Obtaining monokaryotic and dikaryotic mycelial cultures of two Amazonian strains of Geastrum (Geastraceae, Basidiomycota)

The high diversity of the genus Geastrum and the difficulty of obtaining mycelial cultures impairs the study of the ecophysiology and the exploration of the biotechnological potential of the taxon. In this study, different culture media were tested to obtain mycelial cultures for G. lloydianum and G. subiculosum collected in the Brazilian Amazon. Data on spore germination, and isolation of monokaryotic cultures and in vitro sexual reproduction are presented, as well as a brief morphological description of the cultures obtained. For both species, Potato Dextrose Agar (PDA) was the most promising of the tested culture media. The highest growth in agar culture ever recorded for this genus is reported (4.9 mm per week for G. lloydianum and 7.5 mm for G. subiculosum). In the PDA culture medium, spores germinated after 35-40 days of incubation and the isolation of monokaryotic cultures of the two species, as well as in vitro sexual crosses, were successfully performed. (AU)

Chemical composition and antifungal activity of propolis on Aspergillus flavus / Composição química e atividade antifúngica de própolis sobre Aspergillus flavus

Research has indicated the antifungal activity of ethanol extracts from propolis (EEP) on fungi of the genera Phakopsora, Colletotrichum, and Cercospora. Here, chemical compositions and antioxidant activity of three EEP (propolis from Scaptotrigona polysticta stingless bee and two types produced by Apis mellifera - red and brown) were evaluated and their action against Aspergillus flavus was investigated. Ash, dry extract, total phenolic and total flavonoid contents were determined. Phenolic composition was evaluated by high performance liquid chromatography, with using the following reference substances: gallic acid, caffeic acid, -coumaric acid, ferulic acid, quercetin, kaempferol, and apigenin. Tests on mycelial growth, sporulation, and germination of spores were performed to assess the biological activity of the EEP on A. flavus. All EEP showed low dry extract content (<11%) with adequate amounts of ash (<5%). The red EEP (Apis mellifera) showed the highest contents of total phenolic and total flavonoid (5.38 and 2.77 g 100 g-1), while the highest recorded antioxidant activity was exhibited by brown EEP (92.9%). The EEP of S. polysticta presented higher levels of -coumaric acid (10.99 mg g-1), while red and brown extracts from A. mellifera stood out with the highest levels of quercetin (27.26 mg g-1) and gallic acid (5.88 mg g-1), respectively. No extract was effective in inhibiting mycelial growth and sporulation of A. flavus, but red EEP inhibited spore germination. On the basis of these results, it is suggested that the inhibitory effect of red EEP on spore germination may be associated with increased levels of flavonoids found in it as compared with the other extracts investigated here.

Outros estudos apontaram a ação antifúngica dos extratos etanólicos de própolis (EEP) sobre fungos do gênero Phakopsora, Colletotrichum e Cercospora. Neste estudo, avaliou-se a composição química e a atividade antioxidante de três EEP (própolis produzido pela abelha sem ferrão Scaptotrigona polysticta e dois tipos de própolis produzida pela Apis melífera: marrom e vermelha) e sua ação contra o Aspergillus flavus. Foram determinados os teores de cinzas, extrato seco, fenólicos e flavonoides totais nos EEP. A composição fenólica foi determinada por cromatografia líquida de alta eficiência, empregando-se as seguintes substâncias de referência: ácido gálico, ácido p-cumárico, ácido ferrúlico, quercitina, kaempferol e apigenina. Para avaliar a atividade biológica dos EEP sobre A. flavus foram realizados os testes de crescimento micelial, esporulação e germinação de esporos. Todos os EEP apresentaram baixos teores de extrato seco (< 11%), porém quantidades de cinzas adequadas (< 5%). O EEP vermelha apresentou os maiores teores de fenólicos e flavonoides totais (5,38 e 2,77 g 100g-1), enquanto que a maior atividade antioxidante registrada foi do EEP marrom (92,9%). O EEP de S. polysticta apresentou maiores teores de ácido p-cumárico (10,99 mg g-1), enquanto que os de A. mellifera, vermelha e marrom, destacaram-se com os maiores níveis de quercetina (27,26 mg g-1) e de ácido gálico (5,88 mg g-1), respectivamente. Nenhum extrato foi eficaz na inibição do crescimento micelial e esporulação de A. flavus, porém observou-se inibição na germinação de esporos pelo EEP vermelha. Sugere-se que o efeito inibitório na germinação de esporos pode estar relacionado com os maiores níveis de flavonoides encontrados na própolis vermelha, quando comparado com os outros extratos investigados.

Palynological analysis of Dennstaedtiaceae taxa from the Paranaense Phytogeografic Province that produce monolete spores and its systematic implications (I): Blotiella lindeniana, Histiopteris incisa and Paesia glandulosa

ABSTRACT The genera of Dennstaedtiaceae have sporophytes with very different morphological characteristics between each other, and this feature has made difficult the systematic circumscription of the family. This reason makes necessary the study of new characters that allow a better understanding of the relations within the group. The spore morphology and wall structure of Blotiella lindeniana, Histiopteris incisa and Paesia glandulosa from the Paranaense Phytogeographic Province were studied using light microscope, and scanning and transmission electron microscope. The exospore has two layers and, according to the species, the exospore surface bears pila, echinae, verrucae, bacula and tubercles. The perispore has two or three layers and its surface is psilate, baculate or rugulate. The variability found in the sculpture of the spores and their stratification and ultrastructure of perispore reflects the morphological differences observed in the sporophyte of the species studied. Additionally, while the stratification and ultrastructure of the exospore is shared by the Dennstaedtiaceae species, their ornamentation could be a character to distinguish species into the clade "hypolepidoide". The finding of spores with similar characteristics in phylogenetically unrelated families allows us to suggest that palynological features do not have an evolutionary value to establish relationships between groups above the genus level.

ÁNTRAX: enfermedad aún vigente / ANTHRAX: disease still current

El ántrax es una zoonosis producida por Bacillus anthracis, único miembro del género Bacillus que es capaz de causar enfermedad epidémica en humanos y otros mamíferos. Afecta principalmente a los animales herbívoros domésticos y silvestres. Los humanos son hospederos accidentales y se infectan por contacto directo o indirecto con animales o productos contaminados. Las esporas pueden vivir en el suelo por años y los humanos pueden contraer el ántrax al tener contacto con animales infectados, productos provenientes de estos que al consumir carne infectada; esto se presenta principalmente en países poco desarrollados donde los niveles de vacunación animal contra esta enfermedad son bajos. Este escrito tiene por objetivo presentar una revisión sobre el tema, especialmente sobre aspectos como el modo de infección, manifestaciones clínicas, diagnóstico y tratamiento de la enfermedad.

Anthrax is a zoonosis produced by Bacillus anthracis, the only member of the genus Bacillus that is capable of causing epidemic disease in humans and other mammals. It mainly affects wild and domestic animals. Humans are accidental hosts and are infected through direct or indirect contact with animals or contaminated animal products. B. anthracisspores can live in the soil for many years and humans can become infected with anthraxby contact with infected animals or products contaminated from eating meat infected. This disease occurs mainly in developing countries where vaccine levels are low.The objective of this paper is to present a review on thesubject, especially on aspects such as mode ofinfection, clinical features diagnostic assessment and treatment of thedisease.

Criptococosis congénita en un neonato expuesto a VIH: presentación de un caso / Congenital cryptococcosis in a newborn exposed to HIV: A case report

La criptococosis puede afectar niños de todas las edades, especialmente aquellos inmunocomprometidos. Usualmente se adquiere a través de la inhalación de esporas del medio ambiente, aunque existen otras formas de transmisión. Describimos un caso de criptococosis congénita adquirido de una madre con síndrome de inmunodeficiencia adquirida (SIDA) y tratado en forma exitosa con combinación de antimicoticos.

