Dermal toxicology of Lyngbya majuscula, from Moreton Bay, Queensland, Australia

Lyngbya majuscula is a filamentous marine cyanobacterium with a worldwide distribution in temperate and tropical regions to a depth of 30 m. Over 70 chemicals have been isolated and characterised from this organism, many of which are biologically active. Previously, L. majuscula has been reported as implicated in negative health outcomes only in Hawaii and Okinawa. Recently large blooms of L. majuscula have occurred with increasing repetition in the Moreton Bay region as well as other areas along the Australian coastline.Lyngbya toxin A (LA) and debromoaplysiatoxin (DAT) were found in samples of L. majuscula collected from Eastern Moreton Bay and North Deception Bay, Queensland, Australia, respectively. Samples of L. majuscula were also obtained from West Maui, Hawaii and the freshwater Lyngbya wollei from Florida. A quantitative measure of the irritant effects of the chemicals found in L. majuscula was made using a mouse ear swelling test. The relative toxicities of two purified toxins, LA and DAT, were examined. These were found to produce swelling to a similar extent. The time course of inflammation and histopathological results were also similar for the two purified toxins. Less than 1 μg per ear of either toxin or a mixture (1:1) of the two toxins caused a measurable increase in ear thickness. When toxins were combined (1:1) there was an additive, not synergistic effect. Increases in ear thickness occurred within 15 min. Crude extracts of L. majuscula from Moreton Bay were also applied to mice ears. The effect of crude extracts from Eastern Moreton Bay was not fully explained by the measured LA content, suggesting other toxin(s) and/or modulating factors were present. The toxic effects of L. majuscula containing DAT from North Deception Bay were explained by the concentrations measured. Some samples of L. majuscula containing no measurable quantities of LA or DAT were found to exert an inflammatory response. This response had a different time course to the response produced by LA or DAT.     

A bloom of Lyngbya majuscula in Shoalwater Bay, Queensland, Australia: An important feeding ground for the green turtle (Chelonia mydas)

Lyngbya majuscula, a toxic cyanobacterium, was observed blooming during June–July (winter) 2002 in Shoalwater Bay, Queensland, Australia, an important feeding area for a large population of green turtles (Chelonia mydas). The bloom was mapped and extensive mats of L. majuscula were observed overgrowing seagrass beds along at least 18 km of coast, and covering a surface area of more than 11 km². Higher than average rainfall preceded the bloom and high water temperatures in the preceding summer may have contributed to the bloom. In bloom samples, lyngbyatoxin A (LA) was found to be present in low concentration (26 μg kg⁻¹_{(dry weight)}), but debromoaplysiatoxin (DAT) was not detected. The diet of 46 green turtles was assessed during the bloom and L. majuscula was found in 51% of the samples, however, overall it contributed only 2% of the animals’ diets. L. majuscula contribution to turtle diet was found to increase as the availability of the cyanobacterium increased. The bloom appeared to have no immediate impact on turtle body condition, however, the presence of a greater proportion of damaged seagrass leaves in diet in conjunction with decreases in plasma concentrations of sodium and glucose could suggest that the turtles may have been exposed to a substandard diet as a result of the bloom. This is the first confirmed report of L. majuscula blooming in winter in Shoalwater Bay, Queensland, Australia and demonstrates that turtles consume the toxic cyanobacterium in the wild, and that they are potentially exposed to tumour promoting compounds produced by this organism.     

Monitoring toxic cyanobacteria Lyngbya majuscula (Gomont) in Moreton Bay, Australia by integrating satellite image data and field mapping

