Effects of Chemical Deicers on Amphibian Communites

Roads and highways present a unique challenge to wildlife as they exhibit substantial impacts on the surrounding ecosystem through the interruption of a number of ecological processes. With new roads added to the national highway system every year, an understanding of these impacts is required for effective mitigation of potential environmental impacts. A major contributor to these negative effects is the deposition of chemicals used in winter deicing activities to nearby surface waters. These chemicals often vary in composition and may affect freshwater species differently. The negative impacts of widespread deposition of sodium chloride (NaCl) have prompted a search for an `environmentally friendly' alternative. However, little research has investigated the potential environmental effects of widespread use of these alternatives. Herein, I detail the results of laboratory tests and field surveys designed to determine the impacts of road salt (NaCl) and other chemical deicers on amphibian communities in Michigan's Upper Peninsula. Using larval amphibians I demonstrate the lethal impacts of a suite of chemical deicers on this sensitive, freshwater species. Larval wood frogs (Lithobates sylvatica) were tolerant of short-term (96 hours) exposure to urea (CH4N2O), sodium chloride (NaCl), and magnesium chloride (MgCl2). However, these larvae were very sensitive to acetate products (C8H12CaMgO8, CH3COOK) and calcium chloride (CaCl2). These differences in tolerance suggest that certain deicers may be more harmful to amphibians than others. Secondly, I expanded this analysis to include an experiment designed to determine the sublethal effects of chronic exposure to environmentally realistic concentrations of NaCl on two unique amphibian species, L. sylvatica and green frogs (L. clamitans). L. sylvatica tend to breed in small, ephemeral wetlands and metamorphose within a single season. However, L. clamitans breed primarily in more permanent wetlands and often remain as tadpoles for one year or more. These species employ different life history strategies in this region which may influence their response to chronic NaCl exposure. Both species demonstrated potentially harmful effects on individual fitness. L. sylvatica larvae had a high incidence of edema suggesting the NaCl exposure was a significant physiologic stressor to these larvae. L. clamitans larvae reduced tail length during their exposure which may affect adult fitness of these individuals. In order to determine the risk local amphibians face when using these roadside pools, I conducted a survey of the spatial distribution of chloride in the three northernmost counties of Michigan. This area receives a relatively low amount of NaCl which is confined to state and federal highways. The chloride concentrations in this region were much lower than those in urban systems; however, amphibians breeding in the local area may encounter harmful chloride levels arising from temporal variations in hydroperiods. Spatial variation of chloride levels suggests the road-effect zone for amphibians may be as large as 1000 m from a salt-treated highway. Lastly, I performed an analysis of the use of specific conductance to predict chloride concentrations in natural surface water bodies. A number of studies have used this regression to predict chloride concentrations from measurements of specific conductance. This method is often chosen in the place of ion chromatography due to budget and time constraints. However, using a regression method to characterize this relationship does not result in accurate chloride ion concentration estimates.

Accumulation of the pathogenic fungus Batrachochytrium dendrobatidis on the regressing tail of midwife toads Alytes obstetricans undergoing metamorphosis

Batrachochytrium dendrobatidis (Bd) is a fungus infecting the skin of amphibians. On metamorphosing animals the infection is difficult to detect because of the limited information concerning the location of Bd on the animals during this stage. Histological investigation revealed that Bd accumulated on the reabsorbing tail of metamorphosing animals. This observation may facilitate the detection of Bd in metamorphosing amphibians.