Cryptococcosis may affect children of all ages, specially those who are inmunocompromised. It is usually acquired from the inhalation of environmental spores, although other sources of transmission exist. We describe a case of congenital cryptococcosis transmitted from a mother with acquired immunodeficiency syndrome (AIDS), which was successfully treated with combination antifungal agents.

Cumplimiento de normas microbiológicas en productos alimenticios comercializados en la red de mercados de alimentos del estado Nueva Esparta, Venezuela (período 2006-2014) / Compliance of microbiological norms in food products commercialized in the markets of Nueva Esparta state, Venezuela during the years 2006 to 2014

El suministro de suficientes alimentos es prioritario para garantizar la seguridad alimentaria de una población. También es importante corroborar que sean de calidad e inocuos, para que no causen daño al consumirlos cuando se preparen de acuerdo al uso previsto. Esta investigación verifica si 101 lotes (370 unidades de muestra) de alimentos envasados (harina de maíz, leche en polvo, pasta alimenticia, mortadela, salchicha cocida, carne congelada, pollo congelado, leche UHT, atún enlatado y carne de almuerzo) comercializados en el Estado Nueva Esparta durante los años 2006 a 2014, cumplían requisitos microbiológicos indicados por normas venezolanas. Las muestras fueron captadas por Inspectores de Salud Pública (MPPS). Para los análisis se aplicaron procedimientos de normas COVENIN y FONDONORMA. Cumplieron los requisitos considerados todas las muestras de pasta alimenticia, mortadela, pollo congelado, atún en conserva y carne de almuerzo. Sin embargo, dos lotes de leche UHT y otros dos de carne congelada presentaron recuentos de aerobios mesófilos superiores a las estipulaciones en alguna de las unidades analizadas. Por otra parte, tres de los nueve lotes de salchichas cocidas incumplieron alguno de los requisitos con carácter de recomendación, igualmente 5 de los 16 lotes de harina de maíz (recuentos de mohos) y 10 de los 17 de leche en polvo (recuentos de esporas termófilas). Se concluye que estos incumplimientos pudieran estar ocasionados por fallas de control durante la cadena de producción, re-envasado, almacenamiento o comercialización bien sea de materias primas o de productos terminados.

Enough food supply is a priority to guarantee the food security of a country´s residents. It is also important to supervise that they are harmless for human consumption. This study verifies if 370 packed foods (corn flour, powdered milk, spaghetti, bologna, frozen meat and chicken, pork sausage, UHT milk, canned tuna and luncheon meat), that belonged to 101 production lots commercialized during the years 2006 to 2014 in Nueva Esparta state (Venezuela), fulfill microbiological specifications indicated by Venezuelan norms. Samples were picked up by public health inspectors. For analysis the COVENIN and FONDONORMA procedures were used. All the spaghetti, bologna, frozen chicken, canned tuna and luncheon meat samples complied with the norms. Nevertheless, two UHT milk´s production lots and other two of frozen meat showed total aerobic plate counts higher than the recommended limits. By the other hand, 9 pork sausage´s production lots failed to fulfill a few norms, just as 5 of 16 corn flour lots (molds counts), and 10 of 17 powdered milk production lots (thermophiles spore counts). In conclusion, the nonfulfillment of norms detected, particularly in corn flour and powdered milk samples, are caused by not performing proper controls during the production chain and commercialization of raw materials or finished products.

Frações de fósforo e simbiose micorrízica em floresta secundária em resposta a disponibilidade de água e nutrientes na Amazônia oriental / Soil phosphorus fractions and mycorrhizal symbiosis in response to the availability of moisture and nutrients at a secondary forest in eastern Amazonia

The availability of soil nutrients (especially phosphorus) and soil water strongly influence mycorrhizal symbiosis in forest ecosystems. The aim of this study was to evaluate the impact of litter removal and increased soil moisture availability on the spore density and mycorrhizal colonization of apogeotropic and non-apogeotropic roots of a secondary forest stand in eastern Amazonia. We determined the percentage of mycorrhizal colonization of apogeotropic and non-apogeotropic (0-10 cm soil depth) fine roots (diameter 2 mm), spore density, glomalin concentration, and soil phosphorus availability. Litter removal did not reduce soil phosphorus availability. The spore density was not affected by litter removal. In general, the variables varied on a seasonal basis, except the mycorrizal colonization of non-apogetropic roots and glomalin concentration, but were not affected by irrigation. Mechanical damage to the apogeotropic root system, inherent to the fortnightly litter removal, may have contributed to decrease mycorrhizal colonization of both apogeotropic and non-apogeotropic roots and, consequently, soil glomalin. Our results suggest that the reduction of soil cover may have negative impact over the fungus-plant symbiosis.

A disponibilidade de nutrientes, especialmente fósforo, e de água no solo influenciam fortemente a simbiose micorrízica em ecossistemas florestais. O objetivo do estudo foi avaliar o impacto da remoção de serapilheira e do aumento da disponibilidade de água no solo sobre a densidade de esporos e a colonização micorrízica em raízes apogeotrópicas e não-apogeotrópicas em floresta secundária na Amazônia oriental. Foram analisadas a porcentagem de colonização micorrízica de raízes finas (diâmetro ≤ 2 mm) apogeotrópicas e não-apogeotrópicas (presentes na camada de 0-10 cm do solo), a densidade de esporos, o teor de glomalina e a disponibilidade de fósforo no solo. A remoção de serapilheira não reduziu a disponibilidade de fósforo no solo. A densidade de esporos também não foi afetada pela remoção de serapilheira. De forma geral, as variáveis estudadas variaram sazonalmente, com exceção da colonização micorrízica em raízes não-apogeotrópicas e do teor de glomalina, mas não foram afetadas pela alteração na disponibilidade de água no solo decorrente da irrigação. Danos mecânicos ao sistema radicular apogeotrópico, inerentes à remoção quinzenal da serapilheira, devem ter contribuído para reduzir a colonização micorrízica em raízes apogeotrópicas e não-apogeotrópicas e, consequentemente, o teor de glomalina no solo. Os resultados deste estudo sugerem que a redução da cobertura do solo pode impactar negativamente a simbiose fungo-planta.

Lead tolerance and accumulation in initial sporophytes of Regnellidium diphyllum Lindm. (Marsileaceae) / Tolerância e acumulação de chumbo em esporófitos jovens de Regnellidium diphyllum Lindm. (Marsileaceae)

Regnellidium diphyllum Lindm. is a heterosporous fern which grows in shallow waters and wetlands, and water pollution contributes to its vulnerability. Environmental lead contamination is mostly caused by industrial and agricultural residues as well as domestic sewage. Given its persistence in the environment, lead can cause important toxicity in living organisms. Megaspore germination and the initial growth of R. diphyllum sporophytes were assessed in Meyer's solution with lead nitrate (Pb(NO3)2) concentrations of 0 (control), 1, 5, 10 and 50 mg L–1. The study was conducted in a growth chamber at 25±1°C and a 12 hour photoperiod with a nominal irradiance of 100 μmol m–2 s–1, for 28 days. Lead concentration in sporophytes was assessed using atomic absorption spectrometry. In the absence of lead, 74% of spores germinated, while significantly lower germination percentages were observed in Pb(NO3)2 concentrations of 1, 10 and 50 mg L–1. The presence of lead did not significantly influence root growth. At 28 days, primary leaf development was significantly lower in Pb(NO3)2 concentrations of 5 mg L–1 and higher in relation to the control. The length of secondary leaves did not significantly differ between sporophytes exposed to different concentrations of lead and those of the control at 28 days. Sporophytes exposed to 10 and 50 mg L–1 Pb(NO3)2 accumulated 1129 mg kg–1 and 5145 mg kg–1 of Pb, respectively. The presence of high levels of lead in R. diphyllum sporophytes did not prevent initial development. Future studies should investigate the ability of the species to accumulate and tolerate high levels of lead in advanced stages of its development and in environmental conditions.

