Temperature and land use change are associated with Rana temporaria reproductive success and phenology.

Chemical pollution, land cover change, and climate change have all been established as important drivers of amphibian reproductive success and phenology. However, little is known about the relative impacts of these anthropogenic stressors, nor how they may interact to alter amphibian population dynamics. Addressing this gap in our knowledge is important, as it allows us to identify and prioritise the most needed conservation actions. Here, we use long-term datasets to investigate landscape-scale drivers of variation in the reproductive success and phenology of UK Common frog (Rana temporaria) populations. Consistent with predictions, we found that increasing mean temperatures resulted in earlier initialisation of spawning, and earlier hatching, but these relationships were not consistent across all sites. Lower temperatures were also linked to increased spawn mortality. However, temperature increases were also strongly correlated with increases in urban area, arable area, and nitrate levels in the vicinity of spawning grounds. As with spawning and hatching, there was marked spatial variation in spawn mortality trends, where some sites exhibited steady increases over time in the proportion of dead or diseased spawn. These findings support previous work linking warming temperatures to shifts in timing of amphibian breeding, but also highlight the importance of assessing the effect of land use change and pollution on wild amphibian populations. These results have implications for our understanding of the response of wild amphibian populations to climate change, and the management of human-dominated landscapes for declining wildlife populations.

The optimization and operation of multi-energy-coupled microgrids by the improved fireworks algorithm-shuffled frog-leaping algorithm.

This study aims to address optimization and operational challenges in multi-energy coupled microgrids to enhance system stability and reliability. After analyzing the requirements of such systems within comprehensive energy systems, an improved fireworks algorithm (IFWA) is proposed. This algorithm combines an adaptive resource allocation strategy with a community genetic strategy, automatically adjusting explosion range and spark quantity based on individual optimization status to meet actual needs. Additionally, a multi-objective optimization model considering active power network losses and static voltage is constructed, utilizing the shuffled frog-leaping algorithm (SFLA) to solve constrained multi-objective optimization problems. Through simulation experiments on a typical northern comprehensive energy system, conducted with a scheduling period of T = 24, the feasibility and superiority of IFWA-SFLA are validated. Results indicate that IFWA-SFLA performs well in optimizing microgrid stability, managing electrical energy flow effectively within the microgrid, and reducing voltage fluctuations. Furthermore, the circuit structure and control strategy of microgrid energy storage bidirectional inverters based on IFWA are discussed, along with relevant simulation results.

Comparative muscle anatomy of the anuran pelvis and hindlimb in relation to locomotor mode.

Frogs have a highly conserved body plan, yet they employ a diverse array of locomotor modes, making them ideal organisms for investigating the relationships between morphology and locomotor function, in particular whether anatomical complexity is a prerequisite for functional complexity. We use diffusible iodine contrast-enhanced microCT (diceCT) imaging to digitally dissect the gross muscle anatomy of the pelvis and hindlimbs for 30 species of frogs representing five primary locomotor modes, including the first known detailed dissection for some of the world's smallest frogs, forming the largest digital comparative analysis of musculoskeletal structure in any vertebrate clade to date. By linking musculoskeletal dissections and phylogenetic comparative methods, we then quantify and compare relationships between anatomy and function across over 160 million years of anuran evolution. In summary, we have found that bone lengths and pelvic crest sizes are generally not reliable predictors of muscle sizes, which highlights important implications for future palaeontological studies. Our investigation also presents previously unreported differences in muscle anatomy between frogs specialising in different locomotor modes, including several of the smallest frog hindlimb muscles, which are extremely difficult to extract and measure using traditional approaches. Furthermore, we find evidence of many-to-one and one-to-many mapping of form to function across the phylogeny. Additionally, we perform the first quantitative analysis of how the degree of muscle separation can differ between frogs. We find evidence that phylogenetic history is the key contributing factor to muscle separation in the pelvis and thigh, while the separation of shank muscles is influenced more strongly by locomotor mode. Finally, our anatomical 3D reconstructions are published alongside this manuscript to contribute towards future research and serve as educational materials.

