Symbiosis and Its Influence on Animal Physiology

I. Types of Symbioses - Definitions

Symbiosis: In strict terms, a persistent, mutualistic interaction between two or more species. More generally, the term is often applied to any type of long-term interaction among species, including mutualistic, commensalistic, or parasitic relationships.
Symbiosis profoundly impacts the physiology of the participants and is recognized as a significant selective force in the evolution of organisms.
Mutualism: A persistent interaction between species in which both individuals benefit.
Endosymbiosis: A symbiotic relationship in which one symbiont lives either within the cells or the extracellular space of the other.
Ectosymbiosis: A symbiotic relationship in which the symbiont lives on the body surface of the host. In this relationship, the symbiont may also occupy the lumen of the digestive tract or exocrine glands.
Classic examples of animal-unicellular symbioses:
- Algae with cnidarians: Corals, sea anemones
- Bacteria with molluscs: Shipworms
- Bacteria/protozoans with insects: Termites

II. Endosymbiosis in the Deep Sea

A. Food Availability Below the Euphotic Zone

The sparse food supply of the deep sea generally precludes a high biomass in this environment.
Biomass decreases rapidly with increasing distance from the euphotic zone of primary productivity (about 200 m in depth).
Metabolic rates of deep-living animals are only a few percent of those for shallow-living species.
These concepts changed with the discovery of hydrothermal vent communities.
Focus is on an endosymbiosis in the deep sea that involves animals and sulfur-oxidizing bacteria, which fundamentally changed our views of the evolution of animal communities in the absence of sunlight.

B. Hydrothermal Vent Environments: A Hydrogen Sulfide-Based Ecosystem

Hydrothermal vents are located at crustal plate spreading zones, approximately spreading at a rate of $10 \, \text{cm/year}$ .
Mixing zone at hydrothermal vents involves simultaneous exposure to oxygen, sulfide, carbon dioxide, and nitrate.
Depth: 2,450-2,600 meters (about 1.6 miles below the ocean surface).

C. Chemolithoautotrophy

Energy source: Chemo (chemical reactions), Photo (electromagnetic radiation/photons, as in photosynthesis).
Electron donor: Litho (inorganic chemicals), Organo (organic chemicals).
Carbon source: Auto (inorganic carbon, e.g., $CO_2$ ), Hetero (organic carbon).

1. Calvin-Benson Cycle

$CO_2$ fixation for synthesis of carbohydrate.

2. Sulfur Metabolism

For synthesis of ATP and reducing power.

3. Nitrogen Reduction

Ammonium production for protein synthesis.

D. Location of Endosymbiotic Bacteria in Invertebrate Hosts

Examples include Riftia pachyptila (tubeworm), Calyptogena magnifica (vent clam), and Bathymodiolus thermophilus (vent mussel).

E. Types and Capacities for Energy Metabolism

Initial Hydrothermal Vent Expeditions:
- 1977 – Discovered by geological oceanographers (Galapagos Rift Zone).
- 1979 – First viewed by biologists (Galapagos Rift Zone).
- 1982 – ‘Oasis Expedition’: First major biological expedition (21o N Latitude Site, Baja California).
- 1985 – Major biological expedition returned to the Galapagos Rift Zone.

F. Sulfide Toxicity and Protection

Vent animals display the capacity for aerobic metabolism, despite $HS^-$ concentrations several orders of magnitude higher than those needed to inhibit cytochrome c oxidase (COX).
Mechanisms for protection from sulfide poisoning include:
- Sulfide binding by blood proteins (free concentration of sulfide kept low).
- Oxidation to thiosulfate (non-toxic).

G. Other Types of Deep-Sea Vents: Methane and Hydrogen

III. Symbioses in Shallow-Water/Non-Vent Environments

A. Lucina floridana in Seagrass Beds

B. Solemya reidi at Sewage Outfall Areas Along Pacific Coast

C. The Balancing Act of Sulfide and Mitochondria

1. Sulfide Oxidation and Detoxification by Mitochondria

2. ATP Synthesis from Sulfide by Mitochondria

3. Biomedical Application of Findings

IV. The August Krogh Principle

V. The Comparative Approach and Paradigms in Science

Key Organisms at Hydrothermal Vents

Riftia pachyptila, vestimentiferan tube worm
Calyptogena magnifica, vesicomyid clam
Bathymodiolus thermophilus, vent mussel
Bythograea thermydron, brachyuran crab
Alvinella pompejana, Pompeii worm (polychaete)
Munidopsis subsquamosa, galatheoid crab, ‘squat lobster’
Alvinocaris lusca, shrimp
Thermarces andersoni, zoarcid fish (eelpout)
Bythites hollisi, bythitid fish
Coryphaenoides armatus, deep-sea rattail fish (not endemic to vent communities)
These organisms contain bacterial endosymbionts.

