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Overexpression of a novel gene (Pt2015) endows the commercial diatom Phaeodactylum tricornutum high lipid content and grazing resistance
Shan Gao1,2 na1,
Lu Zhou1,2,3 na1,
Wenting Yang4,
Lijun Wang1,2,
Xuehua Liu1,2,
Yingchun Gong5,
Qiang Hu6,7 &
Guangce Wang1,2
The marine diatom Phaeodactylum tricornutum is a commercially viable species due to its bioactive substances and lipid productivity. Increasing attention has been paid to the isolation or genetic modification of species or strains with a rapid growth rate and large quantities of lipids. Furthermore, contamination of microzooplankton has been one of the major constraints in P. tricornutum large-scale cultivation, which adversely affects growth and greatly impedes the course of biomass production industrialization.
Here, based on our previous transcriptomics of P. tricornutum, we found a novel gene (ID: 7202015, hereafter called Pt2015) which affects morphotype of P. tricornutum. Pt2015 protein is located in the plastid, which is highly homologous to part of the sequences of exosome component. The morphotype of the Pt2015 knockout strain (termed 2015KO) using CRISPR/Cas9 method is fusiform, but the Pt2015 overexpression strain (termed oeT) demonstrates a majority triradiate morphotype (approximately 95%) which is stable and has been cultured for more than 200 generations. In addition, the oeT strain demonstrated a similar growth rate to the WT and simultaneously accumulated larger lipids droplets that increased by approximately 30% compared to that of the WT. More importantly, the grazing rate of the amoebae cultured in the oeT strain significantly decreased in comparison with that cultured in WT, suggesting that the oeT can effectively avoid being eaten by microzooplankton.
Therefore, the oeT strain not only improves our understanding of morphotype conversion in diatoms but also demonstrates potential applications for large-scale cultivation of P. tricornutum.
Diatoms are unicellular eukaryotic microalgae responsible for approximately 20% of the primary production on Earth and are the dominant primary producers in the ocean [1]. They are highly abundant and diverse, with estimates of more than 100,000 species [2]. Diatoms have been exploited on a commercial scale for decades due to their bioactive substances, silica biomineralization, and lipid productivity [3, 4]. Phaeodactylum tricornutum is one of the most extensively studied diatom species. Its genome has been sequenced [5], and genetic manipulation, including gene silencing and CRISPR/Cas9, has been proven to be efficient in this species [6,7,8,9]. These resources and tools will help investigate gene function and decipher cellular processes in diatoms. Therefore, P. tricornutum has been a model for studying diatom biology at the molecular level. Additionally, due to its robust growth, a well-characterized genome, and a tailored molecular toolbox, P. tricornutum has become a vital microalgal candidate for a cell chassis and plays an important role in synthetic biology.
P. tricornutum is also a commercially important species that is widely used in aquaculture, and is rich in active substances, such as eicosapentaenoic acid and fucoxanthin [10, 11]. Furthermore, P. tricornutum synthesizes and stores neutral lipids (mainly triglycerides) in lipid droplets, which can be converted to biodiesel, and thus, it has potential to be a useful biofuel [11]. A general trend toward lipid accumulation in response to unfavorable environmental or stressful conditions has been observed in P. tricornutum [11]. Nitrogen and phosphorus limitations have been successfully utilized to accumulate lipids in P. tricornutum [12,13,14]. Nevertheless, unfavorable environmental or stressful conditions, including nutrient limitations, profoundly decrease growth rate and biomass production [11]. Therefore, increasing attention has been paid to the isolation or genetic modification of species or strains that can grow rapidly and simultaneously accumulate large quantities of lipids.
Due to its huge commercial value, large-scale cultivation of P. tricornutum also has gained increasing attention [15]. In fact, P. tricornutum is a successful photoautotroph, which has the capacity to grow in seawater and to utilize carbon dioxide as a feedstock. Thus, low cost is a key advantage of large-scale cultivation of P. tricornutum. However, contamination of algivorous microzooplankton has been one of the main constraints in P. tricornutum large-scale cultivation, which adversely affects growth and greatly impedes the course of biomass production industrialization [16,17,18]. Many control strategies, including physical, chemical, and biological methods, have been proposed to overcome the challenges of microzooplankton contamination [17, 19,20,21]. However, these methods are costly and time consuming. Therefore, isolating a new strain with grazing resistance represents an important strategy for overcoming zooplankton contamination [22].
In fact, in natural aquatic systems, changing the morphological features, such as size, cell shape, or geometry of phytoplankton, is the most obvious way to reduce grazing pressure from zooplankton [23,24,25,26,27]. In response to different conditions, P. tricornutum can display at least three distinct morphotypes: fusiform, oval, and triradiate [28,29,30,31,32]. The fusiform morphotype is the most frequent and stable type in cell cultures [30]. The oval morphotype tends to be benthic, having a lower growth rate and higher sedimentation rate [33, 34]. The triradiate morphotype isolated from natural conditions is favored in an unstressed planktonic environment, which possesses specific geometry with three distal arms [30]. Therefore, the triradiate cells of P. tricornutum may be one of the best morphotypes to withstand grazing pressure.
Our previous transcriptomics of P. tricornutum treated with fluctuating light conditions suggested that thousands of genes in P. tricornutum were related to fluctuating light conditions. Among these genes, we chose several genes including Pt2015 (ID: 7202015) in order to investigate their roles under fluctuating light. Unexpectedly, we found that Pt2015 is not close to fluctuating light condition, but it affects morphotypes conversion and lipid metabolisms in P. tricornutum. Moreover, Pt2015 encodes an unknown protein, and to date, there is no knowledge regarding the function of this gene in diatoms. Therefore, to investigate the function of this gene, we constructed Pt2015 knockout and overexpression strains, respectively. We found that overexpression of Pt2015 caused a P. tricornutum triradiate strain called overexpression triradiate (oeT), in which the abundance of triradiate cells was approximately 95% under normal growth condition. The oeT strain was stable, making it insensitive to hyposalinity and low temperature. More importantly, the oeT strain showed a similar growth rate to the WT, accumulated larger lipids droplets, and withstood grazing pressure, demonstrating potential applications for biofuel production.
Axenic P. tricornutum cells were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences, and grown at 20 °C in a photoperiod 16 h:8 h, light:dark. Cells were grown under 80 μmol photons m−2 s−1 and collected during the exponential growth phase.
Phylogenetic analysis and prediction of Pt2015 localization in P. tricornutum
Phylogenetic analysis of Pt2015 proteins was performed using the MEGA v.7.0 platform using the neighbor-joining method. In total, 34 protein sequences were used to perform the phylogenetic analysis. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was shown next to the branches in Fig. 1. Moreover, a comparison of the conserved regions in the Pt2015 protein from diatoms, chrysophytes, dinoflagellates, and other red-tide algae was performed using the MEGA v.7.0 platform. In addition, the cloned Pt2015 sequences were analyzed for the presence of N-terminal signal peptides and cleavage sites using the SignalP v.4.1 Server (www.cbs.dtu.dk/services/SignalP/). The TMHMM Server v.2.0 (www.cbs.dtu.dk/services/TMHMM/) was used to predict transmembrane domains.
Phylogenetic analysis of Pt2015 proteins. In total, 34 protein sequences were used to perform phylogenetic analysis using the MEGA 7.0 platform and the neighbor-joining method. The percentage of replicate trees in which the associated taxa cluster together in the bootstrap test (500 replicates) is shown next to the branches
Construction of Pt2015 knockout strains using CRISPR/Cas9
Pt2015 knockout strains were constructed according to a previous method in which the Cas9 system was delivered into P. tricornutum via conjugation of plasmids from a bacterial donor cell [7, 8]. Briefly, three guide RNAs (gRNAs) targeting Pt2015 (Additional file 1: Table S1) were designed on the website (http://crispor.tefor.net/). To generate gRNA cloning inserts, the designed oligonucleotide corresponding to the predicted gRNA binding site and the complementary oligonucleotide were ordered, and then phosphorylated and annealed. Using Golden Gate assembly (New England Biolabs), cloned gRNA was inserted into the pPtGE35 plasmid obtained from Addgene (107999). The product from the Golden Gate reaction was transformed into Epi300 E. coli cells using heat shock. Then, the properly cloned gRNA plasmid was transformed into DH10B E. coli cells containing the pTA-Mob plasmid [35] obtained from Dr. Rahmi Lale (Norwegian University of Science and Technology, Trondheim, Norway).
Subsequently, according to the published protocol [8], the pPtGE35 plasmid containing gRNA inserts was transferred into P. tricornutum via conjugation from E. coli. Screening for Pt2015 knockouts in P. tricornutum induced by Cas9 was performed as previously described [8] with minor modifications. After 2–3 weeks, ten P. tricornutum exconjugants were randomly selected, inoculated into liquid F/2 medium with 50 μg mL−1 zeocin (Invitrogen), and grown for 5 d. The P. tricornutum exconjugant cells were lysed and used as the template for polymerase chain reaction (PCR) amplification of the gRNA target site using the specific primers shown in Additional file 1: Table S2. The PCR products were sent for Sanger sequencing to verify the deletion length. Subsequently, liquid cultures of P. tricornutum exconjugants that were identified to be edited were diluted to 10–4 and plated onto F/2 medium plates containing 50 μg mL−1 zeocin and grown for 10–14 d to obtain sub-clones.
Construction of vectors for Pt2015 overexpression
The coding sequence (444 bp) of the Pt2015 gene was amplified for overexpression using the primers Pt2015oe-F and Pt2015oe-R, and further digested with EcoRI and KpnI, and then inserted into pPha-T1 vector [36]. Additionally, the coding sequence (444 bp) of the Pt2015 gene for overexpression was amplified with Pt2015eGFP-F and Pt2015eGFP-R, and further digested by EcoRI and KpnI, and then inserted into pPHat_eGFP vector. The vectors containing coding sequences of the Pt2015 gene or the Pt2015-eGFP were introduced into WT P. tricornutum using biolistic transformation with the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, CA, USA) [37], respectively. For the selection of overexpression clones, bombarded cells were plated onto 50% fresh seawater agar plates (1% agar) supplemented with 100 μg mL−1 zeocin (Invitrogen). The plates were placed under conditions of 80 μmol photons m−2 s−1 for 2–3 weeks, and resistant colonies were inoculated into liquid F/2 medium containing 50 μg mL−1 zeocin. The transformants were screened using primers (YzF and YzR, Additional file 1: Table S2).