Regnellidium diphyllum Lindm. é uma samambaia heterosporada que se desenvolve em águas rasas ou em solos úmidos, sendo que a poluição da água contribui para sua vulnerabilidade. A contaminação ambiental por chumbo ocorre principalmente por resíduos industriais e agrícolas, bem como por efluentes domésticos. Devido à sua persistência no ambiente, esse metal pode apresentar importante toxicidade aos organismos vivos. A germinação de megásporos e o desenvolvimento inicial de esporófitos de R. diphyllum foram avaliados em solução de Meyer com concentrações de 0 (controle), 1, 5, 10 e 50 mg L–1 de nitrato de chumbo (Pb(NO3)2). O estudo foi conduzido em câmara de germinação a 25±1 °C e fotoperíodo de 12 horas sob irradiância nominal de 100 μmol m–2 s–1, por 28 dias. A concentração de chumbo em esporófitos foi analisada por espectrometria de absorção atômica. Na ausência de chumbo, 74% dos esporos germinaram, enquanto que porcentagens de germinação significantemente menores foram observadas nas concentrações de 1, 10 e 50 mg L–1 de Pb(NO3)2. A presença de chumbo não influenciou significativamente o crescimento das raízes. O desenvolvimento das folhas primárias foi significativamente menor em relação ao controle a partir de 5 mg L–1 de Pb(NO3)2 aos 28 dias. O comprimento das folhas secundárias não diferiu significativamente entre esporófitos expostos às diferentes concentrações de chumbo e aqueles do controle, aos 28 dias. Esporófitos expostos a 10 e 50 mg L–1 de Pb(NO3)2 acumularam 1129 mg kg–1 e 5145 mg kg–1 de Pb, respectivamente. A presença de altas concentrações de chumbo nos esporófitos de R. diphyllum não impediu seu desenvolvimento inicial. Estudos futuros deverão investigar a capacidade de a espécie acumular e tolerar altas concentrações de chumbo em estádios avançados de desenvolvimento e também em condições ambientais.

Caracterización de la microbiota de leche ultra alta temperatura (UAT, UHT) analizada en Bogotá / Characterization of the microbiota of ultra-high temperatura (UHT) milk scrutinized in Bogotá / Caracterização da microbiota no leite ultra-alta temperatura (UAT) analisada em Bogotá

En los últimos años, las leches ultra alta temperatura (UAT, o UHT por sus siglas en inglés) ­definidas en el Decreto 616 de 2006­ analizadas en el Laboratorio de Salud Pública de Bogotá han venido presentando resultados no satisfactorios respecto a la prueba de esterilidad comercial, debido a que se han detectado microorganismos productores de esporas altamente resistentes a los tratamientos de ultrapasteurización (HHRS). A escala mundial, se informa como principal causa de esta clase de contaminación al Bacillus sporothermodurans. Con este estudio se determinó si las leches UAT de distribución en Bogotá presentan la misma contaminación detectada por bacterias HHRS del género Bacillus, y principalmente B. sporothermodurans. Las muestras de leches UAT que ingresaron al Laboratorio de Salud Pública de Bogotá se utilizaron para análisis microbiológico, como parte de la vigilancia rutinaria realizada por el Hospital de Fontibón durante 2010 y 2011. Como resultado de la prueba de esterilidad comercial aplicada a las leches UAT se recuperaron 36 cepas de muestras analizadas durante 2010-2011, a las que se les realizó la identificación bioquímica con el test BCL de Biomerieux, en el equipo Vitek ®2 Systems. Los microorganismos recuperados morfológicamente correspondieron a bacilos grampositivos, pertenecientes al género Bacillus, así: Bacillus smitii, 66,67 %; Bacillus sphaericus/fusiformis, 8,33 %; Bacillus choshinensis y Geobacillus stearothermophilus, 8,33 %. Esto permite concluir que no toda la contaminación de las leches UAT (UHT) analizadas en Bogotá se debe a una sola especie de Bacillus; por tanto, no se debe enfocar únicamente en B. sporothermodurans como contaminante único de leches higienizadas a escala nacional

In recent years, ultra-high temperature milk categories (enforced by Decree 616 of 2006 and scrutinized by Public Health Laboratory of Bogota) have reported unsatisfactory results regarding a commercial sterility test because of highly heat-resistant spores ­HHRS­ produced by microorganisms. Globally, Bacillus sporothermodurans has reported as the main cause of this kind of pollution. This study established whether these delivery milks in Bogota have the same pollution detected by HHRS bacteria of the genus Bacillus and mainly B. sporothermodurans. For this purpose, we used microbiological samples as a portion of the repetitive surveillance performed by the Hospital de Fontibon during 2010 and 2011, division of Environment and Consumer Surveillance. Because of the commercial sterility test, 36 sample strains were recovered and biochemically identified using Biomerieux BCL test, supported by Vitek ®2 Systems. The morphologically recovered microorganisms were Gram-positive bacilli belonging to Bacillus type, as follows: Bacillus smitii 66,67 %, Bacillus sphaericus/ fusiformis 8,33 %, Bacillus choshinensis and Geobacillus sterarothermophilus 8,33 %. This lets us to conclude that not all of the pollution is due to single species of Bacillus and should not focus only on B. sporothermodurans as a single contaminant of domestic sanitized milks

Nos últimos anos, as categorias de leite ultra-alta temperatura (UAT) ­ definidas no Decreto 616 de 2006 e analisados no Laboratório de Saúde Pública de Bogotá ­ têm vindo apresentando resultados não satisfatórios respeito à prova de esterilidade comercial, isso devido a que se têm afetado microrganismos produtores de esporas altamente resistentes aos tratamentos de ultra- pasteurização (HHRS). Mundialmente reporta-se como principal causa deste tipo de contaminação ao Bacillus sporothermodurans. Com este estudo determinou-se se esses leites de distribuição em Bogotá apresentam a mesma contaminação detectada por bactérias HHRS do gênero Bacillus e principalmente o B. sporothermodurans. Para tal fim, utilizaram-se amostras de análise microbiológico como parte da vigilância costumeira realizada pelo Hospital de Fontibón durante 2010 y 2011 na área de Vigilância do Ambiente e Consumo. Resultados da prova de esterilidade comercial foram recuperadas 36 cepas de amostras nas quais, a seguir, foram realizada a identificação bioquímica com o teste BCL de Biomerieux na equipe Vitek ®2 Systems. Os microrganismos recuperados morfologicamente corresponderam a bacilos Gram positivos, pertencentes ao gênero Bacillus, deste jeito: Bacillus smitii 66.67 %, Bacillus sphaericus/ fusiformis 8.33 %, Bacillus choshinencis e Geobacillus sterarothermophilus 8.33 %. O anterior permite concluir que não toda a contaminação é devida a uma só espécie de bacilos e não deve se enfocar unicamente em B. sporothermodurans como contaminante único nacional dos leites higienizados

Esporogénesis, esporodermo y ornamentaciónde esporas maduras en Lycopodiaceae / Sporogenesis, sporoderm and mature spore ornamentation in Lycopodiaceae

