Prudent female allocation by modular hermaphrodites: female investment is promoted by the opportunity to outcross in cyclostome bryozoans

Many sessile, suspension-feeding marine invertebrates mate by spermcasting: aquatic sperm are spawned and gathered by conspecific individuals to fertilize eggs that are generally retained during development. In two phylogenetically distant examples, a cheilostome bryozoan and an aplousobranch ascidian, the receipt of allosperm has previously been shown to alter sex allocation by triggering female investment in eggs and brooding. Here we report experiments demonstrating that two species of cyclostome bryozoan also show restrained female investment in the absence of mating opportunity. In Tubulipora plumosa, the production of female zooids and progeny is much reduced in reproductive isolation. In Filicrisia geniculata, development of distinctive female zooids (gonozooids) begins but halts in the absence of mating opportunity, and no completed gonozooids or progeny result. Reduced female investment in the absence of a mate thus occurs in at least two orders of Bryozoa, but significant differences in detail exist and the evolutionary history within the phylum of the mechanism(s) by which female investment is initiated might be complex. The broadening taxonomic spectrum of examples where female investment appears restrained until allosperm becomes available may signify a general adaptive strategy among outcrossing modular animals, analogous to similarly adaptive sex allocation typical of many flowering plants.

Prudent female allocation by modular hermaphrodites: female investment is promoted by the opportunity to outcross in cyclostome bryozoans

Many sessile, suspension-feeding marine invertebrates mate by spermcasting: aquatic sperm are spawned and gathered by conspecific individuals to fertilize eggs that are generally retained during development. In two phylogenetically distant examples, a cheilostome bryozoan and an aplousobranch ascidian, the receipt of allosperm has previously been shown to alter sex allocation by triggering female investment in eggs and brooding. Here we report experiments demonstrating that two species of cyclostome bryozoan also show restrained female investment in the absence of mating opportunity. In Tubulipora plumosa, the production of female zooids and progeny is much reduced in reproductive isolation. In Filicrisia geniculata, development of distinctive female zooids (gonozooids) begins but halts in the absence of mating opportunity, and no completed gonozooids or progeny result. Reduced female investment in the absence of a mate thus occurs in at least two orders of Bryozoa, but significant differences in detail exist and the evolutionary history within the phylum of the mechanism(s) by which female investment is initiated might be complex. The broadening taxonomic spectrum of examples where female investment appears restrained until allosperm becomes available may signify a general adaptive strategy among outcrossing modular animals, analogous to similarly adaptive sex allocation typical of many flowering plants.

The neurotoxin β-N-methylamino-L-alanine (BMAA) : Sources, bioaccumulation and extraction procedures

β-methylamino-L-alanine (BMAA) is a neurotoxin linked to neurodegeneration, which is manifested in the devastating human diseases amyotrophic lateral sclerosis, Alzheimer’s and Parkinson’s disease. This neurotoxin is known to be produced by almost all tested species within the cyanobacterial phylum including free living as well as the symbiotic strains. The global distribution of the BMAA producers ranges from a terrestrial ecosystem on the Island of Guam in the Pacific Ocean to an aquatic ecosystem in Northern Europe, the Baltic Sea, where annually massive surface blooms occur. BMAA had been shown to accumulate in the Baltic Sea food web, with highest levels in the bottom dwelling fish-species as well as in mollusks. One of the aims of this thesis was to test the bottom-dwelling bioaccumulation hypothesis by using a larger number of samples allowing a statistical evaluation. Hence, a large set of fish individuals from the lake Finjasjön, were caught and the BMAA concentrations in different tissues were related to the season of catching, fish gender, total weight and species. The results reveal that fish total weight and fish species were positively correlated with BMAA concentration in the fish brain. Therefore, significantly higher concentrations of BMAA in the brain were detected in plankti-benthivorous fish species and heavier (potentially older) individuals. Another goal was to investigate the potential production of BMAA by other phytoplankton organisms. Therefore, diatom cultures were investigated and confirmed to produce BMAA, even in higher concentrations than cyanobacteria. All diatom cultures studied during this thesis work were show to contain BMAA, as well as one dinoflagellate species. This might imply that the environmental spread of BMAA in aquatic ecosystems is even higher than previously thought. Earlier reports on the concentration of BMAA in different organisms have shown highly variable results and the methods used for quantification have been intensively discussed in the scientific community. In the most recent studies, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the instrument of choice, due to its high sensitivity and selectivity. Even so, different studies show quite variable concentrations of BMAA. In this thesis, three of the most common BMAA extraction protocols were evaluated in order to find out if the extraction could be one of the sources of variability. It was found that the method involving precipitation of proteins using trichloroacetic acid gave the best performance, complying with all in-house validation criteria. However, extractions of diatom and cyanobacteria cultures with this validated method and quantified using LC-MS/MS still resulted in variable BMAA concentrations, which suggest that also biological reasons contribute to the discrepancies. The current knowledge on the environmental factors that can induce or reduce BMAA production is still limited. In cyanobacteria, production of BMAA was earlier shown to be negative correlated with nitrogen availability – both in laboratory cultures as well as in natural populations. Based on this observation, it was suggested that in unicellular non-diazotrophic cyanobacteria, BMAA might take part in nitrogen metabolism. In order to find out if BMAA has a similar role in diatoms, BMAA was added to two diatom species in culture, in concentrations corresponding to those earlier found in the diatoms. The results suggest that BMAA might induce a nitrogen starvation signal in diatoms, as was earlier observed in cyanobacteria. However, diatoms recover shortly by the extracellular presence of excreted ammonia. Thus, also in diatoms, BMAA might be involved in the nitrogen balance in the cell.