Cell preparation for electron microscopy
The procedures used for scanning electron microscopy sample preparation were as previously described, with minor modification [30]. The cells were fixed with 5% glutaraldehyde in phosphate buffered saline (PBS, pH 7.4) for 2 h and dehydrated in a series of increasing ethanol concentrations (30%, 50%, 70%, 80%, 90%, and 100%) for 10 min at 4 °C. The cells were treated with isoamyl acetate for 10 min, critical point dried (Hitachi-HCP, Hitachi, Japan), sputter coated with platinum (MC1000, Hitachi), and examined with a scanning electron microscope (S-3400 N, Hitachi).
The ultrastructure of the different samples was examined using transmission electron microscopy as previously described [30]. Cells were fixed with 2.5% glutaraldehyde (Hushi, Shanghai, China) in PBS for 2 h and washed three times for 30 min in 0.1 M PBS at 4 °C. The cells were then post-fixed with 1% osmium tetroxide (Ted Pella, CA, USA) in PBS for 1.5 h and washed three times with 0.1 M PBS at 4 °C. Next, the samples were dehydrated in alcohol (Hushi), infiltrated with acetone (Tieta, Laiyang, China) and an epoxy resin (SPI-CHEM, USA) mixture, embedded, and polymerized in epoxy resin. Ultrathin sections were obtained using a Leica EM UC7 ultramicrotome (Leica Microsystems, Germany) and transferred onto copper grids covered with a Formvar membrane (Electron Microscopy China, Beijing, China). Two-percent uranyl acetate and lead citrate (Ted Pella Inc.) were used for contrast staining. The sections were photographed using a transmission electron microscope (HT7700, Hitachi).
Fluorescent microscopy
Subcellular localization analysis of chlorophyll autofluorescence and the green fluorescent protein (GFP) fusion of Pt2015 was performed using laser-scanning confocal microscopy (LSM710, Carl Zeiss, 40 ×). Chlorophyll autofluorescence was excited at 633 nm and detected at a bandpass filter of 675–740 nm. GFP fluorescence was excited using an excitation wavelength of 488 nm and detected with a bandpass filter of 510–570 nm.
Lipid analysis
For lipid body labeling, the cells (1 × 106 cells/mL) of different strains were cultured for 7 d and incubated with BODIPY 505/515 (Thermo Fisher Scientific) at a concentration of 0.5 mg/mL for 7 min in the dark [38]. For cell imaging, BODIPY 505/515 was excited at 488 nm and fluorescence was collected from 510 to 550 nm. Gravimetric means, a conventional lipid quantification method, was used for total lipid analysis. Algal cultures were harvested after cultivation for 7 d by centrifugation at 2500 × g for 5 min. Algal cells were frozen in liquid nitrogen and then dried in a freeze dryer. Lipids were extracted from 50 mg dried algal powder using a modified chloroform–methanol system [39]. Crude samples were dried under a N2 flow until a constant weight was obtained.
Plastid purification, membrane solubilization, and immunoblotting
Plastids were purified as previously described [40]. Briefly, cells were centrifuged and re-suspended gently in isolation buffer (0.5 M sorbitol, 50 mM HEPES–KOH, 6 mM ethylenediaminetetraacetic acid (EDTA), 5 mM MgCl2, 10 mM KCl, 1 mM MnCl2, 1% (w/v) polyvinylpyrrolidone 40, 0.5% bovine serum albumin (BSA), and 0.1% cysteine; pH 7.2–7.5) and slowly passed through a French Press at 90 MPa. The mixture of broken cells was centrifuged at 300 × g for 8 min to remove intact cells and cell debris. The supernatant was collected and then centrifuged at 2000 × g for 10 min at 4 °C. The pellet containing the plastids was gently re-suspended in washing buffer (0.5 M sorbitol, 30 mM HEPES–KOH, 6 mM EDTA, 5 mM MgCl2, 10 mM KCl, 1 mM MnCl2, 1% polyvinylpyrrolidone 40, 0.1% BSA; pH 7.2–7.5) and loaded onto a discontinuous Percoll gradient (10%, 20%, and 30%) in the same buffer. After centrifugation (SW40Ti rotor, Beckman Coulter, Indianapolis, IN, USA) at 10,000 × g for 35 min, the plastid fraction was recovered in the 20% Percoll layer of the gradient, diluted in washing buffer (without BSA), and subjected to centrifugation at 14,000 × g for 10 min at 4 °C. To separate thylakoids and the stroma, plastids were subjected to osmotic shock (induced by incubation for 5 min in the washing buffer without sorbitol), and centrifugation. The supernatants contained the stroma and the pellets comprised the thylakoids, which were re-suspended in washing buffer without sorbitol.
Thylakoid membrane complexes were solubilized and separated as previously described, with minor modification [41]. The thylakoids were solubilized in a buffer (25 mM HEPES–KOH [pH 7.0], 10 mM NaCl, and 5 mM MgCl2) containing 1% n-dodecyl-β-D-maltoside (β-DM) with a chlorophyll concentration of 0.5 μg/μL for 10 min on ice. After centrifugation at 15,000 × g for 10 min at 4 °C, the supernatant (200 μg chlorophyll) was loaded onto the top of a sucrose density gradient (1.6 M, 1.3 M, 1 M, 0.7 M, 0.4 M, and 0.1 M) containing 25 mM HEPES–KOH (pH 7.0), 10 mM NaCl, 0.02% β-DM, and 5 mM MgCl2. Thylakoid protein complexes were separated by ultracentrifugation for 22 h at 220,000 × g at 4 °C in a SW40 rotor (Beckman Coulter). Ten fractions were extracted with syringes and equal amounts of each fraction were used for further tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis.
Immunoblotting analysis was performed as previously described [41]. Antibodies to PsbA, PsbD, PsaA, CytF, Actin, ribulose bisphosphate carboxylase large subunit, and LhcX (LhcSR of moss) were purchased from Agrisera (Vännäs, Sweden). Peptides were designed to match unique sections of Pt2015 (CREWRCKFEGDKSDSE), chemically synthesized, and then injected into rabbits to generate polyclonal antibodies (PhytoAB, San Jose, CA, USA). Horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (Tian Gen, Beijing, China) were used for detection.
Measurement of chlorophyll fluorescence and P700
In vivo chlorophyll a fluorescence and P700 oxidation signals were measured simultaneously at 25 °C using a Dual-PAM-100 (Heinz Walz, Germany) in P. tricornutum WT, 2015KO, and oeT strains. Before measurements, cells were centrifuged (1500 × g, at 20 °C, 3 min) and concentrated 40-fold, and then dark acclimated for 10 min. A saturating pulse of light (300 ms, 10,000 μmol photons m−2 s−1) was used to determine the maximal fluorescence levels in the dark-adapted state (Fm) and during actinic light illumination (Fm′). The steady-state fluorescence level (Fs) was recorded during actinic light illumination (80 μmol photons m−2 s−1). The quantum yield [Y(II)] of photosystem II (PSII) was calculated as (Fm–Fs)/Fm′. The Y(NO) and Y(NPQ) were calculated using the Dual-PAM software and saved in a report file. Photosystem I (PSI) was measured in the dual-wavelength mode (photodetector set to measure 875 nm and 830 nm pulse-modulated light). The maximum P700 signal, Pm, was determined by the application of the saturation pulse in the presence of far-red light. The P700 signal, P, was determined just before the saturation pulse. Pm′ is the maximum P700 signal induced by combined actinic illumination with the saturation flash. Y(I) was calculated as (Pm′–P)/Pm. The acceptor-side limitation of PSI (Y(NA)) was calculated as (Pm–Pm′)/Pm, while the donor-side limitation of PSI (Y(ND)) was calculated as 1–Y(I)–Y(NA)[42, 43].
Measurements of photosynthetic O2 evolution
Rates of photosynthetic O2 evolution of the WT, 2015KO, and oeT strains under different light conditions and dark respiration were measured using a Clark-type oxygen electrode (Hansatech, Norfolk, UK). Cells were concentrated via centrifugation at 1500 × g for 3 min. After centrifugation, cells were re-suspended in fresh F/2 culture medium. The OD730 value was adjusted to 1, and 2 mL was placed into the electron chamber. Before measurement under different light conditions, the samples were dark acclimated for 10 min. After the dark pre-treatment, the rate of O2 evolution in the chamber was measured over a range of light intensity (50–2800 μmol photons m−2 s−1), provided by an LED light source peaking at 630–640 nm. In addition, after the dark pre-treatment, the rate of O2 evolution of the cells was measured under a light intensity of 100 and 2000 μmol photons m−2 s−1, respectively.
Cultivation of amoebae
The amoebae used in this study belongs to the Heterolobosea class which was isolated from large-scale cultivation of P. tricornutum. The spores of amoebae were centrifugated at 3500 × g for 5 min, and then diluted with WT and oeT cells with the same cell density (5 × 106 cells/ml), respectively. The diluent was cultured in culture plates (6 wells) at 25 °C under the dark condition. After 24 h, the cells concentrations of the WT and oeT were counted.
Salinity and temperature experiments of the oeT strain
For the salinity experiments, three salinity conditions (100%, 50%, and 30% seawater) were tested using F/2 medium. Nutrient trace metals and vitamins were the same as in the F/2 medium, except the salts were diluted to 50% or 30% seawater. The oeT strain, which was cultured in artificial seawater (100% seawater) for at least 2 months, was moved to three salinity conditions (100% (control), 50%, and 30%) at a starting density of 1 × 106 cell mL−1, followed by weekly cell counting for 60 d. All three salinity treatments were performed at 20 °C. Meanwhile, two temperature conditions (20 °C and 10 °C) with the same salinity (100% seawater) were tested at a starting density of 1 × 106 cell mL−1, followed by cell counting weekly for 60 d. During this period, the F/2 medium was supplemented every 3 weeks and the percentage of abundance of different cell morphologies (triradiate and fusiform) was quantified by counting in a counting chamber.