Studies on reproductive aspects, spore morphology and ultrastructure of Lycopodiaceae are not very common in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. In order to complete existing information, adding new and broader contributions on these topics, a comparative analysis of the sporogenesis ultrastructure, with emphasis on cytological aspects of the sporocyte coat development, tapetum, monoplastidic and polyplastidic meiosis, sporoderm ontogeny and ornamentation of the mature spores, was carried out in 43 taxa of eight genera of the Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (including Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea and Pseudolycopodiella growing in the Andes of Colombia and the Neotropics. For this study, the transmission electron microscopy (TEM) samples were collected in Cauca and Valle del Cauca Departments, while most of the spores for scanning electron microscopy (SEM) analysis were obtained from herbarium samples. We followed standard preparation procedures for spore observation by TEM and SEM. Results showed that the sporocyte coat is largely composed by primary wall components; the sporocyte develop much of their metabolic activity in the production of their coat, which is retained until the spores release; protective functions for the diploid cells undergoing meiosis is postulated here for this layer. The abundance of dictyosomes in the sporocyte cytoplasm was related to the formation and development of the sporocyte coat. Besides microtubule activity, the membrane of sporocyte folds, associated with electrodense material, and would early determine the final patterns of spore ornamentation. Monoplastidic condition is common in Lycopodium s.l., whereas polyplastidic condition was observed in species of Huperzia and Lycopodiella s. l.. In monoplastidic species, the tapetum presents abundant multivesicular bodies, while in polyplastidic species, the secretory activity of the tapetum is less intense. Sporoderm development is centripetal, exospore is the first formed layer, then the endospore and, if present, perispore is the final deposited layer. Adult spores of the Lycopodiaceae showed two patterns of ornamentation: negative or caviform (foveolate spores) and positive or muriform ornamentation, the latter with two subtypes (rugate and reticulate spores). The spores of Huperzia are characteristically foveolate, the rugate spores were found in a few species of Huperzia and in all of the Lycopodiella s. l. taxa studied, while Lycopodium s.l. spores bear reticulate ornamentation. Numerous ornamentation traits are diagnostic at the specific level. The types of ornamentation found do not support the recent extreme fragmentation of the family in several genera, but could match, a priori, with the idea of three subfamilies. The findings of sporogenesis, extremely similar in all taxa studied, point more to consider fewer genera, more comprehensive, than the recent, markedsplitting of the family. Rev. Biol. Trop. 62 (3): 1161-1195. Epub 2014 September 01.

Estudios sobre aspectos reproductivos, morfología y ultraestructura de las esporas de Lycopodiaceae no son abundantes en la literatura científica y constituyen información esencial para apoyar las relaciones taxonómicas y sistemáticas en el grupo. Con el fin de completar la información existente, añadiendo contribuciones nuevas y más amplias sobre estos temas, se realizó un análisis comparado de la ultraestructura de la esporogénesis, con énfasis en aspectos citológicos que tienen que ver con la formación de la cubierta de los esporocitos, el tapete, las meiosis monoplastidial y poliplastidial, la ontogenia del esporodermo y la ornamentación de las esporas maduras en 43 táxones de ocho géneros de Lycopodiaceae: Austrolycopodium, Diphasium, Diphasiastrum, Huperzia (incluyendo Phlegmariurus), Lycopodium, Lycopodiella, Palhinhaea y Pseudolycopodiella que crecen en los Andes de Colombia y el Neotrópico. Para estudios con microscopía electrónica de trasmisión (MET) las muestras se recolectaron en los departamentos de Cauca y Valle del Cauca, mientras que la mayoría de las muestras para microscopía electrónica de barrido (MEB) provienen de material herborizado de colecciones. Para la observación de las muestras con MET y MEB se utilizaron protocolos estándar para el procesamiento de esporas. La cubierta de los esporocitos está formada por pared primaria; los esporocitos invierten gran parte de su actividad metabólica en la producción de esa cubierta, que es mantenida hasta la liberación de las esporas y tiene funciones de protección de las células que harán meiosis. La abundancia de dictiosomas en los esporocitos se relacionó con la formación y desarrollo de la cubierta. Además de la actividad de los microtúbulos, la presencia de sinuosidades y plegamientos asociados con material electro denso en la membrana de los esporocitos determinarían tempranamente los patrones de ornamentación de las esporas. La condición monoplastidial es común en Lycopodium s.l.y la poliplastidial se observó en Huperzia y Lycopodiella s. l. En especies monoplastidiales el tapete presenta abundantes cuerpos plurivesiculares, en las poliplastidiales la actividad secretora del tapete es menos intensa. El desarrollo del esporodermo es centrípeto, el exosporio se forma primero, seguido del endosporio y el perisporio, si está presente, se deposita de último. En las esporas adultas de Lycopodiaceae se encontraron dos patrones de ornamentación: negativo o caviforme (esporas foveoladas) y positivo o muriforme (esporas rugadas y reticuladas). Las esporas foveoladas son características de Huperzia; las rugadas de unas pocas especies de Huperzia y las especies de Lycopodiella s. l., mientras que las reticulada son típicas de Lycopodium s. l.. Numerosos caracteres de la ornamentación resultan diagnósticos en el nivel específico. Los tipos principales no apoyan la extrema fragmentación reciente de la familia en varios géneros, aunque podría coincidir, a priori, con la idea de tres subfamilias. Los hallazgos de la esporogénesis, extremadamente similar en todos los táxones estudiados, apuntan más a la unificación de los géneros en la familia que a su segregación.

Morfología y anatomía de ejes caulinares, licofilos y esporangios de Phlegmariurus phylicifolius: un aporte a la sistemática de las Lycopodiaceae neotropicales / Morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius: a contribution to the systematics of Neotropical Lycopodiaceae

Phlegmariurus is the only genus of Lycopodiaceae with the species grouped in 22 informal groups. Species level relationships within Phlegmariurus are poorly understood and their circumscriptions require a thorough molecular and morphological review. A detailed study of morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius was carried out in order to contribute to the elucidation of species circumscription in the informal group Phlegmariurus phlegmaria. Small pieces of caulinar axes bearing trophophylls, sporophylls and sporangia were fixed, dehydrated, Histowax (paraffin) embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made and stained with the same technique. P. phylicifolius includes slender, pendulous plants up to 40cm long. Shoots heterophyllous, in the basal divisions ca. 10-20(-25)mm in diameter including the trophophylls, then abruptly constricted to (l-) 1.5-2(-2.5)mm in diameter including the imbricate, reduced sporophylls. Trophophylls are borne in alternating whorls of three, or decussate, subdecussate, or alternate, widely spaced in alternate leaved caulinar axes portions, perpendicular to the caulinar axes to falcately ascending, lanceolate to linear-lanceolate, with flat to slightly revolute margins. Each lycophyll is supplied by a single central vascular bundle, connected to a protoxylem pole in the stele. At the site of leaf-trace departure, no leaf (lycophyll) gap is present. Caulinar axes excluding leaves 0.7-1.2mm thick at the base, upward tapering to ca. 0.5mm. Caulinar axes present unistratified epidermis and endodermis, the cortex is characterized by the presence of a trabecular structure of lisigenous origin formed in the parenchimatous tissue next to the endodermis. The vascular tissue occupies the central part of the caulinar axes, forming a plectostele of subradiate organization, with five poles of protoxylem. The epidermal cells present sinuous anticlinal walls; invaginations in the inner side of external periclinal wall of the epidermal cells could be probably adaptive morphological feature of a water deficient environment. Leaves of constricted terminal divisions are decussate, or subdecussate, continuously or discontinuously sporangiate, appressed, abaxially rounded to carinate, widely lanceolate to widely ovate or subcordate, acute to mucronate or cuspidate, shorter than the sporangia. Each sporangium originates from a group of epidermal cells, axilar to the sporophylls. The cell walls of epidermal cell of the sporangia are Huperzioideae type. The morphological studies of trophophylls contribute to confirm the differences between P. phylicifolius and P. subulatus. Rev. Biol. Trop. 62 (3): 1217-1227. Epub 2014 September 01.