A comprehensive evaluation of the frog effect on drivers in mountain highway tunnels - The effect of low-volume intermittent information.

OBJECTIVE: The driver's inability to fully absorb and react to operational cues while driving is like boiling a frog in warm water. With intermittent, low-volume information, drivers can underreact by ignoring these minor but continuous changes. This paper aims to provide an opportunity to test the effects of intermittently occurring low-volume information on drivers. METHODS: A real vehicle test with naturalistic driving was used to collect driving speed data from 40 drivers on a highway tunnel section in Chongqing, China, where nine tunnels are located. Drivers were classified into three categories according to the degree of compliance of their driving speed with the speed limit required by traffic signs, and drivers were analyzed in terms of their sensitivity to traffic signs and their reaction to driving maneuvers. RESULTS: Conservative drivers are the most absorbent of low-volume intermittent information, and the cumulative effect of the frog effect does not exceed 2.00 km; eager drivers tend to ignore this information, and the cumulative effect of the frog effect reaches 2.91 km; and the general type of driver is in the middle of these two types of drivers, and the frog effect gradually penetrates the driving speed in a weakly increasing manner, up to a maximum of 9.8 km. CONCLUSION: At the beginning of a journey, drivers are most sensitive to traffic signs, and low-volume intermittent information can also play a role in guiding driving operations effectively at this time. However, as the driving distance increases, the effect of the frog effect on different types of drivers gradually increases, even exceeding the effect caused by the black-and-white hole effect, especially when driving in tunnel groups. Considering the driving characteristics of different types of drivers to improve the deployment of low-volume intermittent information and reduce the distance of the frog effect can effectively improve driving safety.

Preparation of sanguinarine/glabridin loaded antifungal double-layer nanoemulsion edible coating using arabic gum/WPI for forest frog's oviduct oil preservation.

Forest frog's oviduct oil (FFOO) is highly susceptible to microbial spoilage during storage, which causes serious safety concerns and economic losses. However, little information is available regarding the preservation of it up to now. The aim of this research is to understand the dominant microbial community of FFOO spoilage, and based on this, develop a kind of edible nanoemulsion coating for preserving FFOO. Microbial metagenomic analysis indicated that the Aspergillus genus increased significantly during storage. In the present study, gum arabic and whey protein isolate were chosen as the coating matrix, the natural compounds sanguinarine and glabridin were selected as antimicrobial agents to prepare double-layer nanoemulsion edible coating. When the ratio of sanguinarine and glabridin in the nanoemulsion was 1:3, it exhibited strongest storage stability and antifungal activity. The mycelial inhibition rate of 1:3 nanoemulsion against dominant microbial community (Aspergillus niger and Aspergillus glaucus) reached 88.89 ± 1.37 % and 89.68 ± 1.37 %, respectively. The experimental results indicated that the edible nanoemulsion coating not only had outstanding antifungal activity, but also had excellent fresh-keeping effect on FFOO. This nanoemulsion coating could be a promising and potential candidate for food preservation.

Gastrotheca Fitzinger, 1843 tadpole morphology: Larval cranium description and its evolutionary implications (Amphibia: Anura: Hemiphractidae).

Hemiphractids have a singular mode of reproduction that involves maternal care. The Andean-endemic Gastrotheca marsupiata species group includes direct-developing and tadpole-bearing species, the latter trait being unique among Gastrotheca. Larval morphology has proven to be a valuable source of evidence to understand the taxonomy and evolution of frogs but remains understudied in Hemiphractids. Herein, we redescribe the larval cranium of G. espeletia, G. gracilis, G. marsupiata, G. peruana, G. pseustes, and G. riobambae, and describe those of G. aureomaculata, G. chrysosticta, G. litonedis, G. monticola and G. psychrophila. Additionally, based on the data gathered, we explore their phylogenetic significance, expanding the knowledge regarding Gastrotheca larval internal morphology. We suggest that the presence of the posterolateral process of crista parotica, the concave palatoquadrate, the quadratoorbital commissure, and the proximal commissures II and III are putative synapomorphies for Gastrotheca. Furthermore, we suggest the long pseudopterygoid process as a putative synapomorphy for Hemiphractyidae.

