Fig. 6

Microscopy of the tentacles of Ostiactis pearseae. a Whole image of specimen SO194-S2, SIO-BIC Co3067. b Light microscopy of 3-μm sections embedded in Steedman’s resin. c–f Fluorescent in situ signal amplification via hybridization chain reaction-FISH (HCR-FISH) microscopy of Ostiactis pearseae tentacles. An unlabeled probe (Anem_SUP05), with a specific sequence initiator tag, was designed to be an exact match to the putative thiotrophic symbiont (related to the SUP05 clade). This probe was then amplified via HCR-FISH using DNA hairpins labeled with Alexa488, shown in green. DAPI-stained nuclei of host cells are shown in blue. f Bacteria can be seen within the epidermis (arrows), in and amongst nuclei, positioned just above or immediately adjacent to cnidocysts. g Fluorescent in situ signal amplification via HCR-FISH microscopy of Ostiactis pearseae tentacles without a probe. No bacteria are observed within the epidermis (arrow indicates the same region as in f). Note: future microscopy using non-specific probes would help elucidate the extent of the symbiont layer. h, i Light microscopy of O. pearseae tentacles. j–l Transmission electron (TEM) microscopy of O. pearseae tentacles. i Bacteria are concentrated in the periphery of elongated epidermal cells (designated by the orange box, which corresponds to the area of TEM imagery), and positioned near cnidae, shown in pink arrowheads. No bacteria were observed in the mesogloea or gastrodermis. j Close-up of bacteria near a cnidocyst capsule, with an enclosed tubule. k Close-up showing clear membranes surrounding the bacterial cells, designated by orange arrowheads. l Arrowheads (in green) point to bacteria possibly being endocytized via clathrin-coated pits, as well as nearby clusters of bacterial cells within the elongated epidermal cells of O. pearseae. nu, nucleus; bac, bacteria; cni, cnidae; meso, mesoglea; gastro, gastrodermis; epi, epidermis. Scale bars are 5 mm (a), 2 mm (b), 50 μm (c), 1 μm (d), 10 μm (e–g), 250 μm (h), 25 μm (i), and 1 μm (j–l)