Flow and water budgets in the C-111 and L-31W canals near the Everglades National Park, Dade County, Florida

Acoustic velocity meter (AVM) sites, located both distant and adjacent to canal water control structures, were constructed and calibrated in L-31W borrow canal and Canal 111 (C-111) to measure canal water velocity. Data were used to compute monthly discharge volumes and overall water budgets for several canal reaches from August 1994 to May 1996. The water budgets indicated extensive aquifer inflows in L-31W associated, in part, with S-332 pump station return flows. Canal and groundwater piezometer data showed 5 distinct hydrologic scenarios (distinguished by the direction and magnitude of hydraulic gradients) in the important Frog Pond area on the eastern boundary of the Everglades National Park. Most of the water lost from C-111 was via surface water losses near the outlet of the system, close to Florida Bay. The distribution of flows during the study suggest an alteration of the present South Dade Conveyance System modification plan to improve water deliveries to Taylor Slough and the Eastern Panhandle of the Everglades National Park. ^

Ecological correlates of successful colonization in the life history of the Cuban treefrog, {\it Osteopilus septentrionalis\/} (Anura: Hylidae)

Ten correlates of successful colonization were tested and met in the life history of the Cuban treefrog in Florida and the Caribbean. Like many successful colonizing species of animals, the Cuban treefrog was highly fecund; reproduction was possible at a small body size in males (27.0 mm) and females (45.0 mm), and large females could lay large clutches and eggs throughout the year. Generation times were short in this species thereby accelerating the colonization process. Tadpoles and post-metamorphic individuals could exploit a wide range of physical conditions with respect to weather conditions and structure of the habitat. The Cuban treefrog occupied the terrestrial-arboreal niche which was only marginally exploited by other species in Florida. Habitat preference of the Cuban treefrog was for mesophytic forests and disturbed areas, and both habitats were found in native and introduced ranges. The ability to coexist with man further enabled the Cuban treefrog to expand its geographic range. A broad diet enabled the Cuban treefrog to exploit a wide range of prey species and sizes thereby alleviating an important constraint to colonization success. The Cuban treefrog was gregarious and vagile, thereby accelerating the process of dispersal which is crucial to the colonization process. Thus, many features in its life history enabled the Cuban treefrog to rapidly disperse and colonize, often in high population densities, many kinds of sites in its native and introduced range. Conformity to these correlates by the Cuban treefrog ultimately provides predictive power regarding the future colonization of this tropical frog. ^

The relationship between alkaloid-based chemical defenses and diet in dendrobatid poison frogs

Chemical defenses are common among organisms and represent some of the most complex adaptations for avoiding predation, yet our understanding of the ecological nature of these systems remains incomplete. Poison frogs are a group of chemically defended organisms that are dependent entirely on diet for chemical defense. In this study, I identified the dietary arthropods responsible for chemical defense in poison frogs, described spatial and temporal patterns in alkaloid composition of poison frogs, and established links between patterns of variation in alkaloid defense and arthropod diet in poison frogs. Identifying dietary sources and studying patterns of variation in alkaloid composition is fundamental to understanding the ecology and evolution of chemical defense in poison frogs. ^ The dendrobatid poison frog Oophaga pumilio shares many alkaloids in common with other poison frogs and is known to vary in alkaloid composition throughout its geographic range. I designed my dissertation to take advantage of these characteristics and use O. pumilio as a model species for the study of chemical defense in poison frogs. Here, I identified siphonotid millipedes as a source for spiropyrrolizidine alkaloids, formicine ants as a source for pumiliotoxin alkaloids, and oribatid mites as dietary sources for the majority of alkaloids found in poison frogs. I found that alkaloid composition varied spatially and temporally, on both small and large scales, within and among populations of O. pumilio. Alkaloid variation between populations was related to geographic distance, and closer populations tended to have alkaloid compositions more similar to each other than to distant populations. ^ The findings of my study suggest that oribatid mites are the most important dietary source of alkaloids in poison frogs. However, overall alkaloid defense in poison frogs is based on a combination of dietary arthropods, including mites, ants, millipedes, and beetles. Variation in chemical defenses of poison frogs is due to (1) spatial and temporal differences in the presence of alkaloids in certain arthropods and (2) differences in the availability of certain alkaloid-containing arthropods, which are likely the result of differences as well as successional changes in forest structure among locations and through time. ^

