Sub-lethal coral stress: detecting molecular responses of coral populations to environmental conditions over space and time.

In order for sessile organisms to survive environmental fluctuations and exposures to pollutants, molecular mechanisms (i.e. stress responses) are elicited. Previously, detrimental effects of natural and anthropogenic stressors on coral health could not be ascertained until significant physiological responses resulted in visible signs of stress (e.g. tissue necrosis, bleaching). In this study, a focused anthozoan holobiont microarray was used to detect early and sub-lethal effects of spatial and temporal environmental changes on gene expression patterns in the scleractinian coral, Montastraea cavernosa, on south Florida reefs. Although all colonies appeared healthy (i.e. no visible tissue necrosis or bleaching), corals were differentially physiologically compensating for exposure to stressors that varied over time. Corals near the Port of Miami inlet experienced significant changes in expression of stress responsive and symbiont (zooxanthella)-specific genes after periods of heavy precipitation. In contrast, coral populations did not demonstrate stress responses during periods of increased water temperature (up to 29°C). Specific acute and long-term localized responses to other stressors were also evident. A correlation between stress response genes and symbiont-specific genes was also observed, possibly indicating early processes involved in the maintenance or disruption of the coral-zooxanthella symbiosis. This is the first study to reveal spatially- and temporally-related variation in gene expression in response to different stressors of in situ coral populations, and demonstrates that microarray technology can be used to detect specific sub-lethal physiological responses to specific environmental conditions that are not visually detectable.

Rapid evolution of sex frequency and dormancy as hydroperiod adaptations.

Dormancy can serve as an adaptation to persist in variable habitats and often is coupled with sex. In cyclically parthenogenetic rotifers, an asexual phase enables rapid population growth, whereas sex results in diapausing embryos capable of tolerating desiccation. Few studies have experimentally tested whether sex-dormancy associations in temporary waters reflect evolution in response to the short hydroperiod selecting for diapause ability. Here, we demonstrate evolution of higher propensity for sex and dormancy in ephemeral rotifer cultures mimicking temporary ponds, and lower propensity in permanent cultures. Results are consistent with rapid evolution, with evolutionary changes occurring in a short timeframe (385 days, ≤ 84 generations). We also provide insight into mechanisms for rapid evolution in basal metazoans, discussing potential roles of new mutations, recombination and clonal selection.

Plasma concentrations, behavioural and physiological effects following intravenous and intramuscular detomidine in horses.

REASONS FOR PERFORMING STUDY: Detomidine hydrochloride is used to provide sedation, muscle relaxation and analgesia in horses, but a lack of information pertaining to plasma concentration has limited the ability to correlate drug concentration with effect. OBJECTIVES: To build on previous information and assess detomidine for i.v. and i.m. use in horses by simultaneously assessing plasma drug concentrations, physiological parameters and behavioural characteristics. HYPOTHESIS: Systemic effects would be seen following i.m. and i.v. detomidine administration and these effects would be positively correlated with plasma drug concentrations. METHODS: Behavioural (e.g. head position) and physiological (e.g. heart rate) responses were recorded at fixed time points from 4 min to 24 h after i.m. or i.v. detomidine (30 microg/kg bwt) administration to 8 horses. Route of administration was assigned using a balanced crossover design. Blood was sampled at predetermined time points from 0.5 min to 48 h post administration for subsequent detomidine concentration measurements using liquid chromatography-mass spectrometry. Data were summarised as mean +/- s.d. for subsequent analysis of variance for repeated measures. RESULTS: Plasma detomidine concentration peaked earlier (1.5 min vs. 1.5 h) and was significantly higher (105.4 +/- 71.6 ng/ml vs. 6.9 +/- 1.4 ng/ml) after i.v. vs. i.m. administration. Physiological and behavioural changes were of a greater magnitude and observed at earlier time points for i.v. vs. i.m. groups. For example, head position decreased from an average of 116 cm in both groups to a low value 35 +/- 23 cm from the ground 10 min following i.v. detomidine and to 64 +/- 24 cm 60 min after i.m. detomidine. Changes in heart rate followed a similar pattern; low value of 17 beats/min 10 min after i.v. administration and 29 beats/min 30 min after i.m. administration. CONCLUSIONS: Plasma drug concentration and measured effects were correlated positively and varied with route of administration following a single dose of detomidine. POTENTIAL RELEVANCE: Results support a significant influence of route of administration on desirable and undesirable drug effects that influence case management.