A study of xlcaax-1: Patterns of localization, possible function and usefulness as a developmental marker

Morphogenesis is the process by which the 3-dimensional structure of the developing embryo takes shape. We are studying xlcaax-1, a gene whose product can be used as a molecular marker for several morphogenetic events. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole mount immunocytochemistry and immunoperoxidase staining of tissue sections showed that during development the xlcaax-1 protein accumulation was coincident with the differentiation of the epidermis, pronephros and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein was localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein served as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. The xlcaax-1 protein was most abundant in kidney and immunogold EM analysis showed that the xlcaax-1 protein was highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. The xlcaax-1 protein was also localized in other ion transporting epithelia. The localization pattern and preliminary functional assays of xlcaax-1 suggest that the protein may function in association with an ion transport channel or pump.^ Cell migration and cell-cell interactions play important roles in numerous processes during morphogenesis. One of these is the formation of the pronephric (wolffian) duct (PD), which connects the pronephros to the cloaca. It is currently accepted that in most amphibians the pronephric duct is formed by active migration of the pronephric duct rudiment (PDR) cells along a pre-determined pathway. However, there is evidence that in Xenopus, the PD may be formed entirely by in situ segregation of cells out of the lateral mesoderm. In this study, we showed, using PDR ablation and X. laevis - X. borealis chimeras, that PD elongation in Xenopus required both active cell migration and an induced recruitment of cells from the posterior lateral plate mesoderm. We also showed that PDR cell migration was limited to only a few stages during development and that this temporal control is due, at least in part, to changes in the competence of the PD pathway to support cell migration. In addition, our data suggested that an alkaline phosphatase (APase) adhesion gradient may be involved in determining this competence. ^

H2B histone gene expression during {\it Xenopus laevis\/} development

Histone gene expression is replication-independent during oogenesis and early embryogenesis in amphibians; however, it becomes replication-dependent during later embryogenesis and remains replication-dependent through adulthood. In order to understand the mechanism for this switch in transcriptional regulation of histone gene expression during amphibian development, linker-scanning mutations were made in a Xenopus laevis H2B histone gene promoter by oligonucleotide site-directed mutagenesis and assayed by microinjection into oocytes and embryos. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CCAAT, and ATF motifs, required for maximal transcription in both oocytes and embryos. Factors binding to the CCAAT and ATF motifs are present in oocytes and embryos and increase slightly in abundance during early development. A sequence (CTTTACAT) in the frog H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is additionally required for maximal H2B transcription in frog embryos. Oocytes and embryos contain multiple octamer-binding proteins that are expressed in a sequential manner during early development. Sequences encoding three novel octamer-binding proteins were isolated from Xenopus cDNA libraries by virtue of their similarity with the DNA binding (POU) domain of the ubiquitously expressed transcription factor Oct-1. The protein encoded by one of these genes, termed Oct-60, was localized mainly in the cytoplasm of oocytes and was also present in early embryos until the gastrula stage of development. Proteins encoded by the other two genes, Oct-25 and Oct-91, were present in embryos after the mid-blastula stage of development and decreased by early neurula stage. The activity of the Xenopus H2B octamer motif in embryos is not specifically associated with increased binding by Oct-1 or the appearance of novel octamer-binding proteins but requires the presence of an intact CCAAT motif. We found that synergistic interactions among promoter elements are important for full H2B promoter activity. The results suggest that transcription of the Xenopus H2B gene is replication-dependent when it is activated at the mid-blastula stage of development and that replication-dependent H2B transcription is mediated by Oct-1. ^

CYTOPLASMIC MALATE DEHYDROGENASE IS THE AROMATIC ALPHA-KETO ACID REDUCTASE IN MAN (PHENYLKETONURIA, PKU, TYROSINE)

This dissertation presents evidence to support the hypothesis that cytoplasmic malate dehydrogenase (MDH-1) is the enzyme in humans which catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, and the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; E.C. 1.1.1.96) is actually a secondary activity of cytoplasmic malate dehydrogenase.^ Purified MDH and purified KAR have the same molecular weight, subunit structure, heat-inactivation profile and tissue distribution. After starch gel electrophoresis, and using p-hydroxyphenylpyruvic acid (HPPA) as substrate, KAR activity co-migrates with MDH-1 in all species studied except some marine animals. Inhibition with malate, the end-product of malate dehydrogenase, substantially reduces or totally eliminates KAR activity. Purified cytoplasmic MDH from human erythrocytes has an alpha-keto acid reductase activity with identical mobility. All electrophoretic variants of MDH-1 seen in the fresh-water bony fish Xiphophorus, the amphibians Rana and humans exhibited identical variation for KAR, and the two traits co-segregated in the small group of offspring from one Rana heterozygote studied. Both enzymes show almost no electrophoretic variation among humans from many ethnic groups, and among several inbred strains of mice both MDH-s and KAR co-migrate with no variation. MDH-1 and KAR in mouse and Chinese hamster fibroblasts show identical mobility differences between species. Antisera raised against purified chicken cytoplasmic MDH totally inhibited both MDH-1 and KAR in chickens and humans. Mitochondrial MDH from tissue homogenates has no detectable KAR activity but purified MDH-2 does.^ The previous claim that the gene for KAR is on human chromosome 12 is disputed because both MDH-1 and LDH bands appear with slightly different mobility approximately midway between the human and hamster controls in somatic cell hybrid studies, and the meaning of this artifact is discussed. ^