Karyotypic description and comparison of Litoria (L.) paraewingi (Watson et al., 1971), L.ewingii (Duméril et Bibron, 1841) and L.jervisiensis (Duméril et Bibron, 1841) (Amphibia, Anura).

The karyotype of Litoria (L.) paraewingi (Watson et al., 1971) (Big River State Forest, Victoria) is described here for the first time. It is prepared following tissue culture of toe clipping macerates, cryopreservation, reculture and conventional 4',6-diamidino-2-phenylindole (DAPI) staining. The L.paraewingi karyotype is then compared to similarly processed IUCN (International Union for the Conservation of Nature) least concern members L.ewingii (Duméril et Bibron, 1841) (southern Victoria) and L.jervisiensis (Duméril et Bibron, 1841) (Myall Lakes National Park, New South Wales), all members of the same L.ewingii complex/group. The L.paraewingi diploid number is 2n = 26, the same as for the other two species. Litoriaparaewingi chromosomes 1, 2, 6 and 7 are submetacentric, chromosomes 3 and 5 are subtelocentric and the remainder are metacentric. No secondary constriction or putative nucleolus organiser region (NOR) was readily identifiable following conventional DAPI staining in any scored L.paraewingi metaphase spread. Conversely, a putative NOR was readily identifiable on the long arm of chromosome 1 in all examined metaphase spreads for the other two species. The karyotypes of L.ewingii and L.jervisiensis here further differ from L.paraewingi with chromosome 1 being metacentric and chromosomes 8 and 10 being submetacentric for both former species. The L.jervisiensis karyotype differs from those of L.ewingii and L.paraewingi by DAPI staining with: (i) apparent relative length inversion of subtelocentric chromosome 3 and metacentric chromosome 4 and (ii) chromosome 6 being metacentric rather than submetacentric. All three species have a highly conserved chromosome morphology with respect to chromosomes 2, 5, 7, 9, 11, 12 and 13. The greatest gross morphological difference karyotypically is observed between L.paraewingi and L.jervisiensis. These karyotype data support the previous phylogenetic separation of these three species based upon genetic compatibility and behavioural, biochemical and molecular genetic analyses.

Developmental sequence of the chondrocranium in the obligate carnivorous larvae of Lepidobatrachus laevis (Amphibia: Ceratophryidae).

The vertebrate head and its skull represent a significant innovation that has played a key role in the evolutionary and ecological success of vertebrates. For a global and integral understanding of the evolution of the head skeleton, it is essential to have reliable information on the development of chondrocranium in a wide range of vertebrate species. Therefore, we studied the cranial chondrogenesis of the larva of the Budgett frog, Lepidobatrachus laevis (Ceratophryidae, Neobatrachia). We studied the development using several methods, including histological preparation of transverse sections of the chondrocranium, morphological analysis of three different states of development (mesenchymal aggregation, differentiation, and chondrification), and three-dimensional digital reconstructions. As a result, we observed that the Anlage of the chondrocranium at Gosner stage 19 is laterally compressed, that is, it is initially higher than wide. It gradually flattens, enlarges, and differentiates until reaching a very wide and flat shape at Gosner stage 26. Furthermore, we show that the chondrocranial development of L. laevis takes place in a mosaic pattern, which differs to nontetrapod vertebrates in which an anterior to posterior gradient of chondrification is observed. We identified 19 developmental stages in L. laevis according to the chondrification state of its cranial structures. The first element reaching the differentiation-into-chondroblast stage is the hypobranchial plate of the branchial basket, and chondrification, that is, final differentiation, occurs simultaneously in several structures of the neurocranium and viscerocranium. We hypothesize that the rapid chondrification of L. laevis' chondrocranium is an adaptation to the semi-arid climate conditions from its type locality, El Gran Chaco in South America. Due to the only temporary availability of suitable water accumulations this apparent accelerated development would make sense to ensure the tadpoles are froglets by the time the water body disappears. Here, we provide novel information on chondrocranial development in L. laevis. We compare our results to the developmental sequences, previously known from other vertebrate taxa, particularly within the amphibian phylogeny, in an attempt to understand developmental variability and the evolutionary history of the vertebrate head skeleton.

