Expanding the omics repertoire for model studies on a Chlorella-infecting giant virus

Viruses are the most abundant biological entities in aquatic ecosystems. As top-down controls of plankton abundance and diversity, they are intrinsically linked to biogeochemical cycling, and by proxy, to global climate change. It is thus of great interest for researchers to understand the mechanics of viral infection and persistence among ecologically important phytoplankton assemblages. Viruses which infect eukaryotic algae are observed with diverse nucleic acid types, structures, and sizes, though most isolates to date bear large, dsDNA genomes comprised of genes normally only seen in cellular organisms. The Chlorella viruses are the model system for studying these entities, with many of the ‘omics’ approaches having been used to characterize the biology of this system. Here, we present data generated from epigenomic (i.e. DNA methylation) and metabolomic experiments of the prototype Chlorella virus, PBCV-1. In order to ask questions about virus DNA methylation, we first established a novel protocol for cryopreservation of PBCV-1 to control against epigenomic and genetic drift. This allowed for a baseline characterization of the DNA methylome profile in the prototype chlorovirus, PBCV-1, using PacBio’s single-molecule, real-time (SMRT) sequencing software. The results of this study suggest the possibility of widespread epigenomic modifications, and that DNA methylation by viral restriction-modification associated enzymes is incomplete. Most instances of missing methylation marks are represented as hemimethylated palindromes, which are protected against the types of restriction enzymes encoded by these viruses and thus might represent an epigenomic regulatory function in the virus. Finally, we conducted a non-targeted metabolomics study of PBCV-1 infected Chlorella cells to make some of the first inferences of how viral infection alters the metabolic profile of this host system. Altogether, this work helps to distinguish the baseline epigenomic and metabolomic profiles of the Chlorella-PBCV-1 virus system for future comparison with more ecologically informative treatments (i.e. competition, sub-optimal light, nutrient limitation, etc.). This work will help to uncover general trends specific to algal-giant virus interactions that distinguish themselves from phage-bacteria systems

Piecing Together Prehistoric Life: Scanning and Articulating Gorgosaurus

The Skull bones of a Gorgosaurus Libratus was laser scanned in order to articulate the model into software and 3D print. The model had to be articulated due to some missing bone, making it unrealistic to put together. Using the scanned pieces we articulated the model making a skull of The Gorgosaurus Libratus. This detailed computer skull can be sent anywhere in the world, for anyone to study. These scans could also be used to find out how the Gorgosaurus Libratus bit down or determine the way these animals moved. Prior to laser scanning, a method known as Photogrammetry was used. This method involves taking photos of the model and processing the images on a computer, which slow down the process. Another way used to replicate bones was by making silicone molds. This could damage the bone which makes it a method used less often. Laser scanning is the fastest and safest method in order to scan a bone. After the bones were articulated on the computer they were sent to a 3D printer. Unfortunately, the printer beds could not hold the massive skull. Due to this, the bones were printed half size. In order to 3D print, the holes of the model had to be filled using another program. The holes were caused by the light of the laser scanner not being able to go into all the holes creating shadows that the laser scanner could not pick up. However, after the holes were filled some of the objects were still too big to fit on the printer bed. Therefore, some of the objects were cut in half to fit. The 3D printed models were then printed and assembled

Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host

Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilisNC64A host, and found that virus survivorship was highest (69.5 ± 16.5%) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9–30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts

Consumer feces impact coral health in guild-specific ways

Animal waste products are an important component of nutrient cycles and result in the trophic transmission of diverse microorganisms. There is growing recognition that the feces of consumers, such as predators, may impact resource species, their prey, via physical effects and/or microbial activity. We tested the effect of feces from distinct fish trophic groups on coral health and used heat-killed fecal controls to tease apart physical versus microbial effects of contact with fecal material. Fresh grazer/detritivore fish feces caused lesions more frequently on corals, and lesions were 4.2-fold larger than those from sterilized grazer/detritivore feces; in contrast, fresh corallivore feces did not cause more frequent or larger lesions than sterilized corallivore feces. Thus, microbial activity in grazer/detritivore feces, but not corallivore feces, was harmful to corals. Characterization of bacterial diversity in feces of 10 reef fish species, ranging from obligate corallivores to grazer/detritivores, indicated that our experimental findings may be broadly generalizable to consumer guild, since feces of some obligate corallivores contained ~2-fold higher relative abundances of coral mutualist bacteria (e.g., Endozoicomonadaceae), and lower abundances of the coral pathogen, Vibrio coralliilyticus, than feces of some grazer/detritivores. These findings recontextualize the ecological roles of consumers on coral reefs: although grazer/detritivores support coral reef health in various ways (e.g., promoting coral settlement and herbivory through the removal of detritus and sediments from the algal matrix), they also disperse coral pathogens. Corallivore predation can wound corals, yet their feces contain potentially beneficial coral-associated bacteria, supporting the hypothesized role of consumers, and corallivores in particular, in coral symbiont dispersal. Such consumer-mediated microbial dispersal as demonstrated here has broad implications for environmental management

