Riftia

Riftia pachyptila is a marine invertebrate in the phylum of segmented worms, Annelida, which include the other "polychaete" tube worms commonly found in shallow water marine environments and coral reefs. R. pachyptila lives in the deep sea, growing on geologically active regions of the Pacific Ocean's seafloor, such as near hydrothermal vents. These vents provide a natural ambient temperature ranging from, and emit large amounts of chemicals such as hydrogen sulfide, which this species can tolerate at extremely high levels. These worms can reach a length of, and their tubular bodies have a diameter of.
Historically, the genus Riftia was placed within the phyla Pogonophora and Vestimentifera. It has been informally known as the giant tube worm or the giant beardworm; however, the former name is also used for the largest living species of shipworm, Kuphus polythalamius, which is a type of bivalve.

Discovery

R. pachyptila was discovered in 1977 during an expedition led by geologist Jack Corliss to the Galápagos Rift; the survey was sponsored by the Woods Hole Oceanographic Institution and the National Science Foundation, and the dive was carried out by the American bathyscaphe DSV Alvin. The presence of thermal springs near mid-oceanic ridges was presumed, and the expedition aimed to confirm this.
The discovery of life around hydrothermal vents was unexpected. The vents were assumed to be sterile environments due to high heat emitted from them, thus no biologists were included in the expedition. Many of the species discovered during this expedition had never been seen before, as they are found exclusively near hydrothermal vents. Observed species included "foot-long" bivalves, white crabs, and polychaetes such as R. pachyptila. The discovery was compared to that of Columbus' discovery of the Americas by one of the geologists. Hydrothermal activity at this site was determined to have begun during the early 1970s, with the tubeworms ecologically dominating the site prior to 1979.
Though initially thought to be an isolated phenomenon, this vent ecosystem proved to be the first of many such ecosystems that would later be discovered on other geologically active sections of the sea floor. An expedition in 1985 returned to the Rose Garden site and found that the Riftia were fewer in number, having been displaced by clams and mussels despite having "essentially the same" water chemistry as the initial discovery in 1979. Another returning expedition in 2002 found that it had been destroyed by a lava flow sometime in the prior decade, though a second vent ecosystem was found near the original site, which it was dubbed "Rosebud".

Description

The generic name Riftia alludes to the rift that formed the geothermal vents where the species inhabits, while pachyptila refers to the anterior plume of the worm. The original specimen or holotype, USNM 59951, is held by the National Museum of Natural History.

Anatomy

Isolating the vermiform body from white chitinous tube, a small difference exists from the classic three subdivisions typical of phylum Pogonophora: the prosoma, the mesosoma, and the metasoma.

The first body region is the vascularized branchial plume, which is bright red due to the presence of hemoglobin that contains up to 144 globin chains. These tube worm hemoglobins are remarkable for carrying oxygen in the presence of sulfide, without being inhibited by this molecule, as hemoglobins in most other species are. The soluble hemoglobins, present in the tentacles, are able to bind O₂ and H₂S, which are necessary for chemosynthetic bacteria. Due to the capillaries, these compounds are absorbed by bacteria. The plume provides essential nutrients to bacteria living inside the trophosome. After Paralvinella grasslei, Riftia has the second highest branchial surface area among aquatic animals, having of branchial area per gram of wet mass. Larger, typically mature worms have proportionally smaller branchial areas compared to smaller immature ones. If the tubeworm perceives a threat or is touched, it retracts the plume and the tube is closed due to the obturaculum, a particular operculum that protects and isolates the animal from the external environment. The collagenous obturaculum supports the respiratory lamellae.
The second body region is the vestimentum, formed by muscle bands, having a winged shape, and it presents the two genital openings at the end. The heart, extended portion of dorsal vessel, enclose the vestimentum.
In the middle part, the trunk or third body region, is full of vascularized solid tissue, and includes body wall, gonads, and the coelomic cavity. Here is also located the trophosome, a spongy tissue where a billion symbiotic, thioautotrophic bacteria and sulfur granules are found. Since the mouth, digestive system, and anus are missing, the survival of R. pachyptila is dependent on this mutualistic symbiosis. This process, known as chemosynthesis, was recognized within the trophosome by Colleen Cavanaugh.
In the posterior part, the fourth body region, is the opisthosome, which anchors the animal to the tube and is used for the storage of waste from bacterial reactions.