Reversible Histone Modifications Contribute to the Frozen and Thawed Recovery States of Wood Frog Brains.

Epigenetic regulation, notably histone post-translational modification (PTM), has emerged as a major transcriptional control of gene expression during cellular stress adaptation. In the present study, we use an acid extraction method to isolate total histone protein and investigate dynamic changes in 23 well-characterized histone methylations/acetylations in the brains of wood frogs subject to 24-h freezing and subsequent 8-h thawed recovery conditions. Our results identify four histone PTMs (H2BK5ac, H3K14ac, H3K4me3, H3K9me2) and three histone proteins (H1.0, H2B, H4) that were significantly (p < 0.05) responsive to freeze-thaw in freeze-tolerant R. sylvatica brains. Two other permissive modifications (H3R8me2a, H3K9ac) also trended downwards following freezing stress. Together, these data are strongly supportive of the proposed global transcriptional states of hypometabolic freeze tolerance and rebounded thawed recovery. Our findings shed light on the intricate interplay between epigenetic regulation, gene transcription and energy metabolism in wood frogs' adaptive response to freezing stress.

Bioactivity of synthetic peptides from Ecuadorian frog skin secretions against Leishmania mexicana, Plasmodium falciparum, and Trypanosoma cruzi.

Chagas disease, leishmaniasis, and malaria are major parasitic diseases disproportionately affecting the underprivileged population in developing nations. Finding new, alternative anti-parasitic compounds to treat these diseases is crucial because of the limited number of options currently available, the side effects they cause, the need for long treatment courses, and the emergence of drug-resistant parasites. Anti-microbial peptides (AMPs) derived from amphibian skin secretions are small bioactive molecules capable of lysing the cell membrane of pathogens while having low toxicity against human cells. Here, we report the anti-parasitic activity of five AMPs derived from skin secretions of three Ecuadorian frogs: cruzioseptin-1, cruzioseptin-4 (CZS-4), and cruzioseptin-16 from Cruziohyla calcarifer; dermaseptin-SP2 from Agalychnis spurrelli; and pictuseptin-1 from Boana picturata. These five AMPs were chemically synthesized. Initially, the hemolytic activity of CZS-4 and its minimal inhibitory concentration against Escherichia coli, Staphylococcus aureus, and Candida albicans were determined. Subsequently, the cytotoxicity of the synthetic AMPs against mammalian cells and their anti-parasitic activity against Leishmania mexicana promastigotes, erythrocytic stages of Plasmodium falciparum and mammalian stages of Trypanosoma cruzi were evaluated in vitro. The five AMPs displayed activity against the pathogens studied, with different levels of cytotoxicity against mammalian cells. In silico molecular docking analysis suggests this bioactivity may occur via pore formation in the plasma membrane, resulting in microbial lysis. CZS-4 displayed anti-bacterial, anti-fungal, and anti-parasitic activities with low cytotoxicity against mammalian cells. Further studies about this promising AMP are required to gain a better understanding of its activity.IMPORTANCEChagas disease, malaria, and leishmaniasis are major tropical diseases that cause extensive morbidity and mortality, for which available treatment options are unsatisfactory because of limited efficacy and side effects. Frog skin secretions contain molecules with anti-microbial properties known as anti-microbial peptides. We synthesized five peptides derived from the skin secretions of different species of tropical frogs and tested them against cultures of the causative agents of these three diseases, parasites known as Trypanosoma cruzi, Plasmodium falciparum, and Leishmania mexicana. All the different synthetic peptides studied showed activity against one of more of the parasites. Peptide cruzioseptin-4 is of special interest since it displayed intense activity against parasites while being innocuous against cultured mammalian cells, which indicates it does not simply hold general toxic properties; rather, its activity is specific against the parasites.

