Development and application of an eDNA method to detect and quantify a pathogenic parasite in aquatic ecosystems.

Approaches based on organismal DNA found in the environment (eDNA) have become increasingly utilized for ecological studies and biodiversity inventories as an alternative to traditional field survey methods. Such DNA-based techniques have largely been used to establish the presence of free-living organisms, but have much potential for detecting and quantifying infectious agents in the environment, which is necessary to evaluate disease risk. We developed an eDNA method to examine the distribution and abundance of the trematode Ribeiroia ondatrae, a pathogenic parasite known to cause malformations in North American amphibians. In addition to comparing this eDNA approach to classical host necropsy, we examined the detectability of R. ondatrae in water samples subject to different degradation conditions (time and temperature). Our test exhibited high specificity and sensitivity to R. ondatrae, capable of detecting as little as 14 fg (femtograms) of this parasite's DNA (1/2500th of a single infectious stage) from field water samples. Compared to our results from amphibian host necropsy, quantitative PCR was -90% concordant with respect to R. ondatrae detection from 15 field sites and was also a significant predictor of host infection abundance. DNA was still detectable in lab samples after 21 days at 25°C, indicating that our method is robust to field conditions. By comparing the advantages and disadvantages of eDNA vs. traditional survey methods for determining pathogen presence and abundance in the field, we found that the lower cost and effort associated with eDNA approaches provide many advantages. The development of alternative tools is critical for disease ecology, as wildlife management and conservation efforts require reliable establishment and monitoring of pathogens.

Early evolution of limb regeneration in tetrapods: evidence from a 300-million-year-old amphibian.

Salamanders are the only tetrapods capable of fully regenerating their limbs throughout their entire lives. Much data on the underlying molecular mechanisms of limb regeneration have been gathered in recent years allowing for new comparative studies between salamanders and other tetrapods that lack this unique regenerative potential. By contrast, the evolution of animal regeneration just recently shifted back into focus, despite being highly relevant for research designs aiming to unravel the factors allowing for limb regeneration. We show that the 300-million-year-old temnospondyl amphibian Micromelerpeton, a distant relative of modern amphibians, was already capable of regenerating its limbs. A number of exceptionally well-preserved specimens from fossil deposits show a unique pattern and combination of abnormalities in their limbs that is distinctive of irregular regenerative activity in modern salamanders and does not occur as variants of normal limb development. This demonstrates that the capacity to regenerate limbs is not a derived feature of modern salamanders, but may be an ancient feature of non-amniote tetrapods and possibly even shared by all bony fish. The finding provides a new framework for understanding the evolution of regenerative capacity of paired appendages in vertebrates in the search for conserved versus derived molecular mechanisms of limb regeneration.

Congenital malformations of the vertebral column in ancient amphibians.

Temnospondyls, the largest group of Palaeozoic and Mesozoic amphibians, primitively possess rhachitomous vertebrae with multipartite centra (consisting of one horse-shoe-shaped inter- and paired pleurocentra). In a group of temnospondyls, the stereospondyls, the intercentra became pronounced and disc-like, whereas the pleurocentra were reduced. We report the presence of congenital vertebral malformations (hemi, wedge and block vertebrae) in Permian and Triassic temnospondyls, showing that defects of formation and segmentation in the tetrapod vertebral column represent a fundamental failure of somitogenesis that can be followed throughout tetrapod evolution. This is irrespective of the type of affected vertebra, that is, rhachitomous or stereospondylous, and all components of the vertebra can be involved (intercentrum, pleurocentrum and neural arch), either together or independently on their own. This is the oldest known occurrence of wedge vertebra and congenital block vertebra described in fossil tetrapods. The frequency of vertebral congenital malformations in amphibians appears unchanged from the Holocene.

Localized hotspots drive continental geography of abnormal amphibians on U.S. wildlife refuges.