Large-scale blooms of Lyngbya majuscula (Gomont) have occurred throughout Moreton Bay (south-east Queensland) and have been documented since 1997. L. majuscula is a toxic cyanobacteria which fixes nitrogen and is found attached to: seagrass, algae and coral. The toxic and smothering nature of L. majuscula has affected human and environmental health in sensitive coastal ecosystems. To reduce these impacts, monitoring is an essential component of studying the origins and development of L. majuscula blooms. An accurate and cost effective means to map the extent of a bloom and its biophysical properties is needed. This study presents an operational approach for mapping the extent of L. majuscula blooms in the clear and shallow water regions of Moreton Bay, eastern Australia, from a combination of field and remotely sensed data sets. The ability to discriminate L. majuscula from other substrate types over a range of depths was first examined using detailed field reflectance spectra, measured optical properties of Moreton Bay waters and a radiative transfer model (Hydrolight 4.1). A two-stage process was then used to map L. majuscula. The spatial extent of L. majuscula and other major substrate types was first recorded from a boat-based survey by marine park authorities using point-based GPS measurements. This sampling was timed to coincide with an overpass of the Landsat 7 ETM+ sensor. When the results of the boat-based mapping detected more than 25% L. majuscula cover in the study area, a cloud free Landsat 7 ETM+ image was acquired for that date. In the second stage of mapping, selected field survey data provided the basis for a supervised classification of the ETM+ image data to map L. majuscula. Effort and accuracy assessment of both field and image mapping methods indicated a trade-off between areal coverage and mapping accuracy. The Landsat 7 ETM+ based mapping procedure provided 100% areal coverage with 58% accuracy. In contrast, the boat-based field survey method covered only 0.5% of the study area, but with almost 100% mapping accuracy. The approach outlined in this work has been adopted as a standard operating procedure in Moreton Bay. This study illustrates how remote sensing can be combined with field monitoring, to provide marine park authorities with useful information to understand and manage blooms.     

In situ field experiment shows Lyngbya majuscula (cyanobacterium) growth stimulated by added iron, phosphorus and nitrogen

Coastal waters worldwide have experienced an increase in the occurrence of harmful algal blooms. Over the past decade, nuisance blooms of the toxic cyanobacterium Lyngbya majuscula have increased in frequency and severity in south-east Queensland, Australia, with blooms in Moreton Bay hypothesised to be linked to increased inputs of dissolved nutrients and organic carbon from land-based sources. An in situ field experiment was conducted in northern Moreton Bay (Deception Bay), Australia, to determine the growth response of L. majuscula to phosphorus, nitrogen and organically chelated iron additions to the water column. A three-way analysis of variance showed each of these nutrients stimulated L. majuscula biomass under field conditions, with organically chelated iron (FeEDTA) > phosphorus > nitrogen. Concurrent addition of all three nutrients [FeEDTA + P + N] caused the greatest response being 18 times the control after 49 days; this resulted in an additional 928 g_dw m⁻² of L. majuscula biomass over the control (54 g_dw m⁻²). These results show that water borne nutrients, particularly organically chelated iron, phosphorus and nitrogen can promote prolific growth of the bloom-forming cyanobacterium L. majuscula. It is also likely that the addition of iron and/or phosphorus stimulated nitrogen fixation in L. majuscula, based on the large L. majuscula biomass and higher tissue nitrogen content in the iron and phosphorus treatments. Enrichment with poultry litter (sourced from the adjacent catchment) also substantially enhanced L. majuscula biomass. The experiment shows that a precautionary approach to limit or reduce nutrient additions to streams, estuaries and coastal waters is justified; otherwise the magnitude of L. majuscula blooms is likely to increase in Moreton Bay in the future.     

The exposure of green turtles (Chelonia mydas) to tumour promoting compounds produced by the cyanobacterium Lyngbya majuscula and their potential role in the aetiology of fibropapillomatosis

Lyngbya majuscula, a benthic filamentous cyanobacterium found throughout tropical and subtropical oceans, has been shown to contain the tumour promoting compounds lyngbyatoxin A (LA) and debromoaplysiatoxin (DAT). It grows epiphytically on seagrass and macroalgae, which also form the basis of the diet of the herbivorous green turtle (Chelonia mydas). This toxic cyanobacterium has been observed growing in regions where turtles suffer from fibropapillomatosis (FP), a potentially fatal neoplastic disease. The purpose of this study was to determine whether green turtles consume L. majuscula in Queensland, Australia and the Hawaiian Islands, USA, resulting in potential exposure to tumour promoting compounds produced by this cyanobacterium. L. majuscula was present, though not in bloom, at nine sites examined and LA and DAT were detected in variable concentrations both within and between sites. Although common in green turtle diets, L. majuscula was found to contribute less than 2% of total dietary intake, indicating that turtles may be exposed to low concentrations of tumour promoting compounds during non-bloom conditions. Tissue collected from dead green turtles in Moreton Bay tested positive for LA. An estimated dose, based on dietary intake and average toxin concentration at each site, showed a positive correlation for LA with the proportion of the population observed with external FP lesions. No such relationship was observed for DAT. This does not necessarily demonstrate a cause and effect relationship, but does suggest that naturally produced compounds should be considered in the aetiology of marine turtle FP.     

