Revision of Litoria rothii (Anura: Pelodryadidae) from northern Australia.

Litoria rothii is a widespread pelodryadid frog with a charismatic laughing advertisement call, distributed across the Australian Monsoon Tropics and southern New Guinea. Given its large distribution spanning well-known biogeographic barriers, variation in male advertisement calls and the prevalence of unresolved species complexes in the Australian frog fauna, we examine the genetic, morphological and acoustic diversity in the species from across its range. Our analyses reveal the presence of a previously unrecognised species in western parts of the range of L. rothii sensu lato, which we describe herein as a new species. Litoria ridibunda sp. nov. is distinguished from L. rothii on the basis of paraphyly of nuclear gene trees with L. everetti from Indonesia, colour patterns on the posterior thigh and male advertisement calls. Compared to L. rothii, the new species has a less contrasting pattern on the posterior thigh and a male advertisement call with a greater number of notes per call and a greater call duration. In particular, the magnitude of call differences between the species is highest where the ranges of the two species are in proximity in north-western Queensland. Our study further emphasises the undiagnosed diversity that remains in Australian frogs, even in relatively large, charismatic, frequently encountered species that often share human dwellings.

Two new frog species from the Litoria rubella species group from eastern Australia.

The bleating tree frog (Litoria dentata) is one of the more prominent pelodryadid frogs of eastern Australia by virtue of its extremely loud, piercing, male advertisement call. A member of the Litoria rubella species group, L. dentata has a broad latitudinal distribution and is widespread from coastal and subcoastal lowlands through to montane areas. A recent mitochondrial DNA analysis showed a deep phylogeographic break between populations of L. dentata on the mid-north coast of New South Wales. Here we extended the mitochondrial survey with more geographically comprehensive sampling and tested the systematic implications of our findings with nuclear genome wide single-nucleotide polymorphism, morphological and male advertisement call datasets. While similar in appearance and in male advertisement call, our integrative analysis demonstrates the presence of three species which replace each other in a north-south series. We redescribe Litoria dentata, which is restricted to coastal north-eastern New South Wales, and formally describe Litoria balatus sp. nov., from south-eastern Queensland, and Litoria quiritatus sp. nov., from the mid-coast of New South Wales to north-eastern Victoria.

Minimising exposure of amphibians to pathogens during field studies.

Many of the recent global amphibian mass mortalities, declines and extinctions have been attributed to the emerging infectious disease chytridiomycosis. There have been mass mortalities due to ranaviral disease but no major declines or extinctions. Controlling the transmission and spread of disease is of utmost importance, especially where there is the potential for human involvement. We have reviewed current hygiene guidelines for working with wild frogs, identified potential flaws and recommended those most suitable and effective for the field environment. Our within-site hygiene measures aim to reduce the risk of transmission among individuals. These measures encompass the capture, handling and holding of amphibians, skin disinfection before and after invasive procedures, marking frogs, sealing open wounds and treatment of accessory equipment. Our between-site hygiene measures aim to mitigate the risk of pathogen spread among populations. We have designed a risk calculator to help simplify and standardise the decision-making process for determining the level of risk and appropriate risk mitigation strategies to reduce the risk of increasing pathogen spread above background levels. Calculation of an overall risk score for pathogen spread takes into account the prior activity of field workers, the proposed activity, remoteness of the site, presence of known pathogens and the consequences of increased pathogen spread for amphibians in a given area.

Processing, assembly and localization of a Bacillus anthracis spore protein.

