Alessandra Giani

DOES ALLELOPATHY CONTRIBUTE TO CYLINDROSPERMOPSIS RACIBORSKII (CYANOBACTERIA) BLOOM OCCURRENCE AND GEOGRAPHIC EXPANSION?1

Cylindrospermopsis raciborskii (Wołosz.) Seenayya et Subba Raju is a planktonic filamentous cyanobacterium whose sudden worldwide proliferation and ability to produce toxins are a reason for concern. In this paper, we suggest that its ecological dominance might be explained by antagonistic interaction with other phytoplankton species due to production of allelopathic metabolites. To test this hypothesis, experiments were run with exudates of natural phytoplankton and C. raciborskii strains isolated from Lagoa Santa, a small natural lake in southeastern Brazil, where this species has become dominant in recent years. The exudates were added to different algal species obtained from the same environment and maintained in culture. After 24 h incubation, PAM fluorometry was used to compare control and treatment photosynthetic responses (relative electron transport rate) to the dissolved extracellular products. Results indicate that most of the target species were sensitive to C. raciborskii exudates, which showed strong inhibitory effects on their photosynthetic activities. These results provide evidence that allelopathy may offer a competitive benefit to C. raciborskii and contribute to its stable dominance in Lagoa Santa. A potential allelopathic advantage could also help to explain the geographic expansion of this species at midlatitudes.

Effect of nitrate and ammonium on the growth and protein concentration of Microcystis viridis Lemmermann (Cyanobacteria)

Cyanobacteria are a very important group in aquatic systems, particularly in eutrophic waters. Therefore studies about their success in the environment are essential. Many hypotheses have tried to explain the dominance of Cyanobacteria, and several emphasized the importance of various nitrogen sources for the success of the group. In this study, we measured the effect of ammonium and nitrate on the growth and protein concentration of Microcystis viridis (Cyanobacteria). This species is well-known because bloom formation in eutrophic waters. The study was carried out, in experimental batch cultures, using the WC medium with different nitrogen sources: ammonium, nitrate, ammonium + nitrate (50% ammonium + 50% nitrate) and ammonium at different concentrations (to test for possible NH4+ toxicity). Protein, ammonium and nitrate concentrations were measured at end of each experiment, whereas samples for cell counts were taken daily. Results showed that Microcystis viridis grew faster with ammonium (µ = 0.393 day-1) than with nitrate (µ = 0.263 day-1) and ammonium + nitrate (µ = 0.325 day-1). This pattern is explained by the metabolism of ammonium that presents higher uptake and assimilation rates than nitrate. Maximum cell concentration, however, was higher in the ammonium + nitrate treatment, followed by nitrate treatment. Higher protein concentration were observed in the treatment with nitrate. In the ammonium toxicity test, no difference between the control and NH4+ at 50% was found. Thus, the ammonium concentrations used in these experiments were not toxic. Our results suggest that Cyanobacteria is able to grow on both nitrogen sources even if ammonium may allow faster growth and bloom development.

Microcystin Production and Regulation under Nutrient Stress Conditions in Toxic Microcystis Strains

ABSTRACT Microcystin is a common and well-known cyanobacterial toxin whose intracellular role is still under investigation. Increasing knowledge on microcystin gene expression and regulation can contribute to the understanding of its putative cellular function. In this work, reverse transcription-quantitative PCR (RT-qPCR) was used to investigate the transcriptional response of the mcyD gene to nitrogen (nitrate and ammonium) and phosphorus limitation in two toxic Microcystis strains. The existence of a direct correlation between transcripts of mcyD and ntcA genes was also identified. In previous studies, NtcA (global nitrogen regulator) has been described as a potential component in the control of microcystin biosynthesis. This research showed that stress agents linked to nutrient deprivation could lead to a significant increase of microcystin production in both strains studied. The more toxic strain proved to be more resistant to nutrient limitation. The similar outcomes of mcyD regulation observed for all nutrients suggest that this response can be linked to oxidative stress of cells undergoing adverse growth conditions.

Experimental model of microcystin accumulation in the liver of Oreochromis niloticus exposed subchronically to a toxic bloom of Microcystis sp.

