Figure 1.
Anatomy of the shipworm Bankia setacea.
a, anus; c, caecum; f, foot; g, gill; h, heart; i, intestine; v, valve (shell); aa, anterior adductor muscle; pa, posterior adductor muscle; st, stomach. Caecum and intestine are shown in brown and green respectively. Region boxed in blue corresponds to (A) in Figure 2.
Table 1.
Shipworm specimens examined in this study.
Figure 2.
Single probe localization of bacteria in the gills and digestive system of L. pedicellatus.
Confocal micrographs and diagrams demonstrating patterns of hybridization of either a bacteria-domain targeted 16S rRNA directed oligonucleotide probe (Bact338-cy5) or a negative control probe (Non338-cy5, to reveal autofluorescence or non-specific binding) to adjacent tissue sections of L. pedicellatus containing both gill and digestive tissues. Both probes are labeled with the fluorochrome cy5 (red). (A) Tissue section showing gill filaments (upper right), caecum (lower left) and intestine (diagonal across center, upper left to lower right) probed with Bact338-cy5. Inset- Diagram showing detail of tissue boundaries in the tissue section shown in (A). This region corresponds to the blue box in Figure 1. (B–G) Enlarged views of tissue sections hybridized with bacteria specific probe (Bact338-cy5; B–D) and negative control probe (Non338-cy5; E–G). These sections contain: gill bacteriocytes, (B,E), a fecal pellet within intestine (C,F), and caecum contents (D,G). Note that fluorescence signal localizes bacteria in gill bacteriocytes (large arrows) and in fecal pellets (small arrows), but few bacteria are observed in caecum contents. c, caecum; f, fecal pellet; g, gill; i, lumen of intestine. Scale bar in (a), 50 µm; (B–G), 10 µm.
Figure 3.
Dual probe localization of bacteria in the gills and digestive system of L. massa.
Confocal micrographs demonstrating patterns of hybridization of 16S rRNA directed oligonucleotide probes targeted to the domain bacteria (Bact338-cy5, red) and shipworm symbionts (SS1273-cy3, green) and negative control probes (Non338-cy5, red and SSnon-cy3, green to reveal autofluorescence or non-specific binding). Micrographs show tissue sections hybridized with Bact338-cy5 and SS1273-cy3 (A–C) or Non338-cy5 and SSnon-cy3 (D–F). These sections contain: gill bacteriocytes (A, D, dark voids are host nuclei and lysosomal residual bodies), a fecal pellet within the intestine (B,E), and caecum content (C,F). The combinatorial color yellow indicates hybridization of both bacteria- and symbiont-targeted probes within the same bacterial cells or bacteriocytes. The appearance of both red bacteriocytes (small arrows) and yellow bacteriocytes (large arrows) in (A) indicates that bacteria within some bacteriocytes contain the symbiont sequence targeted by probe SS1273 while others contain bacteria that lack this target sequence. Note that hybridization of the symbiont-targeted probe SS1273 was detected in gill bacteriocytes only, and not in other examined tissues. All scale bars are 10 µm.
Figure 4.
Single probe localization of exogenous bacteria added to caecum contents.
Confocal micrographs depicting hybridization of a bacteria-domain specific 16S rRNA directed oligonucleotide probe (Bact338-cy5) to samples on filters. Probe is labeled with the fluorochrome cy5 and displayed in red in images shown. (A) Filter with a mixture of caecum contents and E. coli cells. (B) An equivalent sample of E. coli cells without caecum contents. (C) Equivalent sample of caecum contents without added E. coli cells. Scale bar 10 µm.
Figure 5.
Bacterial morphotypes in the intestine of Bankia setacea visualized by single probe hybridization.
Confocal micrographs depicting hybridization of either a bacteria-domain specific 16S rRNA directed oligonucleotide probe (Bact338-cy5) or a negative control probe (Non338-cy5) to sectioned tissues of Bankia setacea. Both probes are labeled with the fluorochrome cy5 shown in red. (A) Tissue section hybridized with Bact338-cy5, showing a fecal pellet (F) with a small portion of the intestinal wall (I) visible in the lower left corner. Note the presence of multiple cell morphologies including long spirals (large arrow) and short rods or cocci (small arrow). (B) Enlarged view of the boxed region in A. (C) Tissue section adjacent to and comparable in size to that shown in (A) hybridized with Non338-cy5. Scale bars are 10 µm in (A) and (C) and 5 µm in (B).
Figure 6.
Abundance of bacteria observed in contents of intestine and caecum.
Box plot showing number of bacteria observed per field in intestine and caecum of four shipworm species. Forty randomly selected fields (100 µm×100 µm) were examined for each tissue by laser scanning confocal microscopy using the bacteria-targeted probe Bact338 cy5 or the negative control probe Non338 cy5. Vertical lines indicate range of values, horizontal dashed lines and solid circles indicate median and mean values respectively, and open boxes indicate upper (top line) and lower (bottom line) quartiles respectively. Note upper and lower quartiles and median in caecum have zero values and have been omitted for clarity.
Table 2.
Fluorescence labeled oligonucleotide probes used in Fluorescence in situ hybridizations.