SMRT Sequencing of Paramecium Bursaria Chlorella Virus-1 Reveals Diverse Methylation Stability in Adenines Targeted by Restriction Modification Systems

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives

‘On the outside I’m smiling but inside I’m crying’: communication successes and challenges for undergraduate academic writing

Student difficulties with the transition to writing in higher education are well documented whether from a ‘study skills’, an ‘academic socialisation’ or an ‘academic literacies’ perspective. In order to more closely examine the challenges faced by students from widening participation backgrounds and diverse routes into undergraduate study, this project focuses on first-year undergraduate experiences of developing academic literacies on an Education Studies programme at one university in England. It highlights the impact of different support and guidance within and beyond their degree programme where attempts to embed academic literacy development are part of subject modules. The paper reports the findings generated using a mixed methods interpretive approach. Questionnaires were collected at the beginning (n = 48) and end of the students’ first year (n = 44), and interviews and visual data collection methods (n =19) were used at the mid-point of the academic year. Key findings highlight students’ expectations of achievement on entry to university and the influence of the emotional journey of students as they begin to make progress as academic writers. Identifying, selecting and applying academic reading were an enduring concern whilst some students struggled with the digital literacy implicit in undergraduate work. Importantly, some strategies developed to support student transition to academic writing in higher education may have unintended consequences as they progress through the first year

A Student\u27s Guide to giant Viruses Infecting Small Eukaryotes: From Acanthamoeba to Zooxanthellae

The discovery of infectious particles that challenge conventional thoughts concerning “what is a virus” has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning “giant viruses”, with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host–virus systems that fall into this “giant” category, demonstrating that this field of inquiry presents great opportunities for future research

A Search for Technosignatures Around 11,680 Stars with the Green Bank Telescope at 1.15-1.73 GHz

We conducted a search for narrowband radio signals over four observing sessions in 2020-2023 with the L-band receiver (1.15-1.73 GHz) of the 100 m diameter Green Bank Telescope. We pointed the telescope in the directions of 62 TESS Objects of Interest, capturing radio emissions from a total of ~11,680 stars and planetary systems in the ~9 arcminute beam of the telescope. All detections were either automatically rejected or visually inspected and confirmed to be of anthropogenic nature. In this work, we also quantified the end-to-end efficiency of radio SETI pipelines with a signal injection and recovery analysis. The UCLA SETI pipeline recovers 94.0% of the injected signals over the usable frequency range of the receiver and 98.7% of the injections when regions of dense RFI are excluded. In another pipeline that uses incoherent sums of 51 consecutive spectra, the recovery rate is ~15 times smaller at ~6%. The pipeline efficiency affects calculations of transmitter prevalence and SETI search volume. Accordingly, we developed an improved Drake Figure of Merit and a formalism to place upper limits on transmitter prevalence that take the pipeline efficiency and transmitter duty cycle into account. Based on our observations, we can state at the 95% confidence level that fewer than 6.6% of stars within 100 pc host a transmitter that is detectable in our search (EIRP > 1e13 W). For stars within 20,000 ly, the fraction of stars with detectable transmitters (EIRP > 5e16 W) is at most 3e-4. Finally, we showed that the UCLA SETI pipeline natively detects the signals detected with AI techniques by Ma et al. (2023).Comment: 22 pages, 9 figures, submitted to AJ, revise

Cryopreservation of \u3ci\u3eParamecium bursaria Chlorella Virus-1\u3c/i\u3e during an active infection cycle of its host

Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilis NC64A host, and found that virus survivorship was highest (69.5 ± 16.5%) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9–30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes postinfection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts

SMRT Sequencing of Paramecium Bursaria Chlorella Virus-1 Reveals Diverse Methylation Stability in Adenines Targeted by Restriction Modification Systems

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives