Dataset from RNAseq analysis of differential gene expression among developmental stages of two non-marine ostracodes.

We contribute transcriptomic data for two species of Ostracoda, an early-diverged group of small-sized pancrustaceans. Data include new reference transcriptomes for two asexual non-marine species (Dolerocypris sinensis and Heterocypris aff. salina), as well as single-specimen transcriptomic data that served to analyse gene expression across four developmental stages in D. sinensis. Data are evaluated by computing gene expression profiles of the different developmental stages which consistently placed eggs and small larvae (at the stage of instar A-8) similar to each other, and apart from adults which were distinct from all other developmental stages but closest to large larvae (instar A-4). We further evaluated the transcriptomic data with two newly sequenced low-coverage genomes of the target species. The new data thus document the feasibility of obtaining reliable transcriptomic data from single specimens - even eggs - of these small metazoans.

Draft genome sequences of 21 Pedobacter strains isolated from amphibian specimens.

The genomes of 21 Pedobacter strains isolated from the European salamander Salamandra salamandra and different Madagascan frog species were sequenced using Illumina sequencing. Here, we report their draft genome sequences (~4.7-7.2 Mbp in size) to allow comparative genomics and taxonomic assignment of these strains.

FIRST RECORD OF BATRACHOCHYTRIUM DENDROBATIDIS IN THE NORTHERN NETHERLANDS.

Batrachochytrium dendrobatidis (Bd) infects amphibians and has been linked to the decline of hundreds of anuran amphibians all over the world. In the province of Groningen in the Netherlands, this fungal pathogen was not detected before this study. To determine whether Groningen was Bd-free, we surveyed 12 locations in this province in 2020 and 2021. Samples were then used to quantify the presence of Bd with a qPCR assay. In total, 2 out of 110 (∼0.02%) collected in 2020 and 11 out of 249 samples collected in 2021 tested positive for Bd. Infected amphibians were found in 4 out of the 12 sites, and the prevalence of Bd was estimated at 4% for both years combined. Our study provides the first record of Bd in Groningen, and we hypothesize that Bd is present throughout the Netherlands in regions currently considered "Bd-free." Furthermore, we warn scientists and policymakers to be apprehensive when calling a site free from Bd when sampling is limited or not recent.

Skin and gut microbiomes of tadpoles vary differently with host and water environment: a short-term experiment using 16S metabarcoding.

The host-microbiome community is influenced by several host and environmental factors. In order to disentangle the individual effects of host and environment, we performed a laboratory experiment to assess the effects of the exposure to different water sources on the skin and gut microbiome of two amphibian species (Pelophylax perezi and Bufo spinosus). We observed that the bacterial communities greatly varied with water environment and host identity. Tadpoles of B. spinosus collected from a waterbody with poorer bacterial diversity exhibited a more diverse skin and gut microbiome after exposed to a richer water source. Tadpoles of P. perezi, originally collected from a richer water environment, exhibited less marked alterations in diversity patterns independently of the water source but showed alterations in gut composition. These results highlight that environment alterations, such as the water source, combined with the host effect, impact the microbiome of amphibian species in different ways; the population history (e.g., previous water environment and habitat) of the host species may also influence future alterations on tadpole microbiome.

Responsible biosecurity and risk mitigation for laboratory research on emerging pathogens of amphibians.

The increasing study of emerging wildlife pathogens and a lack of policy or legislation regulating their translocation and use has heightened concerns about laboratory escape, species spillover, and subsequent epizootics among animal populations. Responsible self-regulation by research laboratories, in conjunction with institutional-level safeguards, has an important role in mitigating pathogen transmission and spillover, as well as potential interspecies pathogenesis. A model system in disease ecology that highlights these concerns and related amelioration efforts is research focused on amphibian emerging infectious diseases. Whereas laboratory escape of amphibian pathogens has not been reported and may be rare compared with introduction events from trade or human globalization, the threat that novel disease outbreaks with mass mortality effects pose to wild populations warrants thorough biosecurity measures to ensure containment and prevent spillover. Here, we present a case study of the laboratory biosecurity concerns for the emerging amphibian fungal pathogen Batrachochytrium salamandrivorans. We conclude that proactive biosecurity strategies are needed to integrate researcher and institutional oversight of aquatic wildlife pathogens generally, and we call for increased national and international policy and legislative enforcement. Furthermore, taking professional responsibility beyond current regulations is needed as development of legal guidance can be slow at national and international levels. We outline the need for annual laboratory risk assessments, comprehensive training for all laboratory personnel, and appropriate safeguards specific to pathogens under study. These strategies are critical for upholding the integrity and credibility of the scientific community and maintaining public support for research on wildlife diseases.

Wildlife pathogen detection: evaluation of alternative DNA extraction protocols.

Accurate detection of wildlife pathogens is critical in wildlife disease research. False negatives or positives can have catastrophic consequences for conservation and disease-mitigation decisions. Quantitative polymerase chain reaction is commonly used for molecular detection of wildlife pathogens. The reliability of this method depends on the effective extraction of the pathogen's DNA from host samples. A wildlife disease that has been in the centre of conservationist's attention is the amphibian disease Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Here, we compare the efficiency of a spin column extraction kit (QIAGEN), commonly used in Bd DNA extraction, to an alternative spin column kit (BIOKÈ) used in extractions from other types of samples, which is considerably cheaper but not typically used for Bd DNA extraction. Additionally, we explore the effect of an enzymatic pre-treatment on detection efficiency. Both methods showed similar efficiency when extracting Bd DNA from zoospores from laboratory-created cell-cultures, as well as higher efficiency when combined with the enzymatic pre-treatment. Our results indicate that selecting the optimal method for DNA extraction is essential to ensure minimal false negatives and reduce project costs.

