Draft genome assemblies of the avian louse Brueelia nebulosa and its associates using long-read sequencing from an individual specimen.

Sequencing high molecular weight (HMW) DNA with long-read and linked-read technologies has promoted a major increase in more complete genome sequences for nonmodel organisms. Sequencing approaches that rely on HMW DNA have been limited to larger organisms or pools of multiple individuals, but recent advances have allowed for sequencing from individuals of small-bodied organisms. Here, we use HMW DNA sequencing with PacBio long reads and TELL-Seq linked reads to assemble and annotate the genome from a single individual feather louse (Brueelia nebulosa) from a European Starling (Sturnus vulgaris). We assembled a genome with a relatively high scaffold N50 (637 kb) and with BUSCO scores (96.1%) comparable to louse genomes assembled from pooled individuals. We annotated a number of genes (10,938) similar to the human louse (Pediculus humanus) genome. Additionally, calling phased variants revealed that the Brueelia genome is more heterozygous (∼1%) then expected for a highly obligate and dispersal-limited parasite. We also assembled and annotated the mitochondrial genome and primary endosymbiont (Sodalis) genome from the individual louse, which showed evidence for heteroplasmy in the mitogenome and a reduced genome size in the endosymbiont compared to its free-living relative. Our study is a valuable demonstration of the capability to obtain high-quality genomes from individual small, nonmodel organisms. Applying this approach to other organisms could greatly increase our understanding of the diversity and evolution of individual genomes.

In Silico and Cellular Differences Related to the Cell Division Process between the A and B Races of the Colonial Microalga Botryococcus braunii.

Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.

Draft Nuclear Genome Sequence of the Liquid Hydrocarbon-Accumulating Green Microalga Botryococcus braunii Race B (Showa).

Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism.

High-throughput droplet microfluidics screening platform for selecting fast-growing and high lipid-producing microalgae from a mutant library.

Biofuels derived from microalgal lipids have demonstrated a promising potential as future renewable bioenergy. However, the production costs for microalgae-based biofuels are not economically competitive, and one strategy to overcome this limitation is to develop better-performing microalgal strains that have faster growth and higher lipid content through genetic screening and metabolic engineering. In this work, we present a high-throughput droplet microfluidics-based screening platform capable of analyzing growth and lipid content in populations derived from single cells of a randomly mutated microalgal library to identify and sort variants that exhibit the desired traits such as higher growth rate and increased lipid content. By encapsulating single cells into water-in-oil emulsion droplets, each variant was separately cultured inside an individual droplet that functioned as an independent bioreactor. In conjunction with an on-chip fluorescent lipid staining process within droplets, microalgal growth and lipid content were characterized by measuring chlorophyll and BODIPY fluorescence intensities through an integrated optical detection system in a flow-through manner. Droplets containing cells with higher growth and lipid content were selectively retrieved and further analyzed off-chip. The growth and lipid content screening capabilities of the developed platform were successfully demonstrated by first carrying out proof-of-concept screening using known Chlamydomonas reinhardtii mutants. The platform was then utilized to screen an ethyl methanesulfonate (EMS)-mutated C. reinhardtii population, where eight potential mutants showing faster growth and higher lipid content were selected from 200,000 examined samples, demonstrating the capability of the platform as a high-throughput screening tool for microalgal biofuel development.

ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent.

We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.

Stress responses of the oil-producing green microalga Botryococcus braunii Race B.

Plants react to biotic and abiotic stresses with a variety of responses including the production of reactive oxygen species (ROS), which may result in programmed cell death (PCD). The mechanisms underlying ROS production and PCD have not been well studied in microalgae. Here, we analyzed ROS accumulation, biomass accumulation, and hydrocarbon production in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. We also identified and cloned a single cDNA for the B. braunii ortholog of the Arabidopsis gene defender against cell death 1 (DAD1), a gene that is directly involved in PCD regulation. The function of B. braunii DAD1 was assessed by a complementation assay of the yeast knockout line of the DAD1 ortholog, oligosaccharyl transferase 2. Additionally, we found that DAD1 transcription was induced in response to SA at short times. These results suggest that B. braunii responds to stresses by mechanisms similar to those in land plants and other  organisms.