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Characterization of bacterial ectosymbionts colonizing gills and endophragm of two crabs: Aratus pisonii and Uca rapax

Abstract : Here we present the first description of interactions between bacterial ectosymbionts and two mangrove crabs: Aratus pisonii (Sesarmidae) and Minuca rapax (Ocypodidae). These crabs belong to the order Decapoda and to the infra-order Brachyura known as "real crab". Specimens were collected in Guadeloupe on the mangrove trees Rhizophora mangle for A. pisonii and from the mangrove mud for M. rapax. To observe ectosymbionts on Electron Microscopy (Scanning and Transmission, SEM and TEM), gills were fixed in a 2.5% glutaraldehyde solution in 0.8x PBS buffer for 24h at 4°C. Then they were rinsed in the same buffer and dehydrated in graded concentration of acetone. Finally, they were critical point dried and sputter coated with gold before observation on a Quanta 250 SEM. For TEM observations, gills were fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer for 45 minutes at RT in the dark. Then, they were rinsed in the same buffer then smaller pieces of gills were fixed, for 1 h in 1% osmium tetroxide in the same buffer before a post-fixation with 2% aqueous uranyl acetate for 1 hour at RT. Finally, they were rinsed in H2O and dehydrated in ethanol before embedding in LR White resin. A couple of ultrathin sections (60nm thick) were observed for each crab species analyzed by using a FEI Tecnai G20 at 200 kV. SEM and TEM observations showed that ectosymbionts colonize gills and endophragm (axial skeleton related to legs) for all individuals investigated (Figure 1). For both species studied, several different bacterial morphotypes (cocci, rods, but no filaments) were observed throughout the surface of gill discs and endophragm according to SEM views (Figure 1A). Symbionts did not cover the entire surface of gill discs. They formed patches irregularly distributed while they formed a uniform bacterial biofilm which covers the entire endophragm (data not shown). Moreover, no intracellular bacteria could be observed according to TEM views (Figure 1B). Whole DNA was extracted from gills and PCR using universal primers to amplify the V3-V4 region of the 16S rRNA-encoding gene were performed. The PCR products sequences have been analysed confirming that several bacterial species are involved per crab species. Five main bacterial species are detected for the two crabs' species. However, the bacterial community composition is totally different between Aratus pisonii and Minuca rapax. Moreover, the proportion of the main bacterial species involved in this symbiotic relationship varies according to individuals within the same crab species. Such bacteria belong to Alpha-proteobacteria, Actinobacteria, Flavobacteria, and Bacteroidetes phyla. Most of the bacteria involved represent either a new genus or a new species based on the sequence of a widely accepted marker gene, namely 16S rRNA.
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Béziat Naéma, Sébastien Duperron, Olivier Gros. Characterization of bacterial ectosymbionts colonizing gills and endophragm of two crabs: Aratus pisonii and Uca rapax. Caribbean Science and Innovation Meeting 2019, Oct 2019, Pointe-à-Pitre (Guadeloupe), France. ⟨hal-02423817⟩

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