Phlegmariurus es el único género de Lycopodiaceae con las especies reunidas en 22 grupos informales. Las relaciones a nivel de especie dentro de Phlegmariurus están pobremente estudiadas y la circunscripción de las mismas requiere profundos exámenes moleculares y morfológicos. Se ha llevado a cabo un estudio detallado de la morfología y la anatomía de ejes caulinares, licofilos y esporangios de P. phylicifolius, con el fin de contribuir al esclarecimiento en la delimitación de las especies en el grupo Phlegmariurus phlegmaria. Segmentos de ejes caulinares con trofofilos, esporofilos y esporangios fueron fijados, deshidratados, incluidos en Histowax (parafina), cortados con un micrótomo rotatorio y coloreados usando la técnica tradicional Safranina O-Verde Rápido; además se hicieron cortes a mano alzada y se colorearon con la misma técnica. P. phylicifolius incluye plantas colgantes y péndulas de hasta 40cm de longitud. Los ejes son heterofilos, de aproximadamente 10-20(-25)mm de diámetro en las divisiones basales incluyendo los trofofilos, luego abruptamente reducidos a (l-) 1.5-2(-2.5)mm de diámetro incluyendo los esporofilos reducidos e imbricados. Los trofofilos están dispuestos en anillos alternantes de a tres, o decusados, subdecusados o alternos, dispuestos en forma espaciada en los ejes caulinares, perpendiculares al tallo hasta falcadamente ascendentes, lanceolados a lineal-lanceolados, con márgenes lisos o levemente revolutos. Cada licofilo está provisto de un haz vascular simple y central, conectado a un polo de protoxilema de la estela y sin laguna foliar. Los tallos poseen un ancho de 0.7-1.2mm en la base, excluyendo los licofilos, estrechándose hasta cerca de 0.5mm hacia el ápice. Los ejes caulinares presentan una epidermis uniestratificada y endodermis, la corteza se caracteriza por la presencia de una estructura trabecular de origen lisígeno formada en el tejido parenquimático próximo a la endodermis. El tejido vascular ocupa la parte central del eje caulinar, formando una plectostela de organización subradiada, con cinco polos de protoxilema. Las células epidérmicas presentan paredes anticlinales sinuosas; las invaginaciones en la cara interna de la pared periclinal externa podrían ser probablemente un característica morfológica adaptativa a un ambiente con períodos de sequía. Las hojas de las porciones apicales son decusadas o subdecusadas, con esporangio de disposición continua o discontinua, adpresas, abaxialmente redondeadas a carinadas, ampliamente lanceoladas a ovadas o subcordadas, ápice agudo a mucronado o cuspidado, más corto que el esporangio. Cada esporangio se origina de un grupo de células epidérmicas, en la axila de los esporofilos con el eje caulinar. Las paredes celulares de las células epidérmicas del esporangio son de tipo Huperzioideae. El estudio de la morfología de los trofofilos contribuye a confirmar las diferencias entre P. phylicifolius y P. subulatus.

Ontogenia de los esporangios, formación y citoquímica de esporas en licopodios (Lycopodiaceae) colombianos / Ontogeny of the sporangium, spore formation and cytochemistry in Colombian Lycopodials (Lycopodiaceae)

Studies on reproductive aspects of Lycopodiaceae are not very abundant in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. Here we present a detailed study of the ontogeny of sporangia and sporogenesis, and the chemical determination of several compounds generated during spore formation. The analyses were performed in 14 taxa of six genera of the family, Diphasiastrum, Diphasium, Huperzia (a genus which is treated here including Phlegmariurus), Lycopodiella, Lycopodium and Palhinhaea. Specimens were collected in three departments from the Colombian Andes between 1 454-3 677m altitude. Ontogeny was studied in small, 1cm long pieces of strobili and axis, which were fixed in glutaraldehyde or FAA, dehydrated in alcohol, embedded in LR White, sectioned in 0.2-0.5μm and stained with toluidine blue (TBO), a metachromatic dye that allows to detect both sporopollenin and lignin or its precursors, during these processes. For other studies, paraplast plus-embedded sections (3-5μm) were stained with safranin-fast green and alcian blue-hematoxylin. Chemical tests were also conducted in sections of fresh sporangia at different stages of maturity using alcian blue (mucopolysaccharides), Lugol solution (starch), Sudan III (lipids), phloroglucinol (lignin) and orcein (chromosomes). Sections were observed with photonic microscope equipped with differential interference contrast (DIC) and fluorescence microscopy (for spore and sporangium walls unstained). Strobili and sporangia were dehydrated with 2.2 dimethoxypropane, critical point dried and coated with gold for scanning electron microscopy (SEM). Our results indicated that the ontogeny of sporangia and sporogenesis were very similar to the previously observed in Huperzia brevifolia. Cutinisation occurs in early stages of development of sporangium cell walls, but in their final stages walls become lignified. As for the sporoderm development, the exospore is the first layer formed, composed by sporopollenin. The endospore deposits as a thin inner layer composed of cellulose, pectin and carboxylated polysaccharides. The perispore, if present, deposits at last. Mucopolysaccharides were found on the sporocyte coat and its abundance in sporangial cavity persists up to the immature tetrads stage, and then disappears. The lipids were abundant in the sporocytes, tetrads and spores, representing the main source of energy of the latter. In contrast, starch is not detected in the spores, but is abundant in premeiotic sporocytes and immature tetrads, developmental stages of high cellular metabolic activity. Intrinsic fluorescence corroborates the presence of lignin and cutin in the sporangium wall, while the sporopollenin is restricted to the exospore. The transfusion cells and the perispore are not always present. However, the processes of ontogeny and sporogenesis are extremely similar throughout the taxa studied, suggesting that they represent conservative family traits, nonspecific or generic.

