Joana Sabino-Pinto

Expanding Distribution of Lethal Amphibian Fungus Batrachochytrium salamandrivorans in Europe.

Emerging fungal diseases can drive amphibian species to local extinction. During 2010–2016, we examined 1,921 urodeles in 3 European countries. Presence of the chytrid fungus Batrachochytrium salamandrivorans at new locations and in urodeles of different species expands the known geographic and host range of the fungus and underpins its imminent threat to biodiversity.

First detection of the emerging fungal pathogen Batrachochytrium salamandrivorans in Germany

The emerging infectious disease chytridiomycosis is one of the major factors triggering global amphibian declines. A recently discovered species of chytrid fungus, Batrachochytrium salamandrivorans (Bsal), likely originated in East Asia, has led to massive declines in populations of fire salamanders (Salamandra salamandra) after its apparent introduction to the Netherlands and Belgium. Here, we report the first detection of this pathogen in Germany where it caused mass mortality of fire salamanders in a captive collection. Salamanders from this collection showed an almost 100% prevalence of infection with Bsal. Supposed Bsal-induced mortality occurred in multiple Salamandra species (S. salamandra, S. algira, S. corsica, and S. infraimmaculata), while Bsal infection was confirmed in nine subspecies of S. salamandra and in S. algira. Our study indicates that this pathogen can potentially infect all fire salamander species and subspecies. If Bsal spreads from captive collections to wild populations, then a similar devastating effect associated with high mortality should be expected.

Composition of the Cutaneous Bacterial Community in Japanese Amphibians: Effects of Captivity, Host Species, and Body Region

The cutaneous microbiota plays a significant role in the biology of their vertebrate hosts, and its composition is known to be influenced both by host and environment, with captive conditions often altering alpha diversity. Here, we compare the cutaneous bacterial communities of 61 amphibians (both wild and captive) from Hiroshima, Japan, using high-throughput amplicon sequencing of a segment of the 16S rRNA gene. The majority of these samples came from a captive breeding facility at Hiroshima University where specimens from six species are maintained under highly standardized conditions for several generations. This allowed to identify host effects on the bacterial communities under near identical environmental conditions in captivity. We found the structure of the cutaneous bacterial community significantly differing between wild and captive individuals of newts, Cynops pyrrhogaster, with a higher alpha diversity found in the wild individuals. Community structure also showed distinct patterns when comparing different species of amphibians kept under highly similar conditions, revealing an intrinsic host effect. Bacterial communities of dorsal vs. ventral skin surfaces did not significantly differ in most species, but a trend of higher alpha diversity on the ventral surface was found in Oriental fire-bellied toads, Bombina orientalis. This study confirms the cutaneous microbiota of amphibians as a highly dynamic system influenced by a complex interplay of numerous factors.

Impact of asynchronous emergence of two lethal pathogens on amphibian assemblages

Emerging diseases have been increasingly associated with population declines, with co-infections exhibiting many types of interactions. The chytrid fungus (Batrachochytrium dendrobatidis) and ranaviruses have extraordinarily broad host ranges, however co-infection dynamics have been largely overlooked. We investigated the pattern of co-occurrence of these two pathogens in an amphibian assemblage in Serra da Estrela (Portugal). The detection of chytridiomycosis in Portugal was linked to population declines of midwife-toads (Alytes obstetricans). The asynchronous and subsequent emergence of a second pathogen - ranavirus - caused episodes of lethal ranavirosis. Chytrid effects were limited to high altitudes and a single host, while ranavirus was highly pathogenic across multiple hosts, life-stages and altitudinal range. This new strain (Portuguese newt and toad ranavirus – member of the CMTV clade) caused annual mass die-offs, similar in host range and rapidity of declines to other locations in Iberia affected by CMTV-like ranaviruses. However, ranavirus was not always associated with disease, mortality and declines, contrasting with previous reports on Iberian CMTV-like ranavirosis. We found little evidence that pre-existing chytrid emergence was associated with ranavirus and the emergence of ranavirosis. Despite the lack of cumulative or amplified effects, ranavirus drove declines of host assemblages and changed host community composition and structure, posing a grave threat to all amphibian populations.

Temporal changes in cutaneous bacterial communities of terrestrial- and aquatic-phase newts (Amphibia): Temporal changes in skin microbial communities

Animal-associated bacterial communities play essential roles for their hosts ecology, physiology and health. Temporal dynamics of these communities are poorly understood, but might be of high relevance for amphibians with a well-expressed biphasic biology of adults where the structure of their skin changes drastically between the aquatic and terrestrial phases. Here, we investigated the temporal dynamics of cutaneous bacterial communities of Lissotriton boscai and Triturus marmoratus by monthly sampling populations from a pond and surrounding terrestrial habitats near A Coruña, Spain. These communities were characterized by 16S rRNA gene amplicons from DNA isolated from skin swabs. Newt bacterial communities displayed variation at three levels: between larvae and aquatic adults, between adult life phases (terrestrial versus aquatic), and temporally within life phases. The skin bacterial communities tended to differ to a lesser extent temporally and between larvae and adults, and more strongly between life phases. Larvae had a higher proportion of reads associated with antifungal taxa compared with adults, while no differences were found among adult life phases. Terrestrial specimens exhibited the highest community diversity. The regular transitions of adult newts between aquatic and terrestrial environments might contribute to the diversity of their skin microbiota and could increase disease resistance.

