Barney J. Venables

A non-invasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies:

We have compared two methods for collecting earthworm leukocytes (coelomocytes) with respect to cell yield, viability and behaviour in immunoassays. Non-invasive extrusion was more efficient than puncturing the coelomic cavity. Extrusion does not produce trauma to earthworms maintained under long term laboratory conditions. Neither technique modified immune assays, as determined by erythrocyte and secretory rosette formation, and phagocytosis, since all 3 were functionally equivalent. After an initial extrusion, sequential leukocyte collections by extrusion are possible at intervals of 6 weeks without affecting total and differential cell counts and rosette formation.

Signaling by small metabolites in systemic acquired resistance

Plants can retain the memory of a prior encounter with a pest. This memory confers upon a plant the ability to subsequently activate defenses more robustly when challenged by a pest. In plants that have retained the memory of a prior, localized, foliar infection by a pathogen, the pathogen-free distal organs develop immunity against subsequent infections by a broad-spectrum of pathogens. The long-term immunity conferred by this mechanism, which is termed systemic acquired resistance (SAR), is inheritable over a few generations. Signaling mediated by the phenolic metabolite salicylic acid (SA) is critical for the manifestation of SAR. Recent studies have described the involvement of additional small metabolites in SAR signaling, including methyl salicylate, the abietane diterpenoid dehydroabietinal, the lysine catabolite pipecolic acid, a glycerol-3-phosphate-dependent factor and the dicarboxylic acid azelaic acid. Many of these metabolites can be systemically transported through the plant and probably facilitate communication by the primary infected tissue with the distal tissues, which is essential for the activation of SAR. Some of these metabolites have been implicated in the SAR-associated rapid activation of defenses in response to subsequent exposure to the pathogen, a mechanism termed priming. Here, we summarize the role of these signaling metabolites in SAR, and the relationship between them and SA signaling in SAR.

Manipulation of Arabidopsis fatty acid amide hydrolase expression modifies plant growth and sensitivity to N-acylethanolamines.

In vertebrates, the endocannabinoid signaling pathway is an important lipid regulatory pathway that modulates a variety of physiological and behavioral processes. N-Acylethanolamines (NAEs) comprise a group of fatty acid derivatives that function within this pathway, and their signaling activity is terminated by an enzyme called fatty acid amide hydrolase (FAAH), which hydrolyzes NAEs to ethanolamine and their corresponding free fatty acids. Bioinformatic approaches led to the identification of plant homologues of FAAH that are capable of hydrolyzing NAEs in vitro. To better understand the role of NAEs in plants, we identified T-DNA knockouts to Arabidopsis FAAH (AtFAAH; At5g64440) and generated plants overexpressing AtFAAH. Here we show that seeds of AtFAAH knockouts had elevated levels of endogenous NAEs, and seedling growth was hypersensitive to exogenously applied NAE. On the other hand, seeds and seedlings of AtFAAH overexpressors had lower endogenous NAE content, and seedlings were less sensitive to exogenous NAE. Moreover, AtFAAH overexpressors displayed enhanced seedling growth and increased cell size. AtFAAH expression and FAAH catalytic activity increased during seed germination and seedling growth, consistent with the timing of NAE depletion during seedling establishment. Collectively, our results show that AtFAAH is one, but not the only, modulator of endogenous NAE levels in plants, and that NAE depletion likely participates in the regulation of plant growth.

Triclosan in a north Texas wastewater treatment plant and the influent and effluent of an experimental constructed wetland.

The antimicrobial triclosan was analyzed in unfiltered samples from influent, effluent, and receiving stream and before and after a pilot-scale constructed wetland at a North Texas municipal wastewater treatment plant. Triclosan concentrations were reduced by 97 to 99% by the activated sludge treatment plant. Effluent concentrations were further reduced by passage through the constructed wetland, but receiving stream concentrations were not statistically significantly different from effluent concentrations. Effluent concentrations of triclosan were seasonal with highest concentrations occurring during the summer months. The effluent-dominated receiving stream maximum concentrations during summer months were below reported algal no-observed-effect concentrations based on biomass and growth rate but exceeded concentrations reported to cause shifts in algal community structure.

Bioconcentration of ibuprofen in fathead minnow (Pimephales promelas) and channel catfish (Ictalurus punctatus)

Pharmaceutical products and their metabolites are being widely detected in aquatic environments and there is a growing interest in assessing potential risks of these substances to fish and other non-target species. Ibuprofen is one of the most commonly used analgesic drugs and no peer-reviewed laboratory studies have evaluated the tissue specific bioconcentration of ibuprofen in fish. In the current study, fathead minnow (Pimephales promelas) were exposed to 250 μg L(-1) ibuprofen for 28 d followed by a 14 d depuration phase. In a minimized bioconcentration test design, channel catfish (Ictalurus punctatus) were exposed to 250 μg L(-1) for a week and allowed to depurate for 7 d. Tissues were collected during uptake and depuration phases of each test and the corresponding proportional and kinetic bioconcentration factors (BCFs) were estimated. The results indicated that the BCF levels were very low (0.08-1.4) implying the lack of bioconcentration potential for ibuprofen in the two species. The highest accumulation of ibuprofen was observed in the catfish plasma as opposed to individual tissues. The minimized test design yielded similar bioconcentration results as those of the standard test and has potential for its use in screening approaches for pharmaceuticals and other classes of chemicals.