Amphibians with missing, misshapen, and extra limbs have garnered public and scientific attention for two decades, yet the extent of the phenomenon remains poorly understood. Despite progress in identifying the causes of abnormalities in some regions, a lack of knowledge about their broader spatial distribution and temporal dynamics has hindered efforts to understand their implications for amphibian population declines and environmental quality. To address this data gap, we conducted a nationwide, 10-year assessment of 62,947 amphibians on U.S. National Wildlife Refuges. Analysis of a core dataset of 48,081 individuals revealed that consistent with expected background frequencies, an average of 2% were abnormal, but abnormalities exhibited marked spatial variation with a maximum prevalence of 40%. Variance partitioning analysis demonstrated that factors associated with space (rather than species or year sampled) captured 97% of the variation in abnormalities, and the amount of partitioned variance decreased with increasing spatial scale (from site to refuge to region). Consistent with this, abnormalities occurred in local to regional hotspots, clustering at scales of tens to hundreds of kilometers. We detected such hotspot clusters of high-abnormality sites in the Mississippi River Valley, California, and Alaska. Abnormality frequency was more variable within than outside of hotspot clusters. This is consistent with dynamic phenomena such as disturbance or natural enemies (pathogens or predators), whereas similarity of abnormality frequencies at scales of tens to hundreds of kilometers suggests involvement of factors that are spatially consistent at a regional scale. Our characterization of the spatial and temporal variation inherent in continent-wide amphibian abnormalities demonstrates the disproportionate contribution of local factors in predicting hotspots, and the episodic nature of their occurrence.

The continuing challenge of understanding, preventing, and treating neural tube defects.

Human birth defects are a major public health burden: The Center for Disease Control estimates that 1 of every 33 United States newborns presents with a birth defect, and worldwide the estimate approaches 6% of all births. Among the most common and debilitating of human birth defects are those affecting the formation of the neural tube, the precursor to the central nervous system. Neural tube defects (NTDs) arise from a complex combination of genetic and environmental interactions. Although substantial advances have been made in the prevention and treatment of these malformations, NTDs remain a substantial public health problem, and we are only now beginning to understand their etiology. Here, we review the process of neural tube development and how defects in this process lead to NTDs, both in humans and in the animal models that serve to inform our understanding of these processes. The insights we are gaining will help generate new intervention strategies to tackle the clinical challenges and to alleviate the personal and societal burdens that accompany these defects.

Physiological response of American bullfrog tadpoles to stressor conditions of capture and hypoxia / Resposta fisiológica de girinos de rã-touro americana submetidos aos mecanismos estressores de captura e hipóxia

The American bullfrog (Rana catesbeiana), recently named Lithobates catesbeianus is currently farmed for commercial purposes throughout various regions of Brazil. Stressful situations such as problems of management, inadequate facilities and environmental changes with consequent reduction of immunity are common in intensive production. The assessments of these situations of stress allow us detect these problems decreasing the injuries causes by confinement. The main objective of this study was to use the biological markers of plasma cortisol and glucose level and hematological parameters to evaluate the response of bullfrog tadpoles submitted to stressed mechanisms of capture and hypoxia. The animals were subjected to three treatments: stress due to individual capture with a hand net; stress due to batch capture with a hand net; and stress due to capture by emptying. The results obtained demonstrated that there were no statistically significant differences in the parameters tested when comparing the treatments with and without exposure to air (normoxia and hypoxia). Based on these results we can conclude that the stressful stimuli tested were not adequate to alter the biomarker tested. For the cortisol, probably this should have occurred due to the synergistic action between this hormone and thyroxin, which induces metamorphosis in these animals.