Pond-Breeding Amphibian Species Distributions in a Beaver-Modified Landscape, Acadia National Park, Mount Desert Island, Maine

In order to maintain pond-breeding amphibian species richness, it is important to understand how both natural and anthropogenic disturbances affect species assemblages and individual species distributions both at the scale of individual ponds and at a larger landscape scale. The goal of this project was to investigate what characteristics of ponds and the surrounding wetland landscape were most effective in predicting pond-breeding species richness and the individual occurrence of wood frog (Rana sylvatica), bullfrog (Rana catesbeiana) and pickerel frog (Rana palustris) breeding sites in a beaver-modified landscape and how this landscape has changed over time. The wetland landscape of Acadia National Park was historically modified by the natural disturbance cycles of beaver (Castor cazadensis), and since their reintroduction to the island in 1921, beaver have played a large role in creating and maintaining palustrine wetlands. In 2000 and 2001, I studied pond-breeding amphibian assemblages at 71 palustrine wetlands in Acadia National Park, Mount Desert Island, Maine. I determined breeding presence of 7 amphibian species and quantified 15 variables describing local pond conditions and characteristics of the wetland landscape. I developed a priori models to predict sites with high amphibian species and used model selection with Akaike's Information Criterion (AIC) to identify important variables. Single species models were also developed to predict wood frog, bullfrog and pickerel frogs breeding presence. The variables for wetland connectivity by stream corridors and the presence of beaver disturbance were the most effective variables to predict sites with high amphibian richness. Wood frog breeding was best predicted by local scale variables describing temporary, fishless wetlands and the absence of active beaver disturbance. Abandoned beaver sites provided wood frog breeding habitat (70%) in a similar proportion to that found in non beaver-influenced sites (79%). In contrast, bullfrog breeding presence was limited to active beaver wetlands with fish and permanent water, and 80% of breeding sites were large (≥2ha in size). Pickerel frog breeding site selection was predicted best by the connectivity of sites in the landscape by stream corridors. Models including the presence of beaver disturbance, greater wetland perimeter and greater depth were included in the confidence set of pickerel frog models but showed considerably less support. Analysis of historic aerial photographs showed an 89% increase in the total number of ponded wetlands available in the landscape between the years of 1944 and 1997. Beaver colonization generally converted forested wetlands and riparian areas to open water and emergent wetlands. Temporal colonization of beaver wetlands favored large sites low in the watersheds and sites that were impounded later were generally smaller, higher in the watershed, and more likely to be abandoned. These results suggest that beaver have not only increased the number of available breeding sites in the landscape for pond-breeding amphibians, but the resulting mosaic of active and abandoned beaver wetlands also provides suitable breeding habitat for species with differing habitat requirements.

