Diversity of Somatic Coliphages in Coastal Regions with Different Levels of Anthropogenic Activity in Sao Paulo State, Brazil

Bacteriophages are the most abundant and genetically diverse viruses on Earth, with complex ecology in both quantitative and qualitative terms. Somatic coliphages (SC) have been reported to be good indicators of fecal pollution in seawater. This study focused on determining the concentration of SC and their diversity by electron microscopy of seawater, plankton, and bivalve samples collected at three coastal regions in Sao Paulo, Brazil. The SC counts varied from < 1 to 3.4 x 103 PFU/100 ml in seawater (73 samples tested), from < 1 to 4.7 x 10(2) PFU/g in plankton (46 samples tested), and from < 1 to 2.2 x 10(1) PFU/g in bivalves (11 samples tested). In seawater samples, a relationship between the thermotolerant coliforms and Escherichia coli and SC was observed at the three regions (P = 0.0001) according to the anthropogenic activities present at each region. However, SC were found in plankton samples from three regions: Baixada Santista (17/20), Canal de Sao Sebastiao (6/14), and Ubatuba (3/12). In seawater samples collected from Baixada Santista, four morphotypes were observed: A1 (4.5%), B1 (50%), C1 (36.4%), and D1 (9.1%). One coliphage, Siphoviridae type T1, had the longest tail: between 939 and 995 nm. In plankton samples, Siphoviridae (65.8%), Podoviridae (15.8%), Microviridae (15.8%), and Myoviridae (2.6%) were found. In bivalves, only the morphotype B1 was observed. These SC were associated with enteric hosts: enterobacteria, E. coli, Proteus, Salmonella, and Yersinia. Baixada Santista is an area containing a high level of fecal pollution compared to those in the Canal de Sao Sebastiao and Ubatuba. This is the first report of coliphage diversity in seawater, plankton, and bivalve samples collected from Sao Paulo coastal regions. A better characterization of SC diversity in coastal environments will help with the management and evaluation of the microbiological risks for recreation, seafood cultivation, and consumption.

Decapod crustacean larvae dynamics in the west continental coast of Portugal: the region adjacent to Aveiro coastal lagoon as model

Tese de dout., Ciências do Mar, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2010

Taxonomy and distribution of taxa in the genus Gomphonema from the Florida Everglades, U.S.A.

Located at a subtropical latitude, the expansive Florida Everglades contains a mixture of tropical and temperate diatom taxa, as well as a unique flora adapted to the calcareous, often excessively hot, seasonally flooded wetland conditions. This flora has been poorly documented taxonomically, although diatoms are recognized as important indicators of environmental change in this threatened ecosystem. Gomphonema is a dominant genus in the freshwater marsh, and is represented by highly variable species complexes, including Gomphonema gracile Ehrenberg, Gomphonema intricatum var. vibrio Ehrenberg sensu Fricke, Gomphonema vibrioides Reichardt & Lange-Bertalot and Gomphonema parvulum (Kützing) Grunow. These taxa have been shown to exhibit wide morphological variation in other regions, resulting in considerable nomenclatural confusion. We collected Gomphonema from 237 sites distributed throughout the freshwater Everglades and used qualitative and quantitative morphological data to identify 20 distinguishable populations. Taxonomie assignments were based on descriptions and/or observations of type material of relevant taxa when possible, but deviations from original morphological range descriptions were common. We then compared morphological variation in Everglades Gomphonema taxa to that reported for the same taxa in other regions and suggest revisions of taxonomie concepts when necessary.

Morphological descriptions from the recent bivalve of the genus Calyptogena (Vesicomyidae)