RNA extraction, sequencing, and bioinformatics analysis
Total RNA was extracted using the TRIzol reagent kit (Invitrogen), according to the manufacturer's protocol, and was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and electrophoresis. The enriched mRNA was fragmented into short fragments and reverse transcribed into cDNA. The cDNA fragments were purified, end repaired, and a poly(A) tail was added. Then the fragments were ligated to Illumina sequencing adapters. The ligation products were size selected using electrophoresis, PCR-amplified, and sequenced using an Illumina Novaseq 6000 by Gene Denovo Biotechnology Co. Ltd. (Guangzhou, China).
To obtain high quality clean reads, reads were further filtered using FASTP (version 0.18.0) (version 0.18.0) [44]. Ribosome RNA reads were removed after alignment with Bowtie2 (version 2.2.8) [45]. The remaining clean reads were assembled and mapped to the P. tricornutum genome (National Center for Biotechnology Information—Assembly: ASM15095v2) using HISAT2 2.4 under default parameters [46]. The mapped reads of each sample were assembled using StringTie v1.3.1 [47]. For each transcription region, a fragment per kilobase of transcript per million mapped reads value was calculated to quantify its expression abundance and variation. Principal component analysis was performed using R package gmodels (http://www.r-project.org/), which is a statistical procedure that converts hundreds of thousands of correlated variables (gene expression) into a set of values of linearly uncorrelated variables called principal components. Differential expression analysis of RNA was performed using DESeq2 software between two different groups (false discovery rate (FDR) < 0.05, absolute fold change ≥ 2) [48]. Gene function classification was annotated using Gene Ontology (GO) and KEGG enrichment (FDR ≤ 0.05) [49].
Tandem Mass Tag (TMT)-based quantitative proteomic analysis
Proteomic analysis of the samples (WT, 2015KO, and oeT strains) was performed by PTM-Bio labs Co. Ltd. (Hangzhou, Zhejiang, China). The samples were ground in liquid nitrogen, and the powder was transferred to a 5 mL centrifuge tube and then sonicated in a lysis buffer (8 M urea, 1% Triton X-100, 10 mM dithiothreitol, 1% protease inhibitor cocktail, 3 μM Trichostatin A, 50 mM N-(9-acridinyl)maleimide, and 2 mM EDTA). The precipitate was then reconstituted with 8 M urea, and the protein concentration was measured using a bicinchoninic acid kit. For trypsin digestion, the protein solution was reduced with 5 mM dithiothreitol at 56 °C for 30 min and alkylated with 11 mM iodoacetamide at room temperature for 15 min in the dark.
After trypsin digestion, the peptides were desalted using a Strata X C18 PSE column (Phenomenex, USA) and freeze dried under vacuum. For TMT labeling, the samples were reconstituted in 0.5 M triethylammonium bicarbonate and processed according to the TMT kit instructions (Thermo-Scientific). The detailed procedures were performed in accordance with previous methods [50, 51].
For high-performance liquid chromatography fractionation, the peptides were fractionated using high-pH reverse-phase high-performance liquid chromatography with an Agilent 300Extend C18 column (Agilent, Santa Clara, CA, USA) (5 mm particles, 4.6 mm internal diameter). The peptides were first separated into 80 fractions over 80 min using a gradient of 2%–60% acetonitrile (pH 9), after which the peptides were combined into 18 fractions and dried by vacuum centrifugation. The peptides were then subjected to a nanospray ionization source followed by tandem mass spectrometry with Q Exactive™ Plus (Thermo-Scientific) coupled online to ultra-performance liquid chromatography.
The tandem mass spectrometry data were processed using the Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against the P. tricornutum UniProt database combined with a reverse decoy database. Trypsin/P was specified as a cleavage enzyme, allowing up to two missing cleavages. The mass tolerance for precursor ions was set at 20 ppm in the first search and 5 ppm in the main search. The mass error was set to 0.02 Da for the precursor ions and fragment ions. Carbamidomethylation on cysteine was specified as a fixed modification and oxidation on methionine was specified as a variable modification. FDR thresholds for protein, peptide, and modification site identification were specified at 1%. TMT-6-plex was selected as the quantification method. All other parameters in MaxQuant were set to default values.
The functional annotation of the differentially expressed proteins (DEPs) was annotated to the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The DEP IDs were converted to UniProt IDs, and then the UniProt IDs were used to match the GO ID, and the corresponding information was retrieved from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/) according to the GO ID. Un-annotated proteins were annotated by InterProScan using the protein sequence alignment method. All annotated proteins were then classified into three categories: biological process (BP), cellular component (CC), and molecular function (MF). The KEGG database (http://www.genome.jp/kegg/) was used to annotate the protein pathway.
Data are expressed as the mean value of four independent experiments (± standard deviation). Data were analyzed using analysis of variance with the SPSS 13.0 statistical software (IBM, Armonk, NY, USA). An independent-samples t-test was used at the α = 0.05 significance level to determine whether significant differences existed between the WT, 2015KO, and oeT strains.
Bioinformatics analysis of Pt2015
Pt2015 (ID: 7202015, PHATRDRAFT_47103) is localized in chromosome 12 of P. tricornutum, and contains one exon and no introns. It encodes a protein consisting of 147 amino acids with a predicated molecular mass of 16 kDa (Additional file 1: Fig. S1A). The program SignalP 4.1 predicted that the cleavage site of the signal peptide in Pt2015 was between the 17th and 18th amino acid (Additional file 1: Fig. S1B). In addition, the TMHMM Server 2.0 predicted that Pt2015 does not possess a transmembrane domain (Additional file 1: Fig. S2). Phylogenetic analysis demonstrated that highly homologous proteins were only found in marine algae, mainly including diatoms, chrysophytes, dinoflagellates, and other red-tide algae, and not in freshwater algae (Chlamydomonas) or higher plants (Arabidopsis) (Fig. 1). A comparison of the conserved regions in Pt2015 proteins from diatoms, chrysophytes, dinoflagellates, and other red-tide algae revealed that five conserved motifs were identified in Pt2015. In particular, the two C-terminal motifs were much more conserved in Pt2015 (Additional file 1: Fig. S3A). Interestingly, Pt2015 was highly homologous with part of the sequence of exosome component 10 in the unicellular red algae Porphyridium purpureum (Additional file 1: Fig. S3B).
Subcellular localization of Pt2015 protein
Subcellular localization of Pt2015 was performed by expressing a C-terminal GFP fusion protein in P. tricornutum. The fluorescence signal associated with the Pt2015:GFP transformant was visible within the plastid (Fig. 2A–D). Additionally, intact plastids were further purified from the P. tricornutum WT strain through a discontinuous Percoll gradient (Additional file 1: Fig. S4). As shown in Fig. 2E, immunoblotting analysis demonstrated that Pt2015 was observed in the plastid, with a localization similar to that of PsbA (a protein located on thylakoids), but different to that of actin (a cytoplasm marker). This finding was consistent with an in silico analysis of the signal peptide sequence in Pt2015 (Additional file 1: Fig. S1). Therefore, it was concluded that Pt2015 resides in the plastid.
Subcellular localization of Pt2015. Fluorescent microscopy of Pt2015 green fluorescence protein (GFP) fusion proteins in a P. tricornutum Pt2015:GFP mutant. A Light image, B Chlorophyll autofluorescence, C GFP fluorescence, D Merged image of (B and C) (Scale bar, 5 μm). E Immunoblotting analysis of Pt2015. The cytoplasm and plastid were extracted from the wild-type (WT) strain. F Immunoblotting analysis of Pt2015. Plastids were extracted from the WT strain and then fractionated into thylakoid membranes and stroma. G The thylakoid membranes have been solubilized using n-dodecyl-β-D-maltoside (β-DM) and subjected to sucrose density gradient ultracentrifugation. The solubilized thylakoid membranes (200 μg chlorophyll) have been loaded on a gradient. The gradient was divided into ten fractions according to band color. H An equal volume of each fraction was used for immunoblotting analysis using antibodies against PsbD, PsaA, CytF, LhcSR, and Pt2015, respectively
Interestingly, although there was no transmembrane domain in Pt2015 (Additional file 1: Fig. S2), immunoblotting results indicated that Pt2015 was located on the thylakoids, with a localization similar to that of PsbD (a protein located on thylakoids), although small signals of both proteins were also detected in the stroma fraction which might be caused by the cross-contamination of the stroma (Fig. 2F). These results suggested that Pt2015 is a peripheral protein of thylakoids in diatoms. Subsequently, thylakoids from the P. tricornutum WT were solubilized with a mild detergent (β-DM), loaded onto a sucrose density gradient, and ultracentrifuged. After centrifugation, the gradient from top to bottom was divided into 10 fractions (10 bands, 1 to 10) according to band color (Fig. 2G). To identify polypeptide composition, each band was analyzed using tricine SDS-PAGE and immunoblotting analysis (Fig. 2H). PSII was detectable in bands 7, 8, and 9, while PSI was mainly present in band 8. LhcX was detected in bands 3, 4, and 5, and LhcX in band 3 was significantly different from that of band 5. It should be noted that the immunoblotting analysis of LhcX identified antibodies against LhcSR of Physcomitrella patens. Pt2015 signals were mainly detected in bands 2–5, especially in band 3. These results were in agreement with the analysis of the two dimensional Blue Native/SDS-PAGE (Additional file 1: Fig. S5).