Predator-Induced Plasticity in Spotted Salamanders (Ambystoma maculatum)

The ability to respond plastically to the environment has allowed amphibians to evolve adaptive responses to spatial and temporal variation in predation threat. However, animals exposed to predators may also show costs of plasticity or tradeoffs. This study examines predator-induced plasticity in larval development, behavior, and metamorphosis in the spotted salamander, Ambystoma maculatum. Salamanders were raised in two treatments: with predator cues (a fish predator, genus Lepomis, on the other side of a divided tank), or without predator cues. During the larval stage the predator treatment group experienced higher mortality rates than the no-predator treatment group. Behavioral trials revealed that predator treatment animals ate less than those not exposed, and that this feeding response was immediately inducible and had lasting effects. Animals in the predator treatment group had smaller tail areas during the mid-larval period. Feeding and body size effects may have contributed to increased mortality in the predator-treatment animals. The timing of metamorphic onset was not affected by the presence of predators, but predator-treatment salamanders had shorter snout/vent lengths at metamorphosis. The duration of metamorphosis showed a potentially adaptive plastic response to the presence of predator cues: metamorphosis was longest in the no-predator treatment group, reduced in the predator treatment group, and even further reduced for animals exposed to predator cues only during metamorphosis. Overall, we found a mix of potentially adaptive and costly plastic responses in spotted salamanders.

Ontogenetic Effects of Hatching Plasticity in the Spotted Salamander (Ambystoma maculatum) due to Egg and Larval Predators

The ability to respond plastically to the environment has allowed amphibians to evolve a response to spatial and temporal variation in predation threat (Benard 2004). Embroys exposed to egg predation are expected to hatch out earlier than their conspecifics. Larval predation can induce a suite of phenotypic changes including growing a larger tail area. When presented with cues from both egg and larval predators, embryos are expected to respond to the egg predator by hatching out earlier because the egg predator presents an immediate threat. However, hatching early may be costly in the larval environment in terms of development, morphology, and/or behavior. We created a laboratory experiment in which we exposed clutches of spotted salamander (Ambystoma maculatum) eggs to both egg (caddisfly larvae) and larval (A. opacum) predators to test this hypothesis. We recorded hatching time and stage and took developmental and morphological data of the animals a week after hatching. Larvae were entered into lethal predation trials with a larval predatory sunfish (Lepomis sp.) in order to study behavior. We found that animals exposed to the egg predator cues hatched out earlier and at earlier developmental stages than conspecifics regardless of whether there was a larval predator present. Animals exposed to larval predator cues grew relatively larger tails and survived longer in the lethal predation trials. However the group exposed to both predators showed a cost of early hatching in terms of lower tail area and shorter survival time in predation trials. The morphological and developmental effects measured of hatching plasticity were transient as there were no developmental or morphological differences between the treatment groups at metamorphosis. Hatching plasticity may be transient but it is important to the development and survival of many amphibians.

Effective acoustical monitoring tools for Mannophryne lamarcai

We performed the field-work during the dry (March 2014) and reainy season (May and June 2014) at the species type locality: Cerro Socopó, located at central-west region between Falcón, Lara and Zulia states, Venezuela. Socopó is a small and isolated mountain (1.571 m) belonging to the Ziruma mountains, and represents a relict of tropical mountain forest surrounded by semi-arid vegetation and grassland. This area is home to 312 species of vertebrates, including endangered and endemic amphibians species like Mannophryne lamarcai, Leptodactylus magistris and Dendropsophus amicorum. These forest and species are severely threatened by cattle ranch and illegal timber extraction, with forest formations only above 1000 meters. Despite this, no legal protected figure has been established in the area. We identified a 2.5 km secondary road transect within the study area based on the following criteria: 1) it cover different habitat types (streams and lagoons); and 2) it within the altitudinal gradient described for the specie (1,040 to 1,363 m). We identified three sampling points throughout the transect located in the vicinity of wetland habits: socopo1, socopo2 and socopo4. We did two types of recordings: 1) high quality recordings to characterize the advertisement call for M. lamarcai, non described to date (socopo4), and 2) recordings in different sampling points to evaluate call detectors performance in different acoustic scenarios (in all three localities).

