New type of dual-channel PAM chlorophyll fluorometer for highly sensitive water toxicity biotests

A new type of dual-channel PAM chlorophyll fluorometer has been developed, which is specialised in the detection of extremely small differences in photosynthetic activity in algae or thylakoids suspensions. In conjunction with standardised algae cultures or isolated thylakoids, the new device provides an ultrasensitive biotest system for detection of toxic substances in water samples. In this report, major features of the new device are outlined and examples of its performance are presented using suspensions of Phaeodactylum tricornutum (diatoms) and of freeze-dried thylakoids of Lactuca sativa (salad). Investigated and reference samples are exposed to the same actinic intensity of pulse-modulated measuring light. The quantum yields are assessed by the saturation pulse method. Clock-triggered repetitive measurements of quantum yield typically display a standard deviation of 0.1%, corresponding to the inhibition induced by 0.02 mug diuron l(-1). Hence, for diuron or compounds with similar toxicity, the detection limit is well below the 0.1 mug l(-1) defined as the limit for the presence of a single toxic substance in water by the European Commission drinking water regulation. The amounts of water and biotest material required for analysis are very small, as a single assay involves two 1 ml samples, each containing ca. 0.5 mug chlorophyll. Both with Phaeodactylum and thylakoids the relationship between inhibition and diuron concentration is strictly linear up to 10% inhibition, with very similar slopes. Apparent inhibition depends on the actinic effect of the measuring light, showing optima at 6 and 4 mumol quanta m(-2) s(-1) with Phaeodactylum and thylakoids, respectively.

Cloud-point extraction and preconcentration of cyanobacterial toxins (microcystins) from natural waters using a cationic surfactant

A new cloud-point extraction and preconcentration method, using a cationic, surfactant, Aliquat-336 (tricaprylyl-methy;ammonium chloride), his-been developed for the determination of cyanobacterial toxins, microcystins, in natural waters. Sodium sulfate was used to induce phase separation at 25 degreesC. The phase behavior of Aliquat-336 with respect to concentration of Na2SO4 was studied. The cloud-point system revealed a very high phase volume ratio compared to other established systems of nonionic, anionic, and cationic surfactants: At pH 6-7, it showed an outstanding selectivity in ahalyte extraction for anionic species. Only MC-LR and MC-YR, which are known to be predominantly anionic, were extracted (with averaged recoveries of 113.9 +/- 9% and 87.1 +/- 7%, respectively). MC-RR, which is likely to be amphoteric at the above pH range, was. not cle tectable in.the extract. Coupled to HPLC/UV separation and detection, the cloud-point extraction method (with 2.5 mM Aliquat-336 and 75 mM Na2SO4 at 25 degreesC) offered detection limits of 150 +/- 7 and 470 +/- 72 pg/mL for MC-LR and MC-YR, respectively, in 25 mL of deionized water. Repeatability of the method was 7.6% for MC-LR and 7.3% for MC-YR: The cloud-point extraction process can be. completed within 10-15 min with no cleanup steps required. Applicability of the new method to the determination of microcystins in real samples was demonstrated using natural surface waters, collected from a local river and a local duck pond spiked with realistic. concentrations of microcystins. Effects of salinity and organic matter (TOC) content in the water sample on the extraction efficiency were also studied.

Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3 H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a blackjack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, greater than or equal to 1 and > 1 M Ug g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or -2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H](+) ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.

Effect of organic amendments and solarisation on Fusarium wilt in susceptible banana plantlets, transplanted into naturally infested soil