Pitipeptolides C-F, antimycobacterial cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula from Guam

Pitipeptolides A (1) and B (2) are cyclic depsipeptides isolated from the marine cyanobacterium Lyngbya majuscula from Piti Bomb Holes, Guam. Additional analogues have now been isolated by revisiting larger collections of the same cyanobacterium. The four identified analogues, pitipeptolides C–F (3–6), are the tetrahydro analogue (3), an analogue with a lower degree of methylation (4) as well as two homologues (5 and 6) of pitipeptolide A. Their structures were elucidated using 2D NMR experiments, chiral HPLC analysis and comparison with pitipeptolide A. The identified analogues showed weaker cytotoxic activities compared to the two major parent compounds, pitipeptolides A (1) and B (2), against HT-29 colon adenocarcinoma and MCF7 breast cancer cells. On the other hand, pitipeptolide F (6) was the most potent pitipeptolide in a disc diffusion assay against Mycobacterium tuberculosis. The latter finding suggests that the structure of pitipeptolides could be optimized for selective antibacterial activity.     

Lagunamide C, a cytotoxic cyclodepsipeptide from the marine cyanobacterium Lyngbya majuscula

Lagunamide C (1) is a cytotoxic cyclodepsipeptide isolated from the marine cyanobacterium, Lyngbya majuscula, from the western lagoon of Pulau Hantu Besar, Singapore. The complete structural characterization of the molecule was achieved by extensive NMR spectroscopic analysis as well as chemical manipulations. Several methods, including the advanced Marfey’s method, a modified method based on derivatization with Mosher’s reagents and analysis using LC–MS, and the use of ³J_H–H coupling constant values, were utilized for the determination of its absolute configuration. Compound 1 is related to the aurilide-class of molecules and it differs mainly in the macrocyclic structure by having a 27 membered ring system due to additional methylene carbon in the polyketide moiety. Lagunamide C displayed potent cytotoxic activity against a panel of cancer cell lines, such as P388, A549, PC3, HCT8, and SK-OV3 cell lines, with IC₅₀ values ranging from 2.1 nM to 24.4 nM. Compound 1 also displayed significant antimalarial activity with IC₅₀ value of 0.29 μM when tested against Plasmodium falciparum. In addition, lagunamide C exhibited weak anti-swarming activity when tested at 100 ppm against the Gram-negative bacterial strain, Pseudomonas aeruginosa PA01.     

Occurrence of okadaic acid in the feeding grounds of dugongs (Dugong dugon) and green turtles (Chelonia mydas) in Moreton Bay, Australia

Okadaic acid (OA) is a diarrhetic shellfish poison (DSP) produced by a number of marine organisms including the benthic dinoflagellate Prorocentrum lima, which are often found on seagrass. As seagrass forms the basis of the diet of dugong (Dugong dugon) and green turtle (Chelonia mydas), these herbivores may potentially be exposed to OA through ingestion of P. lima found on the seagrass. In this study, the abundance of epiphytic P. lima, on seagrass, and the concentration of OA produced by these epiphytic dinoflagellates was measured in Moreton Bay, Queensland, Australia. P. lima and OA were found on all four species of seagrass collected. OA was detected in epiphytic material collected from seagrass, with a maximum of 460 ng OA/kg_(wwtSG) found on Halophila spinulosa. From this information, the estimated maximum daily intake (DI) of OA by an adult dugong consuming 40 kg_(wwtSG)/day was 18,400 ng/day, and an adult turtle consuming 2 kg_(wwtSG)/day was 920 ng/day. Analysis by HPLC/MS/MS of 54 stranded dugongs and 19 stranded turtles did not yield OA above the detection limit of 10,000 ng/kg_{(animal tissue)}. OA was found on seagrass, however it was not detected in the tissue samples of dugongs and turtles.     

