Isodomoic acid C, an unusual amnesic shellfish poisoning toxin from Pseudo-nitzschia australis.

An unusual isomer of domoic acid (1), isodomoic acid C (2), has been found in New Zealand shellfish contaminated by amnesic shellfish poisoning (ASP) toxins and was shown to be produced by a local strain of the pennate diatom Pseudo-nitzschia australis. A bulk culture of this strain was used to isolate 2. The structure was determined from spectroscopic data and was shown to correspond to that of 2 from a Japanese red seaweed, the only other reported occurrence of this compound. The affinity of 2 for GluR6 glutamate receptors was 240-fold lower than for 1, indicating low neurotoxic potential.

Method for detecting classes of microcystins by combination of protein phosphatase inhibition assay and ELISA: comparison with LC-MS.

Depending on the class of microcystin the protein phosphatase inhibition assay shows different sensitivities to different classes of toxin. We have determined that the IC50 values obtained from dose-response curves for the inhibition of the enzyme by micro-cystin LR, nodularin, YR, and RR were 2.2, 1.8, 9 and 175 nM, respectively. When equimolar amounts of these toxins were determined by the ELISA assay with microcystin LR as the standard, the assay showed equivalence in toxin responses. However, when the toxins were determined by the protein phosphatase inhibition assay using microcystin LR as the standard, the ratios of the values determined by PP-2A to ELISA decreased in the order: nodularin (2.23) microcystin LR (1.1)> microcystin YR (0.63)> microcystin RR (0.06). When the ratios for each standard were plotted against the IC50 values, the log-log plot was negative linear, and the lowest value for the IC50 corresponded with the lowest ratio. The differential sensitivity of the PP-2A assay to the various standards was used to establish an indicative toxicity ranking (ITR) where a ranking of 1 (the highest) was assigned to ratios of > or = 0.8 or greater, and 3 (the lowest) to values < or = 0.2. The three ranking classes corresponded to toxin equivalence represented by the four standards. The new method allows not only the determination of microcystin toxins in terms of stoichiometry (ELISA) but also in terms of indicative toxicity. The method can be performed using the same instrument (e.g. multiwell fluorimeter with absorbance capability) and offers an advantage to methods presently used to determine microcystins (e.g. ELISA or LC-MS). The former has the propensity to overestimate toxicity because it measures equivalence to microcystin LR and is a stoichiometric measurement and the latter has the disadvantage in that relatively few of the microcystins that occur naturally are available as standards. The new method was applied to the analysis of sample from lakes and streams from temperate locations and to extracts of cyanobacterial mats from ponds and streams in cold temperature locations.

Influences of pond geochemistry, temperature, and freeze-thaw on terminal anaerobic processes occurring in sediments of six ponds of the McMurdo Ice Shelf, near Bratina Island, Antarctica.

The effects of freeze-thaw, freezing and sediment geochemistry on terminal anaerobic processes occurring in sediments taken from below cyanobacterial mats in meltwater ponds of the McMurdo Ice Shelf in Antarctica were investigated. Depending on the geochemical and physical status of the sediments (i.e., frozen or thawed), as well as passage of sediment through a freeze-thaw cycle, terminal carbon and electron flow shifted in which the proportions of hydrogen and acetate utilized for methanogenesis and sulfate reduction changed. Thus, in low-sulfate (or chloride) sediment which was thawed and incubated at 4 degrees C, total carbon and electron flow were mediated by acetate-driven sulfate reduction and H(2)-driven methanogenesis. When the same sediments were incubated frozen, both methanogenesis and sulfate reduction decreased. However, under these conditions methanogenesis was favored over sulfate reduction, and carbon flow from acetate to methane increased relative to sulfate reduction; >70% of methane was contributed by acetate, and more than 80% of acetate was oxidized by pathways not coupled to sulfate reduction. In high-sulfate pond sediments, sulfate reduction was a major process mediating terminal carbon and electron flow in both unfrozen and frozen incubations. However, as with low-sulfate sediments, acetate oxidation became uncoupled from sulfate reduction with freezing. Geochemical and temperature effects could be expressed by linear models in which the log (methanogenesis to sulfate reduction) was negative log linear with respect to either temperature or the log of the sulfate (or chloride) concentration. From these relationships it was possible to predict the ratio for a given temperature (low-sulfate sediments) or sulfate (chloride) concentration. Small transitory changes, such as elevated sulfate reduction coupled to increased acetate turnover, resulted from application of a freeze-thaw cycle to low-salinity pond sediments. The results demonstrate how ecophysiological processes may change in anaerobic systems under extreme conditions (e.g., freezing) and provide new insights into microbial events occurring under these conditions.

Hindgut fermentation in three species of marine herbivorous fish.

Symbioses with gut microorganisms provides a means by which terrestrial herbivores are able to obtain energy. These microorganisms ferment cell wall materials of plants to short-chain fatty acids (SCFA), which are then absorbed and used by the host animal. Many marine herbivorous fishes contain SCFA (predominantly acetate) in their hindgut, indicative of gut microbial activity, but rates of SCFA production have not been measured. Such information is an important prerequisite to understanding the contribution that gut microorganisms make in satisfying the energy needs of the fish. We have estimated the rates of acetate production in the gut of three species of temperate marine herbivorous fish from northeastern New Zealand: Kyphosus sydneyanus (family Kyphosidae), Odax pullus (family Odacidae), and Aplodactylus arctidens (family Aplodactylidae). Ex vivo preparations of freshly caught fish were maintained with their respiratory and circulatory systems intact, radiolabeled acetate was injected into ligated hindgut sections, and gut fluid was sampled at 20-min intervals for 2 h. Ranges for acetate turnover in the hindguts of the studied species were determined from the slope of plots as the log of the specific radioactivity of acetate versus time and pool size, expressed on a nanomole per milliliter per minute basis. Values were 450 to 570 (K. sydneyanus), 373 to 551 (O. pullus), and 130 to 312 (A. arctidens). These rates are comparable to those found in the guts of herbivorous reptiles and mammals. To determine the contribution of metabolic pathways to the fate of acetate, rates of sulfate reduction and methanogenesis were measured in the fore-, mid-, and hindgut sections of the three fish species. Both rates increased from the distal to proximal end of the hindgut, where sulfate reduction accounted for only a small proportion (<5%) of acetate methyl group transformed to CO(2), and exceeded methanogenesis from acetate by >50-fold. When gut size was taken into account, acetate uptake from the hindgut of the fish species, determined on a millimole per day per kilogram of body weight basis, was 70 (K. sydneyanus), 18 (O. pullus), and 10 (A. arctidens).