Although accumulation of the liver toxin microcystin in phytoplanktivorous fish has been demonstrated in captive fish and in natural ecosystems, the relation between microcystin in ingested algae and the pattern of buildup of microcystin in fish is poorly known. In this month-long study performed at a Brazilian fish farm, 45 mature Oreochromis niloticus were fed daily with fresh seston periodically dominated by toxic Microcystis sp. Microcystin was measured daily in the food and every 5 days in liver and muscle samples. Control fish received a diet of seston that was low in toxic cyanobacteria. Initially, in treatment ponds, microcystin available for fish increased from 6.5 to 66.9 ng microcystin fish(-1)day(-1), which was accompanied by an increase from 5.5 to 35.4 ng microcysting liver(-1). Microcystin in muscle was below our detection limit of 4 ng g tissue(-1) for the entire study. In the bloom phase, available microcystin reached its highest concentration (4450 ng MC fish(-1)day(-1)) then decreased to 910 ng microcystin fish(-1)day(-1) on day 31. During this period, microcystin reached its highest concentration of 81.6 ng MC g liver(-1) and stayed high until the end of the experiment. A model based on rapid uptake, saturation, and exponential loss was built with these experimental results, and verified with data from the literature. Our model showed that accumulation was up to 50% of ingestion at low doses, but at intermediate doses, the onset of elimination led to a decline of liver burden. Although the accumulation rate confirms the high contamination potential of microcystin, it was balanced by a high depuration rate and this efficient systemic elimination may explain the tolerance of these fish to toxic blooms in the wild.

Agricultural land-use affects the nutritional quality of stream microbial communities

We investigated how the lipid composition (fatty acids and sterols) of benthic microbial mats, which represent an important basal food resource for stream food webs, differs between tropical streams located in protected pristine and agricultural Cerrado savannah areas. The total microbial biomass and lipid composition differed significantly between pristine and agricultural streams in parallel with differences in water quality and hydrodynamic characteristics. Agricultural streams exhibited lower total biomass of benthic microbial mats than pristine streams. However, the higher concentrations of essential polyunsaturated fatty acids, such as linoleic acid (LIN, 18:2ω6), α-linolenic acid (ALA, 18:3ω3), and eicosapentaenoic acid (EPA, 20:5ω3), that were observed in agricultural streams suggest enhanced lipid complexity and a higher nutritional quality of the microbial community relative to pristine streams. Meanwhile, pristine stream microbial communities had higher total concentrations of saturated fatty acids and cholesterol than those of agricultural streams, reflecting their heterotrophic microbial communities. Moreover, stream morphotype and associated differences in the hydrodynamic characteristics affected the community composition and thereby also the lipid composition of microbial mats. Land-use-induced changes in the total biomass and lipid composition of microbial communities may affect the trophic transfer of energy in stream food webs, leading to changes in the composition and productivity of primary consumers and their predators, and thereby affecting stream ecosystem functioning.

Profiles of toxic and non-toxic oligopeptides of Radiocystis fernandoii (Cyanobacteria) exposed to three different light intensities.

Cyanobacteria produce a high variety of bioactive oligopeptides, which function, ecological, physiological roles and responses to environmental changes are still unclear. The influence of light intensity on the cell quota and the diversity of oligopeptides of two strains of the cyanobacterium Radiocystis fernandoii were experimentally tested. The peptides were quantified by HPLC and identified by a MALDI-TOF-TOF. Microcystins (MC) were generally more abundant in the treatment with low light. A compensatory mechanism was observed for the different variants of microcystin, whereby MC-RR responses were contrary to those observed for the other three variants and showed higher concentration in the treatment with intermediate light. Two microviridins were also produced at higher amounts at intermediate irradiance. For cyanopeptolins and a third microviridin no significant difference among treatments was found. The absence of a similar response for all peptides suggests that these compounds may have unique cellular functions, which better understanding could help explaining changes in toxicity. Finally, we observed that each chemical profile reflected in physiological differences between strains, strengthening the idea that chemotypes may act as distinct ecotypes in nature.