Besarhanamides A and B from the marine cyanobacterium Lyngbya majuscula

Besarhanamides A (1) and B (2) are fatty acid amides purified from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu, Singapore. The structure determination of these secondary metabolites was carried out using extensive 2D NMR spectral data as well as chemical manipulations including the Marfey's method. In addition, besarhanamide A exhibited moderate toxicity with LD(50) at 13 microM in the brine shrimp toxicity bioassay.     

Some chemical factors regulating the growth of <Emphasis Type="Italic">Lyngbya majuscula</Emphasis> in Moreton Bay,Australia: importance of sewage discharges

The relative importance of the additions of iron (Fe), phosphorus (P) and the chelating agent EDTA in promoting the growth of Lyngbya majuscula in waters collected from the NW region of Moreton Bay was investigated using a series of continuous-flow growth studies. In addition, the possible impact of sewage/waste-water treatment plant (WWTP) discharges on the growth of L. majuscula was investigated in a series of batch and semi-continuous-flow growth studies. A preliminary study on the potential of phytoplankton growth in the receiving waters to affect the productivity of L. majuscula was also conducted. The results from the continuous-flow growth studies show that the growth rates of L. majuscula in the NW Moreton Bay waters were stimulated by the addition of EDTA alone but were not stimulated by the addition of P. The additions of P + EDTA, FeEDTA and P + FeEDTA did not result in higher growth rates than those obtained by the addition of EDTA alone. These results demonstrate that the productivity of L. majuscula in the NW Moreton Bay waters was not limited by P and that the addition of Fe did not affect the productivity. The stimulation effect of EDTA could be due to various reasons but we hypothesise that the principal reason for the stimulation is that EDTA increases the bioavailability of non-labile Fe species already present in the water. The results of the batch and semi-continuous-flow growth studies show that diluted (100:1) WWTP discharge water and the receiving waters impacted by WWTP discharges supported significant growth of L. majuscula and that the addition of EDTA to those waters increased the growth potential of L. majuscula. Also the growth of phytoplankton in a sample of the receiving waters impacted by WWTP discharges significantly reduced the growth rate of L. majuscula but the addition of EDTA restored the growth rate to near its maximum value. These results suggest that phytoplankton growth (and probably that of the associated bacterioplankton) could reduce the bioavailability of trace chemical factors needed for the growth of L. majuscula in Moreton Bay and in particular, could reduce the bioavailability of Fe. Overall the results support the hypothesis that growth of L. majuscula in Moreton Bay is often limited by the bioavailability of Fe and the principal reason for this is the lack of supply of suitable organic ligands/chelators, not the lack of Fe per se. Handling editor: D. Hamilton     

First evidence for the production of cylindrospermopsin and deoxy-cylindrospermopsin by the freshwater benthic cyanobacterium, Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck

Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck is a common mat-forming benthic cyanobacterium from freshwater habitats. We found that two populations from southeast Queensland (Australia) produce the potent cyanotoxin cylindrospermopsin (CYN) and its analogue, deoxy-cylindrospermopsin (deoxy-CYN). The highest concentrations in environmental samples were 20 and 550 μg g⁻¹ dry weight for CYN and deoxy-CYN, respectively. A sub-sample maintained in culture for over 16 months yielded concentrations of 33 and 308 μg g⁻¹ dry weight for CYN and deoxy-CYN, respectively. The concentration of deoxy-CYN in L. wollei was 10–300 times higher than CYN, suggesting that, unlike many other CYN-producing cyanobacteria, the primary compound produced by L. wollei is deoxy-CYN. The production of CYN and deoxy-CYN by L. wollei represents a potential human health risk and an additional source of these toxins in freshwaters. This is the first report of the production of CYN and deoxy-CYN by L. wollei or any species of the Oscillatoriales.     

