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The precise mechanisms that allow animals and phototrophic organisms to form a stable photosymbiotic relationship are still unknown. While previous studies focused on genomic adaptations of the animal host, more recent research looked into the role of bacteria in photosymbiosis. Here, we analyzed the core microbiome of the sea slug Berghia stephanieae and its food source Exaiptasia diaphana to understand if the microbiome and the linked bacterial metabolic pathways differ between unstable and stable photosymbiosis. This sea slug feeds solely on the model cnidarian E. diaphana and steals their photobionts which the slug can only maintain for a week. We additionally examined the influence of light and starvation stress on the slug's bacterial composition, which are common experimental set-ups to elucidate the photosymbiotic relationship in the slugs. Our results show that the core microbiome of B. stephanieae and E. diaphana differed significantly suggesting that the slug's microbiome is not obtained from its food source or the water column and indicates a vertical transmission. Further, differences in metabolic pathways imply that the microbiome of B. stephanieae does not support a stable photosymbiosis due to an insufficient nitrogen cycle on part of the photobiont. Starving the slugs induced a shift towards an increased abundance of potential pathogens and led to a downregulation in the sulphur cycle. Yet, starvation in darkness resulted in the depletion of most bacteria and induced a metabolic switch toward bacterial nitrogen fixation. This emphasizes that different holobiont members contribute to essential nutrient cycles, and it is important to look beyond the photobiont to understand the sea slug holobiont. [ABSTRACT FROM AUTHOR]

The phylogeny of phagotrophic euglenids is widely based on nuclear small subunit ribosomal DNA (18S rDNA) sequence data, but most analyses suffer from weakness in statistical support regarding the 'connecting backbone' between monophyletic clades. Moreover, the position of Entosiphon has remained unclear. Testing the 18S rDNA capability for a phylogeny of phagotrophic euglenids, we isolated sequences of Peranema sp. and Ploeotia edaphica and utilized secondary structure data as a prerequisite for recognition of homologous positions. We found a unique, clade-specific nucleotide substitution in the deduced 18S rDNA helix 44. Since our 18S rDNA phylogenies could only in part resolve positions of phagotrophic lineages, but did not verify that of Entosiphon, we investigated the phagotrophic key taxa Peranema trichophorum, Petalomonas cantuscygni, Ploeotia costata, and Entosiphon sulcatum ultrastructurally. Additionally, we explored the presence or absence of the euglenid reserve carbohydrate paramylon by specific staining with monoclonal anti-β-1,3-glucan antibodies. Paramylon was found to be clearly present in P. trichophorum and E. sulcatum, but was absent in Pt. cantuscygni and Pl. costata. Combined results of our molecular, ultrastructural, and immunocytochemical investigations suggest that Entosiphon sulcatum is the sister taxon of a monophyletic euglenid crown clade, characterized by a helical pellicle, which we propose to rename. This phylogenetic affiliation is confirmed by a clade-specific primary absence of the unique nucleotide substitution in helix 44 and by the common presence of paramylon. [ABSTRACT FROM AUTHOR]

Solar-powered sea slugs (Sacoglossa: Gastropoda) have long captured the attention of laymen and scientists alike due to their remarkable ability to steal functional chloroplasts from their algal food, enslaving them to withstand long starvation periods. Recently, a wealth of data has shed insight into this remarkable relationship; however, the cellular mechanisms governing this process are still completely unknown. This study explores these mechanisms, providing insight into the chloroplast retention and delayed digestion, occurring within the slug's digestive gland. We examine the relationships between functional chloroplast and lysosome abundances during starvation, in live material, for the long-term retaining species Elysia timida, the ambiguous long/short-term retaining Elysia viridis, and the short-term retaining Thuridilla hopei, to elucidate digestive differences that contribute to the development of functional kleptoplasty. Functional chloroplast and lysosome abundance are measured using chlorophyll a autofluorescence and the pH-dependent stain acridine orange. In each species, the number of chloroplasts and lysosomes is indirectly proportional, with the plastid density decreasing when starvation begins. We also present a new FIJI/Image J Plugin, the 3D-Accounting and Measuring Plugin, 3D-AMP, which enables the reliable analysis of large image sets. [ABSTRACT FROM AUTHOR]

Expansion segments within eukaryotic nuclear SSU ribosomal RNA have been characterized in many diverse organisms. So far, only a few studies have examined the evolutionary history of SSU rDNA variable regions for monophyletic groups. A euglenozoan SSU rDNA data set was analyzed combining phylogenetic inference and examination of expansion segment evolution. Although SSU rDNA length expansion could be ascribed to all Euglenozoa, most unusual length variation occurs within primary osmotrophic euglenids, particularly within the genus Distigma. The longest SSU rRNA gene reported to date can be found in D. sennii, comprising more than 4500 bases. RT-PCR analyses revealed that the complete gene is transcribed into RNA without posttranscriptional modifications. Further investigations uncovered that most of the length extension is due to elongated variable regions V2 and V4, but virtually all variable regions except for V3 are extended within primary osmotrophic euglenids. Analyses of secondary structure revealed several insertion points within variable regions, some of which are of phylogenetic importance. Varying GC content has been detected among species and between expansion segments and core regions. Nevertheless, individual expansion segments of one species as well as variable sequence positions within core regions tend to evolve in parallel concerning nucleotide frequencies. The presence of a large internal repeat within V2 of Distigma sennii hints at a possible mechanism for large-scale sequence length expansion. [ABSTRACT FROM AUTHOR]