Impact of Pt2015 knockout and overexpression on cell morphotype and photosynthesis
Using the CRISPR/Cas9 method, a Pt2015 knockout line (named 2015KO) was obtained in which the Pt2015 protein was thoroughly knocked out. As shown in Fig. 3A, in the 2015KO strain, 16 bases were deleted between bases 355 and 370, which is the gRNA3 binding site. In addition, a Pt2015 overexpression strain (oeT) was obtained. Both quantitative reverse transcription polymerase chain reaction (qRT–PCR) and immunoblotting analysis suggested that Pt2015 was knocked out in 2015KO and overexpressed in the oeT strains, respectively (Fig. 3B and C).
Characterization of the Pt2015 knockout strain (2015KO). A Characteristics of the 2015KO. Sequences of the 2015KO strain and wild type (WT) were aligned. The mutation in the Pt2015 locus compared to the WT sequence was indicated, in which 16 bp are deleted. B Quantitative reverse transcription polymerase chain reaction (qRT–PCR) analysis of Pt2015 mRNA levels in the WT, 2015KO, and the Pt2015 overexpression (oeT) strains. Ribosomal protein S (RPS) was used as reference gene. Error bars represent the standard deviation (n = 3). C Immunoblotting analysis of the Pt2015 protein in the WT, 2015KO, and oeT strains. Photosystem II protein D1 (PsbA) protein level was detected as the loading control. Fifteen micrograms protein extracts of the WT, 2015KO, and oeT strains have been loaded
Interestingly, the oeT strain displayed a triradiate morphotype, in which cells were 95% triradiate (the rest were fusiform) (Fig. 4A–C). In contrast, the 2015KO strain demonstrated a fusiform morphotype, which was similar to the WT. Furthermore, the WT, 2015KO, and oeT strains were observed using scanning electron microscopy (Fig. 4D–F). These data suggested that there was no difference in the size and shape of fusiform cells between the 2015KO and WT strains (Fig. 4D and E). In contrast, the triradiate cells in the oeT strain demonstrated nonsymmetrical and unequal arm lengths (Fig. 4F). In addition, the ultrastructure of the WT, 2015KO, and oeT strains was analyzed using TEM (Fig. 4G–I, and J). These data suggested that similar organelles were found in the WT, 2015KO, and oeT strains, including nuclei, plastids, mitochondria, and vacuoles. A large plastid was localized near the nucleus in the WT, 2015KO, and oeT strains. Vacuoles occupied the distal arms of the three strains, which were larger in the WT and 2015KO strains. Mitochondria were observed close to the plastid in the three strains. Furthermore, considerably more Golgi apparatus were observed in the oeT strain (Fig. 4I and J). In addition, lipid droplets in the oeT strain were larger than those in the WT and 2015KO strains (Fig. 4I and J).
Micrographs of the wild type (WT), Pt2015 knockout strain (2015KO), and the Pt2015 overexpression (oeT) strains. Micrographs of the WT (A), 2015KO (B), and oeT (C) strains imaged using differential interference contrast (DIC) optics (all DIC images are shown at the same scale and the scale bar: 5 μm). Scanning electron microscopy (SEM) micrographs of the WT (D), 2015KO (E), and oeT (F) strains. The scale bar is 0.5 μm. Transmission electron microscopy (TEM) micrographs of the WT (G), 2015KO (H), and oeT (I and J) strains. White arrows indicate lipid droplets. The scale bar is 0.5 μm
The photosynthetic electron transport of the WT, 2015KO, and oeT strains was analyzed using a Dual-PAM system (Fig. 5). The Y(II) was lower in the oeT strain compared to the WT and 2015KO strains (Fig. 5A). Y(I) displayed a similar trend as the Y(II) (Fig. 5B). Under high light conditions, the Y(NA) value in the oeT strain was much higher than that of the WT and 2015KO strains, suggesting that there was a limitation in electron acceptance from PSI, especially under high light conditions (Fig. 5C). Moreover, there were no significant differences between Y(ND) and Y(NO) among the oeT, 2015KO, and WT strains (Fig. 5D and E). Nevertheless, the Y(NPQ) in the oeT strain was higher than that in the WT and 2015KO strains (Fig. 5F). Additionally, the photosynthetic oxygen rate of the WT, 2015KO, and oeT strains was analyzed (Fig. 5G–I). The photosynthetic oxygen evolution rate of the oeT strain, particularly under higher light (μmol photons m−2 s−1) conditions (Fig. 5H), was much lower than that of the WT and 2015KO strains (Fig. 5G and H). In contrast, the oxygen evolution rate of the 2015KO strain was even higher than that of the WT strain. This was consistent with the Y(II) data using the Dual-PAM.
Impact of Pt2015 knockout and overexpression on photosynthetic activity. The light intensity dependence of photosynthetic parameters has been monitored in the wild type (WT), Pt2015 knockout strain (2015KO), and the Pt2015 overexpression (oeT) strains. The quantum yield of photosystem I [Y(I)] (A), the quantum yield of photosystem II [Y(II)] (B), Y (NA) (C),Y (ND) (D), Y (NO) (E) and Y (NPQ) (F). G The rate of oxygen evolution in the WT, 2015KO, and oeT strains under 100 μmol photons m−2 s−1. H The rate of oxygen evolution in the WT, 2015KO, and oeT strains under 2000 μmol photons m−2 s−1. I The rate of oxygen evolution in the WT, 2015KO, and oeT strains under increasing light intensity (50, 100, 200, 400, 800, 1600, and 2800 μmol photons m−2 s−1). Data represent the mean ± standard deviation (SD) of four biological replicates. The different letters indicate that there was significant difference between WT, 2015KO, and oeT
The oeT strain possessed a higher lipid content, demonstrated grazing resistance, and was insensitive to hyposalinity and low temperature
The lipid content in the oeT strain was clearly different from that in the WT and 2015KO strains. Spherical lipid bodies (also called lipid droplets) were observed in cells of the WT, 2015KO, and oeT strains labeled with BODIPY 505/515 and were generally distributed close to the plastid (Fig. 6). In the oeT cells, lipid bodies were also observed in the distal arm. Additionally, the oeT cells contained larger and more lipid bodies than WT and 2015KO. Total lipid content in the WT, 2015KO, and oeT strains was further analyzed using gravimetric means. These data showed that total lipid content in the oeT strain was higher than that in the WT and 2015KO strains, which increased by approximately 30% compared to that of the WT strain (Fig. 6B). This was consistent with observations of BODIPY 505/515-labeled cells and the transmission electron microscopy results. Although the morphotypes and lipid content of WT, 2015KO, and oeT changed significantly, the growth characteristics among the WT, 2015KO, and oeT strains did not demonstrate significant differences (Fig. 6C). In addition, to test the resistance of the oeT to microzooplankton, the amoebae (the Heterolobosea class) which was isolated from large-scale cultivation of P. tricornutum was used in this study. It should be noted that the 2015KO strain demonstrated a fusiform morphotype, which was similar to the WT, so in the grazer test the fusiform was used as control. The results suggested that during the co-culture of P. tricornutum and amoebae, after 24 h the cell concentrations of oeT strain were significantly higher than that of WT (Fig. 7 and Additional file 1: Fig. S12).
Lipids in the wild type (WT), Pt2015 knockout strain (2015KO), and the Pt2015 overexpression (oeT) strains. A Localization of lipid bodies in the WT, 2015KO, and oeT strains after staining with BODIPY 505/515. Bright-field micrographs, plastid autofluorescence, BODIPY 505/515 images, and a combination of BODIPY 505/515 images (merged) with plastid autofluorescence are shown. All images are shown at the same scale. The scale bar is 5 μm. B Total lipid content of WT, 2015KO, and oeT strains using gravimetric means. Different letters represent significant differences of total lipid content in the WT, 2015KO, and oeT strains. C Growth curves of WT, 2015KO, and oeT strains at 20 °C under 80 μmol photons m−2 s−1
Changes of cells concentrations of the oeT and WT strains during the cultivation with amoebae. The spores of amoebae were centrifugated at 3500 × g for 5 min, and then the precipitation was diluted with WT and oeT cells at the same cell density (5 × 106 cells/ml), respectively. The diluent was cultured in culture plates (6 wells) at 25 °C under the dark condition. After 24 h, the cells of the oeT and WT were counted. The asterisk indicates that there was significant difference between WT and oeT
It has been reported that the triradiate strain isolated from a natural condition was very sensitive to low salinities and low temperatures [30]. Therefore, to test the effects of hyposalinity and low temperatures on morphotypes of the oeT strain, four different experimental conditions were used: 100% seawater (normal seawater) and 20 °C, 50% seawater and 20 °C, 30% seawater and 20 °C, and 100% seawater and 10 °C. The condition comprising 100% seawater and 20 °C was the control (normal condition). Under the condition of 0% seawater, the oeT cells bleached and eventually died; therefore, this condition was not analyzed in this study. Additionally, during treatment under the above-mentioned conditions, fresh culture medium was supplemented every 3 weeks. As shown in Additional file 1: Fig. S6, under the two hyposalinity conditions (50% and 30% seawater) and long-term culture (60 d), the abundance of fusiform and triradiate morphotypes in the oeT strain did not clearly change and abundance was maintained at 5% and 95%, respectively (Additional file 1: Fig.S6B and C). The trends in these two conditions were like that under normal conditions (Additional file 1: Fig. S6A). Moreover, under low temperature conditions (100% seawater and 10 °C) and long-term culture (60 d), the abundance of fusiform and triradiate morphotypes in the oeT strain was also maintained at a constant level (5% and 95%, respectively) (Additional file 1: Fig. S6D). These data suggested that the oeT strain (triradiate cells) was not sensitive to hyposalinity and low temperature.
Transcriptomic and proteomic analysis of WT, 2015KO, and oeT
To gain insight into the gene expression profiles associated with the WT, 2015KO, and oeT strains, the mRNA transcriptome of these three strains was explored using high-throughput RNA-Sequencing (RNA-Seq). Validation of the RNA-Seq data was conducted using qRT–PCR experiments analyzing the expression of eight genes selected randomly from the list of differently expressed genes (DEG). qRT–PCR data suggested the expression of the selected genes was consistent with the RNA-Seq data, suggesting the transcriptomic data were of high quality (Additional file 1: Fig. S7). Principal component analysis suggested that either Pt2015 knockout or overexpression greatly affected gene expression (Fig. 8A). Furthermore, pairwise comparison of gene expression data of the WT, 2015KO, and oeT strains was performed (Fig. 8B and C). Pairwise comparison between the oeT and WT strains suggested that 153 genes were significantly differentially expressed between the two morphotypes, in which 35 genes were upregulated and 118 genes were downregulated. When comparing the oeT and 2015KO strains, 53 genes were upregulated and 395 genes were downregulated.
