The geographical and seasonal effects on the composition of marine microplastic and its microbial communities: The case study of Israel and Portugal.

Introduction: Floating microplastic debris are found in most marine environments around the world. Due to their low density and high durability, plastic polymers such as polyethylene, polypropylene, and polystyrene serve as stable floating substrates for the colonization of diverse communities of marine organisms. Despite the high abundance of microplastic debris in the oceans, it is not clear how the geographical location and season affect the composition of marine microplastic and its bacterial microbiome in the natural environment. Methods: To address this question, microplastic debris were collected from the sea surface near estuaries in the Mediterranean Sea (Israel) and in the Atlantic Ocean (Portugal) during summer and winter of 2021. The microplastic physical characteristics, including shape, color, and polymer composition, were analyzed and the taxonomic structure of the microplastic bacterial microbiome was characterized using a high-resolution metabarcoding pipeline. Results: Our results, supported by previously published data, suggest that the plastisphere is a highly diverse ecosystem which is strongly shaped by spatial and temporal environmental factors. The geographical location had the highest impact on the plastisphere physical characteristics and its microbiome composition, followed by the season. Our metabarcoding analysis showed great variability between the different marine environments with a very limited microbiome "core." Discussion: This notion further emphasizes the importance of plastisphere studies in different geographical locations and/or seasons for the characterization of the plastisphere and the identification of plastic-associated species.

Different sea urchin RAG-like genes were domesticated to carry out different functions.

The closely linked recombination activating genes (RAG1 and RAG2) in vertebrates encode the core of the RAG recombinase that mediates the V(D)J recombination of the immunoglobulin and T-cell receptor genes. RAG1 and RAG2 homologues (RAG1L and RAG2L) are present in multiple invertebrate phyla, including mollusks, nemerteans, cnidarians, and sea urchins. However, the function of the invertebrates' RAGL proteins is yet unknown. The sea urchins contain multiple RAGL genes that presumably originated in a common ancestral transposon. In this study, we demonstrated that two different RAG1L genes in the sea urchin Paracentrutus lividus (PlRAG1La and PlRAG1Lb) lost their mobility and, along with PlRAG2L, were fully domesticated to carry out different functions. We found that the examined echinoid RAGL homologues have distinct expression profiles in early developmental stages and in adult tissues. Moreover, the predicted structure of the proteins suggests that while PlRAG1La could maintain its endonuclease activity and create a heterotetramer with PlRAG2L, the PlRAG1Lb adopted a different function that does not include an interaction with DNA nor a collaboration with PlRAG2L. By characterizing the different RAG homologues in the echinoid lineage, we hope to increase the knowledge about the evolution of these genes and shed light on their domestication processes.

Guardian of the Genome: An Alternative RAG/Transib Co-Evolution Hypothesis for the Origin of V(D)J Recombination.

The appearance of adaptive immunity in jawed vertebrates is termed the immunological 'Big Bang' because of the short evolutionary time over which it developed. Underlying it is the recombination activating gene (RAG)-based V(D)J recombination system, which initiates the sequence diversification of the immunoglobulins and lymphocyte antigen receptors. It was convincingly argued that the RAG1 and RAG2 genes originated from a single transposon. The current dogma postulates that the V(D)J recombination system was established by the split of a primordial vertebrate immune receptor gene into V and J segments by a RAG1/2 transposon, in parallel with the domestication of the same transposable element in a separate genomic locus as the RAG recombinase. Here, based on a new interpretation of previously published data, we propose an alternative evolutionary hypothesis suggesting that two different elements, a RAG1/2 transposase and a Transib transposon invader with RSS-like terminal inverted repeats, co-evolved to work together, resulting in a functional recombination process. This hypothesis offers an alternative understanding of the acquisition of recombinase function by RAGs and the origin of the V(D)J system.

The Diverse Transformer (Trf) Protein Family in the Sea Urchin Paracentrotus lividus Acts through a Collaboration between Cellular and Humoral Immune Effector Arms.

Sea urchins are long-living marine invertebrates with a complex innate immune system, which includes expanded families of immune receptors. A central immune gene family in sea urchins encodes the Transformer (Trf) proteins. The Trf family has been studied mainly in the purple sea urchin Strongylocentrotus purpuratus. Here, we explore this protein family in the Mediterranean Sea urchin Paracentrotus lividus. The PlTrf genes and predicted proteins are highly diverse and show a typical Trf size range and structure. Coelomocytes and cell-free coelomic fluid from P. lividus contain different PlTrf protein repertoires with a shared subset, that bind specifically to E. coli. Using FACS, we identified five different P. lividus coelomocyte sub-populations with cell surface PlTrf protein expression. The relative abundance of the PlTrf-positive cells increases sharply following immune challenge with E. coli, but not following challenge with LPS or the sea urchin pathogen, Vibrio penaeicida. Phagocytosis of E. coli by P. lividus phagocytes is mediated through the cell-free coelomic fluid and is inhibited by blocking PlTrf activity with anti-SpTrf antibodies. Together, our results suggest a collaboration between cellular and humoral PlTrf-mediated effector arms in the P. lividus specific immune response to pathogens.

Identification of plastic-associated species in the Mediterranean Sea using DNA metabarcoding with Nanopore MinION.

Plastic debris in the ocean form a new ecosystem, termed 'plastisphere', which hosts a variety of marine organisms. Recent studies implemented DNA metabarcoding to characterize the taxonomic composition of the plastisphere in different areas of the world. In this study, we used a modified metabarcoding approach which was based on longer barcode sequences for the characterization of the plastisphere biota. We compared the microbiome of polyethylene food bags after 1 month at sea to the free-living biome in two proximal but environmentally different locations on the Mediterranean coast of Israel. We targeted the full 1.5 kb-long 16S rRNA gene for bacteria and 0.4-0.8 kb-long regions within the 18S rRNA, ITS, tufA and COI loci for eukaryotes. The taxonomic barcodes were sequenced using Oxford Nanopore Technology with multiplexing on a single MinION flow cell. We identified between 1249 and 2141 species in each of the plastic samples, of which 61 species (34 bacteria and 27 eukaryotes) were categorized as plastic-specific, including species that belong to known hydrocarbon-degrading genera. In addition to a large prokaryotes repertoire, our results, supported by scanning electron microscopy, depict a surprisingly high biodiversity of eukaryotes within the plastisphere with a dominant presence of diatoms as well as other protists, algae and fungi.