Study of feeding competition between invasion ctenophore (M. leidyi) and anchovy (C. engrauliformis) in the Southern Caspian Sea (Mazandran Coast)

Mnemiopsis leidyi one species of phylum Ctenophora, is a native species in America. It has most likely moved across the Atlantic in the ballast water of cargo ships to the Black sea in 1982, and then to the Caspian Sea thought the Volga-Don Channel, in Nov 1999. The population of M. leidyi rows rapidly and by end of 2000, the entire sea was teeming with them. This survey was arranged In order to study the relationship between the invasion of M. leidyi and sharp decline in main stocks pelagic fish such as Kilka. Dietary analysis was conducted on Anchovy Killka (Clupeonella engrauliformis) and M. leidyi from August 2001 to October 2002 in two stations, located at the costal water near Babolsar (52.38° E ,36.42° N ) and Noshahar (51.33° E,36.39° N) in the Caspian Sea province of Mazandaran, Iran. M. leidyi was caught by plankton net, at three vertical strata of both station at surface 5 in, 10m, and 15 m the Kilka was caught by fisheries boat at Babolasar fishery harbour. Samples of M. leidyi were not fixed in its common fixative, we used 96% Ethanol In order to study of M. leidyi digestion system some alive samples, directly, were studied by the fluorescence microscope which was connected to a computer prepared specially for this process. In many cases, the light was directly reflected on the sample and microscopic image was prepared in dark background. We found that there were some common organisms in diet of both species. The Schoener index analysis reflected these similarities, as values more than critical level of overlap (>89 in Babolsar samples and >84 in Noshahar samples) were found. Results from this study suggests that M. leidyi and Anchovy have a similar feeding niche and computation between them is one of the reasons to decline in anchovy stocks. Economical effects of M. leidyi s invasion in research area were studied by data on kilka caught before and after introducing M. leidyi.

New Perspective On The Haplosporidian Parasites Of Molluscs

The protist phylum Haplosporidia comprises over 40 described species with representatives infecting a range of mollusc hosts, including several ecologically and economically significant pathogens. Continuing exploration of haplosporidian diversity has added ten new species in recent years and brought the phylogenetics of the group into somewhat clearer focus, with monophyletic Bonamia and Minchinia lineages continuing to be supported. However, the addition of new sequences to phylogenetic analyses has left the paraphyletic genus Haplosporidium’s picture less resolved. It is not clear that even two genera will be enough to accommodate the species presently drawn to the Haplosporidium regions of the haplosporidian tree. In this review, we summarize recent findings in haplosporidian diversity and phylogenetics, and provide a synthesis of our understanding of the life cycles and environmental influences on haplosporidians, with particular emphasis on the important pathogens Haplosporidium nelsoni and Bonamia ostreae. Additionally, we consider the evolution of the “microcell haplosporidian” lifestyle of Bonamia parasites, and suggest that colonization of high-density oyster host populations in relatively stable euhaline marine environments may have been an important development favoring the evolution of the microcell haplosporidian life strategy.

