An in situ digital synthesis strategy for the discovery and description of ocean life.

Revolutionary advancements in underwater imaging, robotics, and genomic sequencing have reshaped marine exploration. We present and demonstrate an interdisciplinary approach that uses emerging quantitative imaging technologies, an innovative robotic encapsulation system with in situ RNA preservation and next-generation genomic sequencing to gain comprehensive biological, biophysical, and genomic data from deep-sea organisms. The synthesis of these data provides rich morphological and genetic information for species description, surpassing traditional passive observation methods and preserved specimens, particularly for gelatinous zooplankton. Our approach enhances our ability to study delicate mid-water animals, improving research in the world's oceans.

Long-Read-Based Genome Assembly Reveals Numerous Endogenous Viral Elements in the Green Algal Bacterivore Cymbomonas tetramitiformis.

The marine tetraflagellate Cymbomonas tetramitiformis has drawn attention as an early diverging green alga that uses a phago-mixotrophic mode of nutrition (i.e., the ability to derive nourishment from both photosynthesis and bacterial prey). The Cymbomonas nuclear genome was sequenced previously, but due to the exclusive use of short-read (Illumina) data, the assembly suffered from missing a large proportion of the genome's repeat regions. For this study, we generated Oxford Nanopore long-read and additional short-read Illumina data and performed a hybrid assembly that significantly improved the total assembly size and contiguity. Numerous endogenous viral elements were identified in the repeat regions of the new assembly. These include the complete genome of a giant Algavirales virus along with many genomes of integrated Polinton-like viruses (PLVs) from two groups: Gezel-like PLVs and a novel group of prasinophyte-specific PLVs. The integrated ∼400 kb genome of the giant Algavirales virus is the first account of the association of the uncultured viral family AG_03 with green algae. The complete PLV genomes from C. tetramitiformis ranged between 15 and 25 kb in length and showed a diverse gene content. In addition, heliorhodopsin gene-containing repeat elements of putative mirusvirus origin were identified. These results illustrate past (and possibly ongoing) multiple alga-virus interactions that accompanied the genome evolution of C. tetramitiformis.

Phagocytosis underpins the biotrophic lifestyle of intracellular parasites in the class Phytomyxea (Rhizaria).

Phytomyxea are intracellular biotrophic parasites infecting plants and stramenopiles, including the agriculturally impactful Plasmodiophora brassicae and the brown seaweed pathogen Maullinia ectocarpii. They belong to the clade Rhizaria, where phagotrophy is the main mode of nutrition. Phagocytosis is a complex trait of eukaryotes, well documented for free-living unicellular eukaryotes and specific cellular types of animals. Data on phagocytosis in intracellular, biotrophic parasites are scant. Phagocytosis, where parts of the host cell are consumed at once, is seemingly at odds with intracellular biotrophy. Here we provide evidence that phagotrophy is part of the nutritional strategy of Phytomyxea, using morphological and genetic data (including a novel transcriptome of M. ectocarpii). We document intracellular phagocytosis in P. brassicae and M. ectocarpii by transmission electron microscopy and fluorescent in situ hybridization. Our investigations confirm molecular signatures of phagocytosis in Phytomyxea and hint at a small specialized subset of genes used for intracellular phagocytosis. Microscopic evidence confirms the existence of intracellular phagocytosis, which in Phytomyxea targets primarily host organelles. Phagocytosis seems to coexist with the manipulation of host physiology typical of biotrophic interactions. Our findings resolve long debated questions on the feeding behaviour of Phytomyxea, suggesting an unrecognized role for phagocytosis in biotrophic interactions.

Transcriptomics of a Greenlandic Snailfish Reveals Exceptionally High Expression of Antifreeze Protein Transcripts.