Development of a Rural Asthma Management Model, RAMM.

INTRODUCTION: The prevalence of asthma in Australia is increasing and places a significant cost burden on the community as well as reducing individuals' quality of life. In the late 1990s, asthma was the sixth National Health Priority in Australia and the prevalence of asthma in the Loddon Mallee region (LMR) of Victoria was approximately 1% higher than the State average. Four LMR local government areas had close to double the State average hospital admission ratios for asthma. AIM: The aim of this project was to develop a Regional Asthma Management Model (RAMM) and strategies for its implementation throughout the LMR, as a tool to implement a major health priority of both the Victorian State and Australian Commonwealth governments: to improve health outcomes for people with asthma. METHODS: A literature review was undertaken to identify best practice in asthma management for use as the basis of questions in workbooks designed to profile and compare current asthma management practice in the LMR. The workbooks were sent to all acute hospitals, community health centres and asthma educators in the LMR. The completed workbooks were returned and respondents elaborated on the workbook data at one of five subregional workshops. A survey was also undertaken to identify the range of asthma management strategies currently used by regional general practitioners (GPs) and to invite their views on ways to improve asthma management in the region. To gain consumer input into the RAMM a semi-structured group interview was held in an urban area and individual interviews were held in two rural areas in the region. A multidisciplinary reference group provided guidance to the project and a documentation design team was convened. RESULTS: Of the 19 workbooks sent to individual acute hospitals, 15 (78.9%) were completed and returned; 13 of 14 workbooks (92.8%) sent to individual community health centres were completed and returned. Fourteen of 15 asthma educators identified in the LMR were employed in the acute hospitals and community health centres that returned the workbooks; one asthma educator worked privately. Of the 215 GP surveys distributed, 38 surveys (17.6%) were returned. The majority of this small sample of GPs supported developing a uniform regional approach to asthma management based on NAC guidelines. Consumers interviewed suggested treating doctors, and/or EDs provide patients and carers with written instructions regarding acute asthma attacks and advice on management strategies for the ensuing 24-48 hours. A regional profile of asthma management practice was produced and compared with identified best practice. Gaps in practice and services were identified and responsive recommendations formulated. The National Asthma Campaign (NAC) guidelines were used as the basis for RAMM documentation, a package which consisted of a Regional Asthma Clinical Pathway and Emergency Department (ED) Package. CONCLUSION: The RAMM developed during the project provides documentation to assist best-practice asthma management by regional EDs and acute hospitals. The methodology and outcomes of the RAMM reflect the geography of the region, with multiple service providers from different locations managing a person with asthma across the primary, secondary and tertiary continuum. The RAMM methodology has the potential to be applied to other diseases and to other rural environments. Although the RAMM was designed for rural areas it could be easily adapted to suit the metropolitan environment. Implementation and evaluation of RAMM documentation is in progress.

Full life-cycle toxicity assessment using rotifer resting egg production: implications for ecological risk assessment.

The majority of standardized toxicity tests incorporate only a fraction of the test organism's life-cycle. However, in natural ecosystems, organisms may be exposed at various times during their life-cycle or throughout their life-cycle. Thus, ecotoxicological data from standardized toxicity tests is of limited ecological relevance. Existing standardized toxicity tests using the freshwater rotifer Brachionus calyciflorus utilize 24-h survival or 48-h asexual reproduction as endpoints, despite evidence that sexual reproduction is more sensitive. A 96-h B. calyciflorus resting egg toxicity test was developed and used to estimate the toxicity of pentachlorophenol (PCP) and copper. Results were compared to a variety of acute and sublethal endpoints for both toxicants. The B. calyciflorus 96-h resting egg production NOEC for PCP of 10 micrograms/l was 20 times lower than the 48-h asexual reproduction no observed effect concentration (NOEC) and 120 times lower than the 24-h acute lethal concentration 50%. The 96-h resting egg production NOEC for copper of 2.8 micrograms/l was 7 times lower than the 48-h asexual reproduction NOEC and nine times lower than the 24-h acute LC50. Resting egg production was a more sensitive indicator of toxicity than several other sublethal endpoints as well. These results indicate that partial life-cycle toxicity tests are not sufficiently sensitive to detect ecologically relevant adverse effects.

