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Shallow water chemosynthetic symbioses

Gutless oligochaetes

Christian Lott, Cecilia Wentrup, Juliane Wippler, Manuel Kleiner, Mario Schimak, Silke Wetzel


Gutless oligochaetes are a unique group of marine worms that have no mouth or gut, and are the only free-living animals known to have also reduced their nephridia (kidney-like excretory organs).

Gutless oligochaetes are bright white because of the elemental sulfur stored in their bacterial endosymbionts.
Gutless oligochaetes are bright white because of the elemental sulfur stored in their bacterial endosymbionts.

Who? 

The diversity of symbionts in gutless oligochaetes is higher than in all other chemosynthetic symbioses, with as many as six 16S rRNA phylotypes co-occurring in a single worm. Despite this remarkable diversity, the associations are highly specific and stable.


What? 

Our metagenomic analyses of the symbionts in Olavius algarvensis have revealed how its consortium of sulfur-oxidizing and sulfate-reducing bacteria uses different energy sources and metabolic pathways to provide it with an optimal energy supply.


How? 

Phylogenetic analyses of the oligochaete symbionts and their hosts suggest that both co-speciation and biogeography played a role in the establishment of these symbioses.
Different transmission modes of the symbionts may influence their evolutionary patterns.

In situ identification of 5 co-occurring bacterial symbionts in O. crassitunicatus. Epifluorescence image of the symbiont-containing region of the worm's body wall. Triple hybridization with the 2 gammaproteobacterial symbionts in red (using the general gammaproteobacterial probe GAM42), the 3 deltaproteobacterial symbionts in blue (using the general deltaproteobacterial probe for bacteria belonging to the Desulfosarcina, Desulfofaba, Desulfococcus, Desulfofrigus, and Desulforhopalus genera DSS658/DSR651), and spirochete symbionts in yellow using the specific probe for the spirochete symbionts of O. crassitunicatus (from Blazejak et al. 2005).

Gutless oligochaetes are small worms of 0.2 mm diameter and 1-2 cm length live in an obligate association with endosymbiotic bacteria. In contrast to intracellular endosymbionts that are not directly exposed to the external environment of the host, the endosymbionts of gutless oligochaetes are extracellular, living just below the "skin" of the worms in a space between the outer cuticle and the internal epidermal cells called the symbiont-containing region. The cuticle is highly permeable to both small charged molecules and uncharged substances so that the symbiotic bacteria have free access to most dissolved compounds in the sediment pore waters surrounding the worms.

Our studies on the phylogeny of the oligochaete bacteria have revealed a remarkable diversity of the symbionts, with up to 5 - 6 different 16S rRNA phylotypes co-occurring in the symbiont-containing region. In all host species, the primary endosymbionts are chemoautotrophic sulfide-oxidizers that belong to the gamma subclass of Proteobacteria and are related to free-living sulfide-oxidizers such as Allochromatium vinosum. In addition to the primary symbionts, up to 5 other bacterial phylotypes belonging to the Alphaproteobacteria (Blazejak et al. 2006), Deltaproteobacteria, or the Spirochaetes can co-exist in the same host species (Dubilier et al. 2006).

For the oligochaete hosts, the association is clearly obligate, given their complete reduction of a digestive and excretory system. For the symbionts, that remain as yet uncultivable, it is not known if they can also survive in a free-living stage in the environment.

Habitat conditions are studied in situ by porewater extraction and microsensor measurements.Our study site for Olavius algarvensis is the Bay of Sant'Andrea at the Island of Elba in the Mediterranean Sea.

Stilbonematid nematodes

Judith Zimmermann and Jillian Petersen

Marine nematodes of the subfamily Stilbonematinae (Desmodorida, Desmodoridae) are highly abundant in tropical and subtropical shallow water sands, but they have also been described from intertidal zones of cold temperate regions, deep sea sediments and methane seeps. To date, nine genera have been described worldwide. However, this probably represents only a fraction of the total diversity of these animals, as we've only sampled at a few sites, and we usually discover at least one new species at every new site we visit.

Leptonemella species from the North Sea island Sylt, covered by a thick coat of bacterial ectosymbionts. The white appearance comes from elemental sulfur that is stored in the bacteria.

One characteristic feature shared by all stilbonematid nematodes is a coat of ectosymbiotic bacteria covering their cuticle. Sometimes the head region and the tail tip are symbiont-free. The morphology of these ectosymbiotic bacteria varies for the different host genera. Nematodes of the genus Leptonemella for example are associated with a multilayer of coccoid bacteria, while members of other genera are covered by rods or filamentous bacteria. Molecular analyses of the ectosymbiotic bacteria based on the 16S rRNA gene reflect the morphological observations and show that each nematode host is associated with a specific gammaproteobacterial ectosymbiont. Interestingly, all ectosymbiotic bacteria characterized from stilbonematid nematodes are closely related and form a monophyletic clade with endosymbiotic bacteria of marine gutless oligochaetes and the non-stilbonematid nematodes of the genus Astomonema.

Transverse sections of marine nematodes Leptonemella vicina from Sylt. The ectosymbiotic bacteria are stained with a fluorescent Gammaproteobacteria-specific probe (in green) and DNA is stained with DAPI (in blue).