Additional Notes

Black Smokers: The 'smoke-like' appearance is caused by chemicals/minerals precipitating out of the vent fluid when it mixes with the cold seawater.
Distribution of Vent Sites:
- The size of a given vent site is small and variable (perhaps 10 X 100 meter), but sites are distributed randomly across large stretches of a hydrothermal vent region.
- Hot spots have short lifetimes – 10-20 years or so.
- Larval distribution is key to populating new sites to sustain populations.
Total density of biomass at vent sites has been compared to a cow standing on a square meter of cow pasture.

Transport and Metabolism of Substrates by Tubeworm

Required Substrates for symbiosis:
1. Hydrogen Sulfide
2. Oxygen
3. Carbon Dioxide
4. Nitrate
Approximately $10^9$ bacteria per gram trophosome tissue.
Carbohydrate synthesized by the bacteria is transported to animal tissues for nutrition.

Enzymes Associated with Chemolithoautotrophic Metabolism (inside bacteria)

Calvin-Benson Cycle ( $CO_2$ fixation):
- Ribulose 1,5-bisphosphate Carboxylase (RuBisCo)
- Ribulose-5-Phosphate Kinase
Sulfur Metabolism (ATP and reducing power):
- Rhodanese ( $S2O3^{2-}$ thiosulfate)
- Adenosine Phosphosulfate Reductase ( $2 SO_3^{2-} + 2 AMP \rightarrow 2 APS$ )
- ATP Sulfurylase ( $PP + APS \rightarrow ATP$ )
Nitrogen reduction (ammonium production):
- Nitrate Reductase ( $NO3^- + NADH \rightarrow NO2^- + NAD$ )
- Nitrite Reductase ( $NO2 + reduced \,ferredoxin \rightarrow oxidized \,ferredoxin + NH3$ )

Sulfide Detoxification Mechanisms

Riftia pachyptila (tubeworm)

Sulfide binds to three hemoglobins, which keeps free concentrations very low in blood.
- Two vascular (blood) Hbs, one Hb in coelomic fluid.
- Large hemoglobins: V1 (3,600 kD), V2 (400 kD) and C1 (400 kD).
- C1 hemoglobin has 24 globin chains, 24 heme groups.
- Twelve zinc binding sites per molecule. Zinc ion is involved in the binding mechanism for sulfide (Flores et al., 2005).
- Transports sulfide to the trophosome for symbionts, which require the sulfide.
Sulfide oxidase activity (helps convert sulfide to non-toxic thiosulfate).
- Present in non-symbiont tissues (e.g., worm body wall).

Calyptogena magnifica (vent clam)

Large non-hemoglobin, sulfide binding protein in serum.
- Intracellular and extracellular hemoglobin is present, but does not bind sulfide (cf. Childress et al., 1993; Zal et al., 2000).
High levels of sulfide oxidase activity in foot tissue.
- Likely a considerable amount of sulfur transported as free (non-toxic) thiosulfate in blood.
- Symbionts use both hydrogen sulfide and thiosulfate.

Bathymodiolus thermophilus (vent mussel)

Sulfide oxidase activities present, which help convert sulfide to thiosulfate (non-toxic) before reaching the symbionts.
- Symbionts use only thiosulfate.
- No sulfide binding protein in blood.

Symbiont-free Animals (e.g., Bythograea thermydron, vent brachyuran crab)

Less work done on these species in terms of sulfide detoxification.

Sulfide that enters the body in the Bythograea is efficiently oxidized by sulfide oxidase eventually to thiosulfate (non-toxic) in the hepatopanceas.
- Thiosulfate builds up to millimolar levels in the blood.
- Presumably the thiosulfate is excreted into the environment.

Summary

Pathways for energy metabolism by tissues from hydrothermal vent animals are similar to those of shallow-living species.
- Thus, metabolic rates of vent animals may not be low like other non-vent deep sea animals.
Vent animals display the capacity for aerobic metabolism, despite $HS^-$ concentrations several orders of magnitude higher than those needed to inhibit cytochrome c oxidase (COX).
- COX of vent organisms is very sensitive to $HS^-$ poisoning.
- Protection from inhibition is due to multiple mechanisms depending on the species:
  - Sulfide binding by blood proteins (free concentration of sulfide kept low).
  - Oxidation to thiosulfate (non-toxic).
  - Utilization of sulfide for ATP synthesis by mitochondrion (at LOW, micromolar sulfide concentrations).
    - Present in a number of sulfide- tolerant invertebrates.