Characterization of Achlya americana and A. bisexualis (Saprolegniales, Oomycota) Isolated from Freshwater Environments in Korea

Many members of the Saprolegniales (Oomycete) cause mycoses and disorders of fishes, of which Achlya and Saprolegnia are most ubiquitous genera worldwide. During a survey of the diversity of freshwater oomycetes in Korea, we collected seven isolates of Achlya, for which morphological and molecular phylogenetic analyses enabled them to identify as Achlya americana and Achlya bisexualis. In Korea, only a species of Achlya, A. prolifera, has been previously found to cause seedling rot on rice (Oryza sativa), but none of the two species have been reported yet. Importantly, A. bisexualis was isolated from a live fish, namely rice fish (Oryzias sinensis), as well as freshwater, and this is the first report of Achlya-causing mycoses on freshwater fishes in Korea. The presence of A. americana and A. bisexualis on live fish in Korea should be closely monitored, as considering the well-known broad infectivity of these species it has the potential to cause an important emerging disease on aquaculture industry.

Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus) / Isolamento do oomiceto Saprolegnia aenigmatica e protocolo de infecção experimental em pacu (Piaractus mesopotamicus)

Successful disease treatment depends on molecular studies under indoor conditions with experimental infection protocols that facilitate understanding the disease and the drug`s efficacy. The internal transcribed spacer (ITS) region was sequenced from three isolates, which were identified as Saprolegnia aenigmatica. Subsequently, healthy fish were immunosuppressed with dexamethasone (1.2 mg kg-1) and descaled to the skin using a sharp scalpel. These individuals were isolated in individual aquariums maintained at 22°C. Individuals in one group were subcutaneously inoculated with 9,000 zoospores (DDZ treatment), a second group was exposed to oomycetes in water with three colonized baits (DDB), a third group was maintained in water without zoospores (DD), and a control group (C) consisted of healthy animals. After 48 and 96 hours, two animals from each group were euthanized for fungal reisolation. The fish from groups DD and C did not show clinical signs, and no oomycetes were isolated. The animals from the DDZ and DDB groups showed cotton-wool-like masses on the skin, and S. aenigmatica was re-isolated. Thus, for infection using zoospores or baits parasitized by S. aenigmatica, an immunosuppressor (dexamethasone) and a sharp scalpel can be used effectively to establish an experimental infection in P. mesopotamicus.

O sucesso do tratamento de uma enfermidade depende do estudo de moléculas em condições de laboratório por meio de protocolos de infecção experimental que viabilizam a compreensão da doença e da eficácia dos fármacos. Pela sequência ITS foram identificadas três cepas de Saprolegnia aenigmatica. Dessa forma, pacus sadios foram submetidos à imunossupressão com dexametasona (1,2 mg kg-1), esfoliados com auxílio de bisturi e distribuídos em aquários a 22ºC. Após este procedimento, um grupo de animais foi inoculado com 9.000 zoósporos/peixe subcutâneo (DEZ), a outro foram adicionadas três iscas colonizadas com o oomiceto na água (DEI), um terceiro grupo foi mantido sem contato com o oomiceto (DE) e um quarto grupo, de animais sadios, representaram o controle (C). Após 48 e 96h deste procedimento, foram eutanaziados animais de cada grupo para reisolamento. Os animais do grupo DE e C não apresentaram sinais clínicos e não foi reisolado o oomiceto. Porém, tanto os animais do grupo DEZ quanto como os animais do grupo DEI apresentaram micélio branco na pele e foi reisolado Saprolegnia aenigmatica. Assim, a infecção com zoósporos ou com iscas colonizadas por S. aenigmatica, com o uso de dexametasona e abrasivo epitelial são formas eficazes de infecção experimental em P. mesopotamicus.