Potential effects of traffic noise on anuran call characteristics in Louisiana, USA during winter.

Urban environments expose wildlife to levels of anthropogenic noise they would not experience in rural areas (e.g., traffic noise), and research suggests that many species adjust their acoustic signals for optimal transmission in urban soundscapes. However, our understanding of anuran (order Anura) responses to noise pollution in urban environments of the southeastern United States is limited, particularly for species that can breed during winter. Our goal was to examine how vocal anuran advertisement call characteristics during winter varied with increasing distance from roadways in bottomland hardwoods of Louisiana, USA. We deployed acoustic recording units at two sites (i.e., rural and urban) perpendicular to Interstate 10 at 200-, 400-, and 600-m intervals (i.e., close, middle, and far) from November 2019 to January 2020. We detected Cajun Chorus Frogs (Pseudacris fouquettei) and Cricket Frogs (Acris spp.) at our rural site, and only detected Cricket Frogs at our urban site. At the rural site, Cajun Chorus Frogs produced longer duration notes at the far location compared to the middle location. At the urban site, Cricket Frogs produced higher dominant frequency calls at the close location compared to the far and middle locations and longer duration notes at the far location compared to the close location. We were unable to account for additional factors in our models (e.g., temperature, noise levels), but our results generally align with previous research. Our study provides baseline data for future research to examine the potential effects of traffic noise on winter advertisement calls in locations with similar environmental conditions and species.

UV radiation-induced peptides in frog skin confer protection against cutaneous photodamage through suppressing MAPK signaling.

Overexposure to ultraviolet light (UV) has become a major dermatological problem since the intensity of ultraviolet radiation is increasing. As an adaption to outside environments, amphibians gained an excellent peptide-based defense system in their naked skin from secular evolution. Here, we first determined the adaptation and resistance of the dark-spotted frogs (Pelophylax nigromaculatus) to constant ultraviolet B (UVB) exposure. Subsequently, peptidomics of frog skin identified a series of novel peptides in response to UVB. These UV-induced frog skin peptides (UIFSPs) conferred significant protection against UVB-induced death and senescence in skin cells. Moreover, the protective effects of UIFSPs were boosted by coupling with the transcription trans-activating (TAT) protein transduction domain. In vivo, TAT-conjugated UIFSPs mitigated skin photodamage and accelerated wound healing. Transcriptomic profiling revealed that multiple pathways were modulated by TAT-conjugated UIFSPs, including small GTPase/Ras signaling and MAPK signaling. Importantly, pharmacological activation of MAPK kinases counteracted UIFSP-induced decrease in cell death after UVB exposure. Taken together, our findings provide evidence for the potential preventive and therapeutic significance of UIFSPs in UV-induced skin damage by antagonizing MAPK signaling pathways. In addition, these results suggest a practicable alternative in which potential therapeutic agents can be mined from organisms with a fascinating ability to adapt.

Miliary osteoma cutis in a climbing mantella frog (Mantella laevigata).

A climbing mantella frog (Mantella laevigata) was presented with nodular thickened skin. Histological examination revealed dermal nodules composed of differentiated bone consistent with miliary osteoma cutis, a non-neoplastic condition where bone is abnormally deposited within the skin. This is the first report of idiopathic osteoma cutis in an amphibian.