Assessing coral stress responses using molecular biomarkers of gene transcription.

We present a method for detecting rapid changes in coral gene expression at the messenger ribonucleic acid (mRNA) level. The staghorn coral Acropora cervicornis was exposed to 1 and 10 microg/L permethrin and 25 and 50 microg/L copper for 4 h. Using differential display polymerase chain reaction (PCR), mRNA associated with each toxicant exposure were reverse transcribed into complementary DNA (cDNA) fragments that were subsequently amplified and isolated. Six differentially expressed cDNA fragments were further developed into molecular probes that were used in Northern dot blots to determine the change in transcription levels of target transcripts. Changes in mRNA abundance were quantified by densitometry of chemiluminescence of digoxigenin-labeled probes hybridizing to target mRNA transcripts. The six gene probes showed varying degrees of sensitivity to the toxicants as well as specificity between toxicants. These probes were hybridized in Southern blots to genomic DNA from A. formosa sperm, which lacks zooxanthellae, to demonstrate that the genes coding for the mRNA transcripts produced are found within the coral genome. The gene probes developed in this study provide coral biologists with a new tool for coral assessment. Gene probes are sensitive, toxicant-specific biomarkers of coral stress responses with which gene sequence information can be obtained, providing a mechanism for identifying the stressor altering the gene expression.

Direct and indirect effects of sublethal toxicant exposure on population dynamics of freshwater rotifers: a modeling approach.

Ecotoxicological assessments commonly consider the direct effects of anthropogenic toxicants on the survival, growth, and reproduction of organisms. However, toxicants also may have indirect effects on aquatic communities by altering species interactions such as predation and competition. Such indirect effects are often mediated by changes in animal behavior, and contribute to uncertainty in predicting the environmental effects of toxicants. In an attempt to better understand indirect effects, a modeling approach was used to simulate the population dynamics of the predatory rotifer Asplanchna girodi and six prey species of herbivorous rotifers in response to sublethal concentrations (110, 190 or 330 mg/l) of pentachlorophenol (PCP). Simulations were performed assuming three different toxicant effect scenarios: behavioral toxicity alone, reproductive toxicity alone, or both behavioral and reproductive toxicity. Population growth rates (G(r)), maximum population densities (D(max)), and the percent of carrying capacity at D(max) (%K) were calculated for A. girodi and each prey species for each effect scenario and toxicant concentration. Similar qualitative results were obtained regardless of which prey species was exploited by A. girodi. Toxicant effects on predator and prey population densities appeared to be the dominant cause of both direct and indirect toxicant effects, and behavioral toxicity was a redundant endpoint relative to reproductive toxicity. These results suggest that assessing toxicant effects on the population growth of individual species may provide sufficient data to predict toxicant effects on species interactions as well.

The effects of fluid motion on toxicant sensitivity of the rotifer Brachionus calyciflorus.

Standardized methods for estimating the toxicity of anthropogenic compounds to aquatic organisms frequently fail to consider key elements of the test organisms' environment. Aquatic organisms exist in a fluid environment, and fluid dynamics may have an important influence on the response to toxicants. Rotifers are one of the three major groups of zooplankton and have been increasingly utilized in standardized toxicity testing. However, like other toxicity tests, assays with the species Brachionus calyciflorus are performed under static conditions in the absence of fluid motion. We investigated how fluid motion modifies pentachlorophenol (PCP) toxicity to B. calyciflorus using 24 h acute and 48 h reproductive toxicity tests. Estimates of PCP LC50s and reproduction EC50s in static conditions decreased from 738 and 1082 microg l(-1), respectively, to as low as 262 and 136 microg l(-1), respectively, in fluid motion. Flow analysis indicated that increased toxicant sensitivity can occur at ecologically relevant levels of fluid motion. Mechanistic studies indicated that fluid motion/toxicant interactions may result from the ability of fluid motion to cause shear stress, alter toxicant uptake, and/or alter the bioavailability of food. As fluid motion may have an important effect on the life histories of a wide variety of aquatic organisms, fluid motion/toxicant interactions may be an important consideration in other standard test organisms. These results raise questions about the accuracy of ecological risk assessments based on toxicity data from static conditions.

