3D FISH for the quantification of methane- and sulphur-oxidizing endosymbionts in bacteriocytes of the hydrothermal vent mussel Bathymodiolus azoricus

Abstract

Dual endosymbioses involving methane- and sulphur-oxidizing bacteria occur in the gills of several species of mussels from deep-sea hydrothermal vents and cold seeps. Variations of total and relative abundances of symbionts depending on local environmental parameters are not yet understood, due to a lack of reliable quantification of bacteria in the host tissue. Here, we report the first attempt to quantify volumes occupied by each type of symbiont in bacteriocyte sections from a vent mussel, Bathymodiolus azoricus, using fluorescence in situ hybridization (FISH) coupled to three dimentional microscopy and image analysis carried out by a dedicated software, which we developed. Bacteriocytes from mussels recovered at different vent sites displayed significantly different abundances of bacteria. Specimens kept in aquaria at atmospheric pressure and exposed to an artificial pulse of sulphur displayed an increase in absolute and relative abundance of sulphur oxidizers within their bacteriocytes. Distributions of all measured parameters fitted normal distributions, indicating that bacteriocytes from a specimen tend to display similar behaviours. This study shows that symbiont volume quantification is tractable using 3D FISH, and confirms the impact of local environmental parameters on symbiont abundances.

Influence of CH<sub>4</sub> and H<sub>2</sub>S availability on symbiont distribution, carbon assimilation and transfer in the dual symbiotic vent mussel <i>Bathymodiolus azoricus</i>

“…Although our data must be interpreted with caution as only one individual per experimental condition was analyzed, the fact that observations qualitatively agree with expected results is a strong indication that our set-up is appropriate for the enrichment of each endosymbiont. Recently, a 3D-FISH technique was employed to quantify the effect of a sulphide pulse in a similar experimental set-up, on the volume and relative abundance of symbionts in mussel gills (Halary et al, 2008). We herein provide a first indication that MOB also respond to a methane pulse.…”

“…Duperron et al (2006) found that the MOB 16S rRNA phylotype was also shared between the two mussel species, and that vent chemistry could affect the relative abundance of SOB and MOB. The volume occupied by each type of symbiont present within V. Riou et al: Bathymodiolus azoricus' carbon fixation by chemosynthesis a bacteriocyte, quantified using 3D fluorescence in situ hybridization (FISH) technique, varies from vent site to vent site between B. azoricus specimens (Halary et al, 2008). However, although physiological activity of the symbionts was observed in gills of a hydrothermal vent Bathymodiolid from the Pacific (Fiala-Médioni et al, 1986;Nelson et al 1995), in the Southern MAR B. puteoserpentis (Robinson et al, 1998) and in live specimens of the seep Bathymodiolid from the Gulf of Mexico (Childress et al, 1986;Fisher and Childress, 1992;Kochevar et al, 1992 andLee andChildress, 1995), it has to date not been demonstrated in live specimens of B. azoricus.…”

“…Upon recovery of selected mussel specimens, the most anterior part of their left gill was fixed in 2% formaldehyde (2-4 h, 4 • C), rinsed twice with filtered seawater, and stored at −20 • C in 1:1 ethanol/filtered seawater. In the lab, gill tissue was embedded in polyester wax and 10 µm-thick sections were cut using a microtome (JUNG, Heidelberg, Germany), collected on Superfrost ® Plus slides (Roth, Germany), and hybridized as previously described (Halary et al, 2008) using the ATTO488-labelled ImedM probe (5'-ACCAGGTTGTCCCCCACTAA-3', Duperron et al, 2008) specific for the methane-oxidizing symbiont and the Cy5-labelled BangT-642 probe (5'-CCTATACTCTAGCTTGCCAG-3', Duperron et al, 2005) specific for the sulphide-oxidizing symbiont. Images were acquired using a BX61 microscope (Olympus Optical Co., Tokyo, Japan) under a 10× objective (NA 1.30).…”

“…FISH was performed according to the protocol developed by Halary et al (2008) with a few modifications. Upon recovery of selected mussel specimens, the most anterior part of their left gill was fixed in 2% formaldehyde (2-4 h, 4 • C), rinsed twice with filtered seawater, and stored at −20 • C in 1:1 ethanol/filtered seawater.…”

Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

“…Compared with the specimens of cluster D investigated by Duperron et al (2008), this phylotype 1 was identical to one of the two 16S rRNA partial sequences of one specimen and displayed six differences to that of another. The phylogenetic relationships between symbiont sequences obtained from cluster D and phylotype 1 obtained herein from cluster C were well supported.…”