Characterization of the microbiome of the invasive Asian toad in Madagascar across the expansion range and comparison with a native co-occurring species.

Biological invasions are on the rise, with each invader carrying a plethora of associated microbes. These microbes play important, yet poorly understood, ecological roles that can include assisting the hosts in colonization and adaptation processes or as possible pathogens. Understanding how these communities differ in an invasion scenario may help to understand the host's resilience and adaptability. The Asian common toad, Duttaphrynus melanostictus is an invasive amphibian, which has recently established in Madagascar and is expected to pose numerous threats to the native ecosystems. We characterized the skin and gut bacterial communities of D. melanostictus in Toamasina (Eastern Madagascar), and compared them to those of a co-occurring native frog species, Ptychadena mascareniensis, at three sites where the toad arrived in different years. Microbial composition did not vary among sites, showing that D. melanostictus keeps a stable community across its expansion but significant differences were observed between these two amphibians. Moreover, D. melanostictus had richer and more diverse communities and also harboured a high percentage of total unique taxa (skin: 80%; gut: 52%). These differences may reflect the combination of multiple host-associated factors including microhabitat selection, skin features and dietary preferences.

Erratum: The Role of Plasticity and Adaptation in the Incipient Speciation of a Fire Salamander Population (Genes 2019, 10, 875).

In the original article, there was a mistake in Figure 5 as published. When summarizing the results in the scheme, the treatment groups were mixed, and so some of the symbols for morphological and gene expression traits were not in accordance with the results [...].

A new species of the Spinomantis bertini species complex (Anura: Mantellidae) from Pic d'Ivohibe Special Reserve (Madagascar).

We describe a new frog species of the Spinomantis bertini species complex based on congruent genetic evidence from mitochondrial and nuclear DNA sequences as well as minor morphological differences. A molecular phylogeny derived from the mitochondrial 16S rRNA gene revealed that Spinomantis mirus sp. nov. is sister to a clade containing the other two described species of the species complex, S. bertini and S. beckei, but strongly differentiated from these two species by 8.1-9.8% pairwise distances in this gene. DNA sequences from two nuclear genes revealed that S. mirus sp. nov. shares no alleles with these two species. Phenotypically, S. mirus sp. nov. differs from the other species of the complex by its larger size and some aspects of its coloration, including clear mottling on the flanks, tri-color banding on the legs, and distinct brown dots on the dorsum. The new species is so far only known from Pic d'Ivohibe Special Reserve. Its distribution thus appears to not overlap with those of the other two species, which as far as known are restricted to the Andohahela Massif in the extreme South-East of Madagascar, at a distance of about 250 km from Ivohibe.

The Role of Plasticity and Adaptation in the Incipient Speciation of a Fire Salamander Population.

Phenotypic plasticity and local adaptation via genetic change are two major mechanisms of response to dynamic environmental conditions. These mechanisms are not mutually exclusive, since genetic change can establish similar phenotypes to plasticity. This connection between both mechanisms raises the question of how much of the variation observed between species or populations is plastic and how much of it is genetic. In this study, we used a structured population of fire salamanders (Salamandra salamandra), in which two subpopulations differ in terms of physiology, genetics, mate-, and habitat preferences. Our goal was to identify candidate genes for differential habitat adaptation in this system, and to explore the degree of plasticity compared to local adaptation. We therefore performed a reciprocal transfer experiment of stream- and pond-originated salamander larvae and analyzed changes in morphology and transcriptomic profile (using species-specific microarrays). We observed that stream- and pond-originated individuals diverge in morphology and gene expression. For instance, pond-originated larvae have larger gills, likely to cope with oxygen-poor ponds. When transferred to streams, pond-originated larvae showed a high degree of plasticity, resembling the morphology and gene expression of stream-originated larvae (reversion); however the same was not found for stream-originated larvae when transferred to ponds, where the expression of genes related to reduction-oxidation processes was increased, possibly to cope with environmental stress. The lack of symmetrical responses between transplanted animals highlights the fact that the adaptations are not fully plastic and that some level of local adaptation has already occurred in this population. This study illuminates the process by which phenotypic plasticity allows local adaptation to new environments and its potential role in the pathway of incipient speciation.

Pooling skin swabs does not inhibit qPCR detection of amphibian chytrid infection.

Immediate and reliable pathogen detection in large numbers of samples is essential in wildlife disease monitoring and is often realized by DNA-based techniques. Pooling samples increases processing efficiency and reduces processing costs, and has been suggested as a viable technique for quantitative PCR detection of fungal amphibian pathogens of the genus Batrachochytrium. For these fungi, this diagnostic method has been validated by in vitro set ups that provided controlled test conditions but did not take into account potential effects from amphibian skin compounds (e.g. skin secretions and Microbiota) on the approach. Some of these skin compounds are known to cause PCR inhibition in single sample applications and could lead to false negative reactions and thereby hamper pathogen detection. In this study we examined the effect of skin compounds on the pooled extraction method by swabbing individuals of seven amphibian species (one Anura and six Caudata) prior to the inoculation of the swabs with chytrid zoospores. For each species, swabs were extracted in pools of different sizes (from one to four swabs) with only one swab per pool being inoculated with zoospores. There were no significant differences regarding the ability to detect zoospores when comparing pool sizes for any species, with a tendency for more false negatives when the inoculated swab had been inoculated with a single zoospore. This study provides further in vivo evidence for the viability of the pooled extraction method for DNA-based detection of pathogens.

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.

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.

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

Animal-associated bacterial communities play essential roles for their host's 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.

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.

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.

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.