Saprolegnia parasitica Isolated from Rainbow Trout in Korea: Characterization, Anti-Saprolegnia Activity and Host Pathogen Interaction in Zebrafish Disease Model

Saprolegniasis is one of the most devastating oomycete diseases in freshwater fish which is caused by species in the genus Saprolegnia including Saprolegnia parasitica. In this study, we isolated the strain of S. parasitica from diseased rainbow trout in Korea. Morphological and molecular based identification confirmed that isolated oomycete belongs to the member of S. parasitica, supported by its typical features including cotton-like mycelium, zoospores and phylogenetic analysis with internal transcribed spacer region. Pathogenicity of isolated S. parasitica was developed in embryo, juvenile, and adult zebrafish as a disease model. Host-pathogen interaction in adult zebrafish was investigated at transcriptional level. Upon infection with S. parasitica, pathogen/antigen recognition and signaling (TLR2, TLR4b, TLR5b, NOD1, and major histocompatibility complex class I), pro/anti-inflammatory cytokines (interleukin [IL]-1β, tumor necrosis factor α, IL-6, IL-8, interferon γ, IL-12, and IL-10), matrix metalloproteinase (MMP9 and MMP13), cell surface molecules (CD8⁺ and CD4⁺) and antioxidant enzymes (superoxide dismutase, catalase) related genes were differentially modulated at 3- and 12-hr post infection. As an anti-Saprolegnia agent, plant based lawsone was applied to investigate on the susceptibility of S. parasitica showing the minimum inhibitory concentration and percentage inhibition of radial growth as 200 μg/mL and 31.8%, respectively. Moreover, natural lawsone changed the membrane permeability of S. parasitica mycelium and caused irreversible damage and disintegration to the cellular membranes of S. parasitica. Transcriptional responses of the genes of S. parasitica mycelium exposed to lawsone were altered, indicating that lawsone could be a potential anti-S. parasitica agent for controlling S. parasitica infection.

Novel antimicrobial activity of a dichloromethane extract obtained from red seaweed Ceramium rubrum (Hudson) (Rhodophyta: Florideophyceae) against Yersinia ruckeri and Saprolegnia parasitica, agents that cause diseases in salmonids

Background Enteric red mouth disease and Saprolegniasis, which are caused by the bacteria Yersinia ruckeri and the oomycete Saprolegnia parasitica, respectively, are important illnesses that affect salmonid farming. Sanitary problems in farms are addressed by the prevention of disease outbreaks or by the treatment of diseases with chemicals. Environmental and governmental restrictions, toxicity and high treatment costs limit the use of drugs. Marine organisms, such as algae, sponges and corals, have developed an antimicrobial defense strategy based on the production of bioactive metabolites. Among these organisms, seaweeds offer a particularly rich source of potential new drugs. Hence, many pharmacologically active substances have been isolated from seaweeds. In the Ceramium genus, Ceramium rubrum has been emphasized by several authors for its antimicrobial properties. Based on this background, the present study focused on the antimicrobial activity of a lipophilic extract of C. rubrum on Y. ruckeri and S. parasitica. Results The alga, collected from the Pacific coast of Chile, underwent an ethanol extraction, and the concentrated extract was partitioned between water and dichloromethane. From the dichloromethane extract, fatty acids, fatty acid esters, one hydrocarbon and phytol were identified by Gas Chromatography-Mass Spectrometry (GC/MS) analysis. The antimicrobial study showed that the whole extract was more active than the individual components, which suggests a strong synergistic effect among the components. Conclusions These results may constitute a basis for promising future applied research that could investigate the use of C. rubrum seaweed as a source of antimicrobial compounds against fish pathogens.

First record of Saprolegnia sp. in an amphibian population in Colombia / Primer registro de Saprolegnia sp. en una población de anfibios en Colombia / Primeiro registro de Saprolegnia sp. numa população de anfíbios na Colômbia