Ontogenetic Scaling of Poison Glands in Dendrobates Pumilio

The relationship between granular (poison) gland size and density was examined in an ontogenetic series of the strawberry dart-poison frog, Dendrobates pumilio. Specimens used in this study were collected from the La Selva Biological Station in northeastern Costa Rica. Patches of skin from the dorsal surface of seven frogs, ranging in size from 11 to 23 mm snout-vent length (SVL), were fixed and embedded in paraffin for histological sectioning. Poison gland size and density were quantified microscopically in these sections. Poison glands are uniformly distributed across the skin and mean poison gland diameter increases at a rate faster than snout-vent length from 42.5 at SVL 11mm to 120.0 at SVL 23 mm. Conversely, gland density decreases with body size from 71.9 glands/mm2 to 33.2 glands/mm2 • Due to the positive allometric growth of the poison glands, the percentage of skin surface occupied by poison glands increases from 10.1-22.1% in small frogs (SVL<18 >mm) to 50.0-65.2% in large frogs (SVL>19MM), resulting in more toxin per mm2 in the larger animals. The largest increase in toxicity is correlated temporally with the onset of sexual maturity rather than with changes in aposematic coloring.

Potential impact of invasive anuran species in western Newfoundland

Successful dispersal and establishment of invasive anurans (frogs and toads) may be influenced by competitive exclusion and/or niche differentiation with competing species. I investigated the dispersal of anurans in western Newfoundland using anuran calling surveys and pond-edge visual encounter surveys. The Mink Frog, Lithobates septentrionalis, had dispersed ~50 km northeast from the original (2001) discovery location and ~34 km southwest; displaying spatial separation from Green Frogs, Lithobates clamitans, at landscape and local scales. Visual encounter surveys did not reveal any correlation between adult Mink Frogs and odonate competitors. Additionally, I assessed the impact of varying tadpole densities on removal of epilithic periphyton by providing epilithon covered substrates for American Toad, Anaxyrus americanus, tadpoles raised in laboratory or field enclosures. Higher tadpole densities resulted in smaller tadpoles that removed more periphyton from substrates. As anuran population ranges expand, there may be effects on ecological resources for vertebrate and invertebrate competitors.

Connectivity of fragmented amphibian populations in a Neotropical landscape

A high proportion of amphibian species are threatened with extinction globally, and habitat loss and degradation are the most frequently implicated causes. Rapid deforestation for the establishment of agricultural production is a primary driver of habitat loss in tropical zones where amphibian diversity is highest. Land-cover change affects native assemblages, in part, through the reduction of habitat area and the reduction of movement among remnant populations. Decreased gene flow contributes to loss of genetic diversity, which limits the ability of local populations to respond to further environmental changes. The focus of this dissertation is on the degree to which common land uses in Sarapiquí, Costa Rica impede the movement of two common amphibian species. First, I used field experiments, including displacement trials, and a behavioral landscape ecology framework to investigate the resistance of pastures to movement of Oophaga pumilio. Results from experiments demonstrate that pastures do impede movement of O. pumilio relative to forest. Microclimatic effects on movement performance as well as limited perceptual ranges likely contribute to reduced return rates through pastures. Next, I linked local processes to landscape scale estimates of resistance. I conducted experiments to measure habitat-specific costs to movement for O. pumilio and Craugastor bransfodrii, and then used experimental results to parameterize connectivity models. Model validation indicated highest support for resistance estimates generated from responses to land-use specific microclimates for both species and to predator encounters for O. pumilio. Finally, I used abundance and experiment-derived resistance estimates to analyze the effects of prevalent land uses on population genetic structure of the two focal species. While O. pumilio did not exhibit a strong response to landscape heterogeneity and was primarily structured by distances among sites, C. bransfordii genetic variation was explained by resistance estimates from abundance and experiment data. Collectivity, this work demonstrates that common land uses can offer different levels of resistance to amphibian movements in Sarapiquí and illustrates the value of investigating local scales processes to inform interpretation of landscape-scale patterns.

Researchers and students from Dr. Rehage's lab sampling fish in the Shark River Estuary

http://digitalcommons.fiu.edu/fce_lter_photos/1281/thumbnail.jpg

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilized skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

DNA in Amphibian and Reptile Venom Permits Access to Genomes Without Specimen Sacrifice

Amphibian defensive skin secretions and reptile venoms are rich sources of bioactive peptides with potential pharmacological/pharmaceutical applications. As amphibian and reptile populations are in rapid global decline, our research
group has been developing analytical methods that permit generation of robust molecular data from non-invasive skin secretion samples and venom samples. While previously we have demonstrated that parallel proteome and venom gland
transcriptome analyses can be performed on such samples, here we report the presence of DNA that facilitates the more widely-used applications of gene sequencing, such as molecular phylogenetics, in a non-invasive manner that circumvents specimen sacrifice. From this “surrogate” tissue, we acquired partial 12S and 16S rRNA gene sequences that are presented for illustration purposes. Thus from a single sample of amphibian skin secretion and reptile venom, robust and complementary proteome, transcriptome and genome data can be generated for applications in diverse scientific disciplines.

A Combined Molecular Cloning and Mass Spectrometric Method to Identify, Characterize, and Design Frenatin Peptides from the Skin Secretion of Litoria infrafrenata

Amphibian skin secretions are unique sources of bioactive molecules, particularly bioactive peptides. In this study, the skin secretion of the white-lipped tree frog (Litoria infrafrenata) was obtained to identify peptides with putative therapeutic potential. By utilizing skin secretion-derived mRNA, a cDNA library was constructed, a frenatin gene was cloned and its encoded peptides were deduced and confirmed using RP-HPLC, MALDI-TOF and MS/MS. The deduced peptides were identified as frenatin 4.1 (GFLEKLKTGAKDFASAFVNSIKGT) and a post-translationally modified peptide, frenatin 4.2 (GFLEKLKTGAKDFASAFVNSIK.NH2). Antimicrobial activity of the peptides was assessed by determining their minimal inhibitory concentrations (MICs) using standard model microorganisms. Through studying structure–activity relationships, analogues of the two peptides were designed, resulting in synthesis of frenatin 4.1a (GFLEKLKKGAKDFASALVNSIKGT) and frenatin 4.2a (GFLLKLKLGAKLFASAFVNSIK.NH2). Both analogues exhibited improved antimicrobial activities, especially frenatin 4.2a, which displayed significant enhancement of broad spectrum antimicrobial efficiency. The peptide modifications applied in this study, may provide new ideas for the generation of leads for the design of antimicrobial peptides with therapeutic applications.

Improving interfaces for nerve repair

Autologous nerve grafts are the current gold standard for the repair of peripheral nerve injuries. However, there is a need to develop an alternative to this technique, as donor-site morbidities such as neuroma formation and permanent loss of function are a few of the limitations concerned with this technique. Artificial nerve conduits have therefore emerged as an alternative for the repair of short peripheral nerve defects of less than 30 mm, however they do not surpass autologous nerve grafts clinically. To develop a nerve conduit that supports regeneration over long nerve gaps and in large diameter nerves, researchers have focused on functionalizing of the conduits by studying the components that enhance nerve regeneration such as micro/nano-topography, growth factor delivery systems, supportive cells and extracellular matrix (ECM) proteins as well as understanding the complex biological reactions that take place during peripheral nerve regeneration. This thesis presents strategies to improve peripheral nerve interfaces to better the regenerative potential by using dorsal root ganglions (DRGs) isolated from neonatal rats as an in vitro model of nerve regeneration. The work started off by investigating the usefulness of a frog foam protein Ranaspumin-2 (Rsn2) to coat biomaterials for compatibility, this lead to the discovery of temporary cell adhesion on polydimethylsiloxane (PDMS), which was investigated as a suitable tool to derive cell-sheets for nerve repair. The influence of Rsn2 anchored to specific adhesion peptide sequences, such as isoleucine-lysine-valine-alanine-valine (IKVAV), a sequence derived from laminin proven to promote cell adhesion and neurite outgrowth, was tested as a useful means to influence nerve regeneration. This approach improves the axonal outgrowth and maintains outgrowth long term. Based on the hypothesis that combinational modulation of substrate topography, stiffness and neurotrophic support, affects axonal outgrowth in whole DRGs, dissociated DRGs were used to assess if these factors similarly act at the single cell level. Rho associated protein kinase (ROCK) and myosin II inhibitors, which affect cytoskeletal contractility, were used to influence growth cone traction forces and have shown that these factors work in combination by interfering with growth cone dynamic creating a different response in axonal outgrowth at the single cell level.

Síntese química e avaliação das propriedades antibacterianas e antiparasitárias de análogos de peptídeos antimicrobianos de anuros

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2015.

Inclusão de farinha de resíduos da indústria de filetagem de tilápias na alimentação de girinos de rã-touro (Rana Catesbeiana Shaw,1802)

The objective of this experiment was to evaluate the inclusion of meal of tilapia industrial waster in bullfrog tadpoles' diets (Rana Catesbeina Shaw, 1802). Two hundred fifty tadpoles with initial weight and length of 0,18 +/- 0,05g and 2,50 +/- 0,18 centimeters were used, respectively, they were used and distributed in 25 aquariums of 30 liter each in a complete randomized design with five treatments and five repetitions. The tadpoles were fed four times a day with diets containing 0, 5, 10 and 15% of inclusion of tilapia filleting and another diet with 0% of supplemented tilapia filleting with synthetic methionine, being supplied four times a day at ease. The experiment period was 30 days. The diets with tilapia filleting inclusion and methionine supplementation did not have influence in the final weight and length averages, feed survival and conversion of bullfrog tadpoles. It cam be used until 15% of tilapia filleting inclusion in the bullfrog tadpoles food. The synthetic methionine supplementation in diets for bullfrog tadpoles is not necessary in diets with levels over 0,471/4 of the total methionine.

Effects of rainfall on water quality in six sequentially disposed fishponds with continuous water flow

O estudo foi efetuado durante o período de chuva (dezembro-fevereiro) em seis viveiros de produção semi-intensiva de peixes, a fim de avaliar o efeito da chuva na qualidade da água de viveiros que apresentam fluxo contínuo de água, a qual é passada de um viveiro para outro sem tratamento prévio. Foram amostrados oito pontos de coleta nas saídas dos viveiros. O viveiro P1 (próximo à nascente) apresentou as menores concentrações físicas e químicas da água e as maiores no viveiro P4 (considerado um ponto crítico recebendo material alóctone proveniente de outros viveiros e do escoamento do setor de criação de rãs). A disposição seqüencial dos viveiros estudados promoveu aumento nas concentrações dos nutrientes, clorofila-a e condutividade. As chuvas características desta época do ano aumentaram o fluxo de água nos viveiros e conseqüentemente, carreando material particulado e dissolvido de um viveiro para outro e, promovendo um aumento das variáveis limnológicas em direção do P3 ao P6. Os resultados sugerem que a chuva no período de estudo afetou positivamente a qualidade da água dos viveiros estudados, porém, como os sistemas analisados estão dispostos em distribuição seqüencial e escoamento constante da água de viveiros e tanques paralelos sem tratamento prévio, cuidados devem ser averiguados para que o aumento do fluxo de água provocado pelas chuvas não tenha efeito adverso nos viveiros estudados.