The ectosymbiotic bacteria of stilbonematid nematodes are known to be autotrophic sulfur-oxidizers, gaining energy from oxidizing reduced sulfur compounds and using this energy to fix carbon dioxide into organic matter. Both the symbionts and the hosts need oxygen for respiration. In addition, the symbionts need reduced sulfur compounds as an energy source for chemosynthesis. Stilbonematid nematodes are therefore usually found at the interface between the oxic surface sediment, which is rich in oxygen, and the deeper anoxic sediment, which is rich in sulfide.

Our understanding of the function of stilbonematinid symbioses is still extremely limited. For example, the role of the bacteria is still unclear. One hypothesis postulates that the host benefits nutritionally from the ectosymbionts by grazing on its own coat. We are currently testing this hypothesis with incubation experiments and NanoSIMS. The bacteria may benefit from the mobility of the host through the oxic and anoxic sediment layers, as this would provide them with a regular supply of the reduced and oxidized compounds it needs for its nutrition.

In our group we investigate the following topics:

- Diversity and specificity of the stilbonematid ectosymbioses
- Evolutionary history of the symbioses
- Benefits of the symbiosis for both partners

In this project we work closely together with Joerg Ott, University of Vienna, Austria.

Flatworms

Harald Gruber-Vodicka

Paracatenula is a genus of freeliving catenulid flatworms found in sheltered subtidal sediments of warm temperate and tropic regions. Adult Paracatenula have no mouth or gut. Instead they harbor intracellular endosymbionts in bacteriocytes that are forming a tissue called the trophosome in functional analogy to the trophosome found in the mouthless Siboglinidae (e.g. the giant tube-worm Riftia pachyptila). The worms share habitat with a number of other hosts to sulfur-oxidizing bacteria (SOB) such as nematodes, gutless oligochaetes and lucinid and solemyiid bivalves. By migrating through the redox potential gradient in the uppermost 5-15 cm sediment layer, the millimeter-sized worms can supply chemoautotrophic symbiotic bacteria alternately with spatially separated electron donors and acceptors such as sulfide and oxygen, a strategy that has been described for symbiotic stilbonematid Nematoda and gutless Oligochaeta.
All other known chemoautotrophic and sulfur-oxidizing (thiotrophic) symbionts are either Gamma- or Epsilon-protebacteria, but the Paracatenula symbionts belong to the Alphaproteobacteria. Based on 16S rRNA analyses, all so far studied species of Paracatenula harbor related alphaproteobacterial symbionts that form a family-level clade of Rhodospirillales, which co-evolved with their host for the last 500 million years. The symbionts were named Cand. Riegeria, honoring the late zoologist Reinhard Rieger who described the hosts in the early 1970s together with Wolfgang Sterrer.

Paracatenula urania from Carrie Bow Cay, Belize is one of the largest Paracatenula species, the shown specimen has a length of approximately 9 millimeters.
Paracatenula urania from Carrie Bow Cay, Belize is one of the largest Paracatenula species, the shown specimen has a length of approximately 9 millimeters.

Links for Paracatenula

http://schaechter.asmblog.org/schaechter/2011/12/a-wormful-of-bugs.html

https://www.youtube.com/watch?v=3qMPOLFpAzo

Ciliates

Brandon Seah and Harald Gruber-Vodicka

Symbioses between chemosynthetic bacteria and their eukaryotic hosts are common in shallow-water environments. Although most of the well-known host organisms are animals, several types of ciliates also have symbiotic bacteria. Ciliates are eukaryotic microbes (“protists”) that are common in aquatic environments, and are distinctive for being covered in cilia, which they use for feeding and locomotion. Most species are unicellular (e.g. Paramecium, commonly shown in introductory biology classes) though many are colonial (e.g. Zoothamnium).

The ciliate Kentrophoros has sulfide-oxidizing ectosymbionts, and lives in shallow marine sediments. Like the symbiont-bearing nematodes and gutless oligochaetes, which are also found in the interstitial habitat, they are able to migrate within the sediment to follow gradients of oxygen and sulfide. Despite being single cells, individuals of Kentrophoros can range from a tenth to several millimeters in length.

Top: Live Kentrophoros (about 2-3 mm length) viewed under dissecting microscope. The body appears white because of scattering by sulfur granules within the ectosymbionts.
Bottom: Whole Kentrophoros cell stained with DAPI (fluorescent DNA stain) imaged by confocal microscopy. This individual has very small ectosymbionts (seen as a hazy appearance across the host), five macronuclei (Ma) and a pair of micronuclei (Mi). Scale bar: 20 microns.

Kentrophoros is ribbon-shaped, and is almost entirely covered by its ectosymbionts on one surface. The ectosymbionts also show an unusual mode of cell division. Unlike most bacteria, which divide perpendicular to the long axis of the cell, the Kentrophoros ectosymbionts divide longitudinally. Such longitudinal division is also found in the bacterial ectosymbionts of the nematode Laxus.

These curious organisms have been known to biologists for almost a century, but the phylogenetic identity of the bacterial ectosymbionts are still unknown. Using methods of molecular ecology and phylogenetics, we are investigating the identities of both hosts and symbionts. Our preliminary results show that the ectosymbionts belong to the Gammaproteobacteria but are separate from the clade containing nematode and oligochaete symbionts. We are also interested in testing whether the symbionts are host-specific and co-diversify with their hosts.

Part of a Kentrophoros cell in two different focal planes, showing granules within the cytoplasm (G) and the bacterial ectosymbionts (B) that cover the entire dorsal surface (right panel). Scale bars: 15 microns.
Close-up of ectosymbionts that have fallen off the host cells, showing the Y-shaped appearance of cells that are midway through longitudinal division. Scale bars: 5 microns.