Transcriptomic analysis of the wild type (WT), Pt2015 knockout strain (2015KO), and Pt2015 overexpression (oeT) strains. A Principal components analysis (PCA) of gene expression in the WT, 2015KO, and oeT strains. B Venn diagram of gene expression in the WT, 2015KO, and oeT strains (FPKM > 2). C Pairwise comparison of differently expressed genes (up and downregulated genes) in the WT, 2015KO, and oeT strains. Bar graph showing up- or downregulated gene counts (fold change > 2, false discovery rate [FDR] < 0.05). D Gene ontology (GO) component enrichment analysis of the pairwise comparison between the WT and 2015KO strains. E GO component enrichment analysis of the pairwise comparison between the WT and oeT strains
To characterize the biological process (BP), cellular component (CC), and molecular function (MF) in which the DEGs were involved, a GO analysis was performed. The bar chart of the WT-vs-2015KO and WT-vs-oeT comparisons suggested that many CCs (Fig. 8D and E), BPs (Additional file 1: Fig. S8A and B), and MFs (Additional file 1: Fig. S8C and D) were regulated. The CCs mainly included the plastid part involving the photosynthetic membrane (Fig. 8D and E). Among the BPs in the WT-vs-oeT comparison, the single-organism metabolic process (GO: 0,044,710), DNA metabolic process (GO: 0,006,259), and protein modification process (GO: 0,006,464) were regulated (Additional file 1: Fig. S8B). In addition, similar results were obtained when considering the MF of the WT-vs-oeT comparison, which mainly included molecular transducer activity, tetrapyrrole binding, and oxidoreductase activity (Additional file 1: Fig. S8D). Furthermore, KEGG enrichment analysis of the WT-vs-oeT comparison suggested that nitrogen metabolism and many signaling pathways were regulated (Additional file 1: Fig. S9B). These were also observed in the comparison between the WT and 2015KO strains (Additional file 1: Fig.S8A, S8C and S9A). These data suggested that both the knockout and overexpression of the Pt2015 protein affected the above-mentioned BPs.
To understand the protein levels associated with the WT, 2015KO, and oeT strains, quantitative proteomics were performed. These data suggested that the three strains (WT, 2015KO, and oeT) demonstrated different protein compositions (Fig. 9A). Moreover, 6680 proteins were detected in total, and 730 proteins were differently expressed, while 5950 proteins were not changed significantly. This was mainly consistent with the transcriptomic data. The comparison between the WT and 2015KO strains showed that 257 proteins were significantly differentially expressed. The number of DEPs between the 2015KO and oeT strains was 363 (Fig. 9B). In contrast, 110 proteins were differentially expressed between the WT and oeT strains. Functional classification analysis showed that these regulated (up and down) proteins were mainly located in the plastid, cytoplasm, nucleus, and membrane systems (plasma membrane and vacuolar membrane) upon Pt2015 knockout or overexpression (Fig. 9C–E). Particularly, the percentage of plastid proteins was 38.5% in the 2015KO and oeT comparison (Fig. 9E). KEGG pathways and protein domains were also examined to identify functional information of proteins that were regulated upon Pt2015 knockout or overexpression. Functional enrichment analysis showed that the DEPs were involved in ubiquitin-mediated proteolysis, and inositol phosphate, nitrogen, and ribosome and glycerophospholipid metabolism (Fig. 10A). Moreover, the most highly represented protein domains were a DnaJ C terminal domain, ABC (ATP-binding cassette) transporter, cyclin C (N)-terminal domain, and RNA recognition motif (Fig. 10B). Similar results were obtained when considering BP, CC, and MF (Additional file 1: Fig. S10 and S11) in the pairwise comparisons. Overall, many proteins involving different BPs changed significantly in the triradiate morphotype (oeT) that was obtained from Pt2015 overexpression.
Quantitative proteomic analysis of the wild type (WT), Pt2015 knockout strain (2015KO), and Pt2015 overexpression (oeT) strains. A Principal components analysis (PCA) of the protein composition in the WT, 2015KO, and oeT strains. B Pairwise comparison of differentially expressed proteins (up- and downregulated) in the WT, 2015KO, and oeT strains. C Functional classification analysis between the 2015KO and WT strains. D Functional classification analysis between the oeT and WT strains. E Functional classification analysis between the oeT and 2015KO strains
Functional enrichment analysis of differentially expressed proteins (up- and downregulated) in the wild type (WT), Pt2015 knockout strain (2015KO), and Pt2015 overexpression (oeT) strains. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (A) and protein domain analysis (B) of differentially expressed proteins in the WT, 2015KO, and oeT strains
Pt2015 is a novel gene in P. tricornutum, which has not been reported previously. As shown in Fig. 1 and Additional file 1: Fig. S3, Pt2015 homologs were only found in marine algae, mainly including diatoms, chrysophyta, dinoflagellates, and other red-tide algae, but not in the green lineage (Chlamydomonas and Arabidopsis). Pt2015 is localized to the plastid (Fig. 2), which is highly homologous to part of the sequences of exosome component 10 in the unicellular red algae Porphyridium purpureum (Additional file 1: Fig. S3B). To understand the role of the Pt2015 protein in P. tricornutum, Pt2015 knockout and overexpression strains were constructed. Interestingly, the Pt2015 overexpression strain (oeT) displayed a triradiate morphotype, but the Pt2015 knockout did not affect the morphotype (Fig. 4). Therefore, we believe that Pt2015 positively participates in the fusiform to triradiate morphotype transformation.
To understand the pleomorphic characteristic of P. tricornutum, studies have reported comparative structural and physiological characteristics, and transcriptomes of fusiform, triradiate, and oval P. tricornutum morphotypes [28,29,30, 33, 52]. Nevertheless, the triradiate and oval cells used in those studies were induced or isolated from different culture conditions, of which the triradiate morphotype, particularly, was not stable. For example, the triradiate strain isolated from a natural condition was very sensitive to low salinities and low temperatures [30]. In contrast, in this study, the oeT strain was stable and has been cultured for more than 200 generations, which makes it insensitive to those conditions (Additional file 1: Fig. S6B, C, and D). The abundance of triradiate cells in the oeT was approximately 95%, which was much higher than the abundance of triradiate cells in the natural triradiate strain (approximately 70%) [32] and the induced triradiate strain (approximately 81%) [28, 33]. These data suggested that the oeT strain obtained from gene manipulation was different from the triradiate strains induced from specific culture conditions. In addition, to gain insight into the molecular characteristics of the oeT strain, transcriptomic and proteomics analysis of the WT, 2015KO, and oeT strains were performed. Both suggested that DEGs and proteins were mainly related to plastids, membrane systems, signaling pathway, and transporters (Figs. 8, 9, and 10). Additionally, the metabolic network of Pt2015 and top 30 genes that related closely to Pt2015 indicated that several genes (ID: 7202857, 7198159, 7197826, 7201627, 7196383, and 7203428) encoded solute carriers, channels, and transporters (Additional file 1: Fig. S13), which play an important role in cell osmoregulation, cell homeostasis, and shape [54]. Furthermore, the 2015KO strain showed a fusiform morphotype like the WT, further demonstrating that the Pt2015 protein might be a positive trigger factor during the transform from fusiform to triradiate morphotype. The Pt2015 protein might affect a series of metabolic processes in P. tricornutum; however, the molecular mechanism of Pt2015 in the fusiform to triradiate morphotype transformation still needs further study.
To date, microzooplankton contamination has been a major constraint in P. tricornutum industrial-scale cultivation, which greatly impedes the course of biomass production industrialization [16, 17, 21]. Although many strategies, including physical methods (e.g., electrical field and ultrasonication) [54, 55] and chemical methods (e.g., rotenone and quinine sulfate) [20, 56], have been trialed, they were costly and impracticable on an industrial scale. In this case, selection of microalgal strains with resistance to microzooplanktons is considered as the most practicable approach [22, 57]. In fact, changing in morphological features, such as size, cell shape, or geometry of phytoplankton, is the most obvious way to reduce grazing pressure from zooplankton in natural ecosystems [23, 24, 27]. Generally, zooplankton have two feeding modes: using mouthparts and endocytosis [27, 58]. The fusiform cells of P. tricornutum might be eaten by zooplankton with small mouthparts at the right angle; however, the triradiate cells with special geometry can avoid being eaten. During the co-culture of P. tricornutum and amoebae, the cell concentrations of oeT strain were significantly higher than that of WT (Fig. 7 and Additional file 1: Fig. S11), suggesting that the triradiate cells (the oeT strain) significantly decreased the grazing rate of the amoebae in comparison with that cultured in WT. Even if the triradiate cells were eaten through larger mouthparts or endocytosis of zooplanktons, the geometry and larger spatial structure of the triradiate cells can not only reduce grazing rates but also adversely affect membrane systems and digestion of zooplankton. Therefore, the triradiate cells of P. tricornutum may be the best morphotype to withstand grazing pressure in comparison with WT during industrial-scale cultivations.
