Studies on the acorn barnacle Balanus amphitrite and its associated bacteria

PhD ThesisDespite being a model organism to study settlement in marine invertebrates, little is known about the genetics of the barnacle Balanus amphitrite. To fill this gap, cDNA libraries representative of different developmental stages were generated and sequenced. Nearly 14,000 genes were annotated, which may represent 2/3 of the species’ total protein coding regions. The database that was created to allow public access to this genetic information will profoundly benefit future research aiming to understand the molecular regulation of development and settlement in this species. Furthermore, a quantitative real-time PCR assay to study gene expression in B. amphitrite was designed and validated. Eleven genes were studied for their ability to normalize qRT-PCR data. Total RNA extracted from seven developmental stages was reverse transcribed and the expression stability of the selected genes was compared. It was found that transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, and their use to normalize gene expression data is recommended. Conflicting evidence exists on the role of bacteria in B. amphitrite settlement. However, there is a paucity of information on the microbial community naturally associated with this barnacle. In order to reveal the existence of stable associations, a 16S rRNA-based, taxon-specific qPCR assay was developed to monitor the preponderance of 5 bacterial phyla and classes. Furthermore, attempts to profile these qPCR products by DGGE were made. This new method was applied to characterise the bacterial communities associated with different B. amphitrite developmental stages and body parts. It was found that the structure of these communities changed throughout the barnacle life cycle in a highly reproducible manner. Furthermore, bacteria isolated from the barnacle shell were capable of inducing settlement of conspecific larvae. The analysis of these communities at a lower taxonomic level should confirm if any of these ecologically important bacteria are vertically transmitted.Natural Environment Research Council, UK: The Boehringer Ingelheim Fonds: The European Community, The Marine Genomics Network: The Office of Naval Research,USA: The Prime Minister Initiative, UK

Construction of an adult barnacle (Balanus amphitrite) cDNA library and selection of reference genes for quantitative RT-PCR studies

De Gregoris TB, Borra M, Biffali E, et al. Construction of an adult barnacle (Balanus amphitrite) cDNA library and selection of reference genes for quantitative RT-PCR studies. BMC Molecular Biology. 2009;10(1):62.BACKGROUND: Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR) data obtained from different developmental stages of this animal. RESULTS: We generated a cDNA library containing expressed sequence tags (ESTs) from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets) were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. CONCLUSION: The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies

The bioelectrogenic column: a tool for bringing microbial ecology and electrochemistry into secondary school

Para transmitir conceptos científicos complejos en un entorno escolar, la utilización de experimentos llamativos representa una herramienta muy útil. En este artículo se presenta la columna bioelectrogénica, un sistema experimental que une la columna de Winogradsky clásica con las más recientes investigaciones en el campo de la microbiología aplicada. A través de su implementación es posible visualizar procesos físicos, químicos y biológicos que forman parte de la enseñanza científica en institutos de secundaria. Además, la columna bioelectrogénica ha sido ideada para demostrar la existencia de bacterias capaces de producir energía eléctrica, un fenómeno que induce curiosidad y estupor, elementos claves para estimular la participación de los estudiantes. Este sistema ha sido presentado en varias actividades de divulgación para estudiantes de secundaria y siempre ha despertado un elevado grado de interés.The use of inspiring experimental demonstrations is a fundamental tool for teaching complex scientific concepts in secondary schools. In this article we present the bioelectrogenic column, an experimental system that joins the classic Winogradsky column to the most recent findings in the field of applied microbiology. With this system we can visualise physical, chemical and biological processes that are at the heart of a sound scientific education. Furthermore, the bioelectrogenic column had been designed to demonstrate the existence of bacteria capable of producing electric energy, an astonishing phenomenon that leaves students highly inquisitives, a key for stimulating their participation. This system has been presented in various scientific outreaching activities for secondary school, always awakening a strong interest in the audience

La columna bioelectrogénica: una herramienta para introducir conceptos de ecología microbiana y electroquímica en la educación secundaria