Cytokinins Reduce Viral Replication and Alter Plaque Morphology of Frog Virus 3 In Vitro.

Cytokinins (CKs) are a group of N6-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, N6-isopentenyladenine (iP), N6-isopentenyladenosine (iPR), N6-isopentenyladenosine-5'monophosphate (iPMP), and 2-methylthiol-N6-isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations.

Effects of Rice-Frog Co-Cropping on the Soil Microbial Community Structure in Reclaimed Paddy Fields.

Utilizing and improving the productivity of reclaimed land are highly significant for alleviating the problem of food production shortage in China, and the integrated rice-frog farming model can improve soil fertility. However, there are few studies on the use of integrated rice-frog farming technology to improve the fertility of reclaimed land and increase its efficiency in food production. Therefore, this study was conducted to evaluate the effects of the rice-frog co-cropping mode on the soil fertility and microbial diversity of reclaimed land. A rice monoculture group (SF), low-density rice-frog co-cropping group (SD, 5000 frogs/mu, corresponds to 8 frogs/m2), and high-density rice-frog co-cropping group (SG, 10,000 frogs/mu, corresponds to 15 frogs/m2) were established and tested. The contents of total nitrogen, soil organic matter, available potassium, and available phosphorus of the soil in the SG group were significantly higher than those in the SF group (p < 0.05) in the mature stage of rice. Compared with the SF group, the SD and SG groups improved the soil microbial diversity and changed the structure of the microbial community. This study indicates that compared with the rice monoculture mode, the rice-frog co-cropping pattern can improve the soil fertility, as well as microbial diversity, of reclaimed land.

Ingestion of polyethylene terephthalate microplastic water contaminants by Xenopus laevis tadpoles negatively affects their resistance to ranavirus infection and antiviral immunity.

Small plastic debris (0.1 µm-5 mm) or microplastics (MPs) have become major pollutants of aquatic ecosystems worldwide and studies suggest that MPs exposure can pose serious threats to human and wildlife health. However, to date the potential biological impacts of MPs accumulating in low amount in tissues during early life remains unclear. Here, for a more realistic assessment, we have used environmentally representative, mildly weathered, polyethylene terephthalate microplastics (PET MPs), cryomilled (1-100 µm) and fluorescently labelled. We leveraged the amphibian Xenopus laevis tadpoles as an animal model to define the biodistribution of PET MPs and determine whether exposure to PET MPs induce perturbations of antiviral immunity. Exposure to PET MPs for 1-14 days resulted in detectable PET MPs biodistribution in intestine, gills, liver, and kidney as determined by fluorescence microscopy on whole mount tissues. PET MPs accumulation rate in tissues was further evaluated via a novel in situ enzymatic digestion and subsequent filtration using silicon nanomembranes, which shows that PET MPs rapidly accumulate in tadpole intestine, liver and kidneys and persist over a week. Longer exposure (1 month) of tadpoles to relatively low concentration of PET MPs (25 µg/ml) significantly increased susceptibility to viral infection and altered innate antiviral immunity without inducing overt inflammation. This study provides evidence that exposure to MPs negatively impact immune defenses of aquatic vertebrates.

Baseline haematological parameters in three common Australian frog species.