The oeT strain not only displayed a stable triradiate morphotype but also accumulated larger lipids droplets than those of the WT and 2015KO strains (Figs. 4 and 6). These characteristics of the oeT strain were like the triradiate strains obtained from specific culture conditions that seemed to possess bigger lipids droplets [33]. In many algal species, an increase in lipid content was often observed during the stationary phase [11]. During this period, the growth rate of cells decreased, and nutrients start to become limited. In this study, the growth rate of the oeT strain was like that of the WT (Fig. 6C), but the lipid content in the oeT strain increased by approximately 30% compared to that of the WT, suggesting that the oeT strain could grow normally and simultaneously accumulate lipids. The transcriptomic and proteomic data demonstrated that the putative proteins (acetyl-CoA carboxylase, aldehyde dehydrogenase, and biotin synthase, etc.) involved lipid metabolism were upregulated in the oeT strain. Furthermore, the heatmap of WT-vs-oeT involving lipid metabolism also suggested that the expression of genes encoding phospholipid synthase and lipid-phosphate phosphatase were upregulated (Additional file 1: Fig. S14). In addition, the GO enrichment analysis of our proteomic data indicated that the lipid metabolisms, including sphingolipid metabolic process (GO:0006665), glycerolipid biosynthetic process (GO:0045017), membrane lipid metabolic process (GO:0006643), and glycerolipid metabolic process (GO:0046486), were upregulated in the oeT strain in comparison with WT (Additional file 1: Fig. S15). All these results explained why the lipid content in the oeT strain was higher than that of the WT.
Overall, the oeT strain obtained from the overexpression of Pt2015 was a stable P. tricornutum triradiate strain with a similar growth rate to the WT and increased lipid accumulation. Due to its specific geometry, the oeT strain can withstand grazing pressure during the co-culture of P. tricornutum and amoebae. Therefore, the oeT strain not only advances our understanding of morphotype conversion in diatoms but also demonstrates its potential applications for biofuel production.
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Mahajan S, Tuteja N. Cold, salinity and drought stresses: an overview. Arch Biochem Biophys. 2006;444:139–58.
Rego D, Redondo L, Geraldes V, Costa L, et al. Control of predators in industrial scale microalgae cultures with pulsed electric fields. Bioelectrochemistry. 2015;103:60–4.
Wang Y, Gong YC, Dai L, Sommerfeld M, Hu Q. Identification of harmful protozoa in outdoor cultivation of Chlorella and the use of ultrasonication to control contamination. Algal Res. 2018;31:298–310.
Van Ginkel SW, Igou T, Hu Z, Narode A, et al. Taking advantage of rotifer sensitivity to rotenone to prevent pond crashes for algal-biofuel production. Algal Res. 2015;10:100–3.
Day JG, Gong YC, Hu Q. Microzooplanktonic grazers—a potentially devastating threat to the commercial success of microalgal mass culture. Algal Res. 2017;27:356–65.
Vanderploeg HA, Pacfenhofer GA. Modes of algal capture by the freshwater copepod Diaptomus sicilis and their relation to food-size selection. Limnol Oceanogr. 1985;30:871–85.
This study was supported by National Key R&D Program of China (2018YFD0901500) and National Natural Science Foundation of China (Nos. 41976097, 42106157). The authors are grateful to Dr. Rahmi Lale (Norwegian University of Science and Technology, Trondheim, Norway) for his gift of DH10B E. coli containing the pTA-Mob plasmid and to Dr. Feng Zhang (Minnan Normal University, China) for his technical exchange.
Shan Gao and Lu Zhou contributed equally to this work
CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega‑Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
Shan Gao, Lu Zhou, Lijun Wang, Xuehua Liu & Guangce Wang
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China
College of Earth Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China
Lu Zhou
College of Life Science, Qingdao Agricultural University, Qingdao, China
Wenting Yang
Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China
Yingchun Gong
CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China
Qiang Hu
Faculty of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China
Shan Gao
Xuehua Liu
Guangce Wang
SG, LZ, WTY, and GCW designed this work. SG, LZ, and WY conducted the experiments. SG, LZ, WY, LW, YG, QH and GW analyzed the data. SG, LZ, WY, LW, YG, QH and GW wrote the manuscript. All the authors read and approved the final manuscript.
Correspondence to Guangce Wang.
The authors have no conflict of interest.
Additional file 1
: Figure S1. In silico analysis of the Phaeodactylum tricornutum Pt2015 gene. (A) Schematic drawing of the Pt2015 gene on chromosome 12, which is annotated as an open reading frame of 444 bp in length corresponding to a predicted protein of 147 amino acids. (B) Analysis of the signal peptide in the Pt2015 protein using SignalP 4.1. The cleavage site of the signal peptide in Pt2015 is between the 17th and 18th amino acid. Figure S2. Transmembrane domain analysis of the Pt2015 protein via the TMHMM Server 2.0. Figure S3. (A) Comparison of the conserved regions in Pt2015 proteins from diatoms, chrysophyta, dinoflagellate, and other red-tide algae using the MEGA 7.0 platform. Conserved motifs are indicated as sequence logos, which are on the top of the sequences. (B) Amino acid sequence alignment of the Pt2015 protein with exosome component 10 in Porphyridium purpureum. The sequences are aligned using BioEdit. Black-boxed and gray-boxed letters represent identical or similar residues, respectively. Figure S4. Chloroplast purification using a discontinuous Percoll gradient (10%, 20%, and 30%) and ultracentrifugation. The chloroplast band is indicated on the right. Figure S5. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) analysis of the thylakoid membrane complex from P. tricornutum wild type (WT). (A) The gel is stained with Coomassie Brilliant Blue. (B) Thylakoid membrane complexes separated by BN-PAGE in A have been further subjected to sodium dodecyl sulfate (SDS)-PAGE, and the proteins are detected with specific antibodies against PsbD, PsaA, and Pt2015, respectively. Figure S6. Effects of salinity and temperature on the Pt2015 overexpression (oeT) strain. Percentage of triradiate abundance (Black square) and fusiform (Red circle) morphotypes of the oeT strain at different salinities and temperatures. (A) 100% sea water and 20 °C; (B) 50% sea water and 20 °C; (C) 30% sea water and 20 °C; and (D) 100% sea water and 10 °C. Data represent the mean ± standard deviation (SD) of four biological replicates. Figure S7. Validation of RNA-Sequencing data using qualitative reverse transcriptase-polymerase chain reaction (qRT–PCR) analysis. The expression of eight genes selected randomly from the list of differently expressed genes (DEGs) is shown, including ID 7,199,712 (A), 7,203,450 (B), 7,195,163 (C), 7,195,518 (D), 7,198,479 (E), 7,204,536 (F), 7,205,131 (G), and 7,198,653 (H). Figure S8. Gene ontology (GO) enrichment analysis (biological processes (A and B) and molecular functions (C and D)) of the wild-type (WT)-vs-Pt2015 knockout strain (2015KO) comparison (A and C) and the WT-vs-Pt2015 overexpression (oeT) strain comparison (B and D) based on RNA-Sequencing data. Figure S9. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the wild-type (WT)-vs-Pt2015 knockout strain (2015KO) comparison (A) and the WT-vs-Pt2015 overexpression (oeT) strain comparison (B) based on RNA-Sequencing data. Figure S10. Functional profiling analysis [biological process (A) and cellular component (B)] of the pairwise comparison of the wild type (WT), Pt2015 knockout strain (2015KO), and Pt2015 overexpression (oeT) strain based on proteomic data. Figure S11. Functional profiling analysis (molecular function) of the pairwise comparison of the wild type (WT), Pt2015 knockout strain (2015KO), and Pt2015 overexpression (oeT) strains based on proteomic data. Figure S12. Micrographs of the amoebae cultivated with the oeT (A) and WT (B) cells. The spores of amoebae were centrifugated at 3500 × g for 5 min, and then the precipitation was diluted with WT and oeT cells at the same cell density (5 × 106 cells/ml), respectively. The diluent was cultured in culture plates (6 wells) at 25 °C under the dark condition. After 24 h, the cells of the oeT and WT were observed. The scale bar is 5 μm. Figure S13. The metabolic network of Pt2015 and top 30 genes that related closely to Pt2015 indicated that several genes encoding solute carriers or channels (ID: 7,202,857, 7,198,159, 7,197,826) and transporters (ID: 7,201,627, 7,196,383 7,203,428) based on RNA-Sequencing data. The green circle represents the Pt2015 and the purple circles represents the genes related closely to Pt2105. The red lines and blues line represent positive correlation and negative correlation, respectively. Figure S14. The heatmap of genes involving lipid metabolisms in the comparison of WT and oeT based on RNA-Sequencing data. Figure S15. The GO enrichment analysis (the upregulated processes) of the oeT strain-vs-WT based on proteomic data. In the biological process, the GO ID of sphingolipid metabolic process, glycerolipid biosynthetic process, membrane lipid metabolic process, and glycerolipid metabolic process is GO:0,006,665, GO:0,045,017, GO:0,006,643, GO:0,046,486, respectively. Table S1. Sequences of gRNAs targeting the Pt2015 gene in P. tricornutum. The predicted gRNA binding site is the underlined sequence, and the complementary oligonucleotide is also shown in the table. Oligonucleotides are designed with a top strand 5'-TCGA-3' and bottom strand 3'-CAAA-5' BsaI restriction cut site overhangs to facilitate cloning into the Cas9 vector. Table S2. Lists of Pt2015 gene and reference gene (RPS) primers.
Gao, S., Zhou, L., Yang, W. et al. Overexpression of a novel gene (Pt2015) endows the commercial diatom Phaeodactylum tricornutum high lipid content and grazing resistance. Biotechnol Biofuels 15, 131 (2022). https://doi.org/10.1186/s13068-022-02221-y
DOI: https://doi.org/10.1186/s13068-022-02221-y
Gene manipulation
Morphotype
Phaeodactylum tricornutum

redpajama_set_name: RedPajamaCommonCrawl


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Suggestions for regional/local, non-flatbed Tyler, Tx area?
Discussion in 'Discuss Your Favorite Trucking Company Here' started by txtodd, Aug 10, 2018.
I've got about 3 years OTR experience hauling reefer. No tickets or reportable accidents (a few scrapes of trailers backing). Have all endorsements, and TWIC card. Wanting a job where I can be home 2-3 times a week, PLUS weekends (any 2 consecutive days off), no hard labor (51 years old & ain't gonna strain in the Texas heat), without losing any money ($63k last year, on pace for $66k this year). Live near I-20 / Tyler, Texas area. Any suggestions? @Chinatown Anyone else?