The genus Calyptogena (Bivalvia: Vesicomyidae) comprises highly specialized bivalves living in symbiosis with sulphur-oxidizing bacteria in reducing habitats. In this study, the genus is revised using shell and anatomical features. The work is based on type material, as well as on the extensive collection of vesicomyids obtained during twelve expeditions to the Pacific and Indian Oceans. Nine Recent species are ascribed to the genus Calyptogena, four of which are new: C. pacifica Dall, 1891, C. fausta Okutani, Fujikura & Hashimoto, 1993, C. rectimargo Scarlato, 1981, C. valdiviae (Thiele & Jaeckel, 1931), C. gallardoi Sellanes & Krylova, 2005, C. goffrediae n. sp., C. starobogatovi n. sp., C. makranensis n. sp. and C. costaricana n. sp. The characteristic features of Calyptogena are: shell up to 90 mm in length, elongate-elliptical or elongate; presence of escutcheon; presence of broad posterior ramus (3b) of right subumbonal cardinal tooth as well as right posterior nymphal ridge; absence of pallial sinus as a result of attachment of intersiphonal septal retractor immediately adjacent to ventral surface of posterior adductor; absence of processes on inner vulva of inhalant siphon; presence of inner demibranch only, with descending and ascending lamellae with interlamellar septa not divided into separate tubes. The most closely related taxa to Calyptogena are probably the genus Isorropodon Sturany, 1896, and the group of species represented by 'Calyptogena' phaseoliformis Métivier, Okutani & Ohta, 1986. These groups have several characters in common, namely absence of pallial sinus, presence of single inner pair of demibranchs and absence of processes on inner vulva of inhalant siphon. The worldwide distribution of the genus Calyptogena suggests that methane seeps at continental margins are the major dispersal routes and that speciation was promoted by geographical isolation. Recent species diversity and fossil records indicate that the genus originated in the Pacific Ocean. Sufficient data to discuss the distribution at species level exist only for C. pacifica, which has a remarkably narrow bathymetric range. Published studies on the physiology of C. pacifica suggest that adaptation to a specific geochemical environment has led to coexisting vesicomyid genera. The bacteria-containing gill of C. pacifica and other Calyptogena species is one of the most specialized in the family Vesicomyidae and may reflect these ecological adaptations.

Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.

Phalangopsidae crickets from the Indian Region (Orthoptera, Grylloidea), with the descriptions of new taxa, diagnoses for genera, and a key to Indian genera

The Indian region is presently the second region after the Neotropics in terms of diversity of phalangopsid crickets. Yet their study is impeded by the lack of necessary taxonomic tools for taxon identification. In the present paper, all generic diagnoses are clarified, using morphological and genitalic characters; female genitalia are described and illustrated for all genera with known females. New taxa are described from southern India: Kempiola flavipunctatus Desutter-Grandcolas n. sp., Opiliosina meridionalis Desutter-Grandcolas n. gen., n. sp., Phalangopsina bolivari Desutter-Grandcolas n. sp., P. chopardi Desutter-Grandcolas n. sp., P. gravelyi Desutter-Grandcolas n. sp., and Speluncasina Desutter-Grandcolas n. gen. The list of phalangopsid crickets from the Indian Region is updated, and a key to phalangopsid genera proposed. A lectotype and a paralectotype are designated to fix the name of Phalangopsina dubia (Bolivar, 1900). Opilionacris annandalei Chopard, 1928, previously transferred to the African genus Phaeophilacris Walker, 1871, is transferred to the genus Speluncasina Desutter-Grandcolas n. gen., while Larandopsis jharnae Bhowmik, 1981 and L. newguineae Bhowmik, 1981 described from New Guinea are transferred to the eneopterine genus Lebinthus Stal, 1877. Finally Luzaropsis confusa Chopard, 1969 is removed from its synonymy with L. ferruginea Walker, 1871.

Descriptions of Oscillatoria agardhii, O.prolifera and O.rubescens only [Translation from: Translation from: Flora Slodkowodna Polski. Tom 2. Cyanophyta, Glaucophyta (ed. K. Starmach) p350-352. Warsaw, Polska Academia Nauk, 1966]

Short morphological observations are given for the Cyanophyceae Oscillatoria Agardhii, Oscillatoria prolifica and Oscillatoria rubescens.

Primitive living chitons: Morphological cladistic analysis as a model for character evaluation

Chitons are often referred to as “living fossils” in part because they are proposed as one of the earliest-diverging groups of living molluscs, but also because the gross morphology of the polyplacophoran shell has been conserved for hundreds of millions of years. As such, the analysis of evolution and radiation within polyplacophorans is of considerable interest not only for resolving the shape of pan-molluscan phylogeny but also as model organisms for the study of character evolution. This study presents a new, rigorous cladistic analysis of the morphological characters used in taxonomic descriptions for chitons in the living suborder Lepidopleurina Thiele, 1910 (the earliest-derived living group of chitons). Shell-based characters alone entirely fail to recover any recognized subdivisions within the group, which may raise serious questions about the application of fossil data (from isolated shell valves). New analysis including characters from girdle armature and gill arrangements recovers some genera within the group but also points to the lack of monophyly within the main genus Leptochiton Gray, 1847. Additional characters from molecular data and soft anatomy, used in combination, are clearly needed to resolve questions of chiton relationships. However, the data sets currently available already provide interesting insights into the analytical power of traditional morphology as well as some knowledge about the early evolution and radiation of this group.