Polar fishes have evolved antifreeze proteins (AFPs) that allow them to survive in subzero temperatures. We performed deep transcriptomic sequencing on a postlarval/juvenile variegated snailfish, Liparis gibbus (Actinopterygii: Scorpaeniformes: Cottoidei: Liparidae), living in an iceberg habitat (-2°C) in Eastern Greenland and report detection of highly expressed transcripts that code for putative AFPs from 2 gene families, Type I and LS-12-like proteins (putative Type IV AFPs). The transcripts encoding both proteins have expression levels among the top <1% of expressed genes in the fish. The Type I AFP sequence is different from a reported Type I AFP from the same species, possibly expressed from a different genetic locus. While prior findings from related adult sculpins suggest that LS-12-like/Type IV AFPs may not have a role in antifreeze protection, our finding of very high relative gene expression of the LS-12-like gene suggests that highly active transcription of the gene is important to the fish in the iceberg habitat and raises the possibility that weak or combinatorial antifreeze activity could be beneficial. These findings highlight the physiological importance of antifreeze proteins to the survival of fishes living in polar habitats.

Organismal and cellular interactions in vertebrate-alga symbioses.

Photosymbioses, intimate interactions between photosynthetic algal symbionts and heterotrophic hosts, are well known in invertebrate and protist systems. Vertebrate animals are an exception where photosynthetic microorganisms are not often considered part of the normal vertebrate microbiome, with a few exceptions in amphibian eggs. Here, we review the breadth of vertebrate diversity and explore where algae have taken hold in vertebrate fur, on vertebrate surfaces, in vertebrate tissues, and within vertebrate cells. We find that algae have myriad partnerships with vertebrate animals, from fishes to mammals, and that those symbioses range from apparent mutualisms to commensalisms to parasitisms. The exception in vertebrates, compared with other groups of eukaryotes, is that intracellular mutualisms and commensalisms with algae or other microbes are notably rare. We currently have no clear cell-in-cell (endosymbiotic) examples of a trophic mutualism in any vertebrate, while there is a broad diversity of such interactions in invertebrate animals and protists. This functional divergence in vertebrate symbioses may be related to vertebrate physiology or a byproduct of our adaptive immune system. Overall, we see that diverse algae are part of the vertebrate microbiome, broadly, with numerous symbiotic interactions occurring across all vertebrate and many algal clades. These interactions are being studied for their ecological, organismal, and cellular implications. This synthesis of vertebrate-algal associations may prove useful for the development of novel therapeutics: pairing algae with medical devices, tissue cultures, and artificial ecto- and endosymbioses.

From intent to implementation: Factors affecting public involvement in life science research.

Public involvement is key to closing the gap between research production and research use, and the only way to achieving ultimate transparency in science. The majority of life science research is not public-facing, but is funded by the public and impacts communities. We undertook an exploratory survey of researchers within the life sciences to better understand their views and perceived challenges to involving the public in their research. As survey response rate could not be determined, interpretation of the results must be cautious. We had a valid response cohort of n = 110 researchers, of whom 90% were primarily laboratory based. Using a mixed methods approach, we demonstrate that a top-down approach is key to motivate progression of life scientists from feeling positive towards public involvement to actually engaging in it. Researchers who viewed public involvement as beneficial to their research were more likely to have direct experience of doing it. We demonstrate that the systemic flaws in the way life sciences research enterprise is organised, including the promotion system, hyper-competition, and time pressures are major barriers to involving the public in the scientific process. Scientists are also apprehensive of being involuntarily involved in the current politicized climate; misinformation and publicity hype surrounding science nowadays makes them hesitant to share their early and in-progress research. The time required to deliberate study design and relevance, plan and build relationships for sustained involvement, provide and undertake training, and improve communication in the current research environment is often considered nonpragmatic, particularly for early career researchers. In conclusion, a top-down approach involving institutional incentives and infrastructure appears most effective at transitioning researchers from feeling positive towards public involvement to actually implementing it.

Antibody Development in Patients Treated Long-Term With OnabotulinumtoxinA for Benign Essential Blepharospasm and Hemifacial Spasm.

