Project description:The marine diatom Guinardia delicatula is a cosmopolitan species that dominates seasonal blooms in the English Channel and the North Sea. Several eukaryotic parasites are known to induce the mortality of this key-stone species. Here, we report the isolation and the characterization of the first viruses that infect G. delicatula. Viruses were isolated from the Western English Channel (SOMLIT-ASTAN station) during the late summer bloom decline of G. delicatula. A combination of laboratory approaches revealed that these lytic viruses (GdelRNAV) are small untailed particles of 35-38 nm in diameter that replicated in the host cytoplasm where both unordered particles and crystalline arrays were formed. GdelRNAV displayed a linear single-stranded RNA genome of ~9 kb, including two open reading frames encoding for replication and structural polyproteins. Phylogenetic relationships based on the RNA-dependent-RNA-polymerase gene marker showed that GdelRNAV were new members of the Bacillarnavirus, a monophyletic genus belonging to the order Picornavirales. GdelRNAV were specific to several strains of G. delicatula, they were produced rapidly (< 12h) and in numbers (9.34 x 104 virions per host cell). We recorded a substantial delay (72 h) between virions release and host cell lysis. Our analysis points to variable viral susceptibilities of the host during the early exponential growth phase. Interestingly, we consistently failed to isolate viruses during spring and early summer while G. delicatula developed rapid and massive blooms. While our study suggests that viruses do contribute to the decline of G. delicatula late summer bloom, they may not be the primary mortality agents during the remaining blooms at SOMLIT-ASTAN. Future studies should focus on the relative contribution of the viral and eukaryotic pathogens to the control of Guinardia blooms to understand the fate of these prominent organisms in marine systems.
Project description:A functional gene microarray was developed and used to investigate phytoplankton community composition and gene expression in the English Channel. Genes encoding the CO2 fixation enzyme RuBisCO (rbcL) and the nitrate assimilation enzyme nitrate reductase (NR) representing several major groups of phytoplankton were included as oligonucleotide probes on the 'phytoarray'. Five major groups of eukaryotic phytoplankton that possess the Type 1D rbcL gene were detected, both in terms of presence (DNA) and activity (rbcL gene expression). Changes in relative signal intensity among the Type 1D rbcL probes indicated a shift from diatom dominance in the spring bloom to dominance by haptophytes and flagellates later in the summer. Because of the limitations of a smaller database, NR probes detected fewer groups, but due to the greater diversity among known NR sequences, NR probes provided higher phylogenetic resolution than did rbcL probes, and identified two uncultivated diatom phylotypes as the most abundant (DNA) and active (NR gene expression) in field samples. Unidentified chlorophytes and the diatom Phaeodactylum tricornutum were detected at both the DNA and cDNA (gene expression) levels. The reproducibility of the array was evaluated in several ways and future directions for further improvement of probe development and sensitivity are outlined. The phytoarray provides a relatively high resolution, high throughput approach to assessing phytoplankton community composition in marine environments. Keywords: seawater natural assemblages, functional gene expression
Project description:A functional gene microarray was developed and used to investigate phytoplankton community composition and gene expression in the English Channel. Genes encoding the CO2 fixation enzyme RuBisCO (rbcL) and the nitrate assimilation enzyme nitrate reductase (NR) representing several major groups of phytoplankton were included as oligonucleotide probes on the 'phytoarray'. Five major groups of eukaryotic phytoplankton that possess the Type 1D rbcL gene were detected, both in terms of presence (DNA) and activity (rbcL gene expression). Changes in relative signal intensity among the Type 1D rbcL probes indicated a shift from diatom dominance in the spring bloom to dominance by haptophytes and flagellates later in the summer. Because of the limitations of a smaller database, NR probes detected fewer groups, but due to the greater diversity among known NR sequences, NR probes provided higher phylogenetic resolution than did rbcL probes, and identified two uncultivated diatom phylotypes as the most abundant (DNA) and active (NR gene expression) in field samples. Unidentified chlorophytes and the diatom Phaeodactylum tricornutum were detected at both the DNA and cDNA (gene expression) levels. The reproducibility of the array was evaluated in several ways and future directions for further improvement of probe development and sensitivity are outlined. The phytoarray provides a relatively high resolution, high throughput approach to assessing phytoplankton community composition in marine environments. Keywords: seawater natural assemblages, functional gene expression Two functional genes, nitrate reductase and RuBisCO, 4 - 8 replicate features per array
Project description:In order to identify gene expression difference between marine and freshwater stickleback populations, we compared the transcriptomes of seven adult tissues (eye, gill, heart, hypothalumus, liver, pectoral muscle, telencephalon) between a marine population sampled from the mouth of the Little Campbell river in British Columbia (LITC) and a freshwater population (Fishtrap Creek, FTC) from northern Washington. For each population, the sampled individuals were the lab-reared progeny of a single pair of wild-caught parents. Four to five fish from each population were used as biological replicates for each of the seven tissues. For each population, the sampled individuals were the lab-reared progeny of a single pair of wild-caught parents. All fish were of similar age and were raised in the same aquarium (salinity: 3.5 ppt), with a plastic divider separating the marine and freshwater groups. One male and four females were sampled from each population. Microarray experiments were performed in a 2-color format on custom Agilent arrays: experimental RNA samples were labeled with Cy5, and the common reference RNA sample was labeled with Cy3. The reference RNA was total RNA isolated from a large number of 7-day-post-hatch embryos from the freshwater population of Bear Paw Lake, Alaska (BEPA). One technical replicate was used for each array, and one of the hypothalamus samples (Hyp_FTC#3) was excluded from further analysis due to poor quality indicators. FTC#1 liver and LITC#2 pectoral muscle samples did not yield RNA of sufficient quality for the microarray experiment, and were also excluded from hybridization.