N-Acylethanolamine Metabolism Interacts with Abscisic Acid Signaling in Arabidopsis thaliana Seedlings

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. In plants, NAEs are generally elevated in desiccated seeds, suggesting that they may play a role in seed physiology. NAE and abscisic acid (ABA) levels were depleted during seed germination, and both metabolites inhibited the growth of Arabidopsis thaliana seedlings within a similar developmental window. Combined application of low levels of ABA and NAE produced a more dramatic reduction in germination and growth than either compound alone. Transcript profiling and gene expression studies in NAE-treated seedlings revealed elevated transcripts for a number of ABA-responsive genes and genes typically enriched in desiccated seeds. The levels of ABI3 transcripts were inversely associated with NAE-modulated growth. Overexpression of the Arabidopsis NAE degrading enzyme fatty acid amide hydrolase resulted in seedlings that were hypersensitive to ABA, whereas the ABA-insensitive mutants, abi1-1, abi2-1, and abi3-1, exhibited reduced sensitivity to NAE. Collectively, our data indicate that an intact ABA signaling pathway is required for NAE action and that NAE may intersect the ABA pathway downstream from ABA. We propose that NAE metabolism interacts with ABA in the negative regulation of seedling development and that normal seedling establishment depends on the reduction of the endogenous levels of both metabolites.

Comparative toxicity of polychlorinated biphenyls to earthworms Eisenia foetida and Lumbricus terrestris

Effects of polychlorinated biphenyls (PCB; Aroclor 1254) in the manure worm, Eisenia foetida, on survival (LC50/LD50), and ability of coelomic leukocytes (also called coelomocytes) to form secretory rosettes (SR) and erythrocyte rosettes (ER) with, and to phagocytose antigenic rabbit red blood cells were determined and compared with those published for the earthworm, Lumbricus terrestris. Using a 5-day filter paper contact exposure protocol, LC50 and LD50 were 30.4 microg cm(-2) and 4500 microg g(-1) dry mass, respectively. Nominal PCB exposure concentrations of 5.0 and 10.0 microg cm(-2) resulted in tissue levels of 1400 and 2900 microg g(-1) dry mass. These body burdens resulted in significant reduction in SR formation by 18 and 52%, respectively. ER formation and phagocytosis were reduced 52 and 61%, respectively, only at the higher tissue concentration. Compared to L. terrestris, E. foetida: (1) accumulated considerably more PCB at each exposure concentration; (2) showed lower LC50, but higher LD50; and (3) exhibited effects on coelmocytes only at tissue PCB concentrations that caused some mortality. In terms of lethality and immunomodulation of SR, ER and phagocytosis, E. foetida appears to be more resistant to PCB than L. terrestris.

Temporal and spatial variability in the estrogenicity of a municipal wastewater effluent.

The estrogenicity of a municipal wastewater effluent was monitored using the vitellogenin biomarker in adult male fathead minnows (Pimephales promelas). The variability in the expression of vitellogenin was evident among the monitoring periods. Significant (alpha< or =0.05) increases in plasma vitellogenin concentrations were detected in March and December, but not in August or June. Additionally, the magnitude of expression was variable. Variability in the spatial scale was also evident during the March and June exposure months. Concurrent exposures in both the creek receiving the effluent from a wastewater treatment plant and an experimental wetland showed estrogenicity to be different with distance from the respective effluent inflow sites. March exposures showed estrogenicity to be somewhat persistent in the receiving creek (>600 m), but to decrease rapidly within the experimental wetland (<40 m). Results are discussed relative to the monitoring season, to the spatial distribution of the response in both receiving systems, and to possible causative factors contributing to the effluent estrogenicity.

Sperm count in earthworms (Lumbricus terrestris) as a biomarker for environmental toxicology: Effects of cadmium and chlordane

Earthworms, Lumbricus terrestris, exposed in artificial soil to sublethal concentrations of technical chlordane (6.25, 12.5, and 25 ppm) and cadmium nitrate (100, 200, and 300 ppm) exhibited significant reduction in spermatozoa from testes and seminal vesicles. The onset time of reduction varied with exposure concentration, but absolute depression in sperm count was independent of exposure concentration or exposure duration after reduction was first manifested, demonstrating a threshold effect. Earthworm sperm counts show potential as a rapid-measurement endpoint biomarker for measuring sublethal effects of chemical pollutants on reproduction.

Overexpression of a fatty acid amide hydrolase compromises innate immunity in Arabidopsis

N-acylethanolamines are a group of lipid mediators that accumulate under a variety of neurological and pathological conditions in mammals. N-acylethanolamine signaling is terminated by the action of diverse hydrolases, among which fatty acid amide hydrolase (FAAH) has been well characterized. Here, we show that transgenic Arabidopsis lines overexpressing an AtFAAH are more susceptible to the bacterial pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. AtFAAH overexpressors also were highly susceptible to non-host pathogens P. syringae pv. syringae and P. syringae pv. tabaci. AtFAAH overexpressors had lower amounts of jasmonic acid, abscisic acid and both free and conjugated salicylic acid (SA), compared with the wild-type. Gene expression studies revealed that transcripts of a number of plant defense genes, as well as genes involved in SA biosynthesis and signaling, were lower in AtFAAH overexpressors than wild-type plants. Our data suggest that FAAH overexpression alters phytohormone accumulation and signaling which in turn compromises innate immunity to bacterial pathogens.