Bioinformatics analysis of the Pedobacter sp. NL19 genome

The last decades of the 20th century defined the genetic engineering advent, climaxing in the development of techniques, such as PCR and Sanger sequencing. This, permitted the appearance of new techniques to sequencing whole genomes, identified as next-generation sequencing. One of the many applications of these techniques is the in silico search for new secondary metabolites, synthesized by microorganisms exhibiting antimicrobial properties. The peptide antibiotics compounds can be classified in two classes, according to their biosynthesis, in ribosomal or nonribosomal peptides. Lanthipeptides are the most studied ribosomal peptides and are characterized by the presence of lanthionine and methylanthionine that result from posttranslational modifications. Lanthipeptides are divided in four classes, depending on their biosynthetic machinery. In class I, a LanB enzyme dehydrate serine and threonine residues in the C-terminus precursor peptide. Then, these residues undergo a cyclization step performed by a LanC enzyme, forming the lanthionine rings. The cleavage and the transport of the peptide is achieved by the LanP and LanT enzymes, respectively. Although, in class II only one enzyme, LanM, is responsible for the dehydration and cyclization steps and also only one enzyme performs the cleavage and transport, LanT. Pedobacter sp. NL19 is a Gram-negative bacterium, isolated from sludge of an abandon uranium mine, in Viseu (Portugal). Antibacterial activity in vitro was detected against several Gram-positive and Gram-negative bacteria. Sequencing and in silico analysis of NL19 genome revealed the presence of 21 biosynthetic clusters for secondary metabolites, including nonribosomal and ribosomal peptides biosynthetic clusters. Four lanthipeptides clusters were predicted, comprising the precursor peptides, the modifying enzymes (LanB and LanC), and also a bifunctional LanT. This result revealed the hybrid nature of the clusters, comprising characteristics from two distinct classes, which are poorly described in literature. The phylogenetic analysis of their enzymes showed that they clustered within the bacteroidetes clade. Furthermore, hybrid gene clusters were also found in other species of this phylum, revealing that it is a common characteristic in this group. Finally, the analysis of NL19 colonies by MALDI-TOF MS allowed the identification of a 3180 Da mass that corresponds to the predicted mass of a lanthipeptide encoded in one of the clusters. However, this result is not fully conclusive and further experiments are needed to understand the full potential of the compounds encoded in this type of clusters. In conclusion, it was determined that NL19 strain has the potential to produce diverse secondary metabolites, including lanthipeptides that were not functionally characterized so far.

Long term repeated fire disturbance alters soil bacterial diversity but not the abundance in an Australian wet sclerophyll forest

Effects of fire on biogeochemical cycling in terrestrial ecosystem are widely acknowledged, while few studies have focused on the bacterial community under the disturbance of long-term frequent prescribed fire. In this study, three treatments (burning every two years (B2), burning every four years (B4) and no burning (B0)) were applied for 38 years in an Australian wet sclerophyll forest. Results showed that bacterial alpha diversity (i.e. bacterial OTU) in the top soil (0-10 cm) was significantly higher in the B2 treatment compared with the B0 and B4 treatments. Non-metric multidimensional analysis (NMDS) of bacterial community showed clear separation of the soil bacterial community structure among different fire frequency regimes and between the depths. Different frequency fire did not have a substantial effect on bacterial composition at phylum level or bacterial 16S rRNA gene abundance. Soil pH and C:N ratio were the major drivers for bacterial community structure in the most frequent fire treatment (B2), while other factors (EC, DOC, DON, MBC, NH 4 +, TC and TN) were significant in the less frequent burning and no burning treatments (B4 and B0). This study suggested that burning had a dramatic impact on bacterial diversity but not abundance with more frequent fire.

Crassaminicella profunda gen. nov., sp nov., an anaerobic marine bacterium isolated from deep-sea sediments