The use of inspiring experimental demonstrations is a fundamental tool for teaching complex scientific concepts in secondary schools. In this article we present the bioelectrogenic column, an experimental system that joins the classic Winogradsky column to the most recent findings in the field of applied microbiology. With this system we can visualise physical, chemical and biological processes that are at the heart of a sound scientific education. Furthermore, the bioelectrogenic column had been designed to demonstrate the existence of bacteria capable of producing electric energy, an astonishing phenomenon that leaves students highly inquisitives, a key for stimulating their participation. This system has been presented in various scientific outreaching activities for secondary school, always awakening a strong interest in the audience.Para transmitir conceptos científicos complejos en un entorno escolar, la utilización de experimentos llamativos representa una herramienta muy útil. En este artículo se presenta la columna bioelectrogénica, un sistema experimental que une la columna de Winogradsky clásica con las más recientes investigaciones en el campo de la microbiología aplicada. A través de su implementación es posible visualizar procesos físicos, químicos y biológicos que forman parte de la enseñanza científica en institutos de secundaria. Además, la columna bioelectrogénica ha sido ideada para demostrar la existencia de bacterias capaces de producir energía eléctrica, un fenómeno que induce curiosidad y estupor, elementos claves para estimular la participación de los estudiantes. Este sistema ha sido presentado en varias actividades de divulgación para estudiantes de secundaria y siempre ha despertado un elevado grado de interés.Palabras clave: Bioelectrogénesis. Columna de Winogradsky. Ecología microbiana. Bioelectroquímica. Bacterias productoras de la electricidad. Geobacter.The bioelectrogenic column: a tool for bringing microbial ecology and electrochemistry into secondary schoolThe use of inspiring experimental demonstrations is a fundamental tool for teaching complex scientific concepts in secondary schools. In this article we present the bioelectrogenic column, an experimental system that joins the classic Winogradsky column to the most recent findings in the field of applied microbiology. With this system we can visualise physical, chemical and biological processes that are at the heart of a sound scientific education. Furthermore, the bioelectrogenic column had been designed to demonstrate the existence of bacteria capable of producing electric energy, an astonishing phenomenon that leaves students highly inquisitives, a key for stimulating their participation. This system has been presented in various scientific outreaching activities for secondary school, always awakening a strong interest in the audience.Keywords: Bioelectrogenesis. Winogradsky column. Molecular ecology. Bioelectrochemistry. Electricity-producing bacteria. Geobacter

Diversity and enzymatic potentialities of Bacillus sp. strains isolated from a polluted freshwater ecosystem in Cuba

Genotypic and phenotypic characterization of Bacillus spp. from polluted freshwater has been poorly addressed. The objective of this research was to determine the diversity and enzymatic potentialities of Bacillus spp. strains isolated from the Almendares River. Bacilli strains from a polluted river were characterized by considering the production of extracellular enzymes using API ZYM. 14 strains were selected and identified using 16S rRNA, gyrB and aroE genes. Genotypic diversity of the Bacillus spp. strains was evaluated using pulsed field gel electrophoresis. Furthermore, the presence of genetic determinants of potential virulence toxins of the Bacillus cereus group and proteinaceous crystal inclusions of Bacillus thuringiensis was determined. 10 strains were identified as B. thuringiensis, two as Bacillus megaterium, one as Bacillus pumilus and one as Bacillus subtilis. Most strains produced proteases, amylases, phosphatases, esterases, aminopeptidases and glucanases, which reflect the abundance of biopolymeric matter in Almendares River. Comparison of the typing results revealed a spatio-temporal distribution among B. thuringiensis strains along the river. The results of the present study highlight the genotypic and phenotypic diversity of Bacillus spp. strains from a polluted river, which contributes to the knowledge of genetic diversity of Bacilli from tropical polluted freshwater ecosystems

Deep sequencing of naupliar-, cyprid- and adult-specific normalised Expressed Sequence Tag (EST) libraries of the acorn barnacle Balanus amphitrite

De Gregoris TB, Rupp O, Klages S, et al. Deep sequencing of naupliar-, cyprid- and adult-specific normalised Expressed Sequence Tag (EST) libraries of the acorn barnacle Balanus amphitrite. Biofouling. 2011;27(4):367-374.In order to improve the genetic characterisation of the barnacle Balanus amphitrite, normalised EST libraries for the developmental stages, viz. nauplius (a mix of instars I and II), cyprid and adult, were generated. The libraries were sequenced independently using 454 technologies and 575,666 reads were generated. For adults, 4843 unique isotigs were estimated and 6754 and 7506 in the cyprid and naupliar stage, respectively. It was found that some of the previously proposed cyprid-specific bcs genes were also expressed during the naupliar and adult stage. Furthermore, as lectins have been hypothesised to influence settlement cue recognition in barnacles, the database was searched for lectin-like isotigs. Two proteins, uniquely expressed in either the cyprid or the adult stage, matched a mannose receptor, and their nucleotide sequences were 33% and 31% identical to a lectin (BRA-3) isolated from Megabalanus rosa. Further characterisation of these genes may suggest their involvement in settlement