Global distributions of epipelagic macrozooplankton abundance and biomass - Gridded data product (NetCDF) - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

Macrozooplankton are an important link between higher and lower trophic levels in the oceans. They serve as the primary food for fish, reptiles, birds and mammals in some regions, and play a role in the export of carbon from the surface to the intermediate and deep ocean. Little, however, is known of their global distribution and biomass. Here we compiled a dataset of macrozooplankton abundance and biomass observations for the global ocean from a collection of four datasets. We harmonise the data to common units, calculate additional carbon biomass where possible, and bin the dataset in a global 1 x 1 degree grid. This dataset is part of a wider effort to provide a global picture of carbon biomass data for key plankton functional types, in particular to support the development of marine ecosystem models. Over 387 700 abundance data and 1330 carbon biomass data have been collected from pre-existing datasets. A further 34 938 abundance data were converted to carbon biomass data using species-specific length frequencies or using species-specific abundance to carbon biomass data. Depth-integrated values are used to calculate known epipelagic macrozooplankton biomass concentrations and global biomass. Global macrozooplankton biomass has a mean of 8.4 µg C l-1, median of 0.15 µg C l-1 and a standard deviation of 63.46 µg C l-1. The global annual average estimate of epipelagic macrozooplankton, based on the median value, is 0.02 Pg C. Biomass is highest in the tropics, decreasing in the sub-tropics and increasing slightly towards the poles. There are, however, limitations on the dataset; abundance observations have good coverage except in the South Pacific mid latitudes, but biomass observation coverage is only good at high latitudes. Biomass is restricted to data that is originally given in carbon or to data that can be converted from abundance to carbon. Carbon conversions from abundance are restricted in the most part by the lack of information on the size of the organism and/or the absence of taxonomic information. Distribution patterns of global macrozooplankton biomass and statistical information about biomass concentrations may be used to validate biogeochemical models and Plankton Functional Type models.

Ingeniería de la conservación: Investigación aplicada para la conservación de la fauna mediterránea amenazada