Morphological cladistic analysis as a model for character evaluation in primitive living chitons (Polyplacophora, Lepidopleurina)

Chitons are often referred to as "living fossils" in part because they are proposed as one of the earliest-diverging groups of living molluscs, but also because the gross morphology of the polyplacophoran shell has been conserved for hundreds of millions of years. As such, the analysis of evolution and radiation within polyplacophorans is of considerable interest not only for resolving the shape of pan-molluscan phylogeny but also as model organisms for the study of character evolution. This study presents a new, rigorous cladistic analysis of the morphological characters used in taxonomic descriptions for chitons in the living suborder Lepidopleurina Thiele, 1910 (the earliest-derived living group of chitons). Shell-based characters alone entirely fail to recover any recognized subdivisions within the group, which may raise serious questions about the application of fossil data (from isolated shell valves). New analysis including characters from girdle armature and gill arrangements recovers some genera within the group but also points to the lack of monophyly within the main genus Leptochiton Gray, 1847. Additional characters from molecular data and soft anatomy, used in combination, are clearly needed to resolve questions of chiton relationships. However, the data sets currently available already provide interesting insights into the analytical power of traditional morphology as well as some knowledge about the early evolution and radiation of this group.

Genetic and morphological characterization of Cladobotryum species causing cobweb disease of mushrooms

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.

Révision taxonomique de la famille des Harrimaniidae (Hemichordata: Enteropneusta) incluant les descriptions de sept espèces de la côte Est du Pacifique

Cette étude comparative est une révision de la famille des Harrimaniidae basée sur les caractères morphologiques d'espèces connues et nouvelles provenant des collections de William E. Ritter, Theodore H. Bullock et Kandula P. Rao rassemblées au cours du 20e siècle. Les descriptions présentées ici portent le total des genres de cinq à neuf par l'ajout de Horstia n. gen., Mesoglossus n. gen., Ritteria n. gen et Saxipendium, un genre auparavant attribué à la famille monospécifique des Saxipendidae. Le nombre d'espèces est porté à 34 par la description de cinq nouvelles espèces du Pacifique oriental: Horstia kincaidi, Mesoglossus intermedius, Mesoglossus macginitiei, Protoglossus mackiei et Ritteria ambigua. La description d'une sixième espèce, Stereobalanus willeyi Ritter et Davis, 1904 (nomen nudum) est présentée ici pour la première fois, ainsi qu'une description abrégée de Saxipendium coronatum. Quatre espèces précédemment attribuées au genre Saccoglossus sont transférées au genre Mesoglossus: M. bournei, M. caraibicus, M. gurneyi, et M. pygmaeus et Saccoglossus borealis est transféré au genre Harrimania. Une hypothèse phylogénétique sur la famille des Harrimaniidae est émise, présentant l'évolution possible des caractères morphologiques au sein du groupe. Finalement, des notes sur la distribution géographique étendue mais discontinue de plusieurs espèces suggère que les entéropneustes auraient pu avoir une distribution ancienne continue et plus grande qui aurait été fragmentée par la suite.

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

Morphological characterisation of five brazilian species of Fidicinoides (Hemiptera: Cicadidae)

The objective of this study was to describe and illustrate the morphological characteristics of some species of Brazilian Fidicinoides to aid in their identification. Descriptions and illustrations are given of the head, thorax, abdomen, right forewing and the last urostemite of the male of the Fidicinoides besti, Fidicinoides brunnea, Fidicinoides duckensis, Fidicinoides pseudethelae and Fidicinoides sucinalae species. The main diagnostic characteristics of these species are markings on pronotum and/or mesonotum and male stemite VIII characteristics.

Comparative descriptions of eggs from three species of Rhodnius (Hemiptera: Reduviidae: Triatominae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Morphological and morphometric comparisons of the first zoea of four species of purse crabs (Decapoda: Brachyura: Leucosiidae) from the Brazilian South Atlantic

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)