I live in your area, can't speak to the actual money, but I know that JBS is hiring for local home every night and home weekly drivers all over this area right now. Just saw an ad in the local newspaper this morning.
win-some-loose-less and txtodd Thank this.
Probably plenty of good tanker jobs around Tyler.
Local CDL Fuel Delivery Truck Driver, Home Daily!!!
CURRENTLY LOOKING FOR A NIGHT TRUCK DRIVER!
slim shady and txtodd Thank this.
Haul diesel & DEF to the truckstops & also has dry vans hauling truck tires to the truckstops.
Let me know when you find this job.
Cause I haven't, not that I've been looking to hard.
The target DC in Tyler.
There off of the weekends though.
Lots of low paying #### jobs where we live.
That's why I work out of dallas.
Maybe Tyson. They have numerous types of driving jobs not too far south.


text:
Spangenberg steht für:

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Siehe auch:
 Spangberg
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redpajama_set_name: RedPajamaWikipedia


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Wellforce names new CIO, chief digital officer
Kelly Gooch - Tuesday, October 6th, 2020 Print	| Email
Burlington, Mass.-based Wellforce has selected Shafiq Rab, MD, as chief digital officer and CIO.
The health system, comprising Boston-based Tufts Medical Center, Lowell (Mass.) General Hospital and Medford, Mass.-based MelroseWakefield Healthcare, said Dr. Rab will lead digital transformation efforts and be responsible for the systemwide IT organization.
"Digital technology has become a part of consumers' everyday lives. They connect with the brands they love in ways most convenient for them, and they expect healthcare providers to offer them a frictionless experience that helps them easily navigate their health needs," Michael Dandorph, president and CEO of Wellforce, said in a news release. "To help Wellforce exceed our patients' expectations, Dr. Rab will accelerate our digital transformation efforts. He will unify our information systems and technology so we can create an unmatched patient experience, lessen the burden on our care teams, and continue to deliver the highest quality healthcare."
Dr. Rab previously served as senior vice president and CIO of Rush University Medical Center in Chicago. Before joining Rush in January 2017, he was CIO at Hackensack (N.J.) University Health Network.
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Dr. Rab currently is chair of the College of Healthcare Information Management Executives foundation's board.
More articles on executive moves:
9 recent hospital, health system resignations, departures
UC Health names new hospital chief administrative officer
Harvard, Brigham leader tapped for new roles in Houston


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Dina Buchbinder
Founder and President, Education for Sharing
Connect on LinkedIn Follow on Facebook
Dina is a social entrepreneur who strongly believes that the power of play is a game changer to experience "why we care", and that it can make a difference in the world, beginning with educational systems.
In 2007, she founded Education for Sharing (E4S) as her response to address systemic inequity and discrimination so that every person can thrive. Since then, she has been committed to educational innovation using play to engage entire communities in building global citizenship.
Dina is an Ashoka Fellow since 2010, selected in 2017 Edmund Hillary Fellow to bring the E4S methodology to New Zealand, obtained the UBS Social Entrepreneur award, was selected as World Economic Forum Global Shaper, BMW Responsible Leader, awarded the Emerging Global Leader Award from the Harvard Kennedy School (2018). In 2020, Dina was invited by Prof. Jeffrey Sachs, UNESCO, and the Ban Ki-moon Center for Global Citizens to be a member of the Mission 4.7 Education Task Force.
Also in 2020, E4S was honored with the World Innovation Summit for Education (WISE) Award by the Qatar Foundation, becoming the first Mexican organization to receive this prestigious recognition. E4S was selected as a member of Ashoka's Changemakers network to work on developing strategies to address the consequences of the COVID-19 pandemic. E4S was recognized as one of the most important innovations in LatinAmerica by HundrED, a global non-profit based in Finland that improves education through impactful, scalable innovations.
Dina shares her voice at several global events, Davos World Economic Forum, UNESCO Youth Forum in Paris, LEGO IDEA Conference in Denmark, Learning for Holistic Wellbeing Summit in India among many others. She has inspired several academic articles, case studies, and papers and has also participated as a TED speaker, in Costa Rica, Mexico, Colombia, and India.
Science for Sharing (Sci4S)
By Dina Buchbinder


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text:
77 Snake Facts That Will Give You Nightmares
"THERE IS NOTHING SO ELOQUENT AS THE RATTLESNAKE'S TAIL."
—UNKNOWN NATIVE AMERICAN
These are 77 facts you may not know snakes…and a lot of these I had no idea either! I am going to have nightmares for months over this list. Our Wildlife specialists are on top of it, however. I tried quizzing them with these facts and they knew every… single… fact. I guess this is why they are the experts! How many facts will you know? Let's see…
The decapitated head of a dead snake can still bite, even hours after death. These types of bites usually contain HUGE amounts of venom.
What is considered the most "dangerous" snake depends on both a specific country's health care and the availability of antivenom following a bite. Based on these criteria, the most dangerous snake in the world is the saw-scaled viper, which bites and kills more people each year than any other snake.
Snakes live on everywhere on Earth except Ireland, Iceland, New Zealand, and the North and South Poles.
Of the approximately 725 species of venomous snakes worldwide, 250 can kill a human with one bite.
Snakes evolved from a four-legged reptilian ancestor—most likely a small, burrowing, land-bound lizard—about 100 million years ago. Some snakes, such as pythons and boas, still have traces of back legs.
SCIENTISTS BELIEVE HUMANS ARE PREDISPOSED TO FEAR SNAKES.
The fear of snakes (ophiophobia or herpetophobia) is one of the most common phobias worldwide. Approximately 1/3 of all adult humans ophidiophobic, which suggests that humans have an innate, evolutionary fear of snakes.
The top 5 most venomous snakes in the world are the inland taipan, the eastern brown snake, the coastal taipan, the tiger snake, and the black tiger snake.
The warmer a snake's body, the more quickly it can digest its prey. Typically, it takes 3–5 days for a snake to digest its meal. For very large snakes, such as the anaconda, digestion can take weeks.
Some animals, such as the Mongoose, are immune to snake venom.
To avoid predators, some snakes can poop whenever they want. They make themselves so dirty and smelly that predators will run away.
The heaviest snake in the world is the anaconda. It weighs over 595 pounds (270 kg) and can grow to over 30 feet (9m) long. It has been known to eat caimans, capybaras, and jaguars.
The Brahminy Blind Snake, or flowerpot snake, is the only snake species made up of sole females and, as such, does not need a mate to reproduce. It is also the most widespread terrestrial snake in the world.
If a person suddenly turned into a snake, they would be about 4 times longer than they are now and only a few inches thick. While humans have 24 ribs, some snakes can have more than 400.
The most advanced snake species in the world is believed to be the black mamba. It has the most highly evolved venom delivery system of any snake on Earth. It can strike up to 12 times in a row, though just one bite is enough to kill a grown man.
WHILE THE INLAND TAIPAN IS THE WORLD'S MOST VENOMOUS SNAKE, IT IS ACTUALLY QUITE SHY AND PLACID.
The inland taipan is the world's most toxic snake, meaning it has both the most toxic venom and it injects the most venom when it bites. Its venom sacs hold enough poison to kill up to 80 people.
The death adder has the fastest strike of any snake in the world. It can attack, inject venom, and go back to a striking position in under 0.15 seconds.
While snakes do not have external ears or eardrums, their skin, muscles, and bones carry sound vibrations to their inner ears.
Some snakes have been known to explode after eating a large meal. For example, a 13-foot python blew up after it tried to eat a 6-foot alligator. The python was found with the alligator's tail protruding from its midsection. Its head was missing.
The word "snake" is from the Proto-Indo-European root *sneg-, meaning "to crawl, creeping thing." The word "serpent" is from the Proto-Indo-European root *serp-, meaning "to crawl, creep."
Rattlesnake rattles are made of rings of keratin, which is the same material as human hair and fingernails. A rattler will add a new ring each time it sheds its skin.
Some snakes have over 200 teeth. The teeth aren't used for chewing but they point backward to prevent prey from escaping the snake's throat.
There are about 500 genera and 3,000 different species of snakes. All of them are predators.
Naturalist Paul Rosolie attempted to be the first person to survive being swallowed by an anaconda in 2014. Though he was wearing a specially designed carbon fiber suit equipped with a breathing system, cameras, and a communication system, he ultimately called off his stunt when he felt like the anaconda was breaking his arm as it tightened its grip around his body.
There are five recognized species of flying snakes. Growing up to 4 feet, some types can glide up to 330 feet through the air.
Scales cover every inch of a snake's body, even its eyes. Scales are thick, tough pieces of skin made from keratin, which is the same material human nails and hair are made from.
The most common snake in North America is the garter (gardener) snake. This snake is also Massachusetts's state reptile. While previously thought to be nonvenomous, garter snakes do, in fact, produce a mild neurotoxic venom that is harmless to humans.
Snakes do not lap up water like mammals do. Instead, they dunk their snouts underwater and use their throats to pump water into their stomachs.
A snake's fangs usually last about 6–10 weeks. When a fang wears out, a new one grows in its place.
A SNAKE'S TONGUE IS A MARVEL OF NATURE.
Because the end of a snake's tongue is forked, the two tips taste different amounts of chemicals. Essentially, a snake "smells in stereo" and can even tell which direction a smell is coming from. It identifies scents on its tongue using pits in the roof of its mouth called the Jacobson's organ.
The amount of food a snake eats determines how many offspring it will have. The Arafura file snake eats the least and lays just one egg every decade.
While smaller snakes, such a tree- or- ground-dwelling snakes, use their tongues to follow the scent trails of prey (such as spiders, birds, and other snakes). Larger snakes, such as boas, have heat-sensing organs called labial (lip) pits in their snouts.
Snakes typically need to eat only 6–30 meals each year to be healthy.
Snakes like to lie on roads and rocky areas because stones and rocks absorb heat from the sun, which warms them. Basking on these surfaces warms a snake quickly so it can move. If the temperature reaches 50° Fahrenheit or below, a snake's body does not work properly.