Los estudios sobre aspectos reproductivos no son muy abundantes en la literatura científica sobre los taxones de Lycopodiaceae y constituyen información esencial para apoyar la taxonomía y relaciones sistemáticas en el grupo. Por lo tanto, se presenta aquí un análisis detallado de la ontogenia de los esporangios y esporogénesis, así como determinaciones químicas de varios compuestos generados durante la formación de las esporas. Los análisis se llevaron a cabo en 14 taxones de seis géneros de la familia: Diphasiastrum, Diphasium, Huperzia (un género que se trata aquí, incluyendo Phlegmariurus), Lycopodiella, Lycopodium y Palhinhaea. Las muestras fueron recolectadas en tres departamentos de los Andes de Colombia entre 1 454-3 677m de altitud. La ontogenia se estudió en trozos de estróbilos y ejes, de 1cm de largo, que se fijaron en glutaraldehido o FAA, se deshidrataron en alcohol, se incluyeron en LR White, se seccionaron en cortes de 0.2-0.5μm y se colorearon con azul de toluidina (TBO), un colorante metacromático que permite detectar tanto esporopolenina como lignina o sus precursores. Para estudios adicionales, secciones de 3-5μm de material incluido en paraplast plus se colorearon con safranina-verde rápido y azul alciánhematoxilina. Las pruebas químicas se llevaron a cabo en secciones de esporangios sin fijar en diferentes etapas de madurez utilizando azul alcián (mucopolisacáridos), solución de Lugol (almidón), Sudán III (lípidos), fluoroglucinol (lignina) y orceína (cromosomas). Las observaciones se efectuaron con microscopio fotónico equipado con contraste diferencial de interferencia (DIC) y microscopía de fluorescencia (para esporas y pared de los esporangios sin colorear). Para observaciones con microscopía electrónica de barrido (MEB), los estróbilos y esporangios se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Los resultados indican que la ontogenia de los esporangios y esporogénesis es muy similar a la observada previamente en Huperzia brevifolia. En las primeras etapas de desarrollo, las paredes celulares de la epidermis del esporangio se cutinizan y en las finales se lignifican. En el desarrollo del esporodermo, la primera capa que se forma es el exosporio, compuesto por esporopolenina. El endosporio es una capa interna delgada compuesta de celulosa, pectina y polisacáridos carboxilados. El perisporio, si está presente, es la última capa que se deposita. Los mucopolisacáridos se encontraron en la cubierta del esporocito, son abundantes en la cavidad esporangial hasta la etapa de tétradas inmaduras y luego desaparecen. Los lípidos son abundantes en esporocitos, tétradas y esporas, y representan la principal fuente de energía de estas. En contraste, el almidón no se detecta en las esporas pero es abundante en esporocitos premeióticos y tétradas inmaduras, ambos con gran actividad metabólica. La fluorescencia intrínseca corrobora la presencia de lignina y cutina en la pared del esporangio, mientras que la esporopolenina se limita al exosporio. Las células de transfusión y el perisporio no siempre están presentes. Sin embargo, los procesos de la ontogenia y esporogénesis son extremadamente similares en todos los taxones estudiados, lo que sugiere que representan rasgos típicos de familia, no específicos ni genéricos.

In vitro propagation of Cyathea atrovirens (Cyatheaceae): spore storage and sterilization conditions / Propagación in vitro de Cyathea atrovirens (Cyatheaceae): almacenamiento de esporas y condiciones de esterilización

Cyathea atrovirens occurs in a wide range of habitats in Brazil, Paraguay, Uruguay and Argentina. In the Brazilian State of Rio Grande do Sul, this commonly found species is a target of intense exploitation, because of its ornamental characteristics. The in vitro cultura is an important tool for propagation which may contribute toward the reduction of extractivism. However, exogenous contamination of spores is an obstacle for the success of aseptic long-term cultures. This study evaluated the influence of different sterilization methods combined with storage conditions on the contamination of the in vitro cultures and the gametophytic development of C. atrovirens, in order to establish an efficient propagation protocol. Spores were obtained from plants collected in Novo Hamburgo, State of Rio Grande do Sul, Brazil. In the first experiment, spores stored at 7oC were surface sterilized with 0.5, 0.8 and 2% of sodium hypochlorite (NaClO) for 15 minutes and sown in Meyer’s culture medium. The cultures were maintained in a growth room at 26±1ºC for a 12-h photoperiod and photon flux density of 100μmol/m²/s provided by cool white fluorescent light. Contamination was assessed at 60 days, and gametophytic development was scored at 30, 60, 120 and 130 days of in vitro culture, analyzing 300 individuals for each treatment. There was no significant difference in culture contamination among the different sodium hypochlorite concentrations tested, and all treatments allowed for the development of cordiform gametophytes at 130 days of culture. In the second experiment, spores stored at 7 and -20°C were divided into two groups. Half of the spores were surface sterilized with 2% of NaClO for 15 minutes and the other half was not sterilized. All spores were sown in Meyer’s medium supplemented with one of the following antibiotics: nystatin, Micostatin® and actidione. The culture conditions and the procedures used for evaluating contamination and gametophytic development were the same described for the first experiment. No contamination was observed in spores stored at -20°C and treated with NaClO and actidione. In all treatments, cordiform gametophytes presenting antheridia were observed at 120 days. The percentages of these gametophytes increased from 120 to 130 days and no significant differences were observed among treatments. Archegonia were observed on cordiform gametophytes at 130 days. The findings provide data relevant to in vitro propagation procedures of this species, which may increase the availability of plants for ornamental purposes, therefore contributing to the reduction of the exploitation of endangered tree ferns species. Rev. Biol. Trop. 62 (1): 299-308. Epub 2014 March 01.

Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) se presenta en una amplia gama de hábitats en Brasil, Paraguay, Uruguay y Argentina. Debido a sus características ornamentales, la especie es objeto de intensa explotación. El cultivo in vitro es una herramienta importante para la propagación lo que puede contribuir a la reducción del impacto de las actividades extractivas. Sin embargo, la contaminación exógena de esporas es un obstáculo para el éxito de cultivos asépticos a largo plazo. Este estudio evaluó la influencia de diferentes métodos de esterilización en combinación con las condiciones de almacenamiento sobre la contaminación de los cultivos in vitro y el desarrollo gametofítico de C. atrovirens. En el primer experimento, las esporas almacenadas a 7°C se esterilizaron superficialmente con 0.5, 0.8 y 2% de hipoclorito de sodio (NaClO) durante 15 minutos y se sembraron en medio de cultivo de Meyer. Aunque no hubo diferencia en la contaminación de lós cultivos entre las concentraciones de hipoclorito de sodio de las diferentes pruebas, en el tratamiento con 2% NaClO se observó un mayor porcentaje de gametofitos cordiformes a los 130 días. En el segundo experimento, las esporas almacenadas a 7 y -20°C fueron divididas en dos grupos. La mitad de las esporas se esterilizaron con 2% de NaClO durante 15 minutos y la otra mitad no fue esterilizada. Todas las esporas se sembraron en medio de Meyer suplementado con uno de los siguientes antibióticos: nistatina, Micostatin® o actidiona. No se observó contaminación de las esporas almacenadas a -20°C y tratadas con NaClO y actidiona. En todos los tratamientos, se observaron gametofitos cordiformes con anteridios y arquegonios. Los resultados proporcionan datos relevantes para la propagación in vitro de C. atrovirens, que pueden aumentar la disponibilidad de las plantas para fines ornamentales, contribuyendo así a la reducción de la exploración de las especies de helechos arborescentes en peligro de extinción.

Esporogénesis y esporas de Equisetum bogotense (Equisetaceae) de las áreas montañosas de Colombia / Sporogenesis and spores of Equisetum bogotense (Equisetaceae) from mountain areas of Colombia

Studies on some reproductive traits in Equisetum species are scarce and valuable to understand species distribution. Therefore, a detailed study of the sporogenesis process and spore development in E. bogotense is presented, with an analysis of the main events during meiosis, maturation of spores, spore wall ultrastructure, orbicules and elaters. Specimens were collected from 500 to 4 500m in Cauca, Colombia. Strobili at different maturation stages were fixed, dehydrated, embedded in resin, and ultra-microtome obtained sections were stained with Toluidine blue. Observations were made with optical microscopy with differential interference contrast illumination technique (DIC), transmission and scanning electron microscopy (TEM and SEM). Ultrathin sections (70-80μm) for TEM observations were stained with uranyl acetate and lead citrate; while samples for SEM observations, were fixed, dehydrated in 2.2-dimethoxypropane and dried at critical point as in standard methods. Strobili have numerous mature sporangiophores, each one with a peltate structure, the scutellum, bearing five-six sessile sporangia attached to the axis of strobilus by the manubrium. Immature sporocytes (spore mother cells) are tightly packed within the young sporangia. The sporocytes quickly undergo meiosis, by passing the stage of archesporium and give origin to tetrads of spores. The tapetum loses histological integrity during early stages of sporogenesis, intrudes as a plasmodial mass into the cavity of the sporangium, partially surrounding premeiotic sporocytes, and then, tetrads and adult spores. The tapetum disintegrates towards the end of the sporogenesis, leaving spores free within the sporangial cavity. Spores present several cytological changes that allow them to achieve greater size and increase the number of plastids, before reaching the adult stage. Sporoderm includes three layers external to the cytoplasmic membrane of the spore cell, and they are pseudoendospore, exospore and perispore. Viewed with SEM, the exospore is smooth to rugulate, with micro perforations, while the perispore is muriform, rugate, with narrow, delicate, discontinuous, randomly distributed folds delimiting incomplete, irregular areolae, externally covered by of different size, densely distributed orbicules. These orbicules are also found all over the external face and margins of the elaters, while the internal face is smooth and lack orbicules. Viewed with TEM, the exospore is a thick layer of fine granular material, while perispore is a thinner layer of dense, separate orbicules. The elaters are composed by two layers of fibrillar material: an inner layer with longitudinally oriented fibrils and an outer, thicker and less dense layer with fibrils transversely fibrils and abundant, external orbicules. It is suggested that the processes of ontogeny and characters of the sporoderm are relatively constant in Equisetum; however, sporogenesis in E. bogotense is synchronous and this condition has been observed so far only in E. giganteum, a tropical genus also found in Colombia.

Los estudios sobre aspectos reproductivos son escasos en Equisetum. Por eso, hemos realizado un análisis detallado del proceso de esporogénesis, desarrollo de las esporas, ultraestructura de procesos que tienen lugar durante la meiosis, formación de la pared esporal, orbículas y eláteres de E. bogotense, en especímenes procedentes del Cauca, Colombia. Los estudios se efectuaron mediante microscopía fotónica, electrónica de transmisión (TEM) y de barrido (SEM). Los estróbilos llevan numerosos esporangióforos maduros, cada uno con un escutelo peltado, unido al eje del estróbilo por el manubrio y portador de 5-6 esporangios sésiles. Los esporocitos experimentan meiosis dando origen a tétradas de esporas. El tapete pierde la integridad histológica en las primeras etapas de esporogénesis y rodea los esporocitos premeióticos, posteriormente a las tétradas y finalmente las esporas inmaduras, que experimentan cambios citológicos y de tamaño antes de alcanzar la etapa adulta. El esporodermo de las esporas adultas de E. bogotense consiste de seudoendosporio, exosporio y perisporio. Vistos con MEB, el exosporio de las esporas adultas es liso a rugulado con microperforaciones y el perisporio es muriforme, rugado, con pliegues delicados, estrechos, discontinuos, que se distribuyen al azar y delimitan aréolas incompletas. Externamente el perisporio está cubierto por orbículas, que se forman también en la cara externa y los márgenes de los eláteres. Vistos con TEM, el exosporio es una capa de material granular fino y el perisporio, una capa mucho más delgada con orbículas discretas. Los eláteres están formados por dos capas de naturaleza fibrilar, orientadas longitudinalmente y transversalmente. La esporogénesis en E. bogotense es sincrónica, similar a la de E. giganteum, otra especie de distribución tropical que también crece en Colombia.

Myxosporidiosis in intensively-reared Piaractus mesopotamicus: Histopathological diagnosis by means of Ziehl-Neelsen staining / Mixosporidiose em Piaractus mesopotamicus de criação intensiva: diagnóstico histopatológico pela coloração Ziehl-Neelsen

Samples of different organs from intensively-reared Piaractus mesopotamicus were collected and processed using routine histological techniques in order to produce thin sections for staining with hematoxylin-eosin and with the Ziehl-Neelsen method. Through examination under an optical microscope, myxosporidians of the genera Henneguya sp. and Myxobolus sp. were identified, respectivelyin the gills and kidneys of P. mesopotamicus. Plasmodia with immature spores of Henneguya sp. were located along the secondary lamellae, with total length of 30.45±4.84µm and width of 3.52±0.33µm. Spores of Myxobolus sp. were located in the kidneys, with total length of 8.94±0.82µm and width of 5.59±0.39µm. Histopathological analysis of the gills showed plasmodia containing spores of Henneguya sp., at intralamellar and intravascular localities, at different stages of development. Spores of Myxobolus sp. were identified in the kidneys, in the peritubular region and in the interstices and glomerulus, surrounded by melanomacrophages. Focal hemorrhage was recorded in a few cases. Ziehl-Neelsen staining allowed to identify particular features of the spores and facilitated biometry and enabled classification in comparison with hematoxylin-eosin, thus demonstrating its usefulness for histopathological diagnosis of the parasitosis.

Amostras de diferentes órgãos de Piaractus mesopotamicus mantidos em criação intensiva foram coletadas e processadas mediante as técnicas histológicas usuais para obtenção de cortes que foram corados com hematoxilina-eosina e pelo método de Ziehl-Neelsen. Ao exame em microscopia de luz foi possível identificar mixosporídeos dos gêneros Henneguya sp. e Myxobolus sp. em brânquia e rim de P. mesopotamicus respectivamente. Plasmódios com esporos imaturos de Henneguya sp. foram localizados ao longo das lamelas secundárias e mensurados (comprimento total 30,45±4,84µm e largura 3,52±0,33µm) e no rim esporos de Myxobolus sp. (comprimento total 8,94±0,82µm e largura 5,59±0,39µm). Na análise histopatológica das brânquias observaram-se plasmódios contendo esporos de Henneguya sp., com localização intralamelar e intravascular, em diferentes estágios de desenvolvimento. No rim identificaram-se esporos de Myxobolus sp., na região peritubular e no interstício e glomérulo, circundados por melanomacrófagos. Em poucos casos foi registrada hemorragia focal. O uso da coloração de Ziehl-Neelsen permitiu identificar particularidades dos esporos, facilitou sua biometria e classificação em comparação com a hematoxilina-eosina, demonstrando sua utilidade no diagnóstico histopatológico da referida parasitose.

Mycorrhizal association in gametophytes and sporophytes of the fern Pteris vittata (Pteridaceae) with Glomus intraradices

Ferns, which are usually colonizing different environments and their roots frequently present mycorrhization, have two adult stages in their life cycle, the sporophytic and the gametophytic phase. This paper describes the experimental mycorrhizal association between Pteris vittata leptosporangiate fern and a strain of Glomus intraradices during the life cycle of the fern, from spore germination to the development of a mature sporophyte. The aim of this study was to compare the colonization pattern of in vitro cultures of G. intraradices along the fern life cycle with those found in nature. For this, mature spores were obtained from fertile P. vittata fronds growing in walls of Buenos Aires city, Argentina. Roots were stained and observed under the light microscope for arbuscular mycorrhizal colonization. Approximately, 75 fern spores were cultured in each pot filled with a sterile substrate and G. intraradices (BAFC N° 51.331) as inoculum on the surface. After germination took place, samples were taken every 15 days until the fern cycle was completed. In order to determine colonization dynamics each sample was observed under optical and confocal microscope after staining. Gametophyte was classified as Adiantum type. Male and female gametangia were limited to the lower face, mycorrhizal colonization started when they were differentiated and took place through the rhizoids. Spores and vesicles were not found in this cycle stage. Paris-type mycorrhizal colonization was established in the midrib and in the embrionary foot. It was colonized by external mycelium. When the first root was developed soil inoculum colonized de novo this structure and Arum-type colonization was observed. This study proves that the type of colonization is determined by the structure of the host, not by the fungus. Both the gametophyte and embryo foot have determined growth and Paris-type colonization, while, sporophyte roots have undetermined growth and Arum-type colonization. The structures found in vitro cultures were highly similar to those found under natural conditions. Rev. Biol. Trop. 60 (2): 857-865. Epub 2012 June 01.

