Molecular phylogenetic analysis of bacterial symbionts of the deep-sea hydrothermal vent scaly snail

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pdfGoffredi SK, Waren A, Orphan VJ, Van Dover CL & Vrijenhoek RC 2004 Novel forms of structural integration between microbes and a hydrothermal vent gastropod from the Indian Ocean. Appl Env Microbiol 70:3082-3090


Questions : 1) Is this snail sustained by autotrophics microbes, and if so who are they?, and 2) is the composition of the bosy scale biofilms unusual and likely to be responsible for the metallic surface coating?

Deep-sea hydrothermal vents are hot springs found deep in the ocean at the boundaries between techtonic plates. These environments are rich in microbial and animal life, subsisting on the oxidation of sulfide and hydrogen coming from the hydrothermal fluid. A wide range of animals have adapte to these unique environments. Many of these fed off of the sulfide-oxiidng Bacteria, or each other, but many of them have formed symbioses with the sulfide-oxidizing Bacteria. Perhaps the most well-known of these is the giant vent tube worm Riftia, which absorbs oxygen, carbon dioxide, and sulfide from the environment with its gills, which are carried to a specialized organ full of endosymbiotic sulfide oxidizing autotrophic Bacteria that provide the worm with all of the nutrients it needs. Riftia, like other vent animals with such symbionts, lacks a digestive tract.

In this paper, the authors describe a new deep-sea hydrothermal vent animal, a snail about 2 inches in diameter. This animal was found in the Indian Ocean in a hydrothermal vent field at a depth of about 2400 meters (1 1/2 mile deep).

This snail lacks the operculum (the plate the snail uses to cover it's opening when retracted into the shell), but instead has a body covered in small scales hardened by a plating of iron sulfides. This animal doesn't retract into its shell - but when it contracts its foot, it is protected by an arsenal of hardened scales.

In addition to its unusual body covering, this snail has only the remnants of a digestive tract - so vestigial that it doesn't seem likely to be functional. The digestive tract never contains anything like food, only scraps of iron sulfides, and the radulus, a sort of a tongue that snails use to scrape up the food they eat, is almost non-existent. On the other hand, the snail has HUGE esophogeal glands; these are the glands that secrete lubricating mucous into the esophagous. The esophageal gland contain what look like bacteriocytes, host cells with vacuoles full of bacteria. The esophageal gland and foot are very heavily vascularized, and presumably sulfide and oxygen are absoerbed by the foot and transported directly to the esophageal gland. (They also looked at the gills, because other vent molluscs that harbor chemoautotrophic symbionts do so in the gills, but apparently not so in this case.)

The first step, then, was to identify the esophageal gland symbiont. Glands were dissected from six frozen animals, DNA extracted, and ssu-rRNA sequences were amplified by PCR. These were cloned and 29 clones tested by RFLP, but it turned out that all of the clones were alike - in other words, as far as they could tell thse glands contain only a single bacterial type. This was sequenced and analyzed phylogenetically (i.e. they built trees), and it was found to be a gamma-proteobacterium related to other sulfide-oxidizing symbionts and related free-living vent species.

(In this Figure, I've colorized the esophageal endosymbiont in red, the scale surface symbionts in green, and the shell surface organisms in blue.)

The next question, then, is whether or not these symbionts really are autotrophic feeders of the animal. This was addressed by examining the isotope fractionation of the animals. Primary production by sulfide-oxidizing results in a large isotope effect; a lower fraction of heavy carbon and nitrogen isotpes in tissues. Metabolic processes eventually reduce this isotope effect, so if the snail gets its carbon and nitrogen directly from sulfide-oxidizing Bacteria, it should be enriched for light isotopes, whereas if it grazes on these and digests them, or if it is a predator, this isotope effect would be reduced. As Table 1 shows, the scaly snail has a greater isotope effect than either grazers (Lepetorilus) or predators (Phymorhynchus) of the same environment. So it really does look like this is a chemoautotrophic snail!

Next, the authors wanted to look at the snails scales, and in particular their amazing iron sulide plating. These scales look like a highly modified operculum, and contain the expected concholin tissue that would normally coat the interior of a snails shell. The scales are covered and permiated with a plating of iron sulfides and this is in turn coated by a microbial biofilm. DNA was extacted from this biofilm, ssu-rDNA was amplified and cloned, yielding 155 sequences originating from two snails. Most of these (67%) were epsilon proteobacteria; this group has been seen to predominate most vent environments. There were also a number (15%) of sequences related to sulfate-reducing delta proteobacteria, and gamma proteobacteria related to known sulfur-oxidizing organisms, many of which are symbionts of vent animals. They also got a few Chloroflexi (GNS), spirochaete, and Bacteroid (CFB) sequences.

The surface of the shell is lightly covered in Bacteria, looking like the stuff covering a wide range of other vent animals. The diversity of Bacteria on the shell is much lower than on the scales, and is a haphazard collection of sulfur-oxidizing or reducing epsilon or gamma proteobacteria. These are probably free-living organisms rather than symbionts, just using the shell for a surface to grow on. Keep in mind that although the shell looks a lot like the scales in color, it's made of regular calcium carbonate (aragonite) and is not coated in iron sulfides.

The external scale symbionts (they use the term epibionts) are unique to the snails scales; they are not common in this environment. Given their relationship to known sulfur oxidizers and reducers, it seems likely that this biofilm, or some organisms in this biofilm, are responsible for this iron sulfide mineralization. Since these same Bacteria could not be found either on the foot or on the shell of the same animals, their growth on the scales must be facilitated somehow by the animal. On the other hand, there is some iron sulfide within the tissue of the cales and the foot, suggesting that maybe the animal itself is capable of this minerization. This seems unlikely, but at this point who's responsible for the minerization is unresolved.

So, in conclusion, this scaly snail is an autotrophic animal, absorbing oxygen and sulfide from its vent environment and feeding this to gamma proteobacterial endosymbionts in an enlarged, specialized esophageal gland. This snail lacks a typical opersulum, and is instead covered in protective scales plated in iron sulfides that are probably mineralized by a complex bacterial biofilm.


chimeraA note about chimeras

Although the authors don't mention it, any time you do this sort of a population analysis, you have to screen your sequences to eliminate any "chimera"s. These types of sequences show up in most rDNA PCR experiments from natural mixed populations. They arise when a molecule of Taq polymerase stalls partway through a PCR round. If the resulting truncated DNA strand ends in a conserved part of the rDNA, it can anneal to another DNA molecule, whether or not it is from the same organism, and serve in the next PCR round as a primer for the synthesis of a DNA molecule that is from one organism at one end and another organism at the other end. Such chimeras can be identified using three standard methods:

  1. Making phylogenetic trees based independently on the 5' half and 3' half of each sequence. if the two trees disagree significantly, it's probably a chimeric sequence.
  2. drawing the secondary structure of the RNA - if the basepairs involving the 5' and 3' sequences don't work, it's probably a chimera.
  3. Using the CHECK_CHIMERA function and the Ribosomal Database Project. This program compares the similarity of a sequence along it's length to other sequences in the database - a "break" in this similarity, where the sequence begins to look less and less like one sequence and more like another, indicates that the sequence is probably a chimera.

Anything that seems to be a chimeric sequence is, of course, discarded from the analysis.