El ser humano nunca ha tenido una capacidad semejante a la actual para alterar el medio en el que vive. A través fundamentalmente de cambios en el uso del hábitat, alteración de los ciclos biogeoquímicos, cambio climático y presencia de especies invasoras, la tasa de extinción de especies se ha acelerado enormemente. Al crecer más rápido la población humana dentro de las áreas en las que se concentra una mayor biodiversidad (biodiversity hotspots), las tasas de transformación son mayores, por lo que dicha biodiversidad se encuentra más amenazada. Entre los principales 25 biodiversity hotspots globales se encuentra la cuenca mediterránea. Las afecciones a la biodiversidad hacen por tanto que en dicha cuenca mediterránea sea necesario poner en práctica iniciativas innovadoras para su conservación. Pero además de una cuidadosa selección, es necesario el seguimiento y la evaluación de la eficacia y la eficienca de dichas medidas, para comprobar su idoneidad y mejorarlas cuando sea posible. Esta es la tarea de la ingeniería de la conservación. Esta Memoria busca analizar y proponer mejoras en cuatro acciones experimentales de conservación de la biodiversidad que tienen un marco común, la conservación de especies subrogadas del monte mediterráneo. En concreto de cinco especies amenazadas: conejo de monte (Oryctolagus cuniculus, especie llave), águila imperial ibérica (Aquila adalberti), águila perdicera (Aquila fasciata), cigüeña negra (Ciconia nigra) y lince ibérico (Lynx pardinus), todas ellas especies paraguas y bandera. Casi todas estas especies se enfrentan a amenazas comunes, como son la pérdida de la calidad del hábitat, la mortalidad de origen humano, la escasez de alimento o la falta de eficiencia en las técnicas de seguimiento. Las acciones destinadas a revertir o minimizar estas amenazas han tenido un marco geográfico y ecológico común, 49 fincas privadas de monte mediterráneo situadas en 9 espacios de la Red Natura 2000 del centro-occidental peninsular, en las que se ha podido trabajar merced a acuerdos de custodia del territorio. Dichas actividades han sido financiadas casi por completo con cargo a fondos LIFE Naturaleza de la Comisión Europea. El presente documento contribuye a aportar el conocimiento científico imprescindible para mejorar la eficacia y eficiencia de las medidas técnicas de gestión y conservación: la ingeniería de la conservación. Como resultados más relevantes se pueden citar que la selección del conejo de las zonas sembradas aumenta con la presencia tanto de isletas de matorral como de refugios y resulta inferior en las zonas de matorral más cerrado; que la electrocución es una de las principales causas de mortalidad de las rapaces amenazadas, así como que se ha detectado que la mortalidad se concentra en determinadas zonas que deben ser prioritarias para su corrección y que éstas deben pasar por las modificaciones estructurales; que la selección de zonas de alimentación por parte de la cigüeña negra se ve afectada por numerosos factores y que una adecuada ubicación y gestión de las charcas para ganadería y caza mayor puede favorecer no sólo la alimentación de esta especie, sino suponer un lugar de cría para diversas especies de anfibios y peces autóctonos, en franco declive; finalmente, hay que destacar que la mejora en el seguimiento de las poblaciones de lince ibérico pasa por la incorporación de métodos de seguimiento novedosos, como las cámaras infrarrojas en letrinas, dada la capacidad de aprendizaje de la especie. Abstract Nowadays the human being has reached its top ability to exchange its environment. Mainly through habitat change, altering biogeochemical cycles, climatic change and alien species, global extinction rate has increased dramatically. Population increases faster in biodiversity hotspots, thus change rates are higher. Mediterranean basin is included within the 25 most valuable biodiversity hotspots. Affections on biodiversity turns necessary to develop innovative actions to maintain the most threatened species in this hotspot. But further than a careful selection and implementarion, survey and evaluation of those practices is necessary to check its suitability. Those are actions for the conservation engineering. This Memory looks for to analyze and to put forward improvements in four experimental biodiversity conservation actions with a common framework: conservation of surrogate Mediterranean species. Those actions were specifically directed to five threatened species: the European wild rabbit (Oryctolagus cuniculus, key species), Spanish imperial eagle (Aquila adalberti) and Bonelli’s eagle (Aquila fasciata, both of the umbrella and flagship species), black stork (Ciconia nigra, umbrella and flagship species) and the Iberian lynx (Lynx pardinus, umbrella and flagship species). Those species have common threats: habitat loss, maninduced mortality, food shortening and lack of surveying efficiency. Actions aimed to minimize those threats also had a common geographical frame: 49 privately owned states within 9 Natura 2000 areas in Southwestern Spain. Actions were developed through lands tewardship agreements, and were even wholly undertaken through European Commision LIFE’s fund. This document contributes to develop cientific knowledge necessary for increasing efficacy and efficiency for implementing technical measures devoted to conservation, the conservation engineering. One of the most outstanding result might be the necessity of including bushy islets or rabbit shelter within sowings when enhacing rabbit populations is the goal. Electrocution is one of the commonest man-induced death causes for raptors, especially for the threatened ones. Areas of concentration of this mortality have been detected, and when mitigation measures have been evaluated, only structural modifications had results. The black stork’s selection of feeding areas is influenced by several factors. An adequate location and management of livestock’s ponds can favour not only this species, but also creating new breeding places for amphibians and indigenous fish species, most of them in a sharp decline. Finally, improving the survey of the Iberian lynx might be achieved through implementing newly developed surveying systems, such as the infrared cameras in latrines.

Allergy to Uncommon Pets: New Allergies but the Same Allergens.

The prevalence of exotic pet allergies has been increasing over the last decade. Years ago, the main allergy-causing domestic animals were dogs and cats, although nowadays there is an increasing number of allergic diseases related to insects, rodents, amphibians, fish, and birds, among others. The current socio-economic situation, in which more and more people have to live in small apartments, might be related to this tendency. The main allergic symptoms related to exotic pets are the same as those described for dog and cat allergy: respiratory symptoms. Animal allergens are therefore, important sensitizing agents and an important risk factor for asthma. There are three main protein families implicated in these allergies, which are the lipocalin superfamily, serum albumin family, and secretoglobin superfamily. Detailed knowledge of the characteristics of allergens is crucial to improvement treatment of uncommon-pet allergies.