Mucus Glycoproteins Selectively Secreted from Bacteriocytes in Gill Filaments of the Deep-Sea Clam <i>Calyptogena okutanii</i>

Abstract

The deep-sea clam Calyptogena okutanii possesses a large gill containing vertically transmitted symbiotic sulfur-oxidizing bacteria. It produces large amounts of highly viscoelastic mucus from the gill, which is thought to be a physical and chemical barrier. The mucus collected from the gill was shown to be composed of glycoproteins having the following sugar composition: Man (17.4%), GlcNAc (16.6%), GalNAc (15%), Glc (1.1%), Gal (29.9%), Xyl (3.0%), Fuc (14.4%), and unknown (2.6%), indicating that it contained mucin-like glycoproteins. In a monoclonal antibody library against the gill tissue, we found a monoclonal antibody (mAb), CokG-B3C10, reacting to the mucus. Western blot analysis using the mAb showed that it reacted to several glycoproteins in the mucus from the gill tissue, but not with those of other tissues such as the mantle, foot, and ovary, where mucus production has been reported in bivalves. Further, immunohistochemical analysis showed the CokG-B3C10 mAb reacting to glycoproteins was detected in the inner area of the gill, which was occupied by many bacteriocytes in the row of gill filaments. Strong mAb signals were found on the outer surface of the bacteriocytes facing the interfilamental space, and in the interfilamental spaces between filaments. Weaker signals were also observed in the bacteriocyte cells. These results indicate that the CokG-B3C10 mAbbinding mucus glycoproteins were produced from cells including bacteriocytes and nonbacteriocyte cells in the inner area of the gill filaments.

Surfing the vegetal pole in a small population: extracellular vertical transmission of an 'intracellular' deep-sea clam symbiont

“…To investigate the localization of the symbiont in the spawned eggs, we conducted WISH analysis using a specific probe designed for the symbiont of C. okutanii [ 12 ]. To avoid the strong autofluorescence of the yolk, and to obtain a complete view of the egg, we employed a probe labelled with DIG, and the conventional NBT/BCIP chromogenic staining.…”

“…The stored eggs were soaked into hybridization buffer (20% FA, 0.9 M NaCl, 20 mM Tris–HCl pH 7.5, 5 mM EDTA, 0.01% SDS) twice for 10 min and hybridized in hybridization buffer containing 0.5 µM of probe at 46°C overnight. Probe sequence for WISH was the same as Cok 16S_1 (5′-AGCTTCGCCACTAAAGGGTACCCCC-3′), which was designed to be specific to 16S rRNA gene of the C. okutanii symbiont [ 12 ], and its 5′ end was labelled with digoxigenin (DIG). For the negative control, the ‘No-bind probe’ (5′-CCTAGTGACGCCGTCGAC-3′) [ 13 ] labelled with DIG was used.…”

“…(5 -AGCTTCGCCACTAAAGGGTACCCCC-3 ), which was designed to be specific to 16S rRNA gene of the C. okutanii symbiont [12], and its 5 end was labelled with digoxigenin (DIG). For the negative control, the 'No-bind probe' (5 -CCTAGTGACGCCGTCGAC-3 ) [13] labelled with DIG was used.…”

Molecular analyses of the gill symbiosis of the bathymodiolin mussel Gigantidas platifrons

“…In shallow-water filter-feeding bivalves, the mucus functions cooperatively with the cilia to capture, process, and transport food particles to the mouth ( Dufour and Beninger, 2001 ; Gómez-Mendikute et al., 2005 ; Beninger and Dufour, 1996 ). In deep-sea symbiotic bivalves with vestigial digestive systems, it is unlikely that mucus is involved in feeding, but it acts as a defensive barrier and/or for the maintenance of the symbiotic bacteria ( Nakamura et al., 2013 ). Interestingly, the gill of G. platifrons expresses a large variety of C1q domain-containing proteins (C1qDCs), including C1q domain-containing protein, complement C1q, and complement C1q tumor-necrosis-factor-related protein, many of which are enriched in WG samples ( Figure S3 ).…”

Effects of a long-term rearing system for deep-sea vesicomyid clams on host survival and endosymbiont retention

“…The fixed gill was embedded in paraffin and sliced into 4-μm transverse sections. The sections were dewaxed and subjected to hybridization at 46 °C using a solution containing 20% formamide, 0.9 M NaCl, 20 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.01% SDS, and 0.5 μM FITC-labeled probe Cok 16S_1 (5′-AGC TTC GCC ACT AAA GGG TAC CCC C-3′), which was designed to be specific to the 16S rRNA of the P. okutanii symbiont [17]. After hybridization, excess probe was washed away twice in a wash solution containing 0.225 M NaCl, 20 mM TrisHCl pH 7.5, 5 mM EDTA, and 0.01% SDS for 30 min at 48 °C, and the sections were then mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA).…”