Cell density-dependent oligopeptide production in cyanobacterial strains

Cyanobacteria can form blooms and in these situations they dominate the phytoplanktonic community, reaching extremely high densities. In the domain Bacteria, high population densities can stimulate a phenomenon known as quorum sensing, which may produce several modifications in the cell physiology. Very little is known about quorum sensing in Cyanobacteria. Because of their planktonic way of life, quorum sensing should be more evident during a bloom event. In this work, we tested whether cell density could shape the production of bioactive compounds produced by Cyanobacteria. The experiments consisted of two treatments, where cultures of Cyanobacteria were maintained at low and high cellular densities through a semi-continuous set-up. Analyses were performed by HPLC-PDA and MALDI-TOF MS. Seventeen peptides were detected and 14 identified, including microcystins, aeruginosins, cyanopeptolins and microviridins. The results showed that cellular density seems to have a significant effect on the peptides production. Most of the compounds had significantly higher cellular quotas in the higher-density treatment, although microviridins and an unknown peptide were produced only at low density. These results may hint at a possible role for quorum sensing in triggering the production of several cyanobacterial peptides.

Algas planctônicas do reservatório da Pampulha (MG): Euglenophyta, Chrysophyta, Pyrrophyta, Cyanobacteria

This paper contains descriptions of 28 taxa of planktonic algae distributed in four divisions (Euglenophyta, Chrysophyta, Pyrrophyta and Cyanobacteria) found in the Pampulha reservoir in Minas Gerais state. Samples from May/1992 to February/1997 were analyzed. The best represented division was Euglenophyta (13 species in one family), followed by Cyanobacteria (eight species), Pyrrophyta (five species) and Chrysophyta (two species). Twenty three taxa are reported for the first time to the State of Minas Gerais.

Crude extract of cyanobacteria (Radiocystis fernandoi, strain R28) induces liver impairments in fish.

Radiocystis fernandoi R28 strain is a cyanobacterium which produces mostly the RR and YR microcystin variants (MC-RR and MC-YR, respectively). The effects of crude extract of the R. fernandoi strain R28 were evaluated on the protein phosphatases and on the structure and ultrastructure of the liver of the Neotropical fish, Hoplias malabaricus, after acute and subchronic exposure. Concomitantly, the accumulation of the majority of MCs was determined in the liver and muscle. The fish were exposed to 120.60 MC-RR+MC-LR kg-fish-1 (=100μg MC-LReq kg-fish-1) for 12 and 96h (one single dose, acute exposure) and 30days (one similar dose every 72h, subchronic exposure). MCs did not accumulate in the muscle but, in the liver, MC-YR accumulated after acute exposure and MC-RR and MC-YR accumulation occurred after subchronic exposure. Protein phosphatase 2A (PP2A) activity was inhibited only after subchronic exposure. Acute exposure induced liver hyperemia, hemorrhage, changes in hepatocytes and cord-like disorganization. At the ultrastructural level, the decreasing of glycogen and lipid levels, the swelling of mitochondria and whirling of endoplasmic reticulum suggested hepatocyte necrosis. Subchronic exposure resulted in a complete disarrangement of cord-like hepatocytes, some recovery of mitochondria and whirling endoplasmic reticulum and extensive connective tissues containing fibrous materials in the liver parenchyma. Despite microcystin toxicity and liver alterations, no tumor was induced by MCs. In conclusion, the increased algal mass of R. fernandoi in tropical freshwater, producing mainly MC-RR and MC-YR variants, results in fish liver impairments.

Avaliação da remoção de saxitoxinas por meio de técnicas de tratamento das águas de abastecimento

O objetivo do trabalho e avaliar a eficiencia da adsorcao com carvao ativado em po (CAP) e da cloracao de calcio na remocao de saxitoxinas. As saxitoxinas dispersas em agua destilada foram produzidas por meio da extracao de celulas viaveis de cianobacterias da especie Cylindrospermopsis raciborskii. Os ensaios foram realizados em equipamento de jar test adaptado e empregando-se tres tipos de CAP e hipoclorito de calcio. Os tempos de detencao aplicados foram de duas horas para adsorcao e 30 e 60 minutos para oxidacao. Os resultados evidenciaram que a eficiencia de remocao, para a adsorcao, esta intrinsecamente relacionada ao tipo de carvao e a dosagem empregada, obtendo maior eficiencia para o CAP de madeira. Para oxidacao, os dois tempos de contato e as dosagens avaliadas apresentaram eficiencia praticamente constante, da ordem de 80%, atendendo ao estabelecido pela Portaria 518.