GABA, 5-HT and amino acids in the rotifers Brachionus plicatilis and Brachionus rotundiformis.

gamma-Aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) have been shown to increase the reproduction of the Brachionus plicatilis (NH3L strain). In the present study, the endogenous presence of GABA and 5-HT in the rotifers B. plicatilis (NH3L and Kamiura strains) and Brachionus rotundiformis (Langkawi strain) were confirmed by dot blot immunoassay and high-performance liquid chromatography (HPLC). HPLC showed that GABA and 5-HT concentrations in the three rotifer strains range from 71 to 188 pmol/mg and from 12 to 64 pmol/mg, respectively. A total of 33 amino acids were also detected in B. plicatilis and B. rotundiformis, with glutamic acid, serine, glycine, taurine, threonine, alanine, arginine, proline, valine and isoleucine in high concentrations relative to other amino acids.

UV-B exposure increases acute toxicity of pentachlorophenol and mercury to the rotifer Brachionus calyciflorus.

Adverse biological effects of ultraviolet-B (UV-B) radiation have been well documented for phytoplankton and zooplankton in both marine and freshwater ecosystems. However, investigations of interactions between UV-B and anthropogenic toxicants have focused primarily on the chemical interactions between UV-B and the toxicant. Here we investigate the potential for UV-B to increase the sensitivity of the rotifer Brachionus calyciflorus to either acute pentachlorophenol (PCP) or mercury toxicity, independent of UV-B effects on these toxicants. UV-B increased the toxicity of PCP and mercury to B. calyciflorus as much as five-fold, depending on duration of UV-B exposure and toxicant concentration. Reductions in the LC(50) of up to 60% were also seen for both toxicants. UV-B alone effectively eliminated B. calyciflorus reproduction and reduced ingestion by up to 90%. These results demonstrate the potential for UV-B to increase rotifer sensitivity to anthropogenic stressors independent of photochemical reactions with toxicants.

A control problem in a polluted environment.

Due to the importance of remediating polluted environments, many mathematical models have been developed to describe population-toxicant interactions. This article considers the situation where the amount of pollution in the environment is restricted to lie below a certain value M. A model is presented and viewed as a control problem in which the toxicant input into the environment is the controllable variable. An explicit formula is computed for the input function that meets certain restrictions. By analytically studying the dynamics of the population, it is possible to provide conditions on how to set M in order to guarantee survival of the population without a significant reduction of its carrying capacity. Numerical and experimental data are described that support these results.

Rapid toxicity assessment using an in vivo enzyme test for Brachionus plicatilis (Rotifera).

A 1-hr in vivo enzyme inhibition assay based on esterase activity has good potential for marine toxicity assessment. A test was developed for the rotifer Brachionus plicatilis based on the nonfluorescent substrate fluorescein diacetate (FDA), which is metabolized by esterases to a fluorescent product. Enzyme inhibition, as determined by reduced fluorescence, can be scored visually or quantified using a fluorometer. Quantification of fluorescence permits the calculation of NOEC, LOEC, chronic value, and IC20. The 1-hr esterase inhibition test has sensitivity comparable to that of 24-hr rotifer acute tests for several compounds. The toxicity of six compounds was examined using the quantified assay. The resulting IC20s were within a factor of 3 of the 24-hour LC50s. IC20 values ranged from 0.017 mg/l for tributyltin to 3.1 mg/l for zinc, with an average coefficient of variation of 17.8%. Electrophoretic analysis of rotifer homogenates suggested that a single C esterase (acetylesterase) was responsible for FDA metabolism in B. plicatilis. Several other aquatic species are capable of metabolizing FDA, including Brachionus calyciflorus, Mysidopsis bahia, Menidia beryllina, Pimephales promelas, Ceriodaphnia dubia, Daphnia pulex, Artemia salina, and Ophryotrocha sp. The esterase inhibition test is an attractive tool for assessing aquatic toxicity because of its speed, simplicity, sensitivity, and applicability to a broad range of aquatic species.