A rã-touro americana (Rana catesbeiana) recentemente denominada Lithobates catesbeianus é cria da com propósito comercial em várias regiões do Brasil. Situações estressantes tais como problemas de manejo, criação inadequada e alterações ambientais com consequente redução da imunidade são comuns em produções intensivas. A avaliação destas situações de estresse permite-nos detectar estes probemas e diminuir as injurias causadas pelo confinamento. O principal objetivo deste estudo foi utilizar os marcadores biológicos de cortisol, glicemia e dados hematológicos para avaliar a resposta de girinos de rã-touro submetidos aos mecanismos estressores de captura e hipóxia. Os animais foram distribuídosem três tratamentos: estresse por captura individual com puçá; estresse por captura em massa com puçá e estresse por captura por escoamento. Os resultados obtidos demostraram não haver diferenças estatisticamente significativas entre os parametros avaliados quando comparou-se os grupos com e sem exposição ao ar (normoxia e hipoxia). Com base nestes resultados pode-se concluir que os estímulos estressores avaliados não foram adequados para alterar os valores plasmáticos dos marcadores biológicos testados. Para o cortisol, isto ocorreu provavelmente em virtude da ação sinérgica deste hormônio e a tiroxina, que induz a metamorfose nestes animais.

Mystery unsolved: missing limbs in deformed amphibians.

Ballengee and Sessions (2009) claim that predatory attacks by small predators such as Sympetrum dragonfly larvae are sufficient to explain amphibian limb deformities in which the limb is partly or completely missing. This deformity type, the most common in nature, is not well explained by Ribeiroia infection which has also been nominated as a mechanism for limb deformities. We argue that the conclusions of the Ballanegee and Sessions study are not well founded. In part this is because the authors have provided no quantitative analysis of the association between limb deformities and predator densities. Our own data on frequencies of limb deformities suggest that missing hind limbs are often extremely rare even when Sympetrum and other small predators are common. While predatory attacks may contribute to observations of limb deformities, further study will be required to elucidate their role; other potential mechanisms deserve study as well. It is premature, and counterproductive, to draw any conclusions regarding the mechanisms behind the most common limb deformities recorded in natural populations.

Terminology of gonadal anomalies in fish and amphibians resulting from chemical exposures.

Given the recent increase in the number of studies describing the ability of chemicals to exert endocrine-disrupting effects, not only in fish but in a variety of other oviparous groups such as amphibians and reptiles, there is an urgent need to harmonize the terminology currently used in describing pathological changes of the gonads. In addition to difficulties in comparing results from different studies, there is also the risk of miscommunication by using terms that imply a certain clinical relevance which may not be true for the species examined. Especially in the case of the recent and controversial issue about potential effects of the triazine herbicide atrazine on amphibians, clinical terminology has been utilized beyond its true meaning by using terms such as "chemical castration" to describe occurrence of TOs or ovarian tissue in the testis of male frogs exposed to environmental chemicals (Hayes 2004). In clinical terminology, castration is defined as the removal of the gonads or their destruction by an external influence, resulting in a nonfertile organism. However, Hayes (2004) did not investigate any possible effects on the fertility of the test animals and thus did not know if these animals were truly castrated. Similarly, terms such as intersex, hermaphrodite, and sex reversal have been used in ways that appear inappropriate with regard to their clinical meaning in a series of different studies with fish or frogs (see previous sections for a detailed discussion). To ensure the appropriate use of certain terminology in a field as controversial and complex as the study of endocrine disruption, we have attempted, in this chapter, to harmonize the terminology used to describe changes in gonadal development of vertebrates such as fish and amphibians, especially frogs (see Table 3). Where appropriate, the terminology suggested was adopted directly from the clinical terminology. However, as outlined here there are substantial differences between the developmental biology of oviparous vertebrates and mammals, and especially humans, that necessitate modification of the definitions of some of the clinical terms. Where appropriate, therefore, the terminology proposed in this manuscript was redefined based on the biological meanings of the terms used in clinical diagnosis. Considering the large increase in research in the area of reproductive endocrine disruption over the past decades, the authors see an increasing need for a harmonization of terms to be used to describe effects observed in the investigated species. Agreement on a common terminology will allow scientists to better communicate and compare their work, and will enable risk assessors to conduct large-scale evaluations of environmental endocrine disruption by fitting the information from individual studies into a synthesis of normal and abnormal conditions of gonadal tissues.

A molecular phylogenetic study of the genus Ribeiroia (Digenea): trematodes known to cause limb malformations in amphibians.

Species of Ribeiroia (Trematoda: Psilostomidae) are known to cause severe limb malformations and elevated mortality in amphibians. However, little is known regarding the number of species in this genus or its relation to other taxa. Species of Ribeiroia have historically been differentiated by slight differences among their larval stages. To better understand the systematics and biogeography of this genus and their potential relevance to the distribution of malformed amphibians, specimens identified as Ribeiroia were collected across much of the known range, including samples from 5 states in the United States (8 sites) and 2 islands in the Caribbean (Puerto Rico and Guadeloupe). A cercaria from East Africa identified as Cercaria lileta (Fain, 1953), with attributes suggestive of Ribeiroia (possibly R. congolensis), was also examined. The intertranscribed spacer region 2 (ITS-2) of the ribosomal gene complex was sequenced and found to consist of 429 nucleotides (nt) for R. ondatrae (United States) and 427 nt for R. marini (Caribbean), with only 6 base differences noted between the 2 species. The ITS-2 region of C. lileta (429 nt) aligned closely with those of the 2 other Ribeiroia species in a phylogenetic analysis that included related trematode genera. This evidence suggests that a third Ribeiroia species exists in tropical Africa. Variation in ITS-2 within R. ondatrae was nonexistent among the 8 populations from North America. Our study further suggests that Ribeiroia spp. originally parasitized Biomphalaria sp., and that a host switch to a closely related snail, Helisoma sp., may have occurred in the lineage represented by R. ondatrae. However, relationships within the Echinostomatidae are not understood well enough to make any robust conclusions at this time.

Amphibian deformities and Ribeiroia infection: an emerging helminthiasis.

Since their widespread appearance in the mid-1990s, malformed amphibians have evoked fear, as well as fascination within the scientific and public communities. Recent evidence from field and laboratory studies has implicated infection by a digenetic trematode--Ribeiroia ondatrae--as an important cause of such deformities. Ribeiroia spp. have a complex life cycle involving planorbid snails, amphibians and water birds. Under natural conditions, malformations might promote parasite transmission by increasing the susceptibility of infected amphibians to predation by definitive hosts. However, with respect to the recent outbreak of deformities, we suggest that exogenous agents (e.g. pesticides, nutrient run-off, introduced fishes) might be interacting with Ribeiroia, resulting in elevated infection levels, and we highlight the need for studies incorporating multiple stressor dynamics to further explore this problem.

Synergism between trematode infection and pesticide exposure: a link to amphibian limb deformities in nature?

The apparently rapid increase in the prevalence of amphibian limb deformities has led to substantial interest from ecologists and public health professionals. Hypotheses proposed to explain the deformities fall into two broad categories: chemical contaminants and trematode infection. Although there are convincing experimental demonstrations that certain factors can lead to some deformities, the causes for recent increases in amphibian malformation remain controversial. Moreover, no experimental studies on amphibian deformities have been conducted in the field, and no studies have attempted to examine the synergistic effects of trematode infection and exposure to chemical contaminants. Here, I present the results of field and laboratory experiments that link increased trematode infection, and increased limb deformities, to pesticide exposure. Field experiments conclusively demonstrated that exposure to trematode infection was required for the development of limb deformities in wood frogs, Rana sylvatica. However, deformities were more common at sites adjacent to agricultural runoff. Laboratory experiments corroborated the association between pesticide exposure and increased infection with pesticide-mediated immunocompetency as the apparent mechanism. Given the conservative contaminant exposure levels used [Environmental Protection Agency (EPA) drinking water standards] and the widespread use of many pesticides, these negative impacts may help to explain pathogen-mediated amphibian declines in many regions.