<Emphasis Type="Italic">Batrachochytrium salamandrivorans</Emphasis> and the Risk of a Second Amphibian Pandemic

Amphibians are experiencing devastating population declines globally. A major driver is chytridiomycosis, an emerging infectious disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Bd was described in 1999 and has been linked with declines since the 1970s, while Bsal is a more recently discovered pathogen that was described in 2013. It is hypothesized that Bsal originated in Asia and spread via international trade to Europe, where it has been linked to salamander die-offs. Trade in live amphibians thus represents a significant threat to global biodiversity in amphibians. We review the current state of knowledge regarding Bsal and describe the risk of Bsal spread. We discuss regional responses to Bsal and barriers that impede a rapid, coordinated global effort. The discovery of a second deadly emerging chytrid fungal pathogen in amphibians poses an opportunity for scientists, conservationists, and governments to improve global biosecurity and further protect humans and wildlife from a growing number of emerging infectious diseases.     

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.     

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.     

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.     

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.     

<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.     

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.     

<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.     

<Emphasis Type="Italic">Erysiphe catalpae</Emphasis> and <Emphasis Type="Italic">Erysiphe elevata</Emphasis> in Europe

The recent epidemic spread of the North American powdery mildew Erysiphe elevata in Europe is described and discussed. Since 2002, this plant pathogenic fungus has been collected on Catalpa bignonioides, C. erubescens and C. speciosa in the Czech Republic, Germany, Hungary, Slovakia and Switzerland. The diagnostically important anamorph of E. elevata, so far unknown, is described and illustrated in detail. Type material of Erysiphe catalpae and two specimens of E. catalpae recently collected in Poland have been examined and compared with E. elevata. The anamorph as well as the teleomorph of E. catalpae proved to be easily distinguishable from E. elevata. The supposition that E. catalpae, introduced in Armenia, was based on immature ascomata of E. elevata proved to be wrong. The origin and distribution of E. catalpae are discussed, and a key to powdery mildew fungi on Catalpa spp. in Europe is provided.     

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.     

<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.     

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">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.     

Chytrid infection reduces thoracic beat and heart rate of <Emphasis Type="Italic">Daphnia</Emphasis><Emphasis Type="Italic">pulicaria</Emphasis>

Zooplankters are hosts to numerous endo- and ectoparasites, some of which have dramatic impacts on their hosts. Epizootics on zooplankton are probably more widespread in lake systems than it is currently known, and few studies have explored the direct and indirect importance of parasitism in aquatic food webs. In addition, our understanding of the sublethal effects of parasitic infections on host organisms and populations is limited. We used a novel electro-chemical based technique to measure in the outflow of the feeding current changes in the beat rate of the thoracic appendages in female Daphnia pulicaria. We observed simultaneously the heart rates and compared chytrid infected animals with uninfected gravid and non-gravid ones. We found in uninfected animals a thoracic beat rate of 3.81 ± 018 Hz and a heart rate of 4.67 ± 0.42 Hz. Gravid daphnids had a 14% lower thoracic beat rate (3.27 ± 0.30 Hz) than non-gravid females while the heart rate did not significantly differ (4.48 ± 0.28 Hz). In contrast, infected animals showed a 22% lower thoracic beat rate (2.96 ± 0.47 Hz) and a 36% lower heart rate (2.98 ± 0.5 Hz) when compared with uninfected non-gravid females. We discuss the ways Daphnia are affected by Polycaryum leave infections on the individual and population level.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.