Despite extensive research since pathogenicity was first established in 1919, no cultural or chemical control strategy has proven effective against Fusarium wilt of bananas. The efficacy of cultural control is attributed to the suppression of pathogen activity. Yet, amending naturally infested soil with aged chicken manure has been shown to enhance disease severity, without any change in the activity of the pathogen Fusarium oxysporum f. sp. cubense (Foc) in the soil. In this study, the effect of amending soil with composted sawdust, and of solarising soil, was compared with the effect of amending soil with chicken manure. Bioassays comparing the activity of Foc in the soil with the extent of invasion of banana pseudostem tissue by Foc were used to investigate why strategies targetting pathogen survival have not proven successful in controlling this disease. The enhancement of Foc invasion of the banana plantlets was reproduced with the addition of chicken manure to the naturally infested soil. However, changes in the activity of Foc in the soil were not associated with changes in the frequency of invasion of the plantlets. Invasion of banana pseudostems in the sawdust and solarisation treatments was not significantly different from invasion in the respective control treatments, despite a reduction in the activity of Foc in the sawdust-amended soil and an enhancement in the solarised soil. Moreover, the increase in Foc activity in the solarised soil recorded during the bioassays occurred despite the effectiveness of solarisation in reducing the survival of Foc in pre-colonised banana root tips buried in the soil. Changes in the frequency of invasion were associated with changes in the availability of mineral nitrogen, particularly ammonium N. These results suggest that the physiological response of banana cultivars to ammonium N may be associated with their susceptibility to Fusarium wilt. Accordingly, cultural strategies for controlling Panama disease will only be effective if they enhance the ability of the host to resist invasion.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography-mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18-23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential. (C) 2003 Elsevier B.V All rights reserved.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.

Extratos vegetais e Bacillus thuringiensis visando o manejo de Duponchelia fovealis Zeller (Lepidoptera: Crambidae)

A lagarta Duponchelia fovealis Zeller (Lepidoptera: Crambidae) foi relatada em plantios comerciais do morangueiro no estado do Espírito Santo ocasionando grandes problemas à cultura. Porém, por ser uma praga recente, não há registro de produtos para o seu controle. A cada dia aumenta a demanda por alimentos e outros produtos livres de resíduos, além da necessidade de uma agricultura mais desenvolvida e sustentável. Pesquisas com agentes de controle biológico e extratos vegetais surgem como alternativa para o manejo desse inseto-praga. Este trabalho teve como objetivo avaliar a eficiência de formulados comerciais à base de Bacillus thuringiensis e a atividade do uso dos extratos aquosos de alho e fumo, visando sua adoção como métodos alternativos de controle de D. fovealis. Nos bioensaios para avaliar a patogenicidade e virulência de duas formulações comerciais à base de B. thuringiensis, Agree® e Dipel WP®, sobre a dieta artificial adaptada à base de farelo de soja, germe de trigo e açúcar, proposta por King e Hartley (1985) para Diatraea saccharalis (Lepidoptera: Crambidae), foram inoculados 70 μL de cada formulado comercial, na concentração 1 x 108 esporos·mL-1. Em seguida, avaliou-se a virulência dos respectivos formulados, isso através da estimativa da concentração letal (CL50) para o estádio de maior suscetibilidade. Em virtude dos resultados encontrados, observou-se que o estágio 1 de desenvolvimento apresentou 95,88% e 86,76% de mortalidades para os produtos Agree® e Dipel WP®, respectivamente, demonstrando patogenicidade e virulência à D. fovealis. No bioensaio para avaliar a atividade dos extratos aquosos de alho e fumo, estes foram aplicados na concentração 10% (m/v). Todos os tratamentos foram pulverizados com Torre de Potter, calibrada a pressão de 15 lb/pol². Posteriormente estimou-se a concentração letal (CL50) do extrato aquoso de fumo, o qual apresentou 95% de mortalidade no teste de suscetibilidade. Desta forma, com os resultados obtidos na presente pesquisa, concluiu-se que a utilização de formulados comerciais à base de B. thuringiensis e extrato de fumo podem ser uma alternativa no manejo fitossanitário de D. fovealis.