Amphibians are experiencing declines globally, with emerging infectious diseases as one of the main causes. Haematological parameters present a useful method for determining the health status of animals and the effects of particular diseases, but the interpretation of differential cell counts relies on knowing the normal ranges for the species and factors that can affect these counts. However, there is very little data on either normal haematological parameters or guides for blood cell types for free-ranging frog species across the world. This study aims to 1) create a visual guide for three different Australian frog species: Litoria paraewingi, Limnodynastes dumerilii, and Crinia signifera, 2) determine the proportions of erythrocytes to leukocytes and 3) differential leukocytes within blood smears from these three species and 4) assess the association between parasites and differential counts. We collected blood samples from free-ranging frogs and analysed blood smears. We also looked for ectoparasites and tested for the fungal disease chytridiomycosis. Overall, we found that the differentials of erythrocytes to leukocytes were not affected by species, but the proportions of different leukocytes did vary across species. For example, while lymphocytes were the most common type of leukocyte across the three species, eosinophils were relatively common in Limnodynastes dumerilii but rarely present in the other two species. We noted chytridiomycosis infection as well as ectoparasites present in some individuals but found no effect of parasites on blood parameters. Our results add baseline haematological parameters for three Australian frog species and provide an example of how different frog species can vary in their differential blood cell counts. More information is needed on frog haematological data before these parameters can be used to determine the health status of wild or captive frogs.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Case report: Disseminated larval trematodiasis caused by Clinostomum marginatum in a green tree frog (Hyla cinerea).

Clinostomum spp. are common parasites of piscivorous birds. Metacercaria are typically observed in the muscles or just under the skin of fish and rarely amphibians. We describe an unusually severe case of Clinostomum marginatum infection in an adult female green tree frog (Hyla cinerea) from Georgia (USA). The frog was found in November 2015 with a high number (>250) of widely disseminated, raised, subcutaneous nodules. The frog died in December. At necropsy, it was emaciated, and the skin was covered in raised uniform, tan-green, subcutaneous, ∼2-3 mm diameter nodules. Each nodule contained 1-3 C. marginatum metacercariae. Microscopically, high numbers of trematodes were within subcutaneous tissues and in coelomic and oral cavities, lung, liver, kidney, ovary, orbit and calvarium. Small to large numbers of lymphocytes and melanomacrophages were in connective tissues and epidermis. A 732 bp region of COI was 98.8-99.8% similar to numerous sequences of C. marginatum and, phylogenetically it grouped with these C. marginatum sequences. The ITS-1 region was 100% similar to a C. marginatum sample from a great egret (Ardea alba) from Mississippi. This report represents a novel finding of severe trematodiasis in a free-ranging amphibian with C. marginatum infection.

Feral frogs, native newts, and chemical cues: identifying threats from and management opportunities for invasive African Clawed Frogs in Washington state.

Invasive species threaten biodiversity globally. Amphibians are one of the most threatened vertebrate taxa and are particularly sensitive to invasive species, including other amphibians. African clawed frogs (Xenopus laevis) are native to Southern Africa but have subsequently become invasive on multiple continents-including multiple parts of North America-due to releases from the pet and biomedical trades. Despite their prevalence as a global invader, the impact of X. laevis remains understudied. This includes the Pacific Northwest of the USA, which now hosts multiple expanding X. laevis populations. For many amphibians, chemical cues communicate important information, including the presence of predators. Here, we tested the role chemical cues may play in mediating interactions between feral X. laevis and native amphibians in the Pacific Northwest. We tested whether native red-legged frog (Rana aurora) tadpoles display an antipredator response to non-native frog (X. laevis) or native newt (rough-skinned newts, Taricha granulosa) predator chemical stimuli. We found that R. aurora tadpoles exhibited pronounced anti-predator responses when exposed to chemical cues from T. granulosa but did not display anti-predator response to invasive X. laevis chemical cues. We also began experimentally testing whether T. granulosa-which produce a powerful neurotoxin tetrodotoxin (TTX)-may elicit an anti-predator response in X. laevis, that could serve to deter co-occupation. However, our short-duration experiments found that X. laevis were attracted to newt chemical stimuli rather than deterred. Our findings show that X. laevis likely poses a threat to native amphibians, and that these native species may also be particularly vulnerable to this invasive predator, compared to native predators, because toxic native newts may not limit X. laevis invasions. Our research provides some of the first indications that native Pacific Northwest species may be threatened by feral X. laevis and provides a foundation for future experiments testing potential management techniques for X. laevis.