi (<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> and <Emphasis Type="Italic">B. salamandrivorans</Emphasis>) in Salamanders from Three Mountain Ranges in Europe and the Americas

Batrachochytrium salamandrivorans (Bsal) is a virulent fungal pathogen that infects salamanders. It is implicated in the recent collapse of several populations of fire salamanders in Europe. This pathogen seems much like that of its sister species, Batrachochytrium dendrobatidis (Bd), the agent responsible for anuran extinctions and extirpations worldwide, and is considered to be an emerging global threat to salamander communities. Bsal thrives at temperatures found in many mountainous regions rich in salamander species; because of this, we have screened specimens of salamanders representing 17 species inhabiting mountain ranges in three continents: The Smoky Mountains, the Swiss Alps, and the Peruvian Andes. We screened 509 salamanders, with 192 representing New World salamanders that were never tested for Bsal previously. Bsal was not detected, and Bd was mostly present at low prevalence except for one site in the Andes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Anti-<Emphasis Type="Italic">Helicobacter pylori</Emphasis> substances from endophytic fungal cultures

The human pathogenic bacterium Helicobacter pylori has been ascertained to be an aetiological agent for chronic active gastritis and a significant determinant in peptic and duodenal ulcer diseases. Endophytic metabolites are being recognized as a versatile arsenal of antimicrobial agents, since some endophytes have been shown to possess superior biosynthetic capabilities owing to their presumable gene recombination with the host, while residing and reproducing inside the healthy plant tissues. A total of 32 endophytic fungi isolated from the medicinal herb Cynodon dactylon(Poaceae) were grown in in vitroculture, and the ethyl acetate extracts of the cultures were examined in vitro for the anti-H. pylori activity. As a result, a total of 16 endophyte culture extracts were identified as having potent anti-H. pyloriactivities. Subsequently, a detailed bioassay-guided fractionation of the extract of the most active endophyte (strain number: CY725) identified as Aspergillussp., was performed to afford eventually four anti-H. pylori secondary metabolites. The four isolated compounds were identified through a combination of spectral and chemical methods (IR, MS, ¹H- and ¹³C-NMR) to be helvolic acid, monomethylsulochrin, ergosterol and 3β-hydroxy-5α,8α-epidioxy- ergosta-6,22-diene with corresponding MICs of 8.0, 10.0, 20.0 and 30.0 μg/ml, respectively. The MIC of ampicillin co-assayed as a reference drug against H. pylori was 2.0 μg/ml. Furthermore, preliminary examination of the antimicrobial spectrum of helvolic acid, the most active anti-H. pylori metabolite characterized from the endophyte culture, showed that it was inhibitory to the growth of Sarcina lutea, Staphylococcus aureusand Candida albicans with MICs of 15.0, 20.0 and 30.0 μg/ml, respectively.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

First Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in China: Discovery of Chytridiomycosis in Introduced American Bullfrogs and Native Amphibians in the Yunnan Province,China

Although the chytrid fungus Batrachochytrium dendrobatidis (Bd), the etiological agent of amphibian chytridiomycosis, has been implicated in mass mortality and population declines on several continents around the world, there have been no reports on the presence of Bd infections in amphibians in China. We employed quantitative PCR and histological techniques to investigate the presence of Bd in introduced North American bullfrogs (Rana catesbeiana) (referred to hereafter as bullfrog) and native amphibians in bullfrog-invaded areas of the Yunnan Province, China. A total of 259 samples at five wild sites were collected between June and September in 2007 and 2008, including bullfrogs and four native amphibian species (Rana pleuraden, Rana chaochiaoensis, Odorrana andersonii, and Bombina maxima). In addition, 37 samples of adult bullfrogs were obtained from a food market. Bd infections were discovered in bullfrogs and three native amphibian species from all of the surveyed sites. Of the 39 Bd-positive samples, 35 were from wild-caught bullfrog tadpoles, postmetamorphic bullfrogs, R. pleuraden, R. chaochiaoensis, and O. andersonii, and four were from adult bullfrogs from the market. Our results provide the first evidence of the presence of Bd in Chinese amphibians, suggesting that native amphibian diversity in China is at risk from Bd. There is an urgent need to monitor the distribution of Bd in amphibians in China and understand the susceptibility of native amphibian species to chytridiomycosis. Strict regulations on the transportation of bullfrogs and the breeding of bullfrogs in markets and farms should be drafted in order to stop the spread of Bd by bullfrogs.