A critical body size for use of pheromones in mate location.

Pheromones have demonstrated importance in mate location in many insect species. Because chemoreception is the most universal sense, it has been assumed that pheromones also are important in aquatic organisms, including bacteria, but few have been found. The physical limits on effective strategies for organisms to come into contact for mating were modeled with assumptions appropriate for organisms less than a millimeter in size in an open aquatic environment. One sex was assumed to be motile, while the other sex was passive or devoted energy to locomotion or to diffusible pheromone production. Assuming spherical organisms, random locomotion by the second sex at the same velocity as the first sex increases the chances of contact by a factor of 4/3 over being passive; this ratio is independent of size. For detection by contact, the effectiveness of searching increases with the third power of the radius of the organisms; for detection by pheromones, search effectiveness increases with the seventh power of the radius above a critical size. Diverting energy from motility to pheromone production is not productive for organisms smaller than the critical size, which corresponds to a radius of 1.8 times the square root of the diffusion coefficient of the pheromone times the threshold concentration for detection divided by the rate of pheromone production per unit volume of organism. Thus, pheromone production is very favorable for organisms much above the critical size, which appears to be between 0.2 and 5 mm in water. On the other hand, bacteria are probably too small to use diffusable pheromones for mate location; most protozoans and rotifers may also be too small.

Acute toxicity tests using rotifers. IV. Effects of cyst age, temperature, and salinity on the sensitivity of Brachionus calyciflorus.

Several aspects of the response to toxicants using a standardized toxicity test with the freshwater rotifer Brachionus calyciflorus are described. Test animals are obtained by hatching cysts which produce animals of similar age and physiological condition. The acute toxicity of 28 compounds is described with 24-hr LC50's. The LC50's span five orders of magnitude, from silver at 0.008 mg.liter-1 to benzene at more than 1000 mg.liter-1. Control mortality in 84 tests averaged 2% with a standard deviation of 3%, indicating very consistent test sensitivity. Only once in 84 trials did a test fail because of excessive control mortality, yielding a failure rate of 1.2%. Cyst age from 0 to 18 months had no effect on the sensitivity of neonates to reference toxicants. Both high and low temperatures increased rotifer sensitivity to reference toxicants. Copper sensitivity was greater at 10, 25, and 30 degrees C compared with results at 20 degrees C. Likewise, sodium pentachlorophenol toxicity was greater at 10 and 30 degrees C compared with results at 20 degrees C. Survivorship curves at 25 degrees C of neonates under control conditions indicated that mortality begins at about 30 hr. This places a practical limit on toxicant exposure for the assay of 24 hr. B. calyciflorus cysts hatch at salinities up to 5 ppt and acute toxicity tests using pentachlorophenol at this salinity yielded LC50's about one-half those of standard freshwater. B. calyciflorus is preferred over Brachionus plicatilis for toxicity tests in salinities up to 5 ppt because it is consistently more sensitive.

Effects of copper and tributyltin on stress protein abundance in the rotifer Brachionus plicatilis.

1. Exposure of the rotifer Brachionus plicatilis to elevated temperature resulted in the synthesis of a number of proteins, including a prominent one of 58,000 Da (SP58). 2. This protein is immunologically crossreactive with the 65,000 Da heat shock protein of the moth Heliothis virescens, which is a member of a highly conserved family of mitochondrial proteins. 3. Exposure of rotifers to sublethal doses of CuSO4 leads to a 4-5-fold increase in abundance of SP58, with maximum increase occurring at a dose that is approximately 5% of the LC50 for that compound. 4. A similar response was seen with tributyl tin (TBT). Kinetics of induction were sigmoidal, with induction occurring in the range of 20-30 micrograms/l. 5. No response was observed when rotifers were exposed to aluminum chloride, mercury chloride, pentachlorophenol, sodium arsenite, sodium azide, sodium dodecyl sulfate, or zinc chloride. 6. These results indicate that changes in stress protein abundance may prove useful as a biomarker of exposure to particular toxicants.

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera).

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.