All Bacillus spores are encased in macromolecular shells. One of these is a proteinacious shell called the coat that, in Bacillus subtilis, provides critical protective functions. The Bacillus anthracis spore is the infectious particle for the disease anthrax. Therefore, the coat is of particular interest because it may provide essential protective functions required for the appearance of anthrax. Here, we analyse a protein component of the spore outer layers that was previously designated BxpA. Our data indicate that a significant amount of BxpA is located below the spore coat and associated with the cortex. By SDS-PAGE, BxpA migrates as a 9 kDa species when extracted from Sterne strain spores, and as 11 and 14 kDa species from Ames strain spores, even though it has predicted masses of 27 and 29 kDa, respectively, in these two strains. We investigated the possibility that BxpA is subject to post-translational processing as previously suggested. In B. subtilis, a subset of coat proteins is proteolysed or cross-linked by the spore proteins YabG or Tgl, respectively. To investigate the possibility that similar processing occurs in B. anthracis, we generated mutations in the yabG or tgl genes in the Sterne and Ames strains and analysed the consequences for BxpA assembly by SDS-PAGE. We found that in a tgl mutant of B. anthracis, the apparent mass of BxpA increased. This is consistent with the possibility that Tgl directs the cross-linking of BxpA into a form that normally does not enter the gel. Unexpectedly, the apparent mass of BxpA also increased in a yabG mutant, suggesting a relatively complex role for proteolysis in spore protein maturation in B. anthracis. These data reveal a previously unobserved event in spore protein maturation in B. anthracis. We speculate that proteolysis and cross-linking are ubiquitous spore assembly mechanisms throughout the genus Bacillus.

Effect of season and temperature on mortality in amphibians due to chytridiomycosis.

OBJECTIVE: To investigate the distribution and incidence of chytridiomycosis in eastern Australian frogs and to examine the effects of temperature on this disease. DESIGN: A pathological survey and a transmission experiment were conducted. PROCEDURE: Diagnostic pathology examinations were performed on free-living and captive, ill and dead amphibians collected opportunistically from eastern Australia between October 1993 and December 2000. We conducted a transmission experiment in the laboratory to investigate the effects of temperature: eight great barred frogs (Mixophyes fasciolatus) exposed to zoospores of Batrachochytrium dendrobatidis and six unexposed frogs were housed individually in each of three rooms held at 17 degrees C, 23 degrees C and 27 degrees C. RESULTS: Chytridiomycosis was the cause of death or morbidity for 133 (55.2%) of 241 free-living amphibians and for 66 (58.4%) of 113 captive amphibians. This disease occurred in 34 amphibian species, was widespread around the eastern seaboard of Australia and affected amphibians in a variety of habitats at high and low altitudes on or between the Great Dividing Range and the coast. The incidence of chytridiomycosis was higher in winter, with 53% of wild frogs from Queensland and New South Wales dying in July and August. Other diseases were much less common and were detected mostly in spring and summer. In experimental infections, lower temperatures enhanced the pathogenicity of B. dendrobatidis in M. fasciolatus. All 16 frogs exposed to B. dendrobatidis at 17 degrees C and 23 degrees C died, whereas 4 of 8 frogs exposed at 27 degrees C survived. However, the time until death for the frogs that died at 27 degrees C was shorter than at the lower temperatures. Infections in survivors were eliminated by 98 days. CONCLUSION: Chytridiomycosis is a major cause of mortality in free-living and captive amphibians in Australia and mortality rate increases at lower temperatures.

Enzymatic autocatalysis of botulinum A neurotoxin light chain.

Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds.

Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America.

Epidermal changes caused by a chytridiomycete fungus (Chytridiomycota; Chytridiales) were found in sick and dead adult anurans collected from montane rain forests in Queensland (Australia) and Panama during mass mortality events associated with significant population declines. We also have found this new disease associated with morbidity and mortality in wild and captive anurans from additional locations in Australia and Central America. This is the first report of parasitism of a vertebrate by a member of the phylum Chytridiomycota. Experimental data support the conclusion that cutaneous chytridiomycosis is a fatal disease of anurans, and we hypothesize that it is the proximate cause of these recent amphibian declines.

Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture.

A substantial proportion of bacteria from five Alexandrium cultures originally isolated from various countries produced sodium channel blocking (SCB) toxins, as ascertained by mouse neuroblastoma assay. The quantities of SCB toxins produced by bacteria and dinoflagellates were noted, and the limitations in comparing the toxicities of these two organisms are discussed. The chemical nature of the SCB toxins in selected bacterial isolates was determined as paralytic shellfish toxins by pre- and postcolumn high-performance liquid chromatography, capillary electrophoresis-mass spectrometry, and enzyme immunoassay.