Soybean growth and yield after single tillage and species mixture of cover plants

The use of cover crops is important for the agricultural crop and soil management in order to improve the system and, consequently, to increase yield. Therefore, the present study analyzed the effect of crop residues of black oat (Avena strigosa Schreb.) (BO) and a cocktail (CO) of BO, forage turnip (Raphanus sativus L.) (FT) and common vetch (Vicia sativa L.) (V) on the emergence speed index (ESI), seedling emergence speed (SES) plant height and soybean yield in different intervals between cover crop desiccation with glyphosate 480 (3 L ha-1) and BRS 232 cultivar sowing. Plots of 5 x 2.5 m with 1 m of border received four treatments with BO cover crops and four with CO as well as a control for each cover crop, at random, with five replications. The plots were desiccated in intervals of 1, 10, 20 and 30 days before soybean seeding. The harvest was manual while yield was adjusted to 13% of moisture content. The experimental design was completely randomized with splitplots and means compared by the Scott and Knott test at 5% of significance. The results showed that CO of cover crops can be recommended for soybean to obtain a more vigorous seedling emergence, from 10 days after cover crop desiccation.

Maize nitrogen fertilization in two crop rotation systems under no-till

The objective of this study was to evaluate split nitrogen (N) fertilization of maize applied in band at sowing and top dressing with and without crop rotation, under no-till. The experiment was conducted with six N rates at sowing (0, 20, 30, 40, 50 and 60 kg ha-1) combined with three rates in top dressing (40, 70, 100 kg ha-1) and two management systems: after five cropping sequences of maize and crop rotation (maize + soybean + oat + soybean + corn) in a randomized block design with four replications. The crop rotation system increased yield in approximately 7% in relation to the area without rotation. The split of nitrogen fertilization, in rates above 39 and 54 kg ha-1 at sowing and 70 and 40 kg ha-1 in top dressing, resulted in yield higher than that obtained with the application of 100 kg ha-1 in top dressing. Grain yield was higher with the rates 50 and 70 kg ha-1 of N compared with that obtained with 20 and 100 kg ha-1 at sowing and top dressing, respectively. The rate 70 kg ha-1 of N resulted in the highest yield at the lowest cost compared with the revenues and costs incurred with the rates 40 and 100 kg ha-1.

Effect of hay on performance of Holstein calves at suckling and post-weaning

The objective of this work was to evaluate the performance of Holstein calves in suckling and post-weaning phases, intensively managed during suckling in the absence or presence of hay. Twenty-four male Holstein calves, at an average age of 15 days and initial weight of 43 kg were used in the experiment. The experimental design was completely randomized, consisting of two treatments and six replications. The treatments were as follows: 1) suckling with milk substitute + initial concentrate for calves, ad libitum + temperate grass hay (oat/ryegrass), ad libitum; 2) suckling with milk substitute + initial concentrate for calves, ad libitum. No significant difference was found between treatments for weight gain and feed conversion. However, the supply of hay caused an increase in daily dry matter intake (2.127 vs 1.894 kg). The intake of hay promoted greater stimulus to consumption of concentrate and greater weight at weaning.

Diversidade e prospecção de fungos endofíticos de Begonia spp. encontradas na Mata Atlântica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Are known cyanotoxins involved in the toxicity of picoplanktonic and filamentous north atlantic marine cyanobacteria?

Eight marine cyanobacteria strains of the genera Cyanobium, Leptolyngbya, Oscillatoria, Phormidium, and Synechococcus were isolated from rocky beaches along the Atlantic Portuguese central coast and tested for ecotoxicity. Strains were identified by morphological characteristics and by the amplification and sequentiation of the 16S rDNA. Bioactivity of dichloromethane, methanol and aqueous extracts was assessed by the Artemia salina bioassay. Peptide toxin production was screened by matrix assisted laser desorption/ionization time of flight mass spectrometry. Molecular analysis of the genes involved in the production of known cyanotoxins such as microcystins, nodularins and cylindrospermopsin was also performed. Strains were toxic to the brine shrimp A. salina nauplii with aqueous extracts being more toxic than the organic ones. Although mass spectrometry analysis did not reveal the production of microcystins or other known toxic peptides, a positive result for the presence of mcyE gene was found in one Leptolyngbya strain and one Oscillatoria strain. The extensive brine shrimp mortality points to the involvement of other unknown toxins, and the presence of a fragment of genes involved in the cyanotoxin production highlight the potential risk of cyanobacteria occurrence on the Atlantic coast.