Primer registro de Saprolegnia sp. en una población de anfibios en Colombia. Objetivo. La mayoría de investigaciones en relación al declive de anfibios se han enfocado en la detección del hongo patógeno Batrachochytrium dendrobatidis. Este es el principal patógeno detectado en el mundo. Sin embargo, otras investigaciones han demostrado la presencia de Saprolegnia ferax como un vector mortal en anfibios de Norteamérica. Este trabajo establece una posible transmisión entre especies causada por la presencia de la trucha arcoíris, sugiriendo que el declive de anfibios no solo se atribuye a la presencia de un solo patógeno, sino que puede ser causado por otros patógenos y factores. Materiales y métodos. El estudio revela la presencia de Saprolegnia sp. en la rana de los Andes Atelopus mittermeieri mediante la técnica de impronta con tinción de azul de lactofenol, que permitió la observación de las estructuras típicas de este hongo. Resultados. La importancia de este descubrimiento es la presencia de dos hongos patógenos, B. dendrobatidis y Saprolegnia, afectando simultáneamente una población de anfibios. Este hallazgo enfoca la atención en la eventual presencia de otros microorganismos que podrían estar involucrados individualmente o colectivamente en el declive de especies de anfibios. Conclusiones. Este registro sugiere una posible transmisión entre la trucha arcoiris (Oncorhynchus mykiss), una especie introducida en las montañas de Colombia, que comparte los mismos habitats con diferentes especies de anfibios en el Santuario de Flora y Fauna de Guanentá en el alto río Fonce en la parte media de la Cordillera Oriental de Colombia...

Objective: Most research related to the decline of amphibians has been focused on the detection of the pathogenic fungus Batrachochytrium dendrobatidis. This fungus is the main pathogen detected around the world. However, research has shown the presence of another fungus, Saprolegnia ferax, as a cause of mortality in amphibians in North America. Our study suggests a possible interspecific transmission caused by the presence of rainbow trout; thus, amphibian declines may not be attributable only to the presence of a single pathogen, but to other organisms and factors. Materials and methods. Our study revealed the presence of Saprolegnia sp. in the Andean frog Atelopus mittermeieri using the imprinting technique with lactophenol blue staining, which allowed the typical structures of this fungus to be observed. Results. The importance of this discovery is the presence of two pathogenic fungi, B. dendrobatidis and Saprolegnia, which affecting simultaneously a population of amphibians. This finding brings attention to the eventual presence of other microorganisms that might be involved individually or collectively in the decline of amphibian species. Conclusions. This record suggests a possible transmission between rainbow trout (Oncorhynchus mykiss), an introduced species in the highlands of Colombia, which shares the same habitats with different species of amphibians in the Sanctuary of Flora and Fauna Guanentá in the upper river Fonce in the mid Cordillera Oriental of Colombia...

Primeiro registro de Saprolegnia sp. numa população de anfíbios na Colômbia. Objetivo. A maioria das pesquisas sobre o declínio dos anfíbios tem-se centrado na detecção do fungo patogênico Batrachochytrium dendrobatidis. Este é o principal patógeno encontrado no mundo. No entanto, outros estudos têm demonstrado a presença de Saprolegnia ferax, como vetor mortal de anfíbios de América do Norte. Esta pesquisa estabelece uma possível transmissão entre espécies causada pela presença de truta arco-íris, afirmando que o declínio de anfíbios não só é atribuído à presença de um único patógeno, pudendo ser causado por outros patógenos e fatores. Materiais e métodos. A pesquisa indica a presença de Saprolegnia sp. em rãs dos Andes, Atelopus mittermeieri, pela técnica de impressão com lactofenol-azul-de-algodão, onde observou-se as estruturas típicas deste fungo. Resultados. A importância desta descoberta é a presença de dois fungos (B. dendrobatidis e Saprolegnia) que simultaneamente afetam uma população de anfíbios, concentrando a atenção na presença de outros microorganismos que podem estar envolvidos individualmente ou coletivamente no declínio das espécies de anfíbios. Conclusões. A truta arco-íris (Oncorhynchus mykiss), uma espécie introduzida nas montanhas da Colômbia, que compartilha o mesmo nicho ecológico com Atelopus mittermeieri, no Santuário de Flora e Fauna de Guanentá no Alto Rio Fonce no centro da Cordilheira Oriental da Colômbia, tem sido sugerida por esta pesquisa como o agente transmissor dos fungos as diferentes espécies de anfíbios...

Detection of Infectious Fungal Diseases of Frogs Inhabiting in Korea

In recent years, there has been a rapid decrease in amphibian populations worldwide, and infectious diseases have been associated with this decline. Diseased frogs inhabiting Korea were collected from fields, and the diseases were identified by morphological and molecular analyses. Two fungal diseases-saprolegniasis and chromomycosis-were detected in the frogs. Saprolegniasis caused by Saprolegnia spp. was found in Rana plancyi chosenica from Gangwon-do and Rana huanrenensis from Chungbuk. Chromomycosis, which is caused by infection with Cladosporium cladosporioides, was detected in Rana catesbeiana from Busan.