Tubes

Riftias tubes possess very thick walls compared to cold-seep tubeworms and pogonophorans, especially at the base. They are composed of chitin associated with proteins, which are secreted out from cup-shaped microvilli-like structures within glands which form crystallite chitin layers over time. These tubes are resistant to enzymatic attack by bacteria, lasting 2.5 years in comparison to degrading in less than 36 days for the exoskeleton of the crab Bythograea thermydron.
The worms are able to remodel both the top and the base of their tubes, which allows for some adaptability in the highly competitive and crowded spaces they grow in. Chitinolytic activity has been detected on Riftias opisthosome; the lysis of chitin is one mechanism which allows tube remodelling. Growth rate of the tubes range from per year. The amount of chitin produced approaches 100 times that of pelagic and benthic ecosystems.

Physiology

Because of the peculiar environment in which R. pachyptila thrives, this species differs greatly from other deep-sea species that do not inhabit hydrothermal vent sites; the activity of diagnostic enzymes for glycolysis, the citric acid cycle and electron transport in the tissues of R. pachyptila is very similar to the activity of these enzymes in the tissues of shallow-living animals. This contrasts with the fact that deep-sea species usually show very low metabolic rates, which in turn suggests that low water temperature and high pressure in the deep sea do not necessarily limit the metabolic rate of animals and that hydrothermal vent sites display characteristics that are completely different from the surrounding environment, thereby shaping the physiology and biological interactions of the organisms living in these sites.
The discovery of bacterial invertebrate chemoautotrophic symbiosis, particularly in vestimentiferan tubeworms R. pachyptila and then in vesicomyid clams and mytilid mussels revealed the chemoautotrophic potential of the hydrothermal vent tube worm. Scientists discovered a remarkable source of nutrition that helps to sustain the conspicuous biomass of invertebrates at vents. Many studies focusing on this type of symbiosis revealed the presence of chemoautotrophic, endosymbiotic, sulfur-oxidizing bacteria mainly in R. pachyptila, which inhabits extreme environments and is adapted to the particular composition of the mixed volcanic and sea waters. This special environment is filled with inorganic metabolites, essentially carbon, nitrogen, oxygen, and sulfur. In its adult phase, R. pachyptila lacks a digestive system. To provide its energetic needs, it retains those dissolved inorganic nutrients into its plume and transports them through a vascular system to the trophosome, which is suspended in paired coelomic cavities and is where the intracellular symbiotic bacteria are found. The trophosome is a soft tissue that runs through almost the whole length of the tube's coelom. It retains a large number of bacteria on the order of 10⁹ bacteria per gram of fresh weight. Bacteria in the trophosome are retained inside bacteriocytes, thereby having no contact with the external environment. Thus, they rely on R. pachyptila for the assimilation of nutrients needed for the array of metabolic reactions they employ and for the excretion of waste products of carbon fixation pathways. At the same time, the tube worm depends completely on the microorganisms for the byproducts of their carbon fixation cycles that are needed for its growth.
Initial evidence for a chemoautotrophic symbiosis in R. pachyptila came from microscopic and biochemical analyses showing Gram-negative bacteria packed within a highly vascularized organ in the tubeworm trunk called the trophosome. Additional analyses involving stable isotope, enzymatic, and physiological characterizations confirmed that the end symbionts of R. pachyptila oxidize reduced-sulfur compounds to synthesize ATP for use in autotrophic carbon fixation through the Calvin cycle. The host tubeworm enables the uptake and transport of the substrates required for thioautotrophy, which are HS⁻, O₂, and CO₂, receiving back a portion of the organic matter synthesized by the symbiont population. The adult tubeworm, given its inability to feed on particulate matter and its entire dependency on its symbionts for nutrition, the bacterial population is then the primary source of carbon acquisition for the symbiosis. Discovery of bacterial–invertebrate chemoautotrophic symbioses, initially in vestimentiferan tubeworms and then in vesicomyid clams and mytilid mussels, pointed to an even more remarkable source of nutrition sustaining the invertebrates at vents.
A wide range of bacterial diversity is associated with symbiotic relationships with R. pachyptila. Many bacteria belong to the phylum Campylobacterota as supported by the recent discovery in 2016 of the new species Sulfurovum riftiae belonging to the phylum Campylobacterota, family Helicobacteraceae isolated from R. pachyptila collected from the East Pacific Rise. Other symbionts belong to the class Delta-, Alpha- and Gammaproteobacteria. The Candidatus Endoriftia persephone is a facultative R. pachyptila symbiont and has been shown to be a mixotroph, thereby exploiting both Calvin Benson cycle and reverse TCA cycle according to availability of carbon resources and whether it is free living in the environment or inside a eukaryotic host. The bacteria apparently prefer a heterotrophic lifestyle when carbon sources are available.
Evidence based on 16S rRNA analysis affirms that R. pachyptila chemoautotrophic bacteria belong to two different clades: Gammaproteobacteria and Campylobacterota that get energy from the oxidation of inorganic sulfur compounds such as hydrogen sulfide to synthesize ATP for carbon fixation via the Calvin cycle. Unfortunately, most of these bacteria are still uncultivable. Symbiosis works so that R. pachyptila provides nutrients such as HS⁻, O₂, CO₂ to bacteria, and in turn it receives organic matter from them. Thus, because of a lack of a digestive system, R. pachyptila depends entirely on its bacterial symbiont to survive.
In the first step of sulfide-oxidation, reduced sulfur passes from the external environment into R. pachyptila blood, where, together with O₂, it is bound by hemoglobin, forming the complex Hb-O₂-HS⁻ and then it is transported to the trophosome, where bacterial symbionts reside. Here, HS⁻ is oxidized to elemental sulfur or to sulfite.
In the second step, the symbionts make sulfite-oxidation by the "APS pathway", to get ATP. In this biochemical pathway, AMP reacts with sulfite in the presence of the enzyme APS reductase, giving APS. Then, APS reacts with the enzyme ATP sulfurylase in presence of pyrophosphate giving ATP and sulfate as end products. In formulas:

AMP + SO3^2- -> APS
APS + PPi -> ATP + SO4^2-

The electrons released during the entire sulfide-oxidation process enter in an electron transport chain, yielding a proton gradient that produces ATP. Thus, ATP generated from oxidative phosphorylation and ATP produced by substrate-level phosphorylation become available for CO₂ fixation in Calvin cycle, whose presence has been demonstrated by the presence of two key enzymes of this pathway: phosphoribulokinase and RubisCO.
To support this unusual metabolism, R. pachyptila has to absorb all the substances necessary for both sulfide-oxidation and carbon fixation, that is: HS⁻, O₂ and CO₂ and other fundamental bacterial nutrients such as N and P. This means that the tubeworm must be able to access both oxic and anoxic areas.
Oxidation of reduced sulfur compounds requires the presence of oxidized reagents such as oxygen and nitrate. Hydrothermal vents are characterized by conditions of high hypoxia. In hypoxic conditions, sulfur-storing organisms start producing hydrogen sulfide. Therefore, the production of in H₂S in anaerobic conditions is common among thiotrophic symbiosis. H₂S can be damaging for some physiological processes as it inhibits the activity of cytochrome c oxidase, consequentially impairing oxidative phosphorylation. In R. pachyptila the production of hydrogen sulfide starts after 24h of hypoxia. In order to avoid physiological damage some animals, including Riftia ''pachyptila are able to bind H₂S to haemoglobin in the blood to eventually expel it in the surrounding environment.
Nitrate and nitrite are toxic, but are required for biosynthetic processes. The chemosynthetic bacteria within the trophosome convert nitrate to ammonium ions, which then are available for production of amino acids in the bacteria, which are in turn released to the tube worm. To transport nitrate to the bacteria, R. pachyptila concentrates nitrate in its blood, to a concentration 100 times more concentrated than the surrounding water. The exact mechanism of R. pachyptila'''s ability to withstand and concentrate nitrate is still unknown.