BACKGROUND: Report the development of onabotulinumtoxinA neutralizing antibodies in patients treated consecutively for 20 years or longer for benign essential blepharospasm (BEB), hemifacial spasm (HFS), and Meige Syndrome. METHODS: Prospective, randomized, cross-sectional study of 12 randomly selected patients from a single clinical practice that have been treated consecutively for 20 or more years with onabotulinumtoxinA for BEB, HFS, or Meige Syndrome. Serum samples were collected from each subject and analyzed for neutralizing antibody formation using the Mouse Protection Assay. RESULTS: None of the tested patients (0%) displayed neutralizing antibodies to onabotulinumtoxinA. The mean duration of treatment was 27.5 years (range 22.1-34.1, SD 3.1, 95% confidence interval 25.45-29.50). Nine of the patients had a diagnosis of BEB, 2 HFS, and one Meige. Eleven of the 12 patients were women. There was no statistically significant difference in treatment dosage or interval over the course of treatment. CONCLUSIONS: The data support previous studies showing low incidence of antibody formation for botulinum A toxins with this subset of long-term treated patients. The results also provide further evidence for studies that have suggested increased onabotulinumtoxinA treatment volumes and/or decreased intervals between treatments are not due to neutralizing antibody formation and secondary non-response, but rather study designs that do not consider the titration phase of initial treatments. This study is specific to long-term treated patients, and the results cannot be generalized to patients naive to treatment.

No evidence of Phago-mixotropy in Micromonas polaris (Mamiellophyceae), the Dominant Picophytoplankton Species in the Arctic.

In the Arctic Ocean, the small green alga Micromonas polaris dominates picophytoplankton during the summer months but is also present in winter. It has been previously hypothesized to be phago-mixotrophic (capable of bacteria ingestion) based on laboratory and field experiments. Prey uptake was analyzed in several M. polaris strains isolated from different regions and depths of the Arctic Ocean and in Ochromonas triangulata, a known phago-mixotroph used as a control. Measuring ingestion of either fluorescent beads or fluorescently labeled bacteria by flow cytometry, we found no evidence of phago-mixotrophy in any M. polaris strain while O. triangulata was ingesting both beads and bacteria. In addition, in silico predictions revealed that members of the genus Micromonas lack a genetic signature of phagocytotic capacity.

Heterotrophic Carbon Fixation in a Salamander-Alga Symbiosis.

The unique symbiosis between a vertebrate salamander, Ambystoma maculatum, and unicellular green alga, Oophila amblystomatis, involves multiple modes of interaction. These include an ectosymbiotic interaction where the alga colonizes the egg capsule, and an intracellular interaction where the alga enters tissues and cells of the salamander. One common interaction in mutualist photosymbioses is the transfer of photosynthate from the algal symbiont to the host animal. In the A. maculatum-O. amblystomatis interaction, there is conflicting evidence regarding whether the algae in the egg capsule transfer chemical energy captured during photosynthesis to the developing salamander embryo. In experiments where we took care to separate the carbon fixation contributions of the salamander embryo and algal symbionts, we show that inorganic carbon fixed by A. maculatum embryos reaches 2% of the inorganic carbon fixed by O. amblystomatis algae within an egg capsule after 2 h in the light. After 2 h in the dark, inorganic carbon fixed by A. maculatum embryos is 800% of the carbon fixed by O. amblystomatis algae within an egg capsule. Using photosynthesis inhibitors, we show that A. maculatum embryos and O. amblystomatis algae compete for available inorganic carbon within the egg capsule environment. Our results confirm earlier studies suggesting a role of heterotrophic carbon fixation during vertebrate embryonic development. Our results also show that the considerable capacity of developing A. maculatum embryos for inorganic carbon fixation precludes our ability to distinguish any minor role of photosynthetically transferred carbon from algal symbionts to host salamanders using bicarbonate introduced to the egg system as a marker.

Ultra-gentle soft robotic fingers induce minimal transcriptomic response in a fragile marine animal.

Tessler et al. demonstrate that a 'soft' robot causes less stress to a jellyfish while handling compared to a traditional 'hard' robot.

The Chytrid Fungus, Batrachochytrium dendrobatidis, is Widespread Among Cuban Amphibians.

The fungus Batrachochytrium dendrobatidis (Bd) is a generalist amphibian pathogen responsible for chytridiomycosis. It was documented for the first time in Cuba in 2007, the apparent cause of the decline in one species of toad. In a recent survey, Bd was reported only for the highlands of Central Cuba. In the present study, we reexamined the geographic distribution and level of impact of Bd in Cuba by conducting an island-wide sampling in 10 localities and collecting skin swabs from 18 species and 28 environmental samples. We report detection of Bd in 60% of sampled sites and in 58% of sampled taxa. We show that Bd is associated with riparian, arboreal and terrestrial species, and it was estimated to occur in approximately 30% of the aquatic habitats we sampled. In addition, we confirmed that a dying individual of the species Eleutherodactylus casparii was severely infected with Bd. We also rise concern about the endanger toad Peltophryne longinasus and about three species of endemic riparian frogs that were not detected during our surveys. This study demonstrates that this pathogen is widespread throughout Cuba and provides relevant evidence to advance our understanding of its detection in amphibians and the aquatic environment in Cuba and about the occurrence of Bd in species with different ecologies. We provide valuable baseline information for Bd risk assessment and decision-making processes to mitigate its negative impact on Cuban amphibians.

Global transcriptome analysis of the aphelid Paraphelidium tribonemae supports the phagotrophic origin of fungi.

Aphelids are little-known phagotrophic parasites of algae whose life cycle and morphology resemble those of the parasitic rozellids (Cryptomycota, Rozellomycota). In previous phylogenetic analyses of RNA polymerase and rRNA genes, aphelids, rozellids and Microsporidia (parasites of animals) formed a clade, named Opisthosporidia, which appeared as the sister group to Fungi. However, the statistical support for the Opisthosporidia was always moderate. Here, we generated full life-cycle transcriptome data for the aphelid species Paraphelidium tribonemae. In-depth multi-gene phylogenomic analyses using several protein datasets place this aphelid as the closest relative of fungi to the exclusion of rozellids and Microsporidia. In contrast with the comparatively reduced Rozella allomycis genome, we infer a rich, free-living-like aphelid proteome, with a metabolism similar to fungi, including cellulases likely involved in algal cell-wall penetration and enzymes involved in chitin biosynthesis. Our results suggest that fungi evolved from complex aphelid-like ancestors that lost phagotrophy and became osmotrophic.

O6-methylguanine-induced transcriptional mutagenesis reduces p53 tumor-suppressor function.

Altered protein function due to mutagenesis plays an important role in disease development. This is perhaps most evident in tumorigenesis and the associated loss or gain of function of tumor-suppressor genes and oncogenes. The extent to which lesion-induced transcriptional mutagenesis (TM) influences protein function and its contribution to the development of disease is not well understood. In this study, the impact of O6-methylguanine on the transcription fidelity of p53 and the subsequent effects on the protein's function as a regulator of cell death and cell-cycle arrest were examined in human cells. Levels of TM were determined by RNA-sequencing. In cells with active DNA repair, misincorporation of uridine opposite the lesion occurred in 0.14% of the transcripts and increased to 14.7% when repair by alkylguanine-DNA alkyltransferase was compromised. Expression of the dominant-negative p53 R248W mutant due to TM significantly reduced the transactivation of several established p53 target genes that mediate the tumor-suppressor function, including CDKN1A (p21) and BBC3 (PUMA). This resulted in deregulated signaling through the retinoblastoma protein and loss of G1/S cell-cycle checkpoint function. In addition, we observed impaired activation of apoptosis coupled to the reduction of the tumor-suppressor functions of p53. Taking these findings together, this work provides evidence that TM can induce phenotypic changes in mammalian cells that have important implications for the role of TM in tumorigenesis.

Publisher Correction: Gene-based predictive models of trophic modes suggest Asgard archaea are not phagocytotic.

In the version of this Article originally published, question marks appeared in Table 1; they should have been tick marks. This has now been corrected in all versions of the Article.

Gene-based predictive models of trophic modes suggest Asgard archaea are not phagocytotic.

With the current explosion of genomic data, there is a greater need to draw inference on phenotypic information based on DNA sequence alone. We considered complete genomes from 35 diverse eukaryotic lineages, and discovered sets of proteins predictive of trophic mode, including a set of 485 proteins that are enriched among phagocytotic eukaryotes (organisms that internalize large particles). Our model is also predictive of other aspects of trophic mode, including photosynthesis and the ability to synthesize a set of organic compounds needed for growth (prototrophy for those molecules). We applied our model to the Asgard archaea, a group of uncultured microorganisms that show close affinities to eukaryotes, to test whether the organisms are capable of phagocytosis, a phenotypic trait often considered a prerequisite for mitochondrial acquisition. Our analyses suggest that members of the Asgard archaea-despite having some eukaryote-specific protein families not found in other prokaryotes-do not use phagocytosis. Moreover, our data suggest that the process of phagocytosis arose from a combination of both archaeal and bacterial components, but also required additional eukaryote-specific innovations.

Genetic instability associated with loop or stem-loop structures within transcription units can be independent of nucleotide excision repair.

Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem-loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them.

Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis.

During embryonic development, cells of the green alga Oophila amblystomatis enter cells of the salamander Ambystoma maculatum forming an endosymbiosis. Here, using de novo dual-RNA seq, we compared the host salamander cells that harbored intracellular algae to those without algae and the algae inside the animal cells to those in the egg capsule. This two-by-two-way analysis revealed that intracellular algae exhibit hallmarks of cellular stress and undergo a striking metabolic shift from oxidative metabolism to fermentation. Culturing experiments with the alga showed that host glutamine may be utilized by the algal endosymbiont as a primary nitrogen source. Transcriptional changes in salamander cells suggest an innate immune response to the alga, with potential attenuation of NF-κB, and metabolic alterations indicative of modulation of insulin sensitivity. In stark contrast to its algal endosymbiont, the salamander cells did not exhibit major stress responses, suggesting that the host cell experience is neutral or beneficial.

Complete mitochondrial genomes of prasinophyte algae Pyramimonas parkeae and Cymbomonas tetramitiformis.

Mitochondria are archetypal eukaryotic organelles that were acquired by endosymbiosis of an ancient species of alpha-proteobacteria by the last eukaryotic common ancestor. The genetic information contained within the mitochondrial genome has been an important source of information for resolving relationships among eukaryotic taxa. In this study, we utilized mitochondrial and chloroplast genomes to explore relationships among prasinophytes. Prasinophytes are represented by diverse early-diverging green algae whose physical structures and genomes have the potential to elucidate the traits of the last common ancestor of the Viridiplantae (or Chloroplastida). We constructed de novo mitochondrial genomes for two prasinophyte algal species, Pyramimonas parkeae and Cymbomonas tetramitiformis, representing the prasinophyte clade. Comparisons of genome structure and gene order between these species and to those of other prasinophytes revealed that the mitochondrial genomes of P. parkeae and C. tetramitiformis are more similar to each other than to other prasinophytes, consistent with other molecular inferences of the close relationship between these two species. Phylogenetic analyses using the inferred amino acid sequences of mitochondrial and chloroplast protein-coding genes resolved a clade consisting of P. parkeae and C. tetramitiformis; and this group (representing the prasinophyte clade I) branched with the clade II, consistent with previous studies based on the use of nuclear gene markers.

Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription.

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N(6)-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N(6)-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N(6)-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N(6)-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N(6)-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.

Comparative Genomics of a Bacterivorous Green Alga Reveals Evolutionary Causalities and Consequences of Phago-Mixotrophic Mode of Nutrition.

Cymbomonas tetramitiformis-a marine prasinophyte-is one of only a few green algae that still retain an ancestral particulate-feeding mechanism while harvesting energy through photosynthesis. The genome of the alga is estimated to be 850 Mb-1.2 Gb in size-the bulk of which is filled with repetitive sequences-and is annotated with 37,366 protein-coding gene models. A number of unusual metabolic pathways (for the Chloroplastida) are predicted for C. tetramitiformis, including pathways for Lipid-A and peptidoglycan metabolism. Comparative analyses of the predicted peptides of C. tetramitiformis to sets of other eukaryotes revealed that nonphagocytes are depleted in a number of genes, a proportion of which have known function in feeding. In addition, our analysis suggests that obligatory phagotrophy is associated with the loss of genes that function in biosynthesis of small molecules (e.g., amino acids). Further, C. tetramitiformis and at least one other phago-mixotrophic alga are thus unique, compared with obligatory heterotrophs and nonphagocytes, in that both feeding and small molecule synthesis-related genes are retained in their genomes. These results suggest that early, ancestral host eukaryotes that gave rise to phototrophs had the capacity to assimilate building block molecules from inorganic substances (i.e., prototrophy). The loss of biosynthesis genes, thus, may at least partially explain the apparent lack of instances of permanent incorporation of photosynthetic endosymbionts in later-divergent, auxotrophic eukaryotic lineages, such as metazoans and ciliates.