Urinary elimination of saxitoxin after intravenous injection.

Paralytic shellfish poisoning is a serious public health concern throughout the world. An analytical method with diagnostic potential was used to isolate and measure saxitoxin, the most potent and studied paralytic shellfish poisoning toxin, in the urine of rats injected i.v. with sublethal doses (2 micrograms/kg) of saxitoxin. Urine was collected at intervals between 4 and 144 hr after injection. Saxitoxin was isolated from urine with an ion-exchange procedure, identified, and measured with a precolumn-oxidation-HPLC procedure coupled with fluorescence detection. The identity of oxidized saxitoxin was confirmed with electrospray ionization mass spectrometry. Four hours after injection, approximately 19% of the injected saxitoxin dose was excreted. By 24 hr, approximately 58% of the administered dose was excreted. Average total urinary excretion of administered saxitoxin was approximately 68% for the full study period. These results demonstrate that small quantities of unmetabolized saxitoxin can be detected in rat urine up to 144 hr after i.v. administration, and that the analytical method may have diagnostic potential for saxitoxin intoxication and paralytic shellfish poisoning.

Fumonisin B1 analysis with capillary electrophoresis-electrospray ionization mass spectrometry.

Fumonisin B1, a mycotoxin produced by a common fungal contaminant of corn, Fusarium moniliforme, was analyzed by capillary electrophoresis-electrospray ionization mass spectrometry. System performance was maximal with uncoated columns. System efficiencies of approximately 44,000 plates/m and reproducible analysis times of about 13 min were obtained. System efficiency with methyl-coated columns was approximately 24,000 plates/m. Reproducible analysis times of about 3.5 min were obtained with these columns. With uncoated columns, the concentration limit of detection was 156 ppb with a s/n ratio of approximately 10. The estimated injected mass at 156 ppb was 1.1 pg. Repeated injections of extracts containing constant fumonisin B1 concentrations showed that peak areas were slightly inconsistent, although generally similar to variations encountered with liquid chromatography-electrospray ionization mass spectrometry. The source of this inconsistency was traced to sample solubility, errors inherent in electrophoresis injections, and electrospray instability. Minimizing these problem areas will produce a technique with peak area reproducibilities comparable to liquid chromatography-electrospray ionization mass spectrometry, but with potentially greater resolving power.

Method for the identification of saxitoxin in rat urine.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning. We used solid-phase extraction (SPE) and prechromatographic oxidation/HPLC with fluorescence detection to isolate, identify, and quantify STX in rat urine. STX recovery from urine with the SPE procedure was approximately 76 +/- 6.5%. The standard curve was linear between 2 and 50 ng/ml. The lower limit of quantification with the method was 2 ng STX/ml of rat urine. Preliminary results with i.v. administration of STX to rats demonstrated that this method can detect and quantify STX in urine.

[3H]-saxitoxinol metabolism and elimination in the rat.

Tritiated saxitoxinol was used to obtain preliminary information on saxitoxin metabolism in the rat. Sublethal doses of tritiated saxitoxinol (18.9-microCi/kg; 3.8 micrograms/kg) were injected i.v. into each of six rats. Urine and fecal samples were collected up to 144 hr post-injection. Within 4 hr, 60% of injected radioactivity was excreted in urine. No radioactivity was found in feces. High performance liquid chromatography analyses of urine showed that saxitoxinol was not metabolized by the rats.

Electrospray ionization of selected low-molecular-weight natural biotoxins.

Electrospray ionization (ESI) spectra were acquired for several low-molecular-weight marine and freshwater biotoxins. Spectra were dominated by ions that represented protonated molecules, contained few other ions, and corresponded well with spectra acquired by other soft ionization techniques. Each biotoxin was successfully analyzed using similar ESI conditions. Nozzle-skimmer differential voltage greatly influenced spectra of several compounds by enhancing collision-induced dissociation in the transport region of the ESI source.

Microcystin-induced activation of prostaglandin synthesis and phospholipid metabolism in rat hepatocytes.

The effects of microcystin-LR, a trichothecene, T-2 and saxitoxin on membrane lipid mediators of inflammatory processes were evaluated in cultured rat hepatocytes using [(14)C]arachidonic acid. Microcystin-LR significantly stimulated the release of prostacyclin, measured as 6-keto PGF(1)alpha, by 38%, and thromboxane B(2) by 50%, in a concentration-dependent manner. The trichothecene toxin T-2 enhanced the release of prostaglandin F(2)alpha by 24% and arachidonic acid by 29%; while saxitoxin did not affect the release prostaglandins or arachidonic acid. Incorporation of arachidonic acid into the lipid pool was reduced by 47% by 1 mum microcystin-LR. Changes in the distribution of radioactivity derived from [(14)C]arachidonic acid within phospholipid classes indicated that prostaglandin formation induced by microcystin-LR was also due to the release of arachidonic acid from the phosphatidylinositol pool. No statistically significant effect of toxin was observed on the contribution of [(14)C]arachidonic acid release by other classes of phospholipids or neutral lipids. These effects may be important in the mechanism of microcystin-LR-induced toxicity in the liver.

Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids.

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.

Effect of toxins on arachidonic acid metabolism in rat cultured pulmonary alveolar macrophages.

The action of a trichothecene (T-2), microcystin-LR and saxitoxin on arachidonic acid metabolism in cultured rat alveolar macrophages was studied. Pulmonary macrophages exposed to T-2 trichothecene were stimulated to synthesize and release large amount of thromboxane B2 (TxB2) and 6-Keto F1 alpha. Microcystin-LR induced significant release of prostaglandins F2 alpha (140%), PGE2 (175%) and TxB2 (169%) compared to controls. Saxitoxin induced TxB2 release by 37%. Arachidonic acid release was stimulated by all three toxins. The release of arachidonic acid and its metabolites in alveolar macrophages exposed to T-2 toxin was partially blocked by fluocinolone (1 microM). These results suggest that macrophages synthesize and release inflammatory mediators in response to toxin exposure, and fluocinolone may protect against T-2 toxicosis.

Structural elucidation of a water-insoluble glucan produced by a cariogenic oral Streptococcus.

The structure of a water-insoluble polysaccharide produced by the D-glucosyl-transferase of Streptococcus mutans 6715 has been elucidated through periodate oxidation, Smith degradation, dextranase digestion, concanavalin A binding studies, and methylation combined with g.l.c.-m.s. analysis. These studies show that the D-glucan is comprised of 67% alpha-(1----3) linkages in a contiguous backbone with the remaining 33% as alpha-(1----6) linkages, possibly as linear residues extending from alpha-(1----6) branch points. Of the residues, 14% are branch points and the ratio of linear alpha-(1----3) residues in the backbone to alpha-(1----6) residues in the side chain was found to be 5:2. Dextranase digestion and Smith degradation both gave rise to a high-molecular-weight fraction that is only alpha-(1----3) linked.

Metabolic profiles of penaied shrimp: dietary lipids and ovarian maturation.

The major impediment to the culture of penaeid shrimp in captivity in the United States has been an inability to obtain ovarian maturation and spawning. Lipid profiles of tissues (gonads, hepatopancreas, and tail muscle) of Penaeus setiferus caught at sea have shown that cholesterol is the dominant sterol and that polyunsaturated fatty acids known to be essential in man comprise a significant portion of the fatty acid fraction. A prioprietary marine ration contains cholesterol, but is devoid of polyunsaturated fatty acids. Ovarian maturation and spawning were obtained when the shrimp diet was supplemented with an annelid rich in lipids containing these compounds. The biochemical significance of these findings is discussed.

2,5-Di-t-amyl-p-benzoquinone: an antioxidant encountered during the analysis of human tissues.

The title compound was found in extracts of several human tissues, including neurological tumors, adult brain and fetal brain. It was also present in adhesive tapes used in the laboratory to label glassware, but not in sufficient quantities to be responsible for contamination of the tissues. No source of contamination was found in the hospital. One possible source is an antioxidant used in rubber manufacture. Analytical data are also given for related benzoquinones and hydroquinones.