Plastic marine debris (PMD) affects spatial scales of life from microbes to whales.However, understanding interactions between plastic and microbes in the "Plastisphere"-the thin layer of life on the surface of PMD-has been technologylimited. Research into microbe-microbe and microbe-substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI-FISH (combinatorial labelling and spectral imaging -fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha-, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell-count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro-metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales. K E Y W O R D S biofilm, CLASI-FISH, confocal microscopy, marine plastic, succession | 621 SCHLUNDT eT aL. and Louis Kerr for help with microscopy and image analysis. AUTH O R CO NTR I B UTI O N S CS and LAA-Z conceived the project. CS, JLMW, LAA-Z, and ERZ designed the research. CS, AMK and ERZ performed research. LAA-Z and JLMW contributed reagents and analytical tools. CS, JLMW, AMK, ERZ and LAA-Z analyzed data. CS, JLMW, ERZ and LAA-Z wrote the paper. All authors read and approved the final draft of the manuscript. O RCI D

1. Despite the large size and economic value of the species, populations of oceanic manta ray (Mobula birostris) are often poorly studied and almost completely undescribed in the Red Sea. Here, photo-identification (photo-ID) was used to provide the first description of M. birostris movement patterns and population demographics for the northern Red Sea. 2. Images collated from social media, researchers, and photo-ID databases from 2004 to 2021 identified 267 individual M. birostris from 395 sightings in Egypt, Israel, Jordan, Saudi Arabia, and Sudan. Sexual parity was observed in the population with 134 females, 111 males, and 22 individuals of undetermined sex. Nearly half of the individuals in this study were first identified through social media searches, highlighting the value of the sightings data collected by the public. 3. The regular presence of juveniles in Sharm el-Sheikh, Egypt, indicates that this area is a potential nursery, which is the second identified for this species globally and the first in the Indo-Pacific. Encounter data from this study recorded the first known movements of juvenile M. birostris and demonstrate that they can travel long distances of at least 525 km. 4. Within the recorded population, 24.7% (n = 67) of individuals were resighted, including 21 oceanic manta rays that were encountered in more than one location. These records reveal long-distance migrations of up to 700 km and confirm the international connectivity of M. birostris in the Red Sea. The lagged identification rate data are best fitted with an open-population model, implying that the northern Red Sea is represented by one well-mixed population. 5. The baseline data presented here should encourage the expansion of directed research and citizen science initiatives for M. birostris. Increased knowledge of movement patterns and site use will be crucial to successfully protecting this

Background Reef manta ray (Mobula alfredi) populations along the Northeastern African coastline are poorly studied. Identifying critical habitats for this species is essential for future research and conservation efforts. Dungonab Bay and Mukkawar Island National Park (DMNP), a component of a UNESCO World Heritage Site in Sudan, hosts the largest known M. alfredi aggregation in the Red Sea. Methods A total of 19 individuals were tagged using surgically implanted acoustic tags and tracked within DMNP on an array of 15 strategically placed acoustic receivers in addition to two offshore receivers. Two of these acoustically monitored M. alfredi were also equipped with satellite linked archival tags and one individual was fitted with a satellite transmitting tag. Together, these data are used to describe approximately two years of residency and seasonal shifts in habitat use. Results Tagged individuals were detected within the array on 96% of monitored days and recorded an average residence index of 0.39 across all receivers. Detections were recorded throughout the year, though some individuals were absent from the receiver array for weeks or months at a time, and generalized additive mixed models showed a clear seasonal pattern in presence with the highest probabilities of detection occurring in boreal fall. The models indicated that M. alfredi presence was highly correlated with increasing chlorophyll-a levels and weakly correlated with the full moon. Modeled biological factors, including sex and wingspan, had no influence on animal presence. Despite the high residency suggested by acoustic telemetry, satellite tag data and offshore acoustic detections in Sanganeb Atoll and Suedi Pass recorded individuals moving up to 125 km from the Bay. However, all these individuals were subsequently detected in the Bay, suggesting a strong degree of site fidelity at this location. Conclusions The current study adds to growing evidence that M. alfredi are highly resident and site-attached to coastal bays and lagoons but display seasonal shifts in habitat use that are likely driven by resource availability. This information can be used to assist in managing and supporting sustainable ecotourism within the DMNP, part of a recently designated UNESCO World Heritage Site.

Filamentous viruses are hypothesized to play a role in stony coral tissue loss disease (SCTLD) through infection of the endosymbiotic dinoflagellates (Family Symbiodiniaceae) of corals. To evaluate this hypothesis, it is critical to understand the global distribution of filamentous virus infections across the genetic diversity of Symbiodiniaceae hosts. We present transmission electron microscopy images demonstrating filamentous virus-like particles (VLPs) are present in 61 and 70% of Symbiodiniaceae cells (genus Cladocopium) within Pacific corals (Acropora hyacinthus, Porites c.f. lobata, respectively); these VLPs are more prevalent in Symbiodiniaceae of in situ colonies experiencing heat stress. Symbiodiniaceae expelled from A. hyacinthus also contain filamentous VLPs, and these cells are more degraded than their in hospite counterparts. Similar to the VLPs reported from SCTLD-affected Caribbean reefs, VLPs range from ~ 150–1500 nm in length and 15–37 nm in diameter and appear to constitute various stages in a replication cycle. Finally, we demonstrate that SCTLD-affected corals containing filamentous VLPs are dominated by diverse Symbiodiniaceae lineages from the genera Breviolum, Cladocopium, and Durusdinium. Although this study cannot definitively confirm or refute the role of filamentous VLPs in SCTLD, it demonstrates that filamentous VLPs are not solely observed in SCTLD-affected corals or reef regions, nor are they solely associated with corals dominated by members of a particular Symbiodiniaceae genus. We hypothesize that filamentous viruses are a widespread, common group that infects Symbiodiniaceae. Genomic characterization of these viruses and empirical tests of the impacts of filamentous virus infection on Symbiodiniaceae and coral colonies should be prioritized.

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