Los helechos presentan dos etapas en su ciclo de vida, una fase esporofítica y una gametofítica. Estos por lo general pueden colonizar diferentes ambientes y frecuentemente presentan raíces micorrizadas. Este estudio describe la asociación experimental entre Pteris vittata, un helecho leptosporangiado y una cepa de Glomus intraradices durante el ciclo de vida del helecho, desde la germinación de las esporas hasta el desarrollo del esporofito maduro. El objetivo de este estudio fue comparar los patrones de colonización de G. intraradices a lo largo de todo el ciclo de vida del helecho con los tipos encontrados en la naturaleza. Las esporas maduras fueron obtenidas de frondes fértiles de P. vittata que crecen sobre las paredes de la ciudad de Buenos Aires, Argentina. Las raíces se tiñeron y fueron observadas bajo microscopio óptico para el estudio de la colonización micorrízica. Aproximadamente 75 esporas de helecho se cultivaron en macetas con un sustrato estéril y con un inóculo de G. intraradices (N° 51.331 BAFC) en la superficie. Después de la germinación, se tomaron muestras cada 15 días hasta que se completó el ciclo de vida del helecho. Con el fin de determinar la dinámica de la colonización, cada muestra se observó con el microscopio óptico y el microscopio de confocal luego de la tinción correspondiente. El gametofito fue clasificado como del tipo “Adiantum”. Los gametangios femeninos y masculinos se desarrollaron en la cara inferior del mismo. La micorrización comenzó cuando los gametangios estaban ya diferenciados y la colonización se produjo a través de los rizoides. Las esporas y las vesículas no se encontraron en esta fase del ciclo. La micorrizacion tipo Paris se observó sobre la línea de la nervadura central. El pie del esporofito fue colonizado por el micelio externo. Cuando la raíz se desarrolló, se colonizó “de novo”, y se observó una colonización de tipo Arum. Este estudio demuestra que el tipo de colonización está determinado por la estructura del helecho y no por el hongo. Tanto el gametofito como el pie del embrión tienen crecimiento definido y colonización tipo Paris, mientras que las raíces del esporofito presentan un crecimiento indeterminado y una colonización tipo Arum. Las estructuras que se encontraron bajo cultivo coinciden con las que se encontraron en condiciones naturales.

Caudex growth and phenology of Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) in secondary forest, southern Brazil / Crescimento do cáudice e fenologia de Cyathea atrovirens (Langsd. & Fisch.) Domin (Cyatheaceae) em floresta secundária, no sul do Brasil

The leaf production and senescence, formation and release of spores of Cyathea atrovirens (Langsd. & Fisch.) Domin were analysed based on the monthly monitoring of 50 plants growing in a secondary forest, in the municipality of Novo Hamburgo, in the state of Rio Grande do Sul, during the year 2004. The caudex height and number of mature and fertile leaves were recorded annually in 2004-09. In 2004, monthly production and senescence of leaves were concomitant, without total leaf abscission. Population synchrony at emergence (Z = 0.86) and leaf senescence (Z = 0.82) increased in spring but did not correlate with temperature and photoperiod. All individuals were fertile and the sporangia production and spore liberation presented higher and equal synchrony (Z = 0.84) respectively in spring and summer. Sporangia production was related with temperature and photoperiod, however taller plants did not produce more fertile leaves. Phenological events analysed were not influenced by precipitation, as expected for forests in non-seasonal climate. Over five years (2004-09), the annual mean caudex growth varied between 1.19 and 2.50 cm.year-1 and the plants appeared to have an ability to maintain a relatively stable amount of leaves throughout this period.

A produção e a senescência de folhas, a formação e a liberação de esporos de Cyathea atrovirens (Langsd. & Fisch.) Domin, durante o ano de 2004, foram analisadas a partir do monitoramento mensal de 50 plantas, crescendo em floresta secundária, no município de Novo Hamburgo, Estado do Rio Grande do Sul, Brasil. A altura do cáudice e o número de folhas foram mensurados anualmente de 2004-2009. Em 2004, a produção e a senescência mensal de folhas foram concomitantes, evitando a abscisão foliar total. A sincronia da população na renovação (Z = 0,86) e na senescência foliar (Z = 0,82) aumentou na primavera, porém esses eventos não se relacionaram com temperatura e fotoperíodo. Todos os indivíduos estavam férteis e a produção de esporângios e a liberação de esporos apresentaram uma sincronia maior e igual (Z = 0,84), respectivamente, na primavera e no verão. A produção de esporângios se relacionou com temperatura e fotoperíodo, porém plantas mais altas não produziram mais folhas férteis. Os eventos fenológicos analisados não foram influenciados pela precipitação, tal como esperado para florestas sob clima não sazonal. Durante cinco anos (2004-2009), a média anual do crescimento do cáudice variou de 1,19 a 2,50 cm.ano-1 e as plantas demonstraram capacidade de manter o número de folhas relativamente estável ao longo do período.

Dermatofitoma en tinea corporis con onicomicosis de pies y manos causados por Trichophyton rubrum / Dermatofitoma in tinea corporis with hand and foot onychomycosis caused by Trichophyton rubrum

Las dermatofitosis son infecciones crónicas de distribución mundial, las cuales pueden presentar ocasionalmente conglomerados de esporas, filamentos o ambos, denominadas ¨dermatofitomas¨. Se presentan un paciente que refiere tinea corporis de brazo derecho automedicado con corticosteroides tópicos, onicomicosis subungueal distal de manos y onicomicosis distrófica total de pies. En la observación microscópica directa se observaron dermatofitomas en el brazo y uñas de manos, y filamentos en las uñas de pies. Se identificó Trichophyton rubrum en brazo, unas de manos y pies.

Isolamento, identificação clássica e análise molecular de bactérias provenientes de coprólitos de sítios arqueológicos brasileiros / Isolation, identification classical and molecular analysis of bacteria from coprolites from archaeological sites in Brazil

A partir de amostras de coprólitos brasileiros datados entre 3490 mais ou menos 120 a 430 mais ou menos 70 anos, recuperados de escavações arqueológicas no Brasil, fomos capazes de isolar bactérias da família Bacillaceae. Objetivou-se com esse estudo testar e padronizar técnicas para detecção, identificação e caracterização molecular de microrganismos bacterianos nessas amostras. Os coprólitos foram inicialmente tratados com radiação ultravioleta, buscando eliminar contaminantes externos e posteriormente perfurados para retirada do material mais interno. Esse material foi cultivado em aerobiose em diferentes meios de cultura, originando o isolamento de bastonetes Gram positivos formadores de endósporos. Esses microrganismos foram submetidos a provas bioquímicas e fisiológicas, de sensibilidade a antimicrobianos, reação em cadeia da polimerase (PCR), clonagem e sequenciamento, buscando sua identificação e caracterização. Medidas para evitar a contaminação por microrganismos modernos foram tomadas, como a utilização de apenas controles negativos nas reações de PCR, duplicação dos experimentos, entre outras. Das 7 amostras de coprólitos utilizadas, apenas 5 originaram algum isolado, sendo estas datadas entre 3490 mais ou menos 120 a 430 mais ou menos 70 anos ou sem datação conhecida . O emprego de diferentes técnicas laboratoriais para identificação de microrganismos antigos ficou evidente. Essa diversificação aumenta as chances de identificação dos isolados, o que possibilitou concluir que se tratava de representantes da família Bacillaceae, do gênero Bacillus e correlatos.