A novel, anaerobic, chemo-organotrophic bacterium, designated strain Ra1766HT, was isolated from sediments of the Guaymas basin (Gulf of California, Mexico) taken from a depth of 2002 m. Cells were thin, motile, Gram-stain-positive, flexible rods forming terminal endospores. Strain Ra1766H(T) grew at temperatures of 25-45 degrees C (optimum 30 degrees C), pH 6.7-8.1 (optimum 7.5) and in a salinity of 5-60 g l(-1) NaCl (optimum 30 g l(-1)). It was an obligate heterotrophic bacterium fermenting carbohydrates (glucose and mannose) and organic acids (pyruvate and succinate). Casamino acids and amino acids (glutamate, aspartate and glycine) were also fermented. The main end products from glucose fermentation were acetate, butyrate, ethanol, H-2 and CO2. Sulfate, sulfite, thiosulfate, elemental sulfur, fumarate, nitrate, nitrite and Fe(III) were not used as terminal electron acceptors. The predominant cellular fatty acids were C-14 : 0, C-16:1 omega 7, C-16:1 omega 7 DMA and C-16:0. The main polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phospholipids. The G +C content of the genomic DNA was 33.7 molo/o. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain Ra1766H(T) was affiliated to cluster XI of the order Clostridia les, phylum Firmicutes. The closest phylogenetic relative of Ra1766H(T) was Geosporobacter subterraneus (94.2% 16S rRNA gene sequence similarity). On the basis of phylogenetic inference and phenotypic properties, strain Ra1766H(T) (=DSM 27501(T)=JCM 19377(T)) is proposed to be the type strain of a novel species of a novel genus, named Crassaminicella pro funda.

Quantification of microbial communities in subsurface marine sediments of the Black Sea and off Namibia

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 10(9) to 10(10) cells/mL at the sediment surface to 10(7)-10(9) cells/mL below one meter depth. Based on CARD FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

Metagenômica comparativa de solo de regiões de mata atlântica e caatinga do estado do Rio Grande do Norte - Brasil

The microorganisms play very important roles in maintaining ecosystems, which explains the enormous interest in understanding the relationship between these organisms as well as between them and the environment. It is estimated that the total number of prokaryotic cells on Earth is between 4 and 6 x 1030, constituting an enormous biological and genetic pool to be explored. Although currently only 1% of all this wealth can be cultivated by standard laboratory techniques, metagenomic tools allow access to the genomic potential of environmental samples in a independent culture manner, and in combination with third generation sequencing technologies, the samples coverage become even greater. Soils, in particular, are the major reservoirs of this diversity, and many important environments around us, as the Brazilian biomes Caatinga and Atlantic Forest, are poorly studied. Thus, the genetic material from environmental soil samples of Caatinga and Atlantic Forest biomes were extracted by direct techniques, pyrosequenced, and the sequences generated were analyzed by bioinformatics programs (MEGAN MG-RAST and WEBCarma). Taxonomic comparative profiles of the samples showed that the phyla Proteobacteria, Actinobacteria, Acidobacteria and Planctomycetes were the most representative. In addition, fungi of the phylum Ascomycota were identified predominantly in the soil sample from the Atlantic Forest. Metabolic profiles showed that despite the existence of environmental differences, sequences from both samples were similarly placed in the various functional subsystems, indicating no specific habitat functions. This work, a pioneer in taxonomic and metabolic comparative analysis of soil samples from Brazilian biomes, contributes to the knowledge of these complex environmental systems, so far little explored

TAXONOMY, MORPHOLOGY, AND RNA-SEQ TRANSCRIPTOMICS OF THE CUBOZOAN ALATINA ALATA, AN EMERGING MODEL CNIDARIAN

Cnidarians are often considered simple animals, but the more than 13,000 estimated species (e.g., corals, hydroids and jellyfish) of the early diverging phylum exhibit a broad diversity of forms, functions and behaviors, some of which are demonstrably complex. In particular, cubozoans (box jellyfish) are cnidarians that have evolved a number of distinguishing features. Some cubozoan species possess complex mating behaviors or particularly potent stings, and all possess well-developed light sensation involving image-forming eyes. Like all cnidarians, cubozoans have specialized subcellular structures called nematocysts that are used in prey capture and defense. The objective of this study is to contribute to the development of the box jellyfish Alatina alata as a model cnidarian. This cubozoan species offers numerous advantages for investigating morphological and molecular traits underlying complex processes and coordinated behavior in free-living medusozoans (i.e., jellyfish), and more broadly throughout Metazoa. First, I provide an overview of Cnidaria with an emphasis on the current understanding of genes and proteins implicated in complex biological processes in a few select cnidarians. Second, to further develop resources for A. alata, I provide a formal redescription of this cubozoan and establish a neotype specimen voucher, which serve to stabilize the taxonomy of the species. Third, I generate the first functionally annotated transcriptome of adult and larval A. alata tissue and apply preliminary differential expression analyses to identify candidate genes implicated broadly in biological processes related to prey capture and defense, vision and the phototransduction pathway and sexual reproduction and gametogenesis. Fourth, to better understand venom diversity and mechanisms controlling venom synthesis in A. alata, I use bioinformatics to investigate gene candidates with dual roles in venom and digestion, and review the biology of prey capture and digestion in cubozoans. The morphological and molecular resources presented herein contribute to understanding the evolution of cubozoan characteristics and serve to facilitate further research on this emerging cubozoan model.