Low infection prevalence of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Chytridiomycetes: Rhizophydiales) in Cuba

The chytrid fungus Batrachochytrium dendrobatidis ( Bd ) is responsible for population declines and extinctions of amphibians worldwide. The distribution and prevalence of Bd in Cuba has remained unknown to date, with only a single report on its presence. We collected 182 samples from wild anuran populations across 21 species and 16 localities and tested for the presence of Bd using qPCRs. Only six Bd positive samples from four species were detected in three very close localities in Central Cuba. Bd prevalence was of 10-20% in the positive localities, and the island-wide prevalence was only 3.2%. These results indicate that Bd occurrence in Cuba might be concentrated in or even restricted to the central Guamuhaya Massif and call for increased conservation and monitoring efforts in these mountains along with additional sampling in areas and species not covered in this study.

Cutaneous microbiota of the Japanese giant salamander (Andrias japonicus), a representative of an ancient amphibian clade

Andrias japonicus, the Japanese giant salamander, is the second largest amphibian species in the world. The biology of this long-lived, fully aquatic salamander is still incompletely known, and studying the threats it experiences is important for conservation management. We used 16S amplicon sequencing to provide the first data on the composition and diversity of the cutaneous microbiome of this species. Skin bacterial communities of adult and larval giant salamanders were composed primarily of taxa belonging to the phyla Proteobacteria and Bacteroidetes, and, their community structure differed significantly from that of two other syntopic amphibians (Cynops pyrrhogaster and Glandirana rugosa). We also found differences between wild A. japonicus and captive individuals, with the latter having an increased bacterial diversity. The fungal pathogen Batrachochytrium dendrobatidis (Bd) was detected only in captive individuals (40% prevalence), and did not correlate with a particular bacterial community structure. We identified eight bacteria that were significantly more abundant on A. japonicus compared to syntopic amphibians, one of which was Janthinobacterium lividum, a bacterial species known to exert Bd-inhibiting effects. Our study provides baseline data for future in-depth studies on the microbial ecology of cutaneous bacteria and the contribution of cutaneous bacteria to Bd resistance in giant salamanders.

Predation on the treefrog Boophis rufioculis (Anura, Mantellidae) by the freshwater crab Hydrothelphusa sp. (Decapoda, Potamonautidae) in Madagascar

GONCALO M. ROSA1,2,3,6), JOANA SABINO-PINTO3), JEAN NOEL4) and FRANCO ANDREONE5) 1)Durrell Institute of Conservation and Ecology, School of Anthropology and Conservation, University of Kent, Canterbury, Kent CT2 7NR, U.K. 2) Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, U.K. 3)Centro de Biologia Ambiental, Faculdade de Ciencias da Universidade de Lisboa, Bloco 2, Piso 5, Campo Grande, 1749-016 Lisbon, Portugal 4)Madagascar Fauna & Flora Group, BP 442, Morafeno, Toamasina 501, Madagascar 5)Museo Regionale di Scienze Naturali, Sezione di Zoologia, Via G. Giolitti, 36, I-10123, Torino, Italy

Asymptomatic infection of the fungal pathogen Batrachochytrium salamandrivorans in captivity

One of the most important factors driving amphibian declines worldwide is the infectious disease, chytridiomycosis. Two fungi have been associated with this disease, Batrachochytrium dendrobatidis and B. salamandrivorans (Bsal). The latter has recently driven Salamandra salamandra populations to extirpation in parts of the Netherlands, and Belgium, and potentially also in Germany. Bsal has been detected in the pet trade, which has been hypothesized to be the pathway by which it reached Europe, and which may continuously contribute to its spread. In the present study, 918 amphibians belonging to 20 captive collections in Germany and Sweden were sampled to explore the extent of Bsal presence in captivity. The fungus was detected by quantitative Polymerase Chain Reaction (qPCR) in ten collections, nine of which lacked clinical symptoms. 23 positives were confirmed by independent processing of duplicate swabs, which were analysed in a separate laboratory, and/or by sequencing ITS and 28 S gene segments. These asymptomatic positives highlight the possibility of Bsal being widespread in captive collections, and is of high conservation concern. This finding may increase the likelihood of the pathogen being introduced from captivity into the wild, and calls for according biosecurity measures. The detection of Bsal-positive alive specimens of the hyper-susceptible fire salamander could indicate the existence of a less aggressive Bsal variant or the importance of environmental conditions for infection progression.

Disruption of skin microbiota contributes to salamander disease

Escalating occurrences of emerging infectious diseases underscore the importance of understanding microbiome–pathogen interactions. The amphibian cutaneous microbiome is widely studied for its potential to mitigate disease-mediated amphibian declines. Other microbial interactions in this system, however, have been largely neglected in the context of disease outbreaks. European fire salamanders have suffered dramatic population crashes as a result of the newly emerged Batrachochytrium salamandrivorans (Bsal). In this paper, we investigate microbial interactions on multiple fronts within this system. We show that wild, healthy fire salamanders maintain complex skin microbiotas containing Bsal-inhibitory members, but these community are present at a remarkably low abundance. Through experimentation, we show that increasing bacterial densities of Bsal-inhibiting bacteria via daily addition slowed disease progression in fire salamanders. Additionally, we find that experimental-Bsal infection elicited subtle changes in the skin microbiome, with selected opportunistic bacteria increasing in relative abundance resulting in septicemic events that coincide with extensive destruction of the epidermis. These results suggest that fire salamander skin, in natural settings, maintains bacterial communities at numbers too low to confer sufficient protection against Bsal, and, in fact, the native skin microbiota can constitute a source of opportunistic bacterial pathogens that contribute to pathogenesis. By shedding light on the complex interaction between the microbiome and a lethal pathogen, these data put the interplay between skin microbiomes and a wildlife disease into a new perspective.