Three new species of Prosorhynchoides (Digenea: Bucephalidae) from Tylosurus gavialoides (Belonidae) in Moreton Bay,Queensland, Australia

We surveyed 30 individuals of Tylosurus gavialoides (Castelnau) (Belonidae) collected from Moreton Bay, Queensland, Australia, and describe three new species of Prosorhynchoides Dollfus, 1929 from them. The new species are morphologically distinct from existing Prosorhynchoides spp. and 28S and ITS-2 ribosomal DNA data further supports our morphological findings. We also conduct the first mitochondrial DNA analysis of species of Prosorhynchoides. The new species from T. gavialoides form a strongly supported clade on the basis of the two ribosomal markers, further supporting the emerging hypothesis that bucephaline clades are strongly associated with host groups. We have not observed any of the new species reported here in over 3500 surveyed individuals of other piscivorous fish in Australia, suggesting that these species are host-specific at least to belonids, if not to only T. gavialoides. Our findings support previous reports that suggest that belonids are exceptional hosts for bucephalids. We predict that further sampling of the numerous other belonid species present in Australian waters, for which nothing is known of the bucephalid fauna, will uncover further bucephalid richness.     

褶皱臂尾轮虫Brachionus plicatilis摄食小球藻Chlorella sp.的碳同化与碳排放

浮游动物在食物链能量流动与物质循环中发挥着重要作用,能将摄入的浮游植物转化为不同形态的碳,在海洋碳循环中发挥重要作用。应用¹⁴C标记示踪方法,定量分析海洋浮游动物褶皱臂尾轮虫Brachionus plicatilis摄入碳的碳同化与碳排放。喂食不同密度小球藻Chlorella sp.（1×10⁵个/mL、5×10⁵个/mL、1×10⁶个/mL）后,褶皱臂尾轮虫对小球藻碳的同化率（AE）为34%-51%,呈现随饵料密度增加而减小的趋势;未被轮虫同化的碳,主要以溶解有机碳（DOC）的形态排放到水体中,DOC占碳排放的比例为37%-51%,随着饵料密度增加而增加;二氧化碳（CO₂）的比例为15%-40%,随着饵料密度增加而减小;颗粒有机碳（POC）占碳排放的比例较少,为23%-34%,随着饵料密度的增加而增加。此外,分析褶皱臂尾轮虫排放DOC的粒径组成,发现低分子量有机碳（LMW,< 3 kDa）的量大于胶体有机碳（COC,3 kDa-0.22 μm）的量,COC占DOC比例为33%-43%;LMW占DOC比例为57%-67%。本研究结果表明,浮游动物可把相当部分食物中的碳转化为DOC,排放到水体中为细菌所利用,在海洋碳循环中发挥重要作用。     

Dietary selectivity for the toxic cyanobacterium Lyngbya majuscula and resultant growth rates in two species of opisthobranch mollusc

Trophodynamics of blooms of the toxic marine cyanobacterium Lyngbya majuscula were investigated to determine dietary specificity in two putative grazers: the opisthobranch molluscs, Stylocheilus striatus and Bursatella leachii. S. striatus is associated with L. majuscula blooms and is known to sequester L. majuscula metabolites. The dietary specificity and toxicodynamics of B. leachii in relation to L. majuscula is less well documented. In this study we found diet history had no significant effect upon dietary selectivity of S. striatus when offered a range of plant species. However, L. majuscula chemotype may alter S. striatus' selectivity for this cyanobacterium. Daily biomass increases between small and large size groups of both species were recorded in no-choice consumption trials using L. majuscula. Both S. striatus and B. leachii preferentially consumed L. majuscula containing lyngbyatoxin-a. Increase in mass over a 10-day period in B. leachii (915%) was significantly greater than S. striatus (150%), yet S. striatus consumed greater quantities of L. majuscula (g day^− 1) and thus had a lower conversion efficiency (0.038) than B. leachii (0.081) based on sea hare weight per gram of L. majuscula consumed day^− 1. Our findings suggest that growth rates and conversion efficiencies may be influenced by sea hare maximum growth potential, acquisition of secondary metabolites or diet type.     