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Protein disulfide isomerase as a target for besnoitiosis therapy : molecular characterization and studies of its role in infection and host immune response

Tese de Doutoramento em Ciências Veterinárias na especialidade de Sanidade Animal

Agents actifs toxiques dans les produits éclaircissants et leurs impacts sur le microbiome cutané humain

Les produits cosmétiques sont des substances utilisées pour entretenir ou modifier l'aspect des parties superficielles du corps humain (telles que la peau, les ongles ou les cheveux). Dans de nombreux pays d’Afrique et d’Asie et dans certaines communautés africaines immigrantes, plusieurs femmes et parfois des hommes utilisent des produits contenant des agents actifs tels que le mercure, l’hydroquinone et le propionate de clobétasol pour éclaircir leur peau. Ces principaux agents sont toxiques et leur présence dans les cosmétiques est règlementée, voire interdite, dans plusieurs pays. Dans notre étude, nous avons déterminé les concentrations de ces ingrédients dans plusieurs produits utilisés en Afrique de l’Ouest et au Canada. Nous avons également exploré l’effet de ces produits sur le microbiome cutané. Nos résultats révèlent que 68 à 84% des crèmes et 7.5 à 65% des savons dépassent les normes lorsqu’on considère l’interdiction de mercure, d’hydroquinone et de propionate de clobétasol et les concentrations déclarées sur les étiquettes ne sont pas souvent fiables. Selon la diversité de Shannon, il semble y avoir plus d’équitabilité, et donc moins de dominance dans le groupe des femmes utilisant les crèmes éclaircissantes que dans le groupe des femmes qui ne les utilisent pas. Par ailleurs, nous n’avons pas trouvé de différences significatives au niveau du microbiome cutané du groupe avec crèmes et sans crèmes au niveau du phylum et du genre. Cependant, d’autres méthodes plus approfondies avec plus d’échantillonnage pourraient révéler à des échelles plus fines (espèces, souches, etc.) l’effet de ces produits sur le microbiome cutané.

Agents actifs toxiques dans les produits éclaircissants et leurs impacts sur le microbiome cutané humain

Les produits cosmétiques sont des substances utilisées pour entretenir ou modifier l'aspect des parties superficielles du corps humain (telles que la peau, les ongles ou les cheveux). Dans de nombreux pays d’Afrique et d’Asie et dans certaines communautés africaines immigrantes, plusieurs femmes et parfois des hommes utilisent des produits contenant des agents actifs tels que le mercure, l’hydroquinone et le propionate de clobétasol pour éclaircir leur peau. Ces principaux agents sont toxiques et leur présence dans les cosmétiques est règlementée, voire interdite, dans plusieurs pays. Dans notre étude, nous avons déterminé les concentrations de ces ingrédients dans plusieurs produits utilisés en Afrique de l’Ouest et au Canada. Nous avons également exploré l’effet de ces produits sur le microbiome cutané. Nos résultats révèlent que 68 à 84% des crèmes et 7.5 à 65% des savons dépassent les normes lorsqu’on considère l’interdiction de mercure, d’hydroquinone et de propionate de clobétasol et les concentrations déclarées sur les étiquettes ne sont pas souvent fiables. Selon la diversité de Shannon, il semble y avoir plus d’équitabilité, et donc moins de dominance dans le groupe des femmes utilisant les crèmes éclaircissantes que dans le groupe des femmes qui ne les utilisent pas. Par ailleurs, nous n’avons pas trouvé de différences significatives au niveau du microbiome cutané du groupe avec crèmes et sans crèmes au niveau du phylum et du genre. Cependant, d’autres méthodes plus approfondies avec plus d’échantillonnage pourraient révéler à des échelles plus fines (espèces, souches, etc.) l’effet de ces produits sur le microbiome cutané.