Removal Efficiency of the Heavy Metals Zn(II), Pb(II) and Cd(II) by Saprolegnia delica and Trichoderma viride at Different pH Values and Temperature Degrees

The removal efficiency of the heavy metals Zn, Pb and Cd by the zoosporic fungal species Saprolegnia delica and the terrestrial fungus Trichoderma viride, isolated from polluted water drainages in the Delta of Nile in Egypt, as affected by various ranges of pH values and different temperature degrees,was extensively investigated. The maximum removal efficiency of S. delica for Zn(II) and Cd(II) was obtained at pH 8 and for Pb(II) was at pH 6 whilst the removal efficiency of T. viride was found to be optimum at pH 6 for the three applied heavy metals. Regardless the median lethal doses of the three heavy metals, Zn recorded the highest bioaccumulation potency by S. delica at all pH values except at pH 4, followed by Pb whereas Cd showed the lowest removal potency by the fungal species and vice versa in case of T. viride. The optimum biomass dry weight production by S. delica was found when the fungus was grown in the medium treated with the heavy metal Pb at pH 6, followed by Zn at pH 8 and Cd at pH 8. The optimum biomass dry weight yield by T. viride amended with Zn,Pb and Cd was obtained at pH 6 for the three heavy metals with the maximum value at Zn. The highest yield of biomass dry weight was found when T. viride treated with Cd at all different pH values followed by Pb whilst Zn output was the lowest and this result was reversed in case of S. delica. The maximum removal efficiency and the biomass dry weight production for the three tested heavy metals was obtained at the incubation temperature 20degrees C in case of S. delica while it was 25degrees C for T. viride. Incubation of T. viride at higher temperatures (30degrees C and 35degrees C) enhanced the removal efficiency of Pb and Cd than low temperatures (15degrees C and 20degrees C) and vice versa in case of Zn removal. At all tested incubation temperatures, the maximum yield of biomass dry weight was attained at Zn treatment by the two tested fungal species. The bioaccumulation potency of S. delica for Zn was higher than that for Pb at all temperature degrees of incubation and Cd bioaccumulation was the lowest whereas T. viride showed the highest removal efficiency for Pb followed by Cd and Zn was the minor of the heavy metals.

Utilização de bioindicadores em diferentes hidrossistemas de uma indústria de papeis reciclados em Governador Valadares - MG / Use of biological indicators in different hydrosystems of an industry of recycled papers in Governador Valadares - MG

A bacia do rio Doce, quinta maior do estado de Minas Gerais, mostra-se bastante degradada com impactos diretos na vegetação, no solo, na biodiversidade e na qualidade das águas. Sabendo-se da importância da bacia em questão, a utilização de bioindicadores aquáticos representa uma das formas mais modernas para se detectar níveis diferenciados de carga orgânica, sendo premente seu estudo e aplicação. Este trabalho propõe a utilização de organismos microscópicos como o fitoplâncton que, aliados a parâmetros físico-químicos, poderiam indicar poluição em seus diferentes níveis. A metodologia utilizada foi estabelecida conforme Sladecek (1973) e Greenberg (1992). Foram amostradas sete estações de coleta no ribeirão Capim, na área de influência de uma empresa de papéis reciclados, objetivando-se detectar possíveis impactos na cadeia trófica. As análises mostraram a ocorrência de 41 taxa, destacando-se a predominância dos gêneros Oscylatoria a Anacystis pertencentes à divisão Cyanophyta nos ambientes de maior estresse, e organismos da divisão Chlorophyta nos demais ambientes amostrados. Observou-se ainda a capacidade de adaptação de alguns gêneros, entre eles Euglena e Navicula, os quais se fizeram presentes em quase todos os ambientes amostrados.

The Rio Doces basin, which is the fifth greatest basin of the state of Minas Gerais, Brazil, is severely degraded by the direct impacts on vegetation, soil, biodiversity, and water quality. Due to the importance of this basin, the use of aquatic bioindicators is one of the most modern methods to detect differentiated levels of organic matter, and its study and application is a relevant issue. This paper proposes the use microscopic organisms such phytoplankton, which combined with physicochemical parameters, would indicate pollution on its different levels. The applied methodology was according to Sladecek (1973) and Greenberg (1992). It was sampled seven collect stations on the Capim stream, in an area under the influence of a recycling paper industry, being the objective to assess the possible impacts on the trophic chain. The analysis presented the existence of 41 taxa, emphasizing the predominance of Oscilatoria and Anacystis genus, that belong to the Cyanophyta division, on more disturbed areas, while organisms from the Chlorophyta division were identified on the other sampled locations. Moreover, it was observed the ability that some genus like the Euglena and Navicula have to adapt to their environment, since these organisms were present on almost all samples.

Effects of Some Amino Acids on Ammonia Secretion and Extracellular Protease Activity by Three Oomycetes in Synthetic Medium with or without Glucose

The effects of different concentrations of three amino acids as carbon and or nitrogen sources on mycelial dry weights, changes in pH values of synthetic medium, ammonia secretion and extracellular protease activity by three zoosporic fungi, pathogens of fish and shellfish, were studied. As compared with the control, the addition of isoleucine and aspartic acid as nitrogen sources were generally stimulative for mycelial dry weight production whereas phenylalanine was inhibitory irrespective to the tested fungal species. When amino acids served as carbon and nitrogen sources, the mycelial dry weights of the three fungi were increased (mostly non-significantly) relative to untreated control but weights were decreased as the concentrations of the three amino acids raised. The addition of individual amino acids as carbon and nitrogen sources to the medium significantly increased pH values of the medium comparable to the control. The addition of each of the three amino acids as carbon and nitrogen sources to the medium significantly induced ammonia secretion by the three species of zoosporic fungi. Ammonia secretion in synthetic medium amended with amino acids as nitrogen source raised by the three zoosporic fungi relative to untreated control except in case of Achlya racemosa treated with isoleucine. Extracellular protease activity was almost promoted in case of Achlya proliferoides and Saprolegnia furcata cultures treated with isoleucine and aspartic acid individually in presence of glucose and vice versa in case of phenylalanine. However, extracellular protease activity of A. racemosa decreased compared with the control at various concentrations of isoleucine and both phenylalanine and aspartic acid assumed inconsistent effects. Extracellular protease activity of the three zoosporic fungi in the medium devoid of glucose varied depending upon zoosporic fungal species, the tested amino acid and the applied concentrations. The values of protease activity were approximately less two folds than that obtained in presence of glucose.

Response of Achlya racemosa, A. proliferoides and Saprolegnia furcata to Sub-lethal Treatments of Amino Acids

The effect of four sub-lethal concentrations (400, 800, 1,200 and 1,600 microg/ml) of three amino acids such as isoluecine, aspartic acid and phenylalanine on vegetative growth and sexual and asexual reproduction of Achlya racemosa, A. proliferoides and Saprolegnia furcata was investigated. The density of vegetative growth and diameters of vegetative colonies of species of the Oomycetes fungi decreased with rising the concentration of the applied amino acid. Vegetative hyphae of treated fungi almost appeared branched in case of S. furcata, thick in case of A. racemosa and distorted in case of A. proliferoides as compared with control. The different treatments with amino acids depressed both sporangial formation and discharge, which were dependent on the tested species of zoosporic fungi, the amino acid and its dosage. Phenylalanine was the most effective amino acid in inhibiting sporulation and S. furcata was the most sensitive fungal species. Aspartic acid and isoleucine stimulated germination of discharged spores through the formation of germlings. Gemmae formation by the three fungi was reduced at the low concentrations of amino acids and nearly missed at high concentrations. Sex organs (oogonia and antheridia) were affected partly; rudiment oogonia were observed at low concentrations (400 and 800 microg/ml) and disappeared at higher concentrations, whereas antheridial branch formation was stimulated as the fungi were treated with isoleucine and to some extent phenylalanine.