Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.

Endocrinology is a useful tool for conservation biologists and animal managers, and measuring glucocorticoids can help understand biological mechanisms associated with species decline and animal welfare. The current study describes the development and optimization of a glucocorticoid enzyme immunoassay (EIA) to non-invasively assess adrenal activity in a variety of taxa. The antiserum (CJM006) was raised in rabbits to a corticosterone-3-CMO-BSA immunogen and used in a standard competitive EIA system. However, the EIA initially produced results with unacceptably high inter-assay variation, attributed to consistent patterns observed within the optical density of developing plates. To determine the cause of this variability, a number of factors were examined using synthetic corticosterone standard and endogenous faecal extract, including: plate type (Nunc MaxiSorp® II versus Immulon IB plates); the use of non-specific secondary antibody; type (artificial versus natural) and presence (light versus dark) of light during incubation; plate loading temperature (4°C versus room temperature); and substrate reagent temperature (4°C versus room temperature). Results indicated that variability was associated with plate location effects, which were not initially detected because control samples were always run in the same positions across plates. Light and temperature were the two major factors that affected EIA reliability. For this assay, the standard protocol required slight modification, with the optimal protocol using Nunc MaxiSorp® plates, room temperature substrate reagents and dark incubation conditions. Following optimization, this EIA was then validated biochemically for 38 species, through parallel displacement curves and interference assessment tests of faecal and urine samples. Additionally, biological validation was performed opportunistically in a subset of species, with use of this EIA demonstrating significant elevations in faecal glucocorticoid metabolites following potentially challenging events. In summary, this glucocorticoid EIA cross-reacts with excreted glucocorticoid metabolites across a wide range of taxa, including ungulates, primates, felids, birds, rodents and amphibians. We conclude that when used with optimal reagent and incubation conditions, this EIA will be useful for non-invasive monitoring of adrenal activity in a wide range of wildlife species.

Assessment and management of risk to wildlife from cadmium.

Cadmium, a nonessential heavy metal that comes from natural and anthropogenic sources, is a teratogen, carcinogen, and a possible mutagen. Assessment of potential risk from cadmium requires understanding environmental exposure, mainly from ingestion, although there is some local exposure through inhalation. Chronic exposure is more problematic than acute exposure for wildlife. There is evidence for bioaccumulation, particularly in freshwater organisms, but evidence for biomagnification up the food chain is inconsistent; in some bird studies, cadmium levels were higher in species that are higher on the food chain than those that are lower. Some freshwater and marine invertebrates are more adversely affected by cadmium exposure than are birds and mammals. There is very little experimental laboratory research on the effects of cadmium in amphibians, birds and reptiles, and almost no data from studies of wildlife in nature. Managing the risk from cadmium to wildlife involves assessment (including ecological risk assessment), biomonitoring, setting benchmarks of effects, regulations and enforcement, and source reduction.

NMR spectroscopic assessment of the structure and dynamic properties of an amphibian antimicrobial peptide (Gaegurin 4) bound to SDS micelles.

The structure and dynamics of a 37-residue antimicrobial peptide gaegurin 4 (GGN4) isolated from the skin of the native Korean frog, Rana rugosa, was determined in SDS micelles by NMR spectroscopy. The solution structure of the peptide in SDS micelles was determined from 352 NOE-derived distance constraints and 22 backbone torsion angle constraints. Dynamic properties for the amide backbone were characterized by (1)H-(15)N heteronuclear NOE experiments. The structural study revealed two amphipathic helices spanning residues 2-10 and 16-32 and that the helices were connected by a flexible loop. An intraresidue disulfide bridge was formed between residues Cys31 and Cys37 near the C-terminus. The loop region (11-15) connecting the two helices are were slightly more flexible than these helices themselves. From the fact that since there is no contact NOEs between two helices, it is implied that the GGN4 peptide shows an independent motion of both helices which has an angle of about 60 degrees -120 degrees from each other.

Urea and amphibian water economy.

Accumulation of urea in the body fluids enables some amphibians to tolerate high ambient salinities (Bufo viridis, Xenopus laevis, Rana cancrivora, Ambystoma tigrinum, Batrachoseps spp.) or to estivate in soil with low water potentials (Scaphiopus spp.). These species are assumed not only to accumulate urea produced in the normal metabolism, but to synthesize urea in response to water shortage. Re-examination of the data did not support the view of an osmoregulatory urea synthesis. Increased urea synthesis on exposure to high salinities in X. laevis, R. cancrivora and Batrachoseps spp. seemed to reflect reactions to an adverse environment. It is suggested that in amphibians, solute concentration in the plasma and rate of excretion of urea are coordinated so that at a certain plasma concentration, urea is excreted at the same rate at which it is produced. The higher the level of urea in the body fluids at balance between production and excretion, the higher the tolerance of the species of low external water potentials. The mechanisms that integrate the relationship between plasma solute concentration and handling of urea by the kidneys are not known.

Distribution of vasotocin- and mesotocin-like immunoreactivities in the brain of typhlonectes compressicauda (Amphibia, gymnophiona): further assessment of primitive and derived traits of amphibian neuropeptidergic systems.

To further assess primitive and derived conditions, we have studied the vasotocinergic (AVT) and mesotocinergic (MST) systems by immmunohistochemistry in the brain of Typhlonectes compressicauda. This species belongs to a separate order of amphibians which differs in several morphological and behavioral aspects from anurans and urodeles which have been studied previously. Nevertheless, the vasotocinergic and mesotocinergic systems of T. compressicauda are largely comparable to those of other amphibians. Apart from a well-developed hypothalamo-hypophyseal system, extrahypothalamic AVT-and MST-immunoreactive groups of cells and extensive networks of fibers were found. A major difference, however, is that neuropeptidergic cells in the caudal hypothalamus and the midbrain tegmentum of T. compressicauda contain MST, whereas those in corresponding locations contain AVT in anurans and urodeles. This suggests that certain neuropeptidergic cell groups in the gymnophionan brain have switched from AVT to MST gene expression, and, thereby, offers a new view on the functional significance of these neuropeptidergic systems.