Flamingomyces and Parvulago, new genera of marine smut fungi (Ustilaginomycotina)

Teliospore walls, teliospore germinations, hyphal septations, cellular interactions, and nucleotide sequences from the D1/D2 region of the nuLSU rRNA gene of the marine smut fungi Melanotaenium ruppiae and Ustilago marina were examined and compared with findings in other Ustilaginomycotina. The data show that Melanotaenium ruppiae belongs to the Urocystaceae and Ustilago marina to the Ustilaginaceae. Within the Urocystaceae, Melanotaenium ruppiae is morphologically similar to Melanustilospora and Vankya. However, according to the molecular results Melanotaenium ruppiae can neither be ascribed to Melanustilospora nor to Vankya. Therefore, the new genus Flamingomyces is proposed for Melanotaenium ruppiae. Ustilago marina differs from the other Ustilaginaceae in the mode of sporulation, which exclusively occurs at the base of the host plant culms. Accordingly, the new genus Parvulago is proposed for Ustilago marina.     

华南吊灯花属(夹竹桃科)植物小志

通过野外调查、标本查阅及文献考证,订正了华南吊灯花属(Ceropegia Linn.)植物,将狭瓣吊灯花(C. angustilimba Merr.)从吊灯花(C. trichantha Hemsl.)的异名中予以恢复,提供了二者的形态描述,确认华南地区记录的柳叶吊灯花(C. salicifolia H. Huber)实为剑叶吊灯花(C. dolichophylla Schltr.)的错误鉴定,并提供了后者的形态描述。     

Detection of Pfiesteria spp. by PCR in surface sediments collected from Chesapeake Bay tributaries (Maryland)

In 1997 blooms of Pfiesteria piscicida occurred in association with fish kills and human health problems in tributaries of the Chesapeake Bay (Maryland) and the scientific and media response resulted in large economic losses in seafood sales and tourism. These events prompted the Maryland Department of Natural Resources (MDNR) to begin monitoring for Pfiesteria spp. in water column samples. Real-time PCR assays targeted to the 18S rRNA gene were developed by our laboratories and utilized in conjunction with traditional microscopy and fish kill bioassays for detection of these organisms in estuarine water samples. This monitoring strategy aided in determining temporal and spatial distribution of motile forms of Pfiesteria spp. (i.e. zoospores), but did not assess resting stages of the dinoflagellates’ life cycle. To address this area, a 3-year study was designed using real-time PCR assays for analysis of surface sediment samples collected from several Chesapeake Bay tributaries. These samples were tested with the real-time PCR assays previously developed by our laboratories. The data reported herein suggest a strong positive association between presence of Pfiesteria spp. in the sediment and water column, based on long-term water column monitoring data. P. piscicida is detected more commonly in Maryland's estuarine waters than Pfiesteria shumwayae and sediment ‘cyst beds’ may exist for these organisms.     

A new fungal genus, Teracosphaeria, with a phialophora-like anamorph (Sordariomycetes, Ascomycota)

The new genus and species Teracosphaeria petroica is described for a perithecial ascomycete and its anamorph occurring on decayed wood collected in New Zealand. The fungus produces immersed, non-stromatic ceratosphaeria-like perithecia in nature, with hyaline, septate ascospores produced in unitunicate, non-amyloid asci. The anamorph produced in vitro is phialophora-like with lightly pigmented phialides terminating in flaring, deep collarettes that are often noticeably brown with conspicuous periclinal thickening. Phylogenetic analysis of LSU rDNA sequence data indicates that this fungus is distinct from morphologically similar fungi classified in the Chaetosphaeriales, the Trichosphaeriales or the Magnaporthaceae. It forms a monophyletic group with recently described, chaetosphaeria-like ascomycetes, such as the pyrenomycete genus Mirannulata, and shows affinity with the anamorphic species Dictyochaeta cylindrospora. The usefulness of describing anamorph genera for morphologically reduced anamorphs, when anamorph characteristics are actually part of the holomorph diagnosis, is discussed. An apparently contradictory example of the so-called Cordana and Pseudobotrytis anamorphs of Porosphaerella spp. is also discussed.