Synopsis of researcher meeting -- Bottlenose Dolphin Health & Risk Assessment Project, February 22-24, 2005

A meeting was convened on February 22-24, 2005 in Charleston, South Carolina to bring together researchers collaborating on the Bottlenose Dolphin Health and Risk Assessment (HERA) Project to review and discuss preliminary health-related findings from captured dolphins during 2003 and 2004 in the Indian River Lagoon (IRL), FL and Charleston (CHS), SC. Over 30 researchers with diverse research expertise representing government, academic and marine institutions participated in the 2-1/2 day meeting. The Bottlenose Dolphin HERA Project is a comprehensive, integrated, multi-disciplinary research program designed to assess environmental and anthropogenic stressors, as well as the health and long-term viability of Atlantic bottlenose dolphins (Tursiops truncatus). Standardized and comprehensive protocols are being used to evaluate dolphin health in the coastal ecosystems in the IRL and CHS. The Bottlenose Dolphin Health and Risk Assessment (HERA) Project was initiated in 2003 by Dr. Patricia Fair at the National Oceanic and Atmospheric Administration/National Ocean Service/Center for Coastal Environmental Health and Biomolecular Research and Dr. Gregory Bossart at the Harbor Branch Oceanographic Institution under NMFS Scientific Research Permit No. 998-1678-00 issued to Dr. Bossart. Towards this end, this study focuses on developing tools and techniques to better identify health threats to these dolphins, and to develop links to possible environmental stressors. Thus, the primary objective of the Dolphin HERA Project is to measure the overall health and as well as the potential health hazards for dolphin populations in the two sites by performing screening-level risk assessments using standardized methods. The screening-level assessment involves capture, sampling and release activities during which physical examinations are performed on dolphins and a suite of nonlethal morphologic and clinicopathologic parameters, to be used to develop indices of dolphin health, are collected. Thus far, standardized health assessments have been performed on 155 dolphins during capture-release studies conducted in Years 2003 and 2004 at the two sites. A major collaboration has been established involving numerous individuals and institutions, which provide the project with a broad assessment capability toward accomplishing the goals and objectives of this project.

Das europäisches Netzwerk zur Koordination der Altersbestimmung bei Fischen: European Fish Ageing Network (EFAN)

Financial support is going to be applied for from the European Commission in order to establish the European Fish Ageing Network (EFAN). The project, which was planned and initiated from Norway (Floedevigen) is supposed to be a concerted action from 14 European Countries involving about 35 institutions. The primary aim of the network is to coordinate the research in age reading, especially the improvement of data bases for reference material, the transformation of research (e.g. daily ring formation in otoliths) to each interested reader. Moreover, the applied financial support is supposed to be preliminary spent for travel of researcher and technicians to other institutes where the same fish species are aged. Reference material is supposed to be sent to the institutes for check-reading. Specific workshops will be held in cases where heterogeneous results occur from check-reading the reference material.

The microbiological quality of water: the nature of the problem

Improvements in methods for the detection and enumeration of microbes in water, particularly the application of techniques of molecular biology, have highlighted shortcomings in the ”standard methods” for assessing water quality. Higher expectations from the consumer and increased publicity associated with pollution incidents can lead to an uncoupling of the cycle which links methodological development with standard-setting and legislation. The new methodology has also highlighted problems within the water cycle, related to the introduction, growth and metabolism of microbes. A greater understanding of the true diversity of the microbial community and the ability to transmit genetic information within aquatic systems ensures that the subject of this symposium and volume provides an ideal forum to discuss the problems encountered by both researcher and practitioner.

A computer software package for assessing and managing risks posed by experiments with genetically modified fish and shellfish

Assessment and management of risk is needed for sustainable use of genetically modified aquatic organisms (aquatic GMOs). A computer software package for safely conducting research with genetically modified fish and shellfish is described. By answering a series of questions about the organism and the accessible aquatic ecosystem, a researcher or oversight authority can either identify specific risks or conclude that there is a specific reason for safety of the experiment. Risk assessment protocols with examples involving transgenic coho salmon, triploid grass carp and hybrid tilapia are described. In case a specific risk is identified, the user is led to consider risk management measures, involving culture methods, facilities design and operations management, to minimize the risk. Key features of the software are its user-friendly organization; easy access to explanatory text, literature citations and glossary; and automated completion of a worksheet. Documented completion of the Performance Standards can facilitate approval of a well designed experiment by oversight authorities.

Quantification of drag and lift imposed by pop-up satellite archival tags and estimation of the metabolic cost to cownose rays (Rhinoptera bonasus)

The recent development of the pop-up satellite archival tag (PSAT) has allowed the collection of information on a tagged animal, such as geolocation, pressure (depth), and ambient water temperature. The success of early studies, where PSATs were used on pelagic fishes, has spurred increasing interest in the use of these tags on a large variety of species and age groups. However, some species and age groups may not be suitable candidates for carrying a PSAT because of the relatively large size of the tag and the consequent energy cost to the study animal. We examined potential energetic costs to carrying a tag for the cownose ray (Rhinoptera bonasus). Two forces act on an animal tagged with a PSAT: lift from the PSATs buoyancy and drag as the tag is moved through the water column. In a freshwater flume, a spring scale measured the total force exerted by a PSAT at flume velocities from 0.00 to 0.60 m/s. By measuring the angle of deflection of the PSAT at each velocity, we separated total force into its constituent forces — lift and drag. The power required to carry a PSAT horizontally through the water was then calculated from the drag force and velocity. Using published metabolic rates, we calculated the power for a ray of a given size to swim at a specified velocity (i.e., its swimming power). For each velocity, the power required to carry a PSAT was compared to the swimming power expressed as a percentage, %TAX (Tag Altered eXertion). A %TAX greater than 5% was felt to be energetically significant. Our analysis indicated that a ray larger than 14.8 kg can carry a PSAT without exceeding this criterion. This method of estimating swimming power can be applied to other species and would allow a researcher to decide the suitability of a given study animal for tagging with a PSAT.

Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .