Phylogenetic Analysis of Algal Symbionts Associated with Four North American Amphibian Egg Masses

Egg masses of the yellow-spotted salamander Ambystoma maculatum form an association with the green alga “Oophila amblystomatis” (Lambert ex Wille), which, in addition to growing within individual egg capsules, has recently been reported to invade embryonic tissues and cells. The binomial O. amblystomatis refers to the algae that occur in A. maculatum egg capsules, but it is unknown whether this population of symbionts constitutes one or several different algal taxa. Moreover, it is unknown whether egg masses across the geographic range of A. maculatum, or other amphibians, associate with one or multiple algal taxa. To address these questions, we conducted a phylogeographic study of algae sampled from egg capsules of A. maculatum, its allopatric congener A. gracile, and two frogs: Lithobates sylvatica and L. aurora. All of these North American amphibians form associations with algae in their egg capsules. We sampled algae from egg capsules of these four amphibians from localities across North America, established representative algal cultures, and amplified and sequenced a region of 18S rDNA for phylogenetic analysis. Our combined analysis shows that symbiotic algae found in egg masses of four North American amphibians are closely related to each other, and form a well-supported clade that also contains three strains of free-living chlamydomonads. We designate this group as the ‘Oophila’ clade, within which the symbiotic algae are further divided into four distinct subclades. Phylogenies of the host amphibians and their algal symbionts are only partially congruent, suggesting that host-switching and co-speciation both play roles in their associations. We also established conditions for isolating and rearing algal symbionts from amphibian egg capsules, which should facilitate further study of these egg mass specialist algae

Map of the geographic range and collection sites for egg masses of four amphibian hosts.

<p>Species range maps are plotted on a map of North America (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#s2" target="_blank">Materials and Methods</a>). The dark green color represents a range overlap between <i>L. sylvatica</i> and <i>A. maculatum</i>, and the pink color represents a range overlap between <i>L. aurora</i> and <i>A. gracile</i>. Numbered locations correspond to higher detail panels below. The maps of collection sites for algae corresponding to egg masses from <i>A. maculatum</i> and L. sylvatica in Nova Scotia, Canada (1), <i>A. gracile</i> in California, USA (2), <i>L. aurora</i> and <i>A. gracile</i> in Vancouver Island, British Columbia, Canada, and <i>A. maculatum</i> in New Jersey and Tennessee of USA (4/5).</p

Maximum likelihood (ML) tree of algal 18S rDNA sequences from egg masses of four amphibian taxa from various North American localities.

<p>The data matrix included 1,653 characters and 180 sequences. Newly obtained sequences are bold-faced. ML and MP bootstrap values greater than 50% are shown at corresponding nodes. Subclades I−IV are collapsed into triangles for visual clarity; an un-collapsed version of the tree can be found as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#pone.0108915.s003" target="_blank">Figure S1</a>. Numbers in parentheses indicates the number of sequences obtained and analyzed for the corresponding sample. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#pone-0108915-t001" target="_blank">Table 1</a> for naming conventions and GenBank accession numbers.</p

Collection details of egg masses from which algae were sampled.

<p>Collection details of egg masses from which algae were sampled.</p

Maximum incidence of non-<i>Oophila</i> taxa in egg capsules, based on a>0.99 cumulative probability of detecting sequences and the actual number of sequences obtained (see equation 2 in Discussion).

<p>Maximum incidence of non-<i>Oophila</i> taxa in egg capsules, based on a>0.99 cumulative probability of detecting sequences and the actual number of sequences obtained (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108915#pone.0108915.e007" target="_blank">equation 2</a> in Discussion).</p

Light microscopic images of cultured strains of <i>A. maculatum</i> algae.

<p>The <i>Oophila</i> strains Hb_cul-rk (A−C) and BB_cul-B (D−F) belong to subclades I and III, respectively. Monotypic cultures displayed at least three different cell types, which include 1) free-swimming biflagellates (A, D), which correspond to zoospores or gametes, 2) cells enclosed within a mother cell wall (B, E), likely representing asexually dividing zoospores, and 3) larger non-motile zygotes (C, F). Scale bars: 10 µm (A−F).</p

Percentage pairwise distances (uncorrected) of 18S rDNA among the <i>Oophila</i> subclades I−IV.

<p>Percentage pairwise distances (uncorrected) of 18S rDNA among the <i>Oophila</i> subclades I−IV.</p

The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect

Summary: Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2. : DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. Off-target RAG cleavage has only been analyzed in the context of DNA repair defects. Here, Hewitt et al. identify a phosphorylation site on RAG2 that controls RAG cleavage to maintain genome stability independent of a repair defect. Keywords: RAG cleavage regulation, genome stability, V(D)J recombination, ATM, RAG2S365, reciprocal translocations, developing lymphocyte

Rapid TCR:Epitope Ranker (RAPTER): a primary human T cell reactivity screening assay pairing epitope and TCR at single cell resolution

Abstract Identifying epitopes that T cells respond to is critical for understanding T cell-mediated immunity. Traditional multimer and other single cell assays often require large blood volumes and/or expensive HLA-specific reagents and provide limited phenotypic and functional information. Here, we present the Rapid TCR:Epitope Ranker (RAPTER) assay, a single cell RNA sequencing (scRNA-SEQ) method that uses primary human T cells and antigen presenting cells (APCs) to assess functional T cell reactivity. Using hash-tag oligonucleotide (HTO) coding and T cell activation-induced markers (AIM), RAPTER defines paired epitope specificity and TCR sequence and can include RNA- and protein-level T cell phenotype information. We demonstrate that RAPTER identified specific reactivities to viral and tumor antigens at sensitivities as low as 0.15% of total CD8+ T cells, and deconvoluted low-frequency circulating HPV16-specific T cell clones from a cervical cancer patient. The specificities of TCRs identified by RAPTER for MART1, EBV, and influenza epitopes were functionally confirmed in vitro. In summary, RAPTER identifies low-frequency T cell reactivities using primary cells from low blood volumes, and the resulting paired TCR:ligand information can directly enable immunogenic antigen selection from limited patient samples for vaccine epitope inclusion, antigen-specific TCR tracking, and TCR cloning for further therapeutic development