The Mozambique spitting cobra can spit venom over 8 feet away. It can spit from any position, including lying on the ground or raised up. It prefers to aim for its victim's eyes.
Snakes cannot chew, so they must swallow their food whole. They are able to stretch their mouths very wide because they have a very flexible lower jaw. Snakes can eat other animals that are 75%–100% bigger than their own bodies.
To keep from choking on large prey, a snake will push the end of its trachea, or windpipe, out of its mouth, similar to the way a snorkel works.
The Gaboon viper has the longest fangs of any snake, reaching about 2 inches (5 cm) long.
Anacondas can hold their breath for up to 10 minutes under water. Additionally, similar to crocodiles, anacondas have eyes and nostrils that can poke above the water's surface to increase their stealth and hunting prowess.
The longest snake ever recorded is the reticulated python. It can reach over 33 feet long, which is big enough to swallow a pig, a deer, or even a person.
Sea snakes with their paddle-shaped tails can dive over 300 feet into the ocean.
If a snake is threatened soon after a meal, it will often regurgitate its food so it can quickly escape the perceived threat. A snake's digestive system can dissolve everything but a prey's hair, feathers, and claws.
SNAKES SLEEP WITH THEIR EYES OPEN.
Snakes do not have eyelids; rather, a single transparent scale called a brille protects their eyes. Most snakes see very well, especially if the object is moving.
The world's longest venomous snake is the king cobra from Asia. It can grow up to 18 feet, rear almost as high as a person, growl loudly, and inject enough venom to kill an elephant.
The king cobra is thought to be one of the most intelligent of all snakes. Additionally, unlike most snakes, who do not care for their young, king cobras are careful parents who defend and protect their eggs from enemies.
Not all snakes have fangs—only those that kill their prey with venom have them. When their fangs are not in use, they fold them back into the roof of the mouth (except for the coral snake, whose fangs do not fold back).
Some venomous snakes have died after biting and poisoning themselves by mistake.
Elephant trunk snakes are almost completely aquatic. They cannot slither because they lack the broad scales in the belly that help other snakes move on land. Rather, elephant trunk snakes have large knobby scales to hold onto slippery fish and constrict them underwater.
The shortest known snake is the thread snake. It is about 4 inches long and lives on the island of Barbados in the Caribbean. It is said to be as "thin as spaghetti" and it feeds primarily on termites and larvae.
In 2009, a farm worker in East Africa survived an epic 3-hour battle with a 12-foot python after accidentally stepping on the large snake. It coiled around the man and carried him into a tree. The man wrapped his shirt over the snake's mouth to prevent it from swallowing him, and he was finally rescued by police after calling for help on his cell phone.
The venom from a Brazilian pit viper is used in a drug to treat high blood pressure.
HUMAN VICTIMS MAY STOP BREATHING JUST 30 MINUTES AFTER BEING BITTEN BY A COBRA.
The word "cobra" means "hooded." Some cobras have large spots on the back of their hood that look like eyes to make them appear intimating even from behind.
Some desert snakes, such as the African rock python, sleep during the hottest parts of the desert summer. This summer sleep is similar to hibernation and is called "aestivation."
The black mamba is the world's fastest snake and the world's second-longest venomous snake in the world, after the king cobra. Found in East Africa, it can reach speeds of up to 12 mph (19kph). It's named not from the color of its scales, which is olive green, but from the inside of its mouth, which is inky black. Its venom is highly toxic, and without anti-venom, death in humans usually occurs within 7–15 hours.
Although a snake's growth rate slows as it gets older, a snake never stops growing.
While a snake cannot hear the music of a snake charmer, the snake responds to the vibrations of the charmer's tapping foot or to the movement of the flute.
Most snakes are not harmful to humans and they help balance the ecosystem by keeping the population of rats, mice, and birds under control.
The largest snake fossil ever found is the Titanoboa. It lived over 60 million years ago and reached over 50 feet (15 meters) long. It weighed more than 20 people and ate crocodiles and giant tortoises.
TWO-HEADED SNAKES ARE RARE.
Two-headed snakes are similar to conjoined twins: an embryo begins to split to create identical twins, but the process does not finish. Such snakes rarely survive in the wild because the two heads have duplicate senses, they fight over food, and one head may try to eat the other head.
Snakes can be grouped into two sections: primitive snakes and true (typical) snakes. Primitive snakes—such as blind snakes, worm snakes, and thread snakes—represent the earliest forms of snakes. True snakes, such as rat snakes and king snakes, are more evolved and more active.
The oldest written record that describes snakes is in the Brooklyn Papyrus, which is a medical papyrus dating from ancient Egypt (450 B.C.).
Approximately 70% of snakes lay eggs. Those that lay eggs are called oviparous. The other 30% of snakes live in colder climates and give birth to live young because it is too cold for eggs outside the body to develop and hatch.
Most snakes have an elongated right lung, many have a smaller left lung, and a few even have a third lung. They do not have a sense of taste, and most of their organs are organized linearly.
The rarest and endangered snake is the St. Lucia racer. There are only 18 to 100 of these snakes left.
Snakes kill over 40,000 people a year—though, with unreported incidents, the total may be over 100,000. About half of these deaths are in India.
In some cultures, eating snakes is considered a delicacy. For example, snake soup has been a popular Cantonese delicacy for over 2,000 years.
In some Asian countries, it is believed that drinking the blood of snakes, particularly the cobra, will increase sexual virility. The blood is usually drained from a live snake and then mixed with liquor.
In the United States, fewer than 1 in 37,500 people are bitten by venomous snakes each year (7,000–8,000 bites per year), and only 1 in 50 million people will die from snake bite (5–6 fatalities per year). In the U.S., a person is 9 times more likely to die from being struck by lightning than to die from a venomous snakebite.
A PERSON IS NINE TIMES MORE LIKELY TO DIE FROM BEING STRUCK BY LIGHTNING THAN TO DIE OF VENOMOUS SNAKEBITE.
Some members of the U.S. Army Special Forces are taught to kill and eat snakes during their survival training, which has earned them the nickname "Snake Eaters."
One of the great feats of the legendary Greek hero Perseus was to kill Medusa, a female monster whose hair consisted of wriggling, venomous snakes.
The symbol of the snake is one of the most widespread and oldest cultural symbols in history. Snakes often represent the duality of good and evil and of life and death.
Because snakes shed their skin, they are often symbols of rebirth, transformation, and healing. For example, Asclepius, the god of medicine, carries a staff encircled by a snake.
The snake has held various meanings throughout history. For example, The Egyptians viewed the snake as representing royalty and deity. In the Jewish rabbinical tradition and in Hinduism, it represents sexual passion and desire. And the Romans interpreted the snake as a symbol of eternal love.
ANACONDAS MATE EVERY OTHER YEAR OR EVEN LESS.
Anacondas mate in a huge "breeding ball." The ball consists of 1 female and nearly 12 males. They stay in a "mating ball" for up to a month.
Depending on the species, snakes can live from 4 to over 25 years.
Snakes that are poisonous have pupils that are shaped like a diamond. Nonpoisonous snakes have round pupils.
Endangered snakes include the San Francisco garter snake, eastern indigo snake, the king cobra, and Dumeril's boa.
A mysterious, new "mad snake disease" causes captive pythons and boas to tie themselves in knots. Other symptoms include "stargazing," which is when snakes stare upwards for long periods of time. Snake experts believe a rodent virus causes the fatal disease.


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Diamond earrings will always be a timeless piece of jewellery. Whether they're subtle or extravagant, they have the power to instantly lift any outfit with their unmistakable sparkle. Take a look at our pick of the best diamond earrings, or find our full collection in our online shop.
These classic round diamond stud earrings will complement any look, and are ideal for both evening and everyday wear. Their simple four-claw setting features brilliant cut diamonds, which are available in a range of carats, clarities and colours. Prices start from £410 for 0.3 carat diamonds, and the earrings are available to buy in platinum, palladium, or in an 18 ct white or yellow gold setting. You can also view our full range of stud earrings online.
These striking cluster earrings feature large princess cut diamonds, surrounded by smaller round diamonds for a dazzling finish. From our contemporary collection, they are sure to add a touch of class to your jewellery collection. The earrings are available in white or yellow 18ct gold, in with a range of different diamond carats and clarities. Prices start from £725 for the earrings with 0.5 carat diamonds. Visit our online shop to view our full range of cluster earrings.
These stylish hoop earrings feature two rows of brilliant cut round diamonds, and will add the perfect finish to any outfit. Their elegant curves are fantastic for both traditional and contemporary looks, and are guaranteed to turn heads, whatever you're wearing. The earrings are available in white or yellow gold, and with a range of different diamond carats and clarities. Prices start from £530 for the earrings with 0.3 carat diamonds. You can find more hoop and drop earrings in our online shop.
These truly chic cluster earrings feature a double halo of micropave diamonds around a brilliant cut diamond, and are guaranteed to bring a touch of sophistication to your wardrobe. Available in white or yellow 18ct gold, the earrings are available with diamonds in a range of different carats and clarities. Prices start from £650 for the earrings with 0.5 carat diamonds. Check out our online shop to see more of our cluster earrings.
If you're looking for something a little different, these gorgeous earrings include a bold sapphire encased in a cluster of diamonds. Ideal for evening wear or special occasions, the midnight blue of the sapphire really brings out the crisp sparkle of the diamonds for a beautiful effect. Prices start from £1,100 for the earrings with 2 carat diamonds, and they are available set in a platinum, or 18ct white or yellow gold setting. You can also view our other gemstone earrings online.
We hope that one of these dazzling pairs of earrings has taken your fancy, but if you'd like to view some more, you'll find our full range of diamond earrings in our online shop.
All jewellery from Diamond Heaven is fully customisable, and is made with ethically sourced diamonds as standard. Our 30 day money back guarantee also means that you're covered, whatever you're spending, so you can shop with confidence. If you'd like some help or advice with your purchase, please book a consultation with one of our experts, who will be on hand to assist you in any way they can.