Duy Thai Nguyen; B.A., M.A.,

Department of Organismic and Evolutionary Biology

Harvard University

Biological Laboratories

16 Divinity Avenue

Cambridge, MA 02138

Abstract 

Introduction 

A. Microorganisms 

In his acclaimed book, The Diversity of Life, E. O. Wilson pays homage to the amazingly vast and still very much understudied world of microorganisms. Wilson (1992) writes, "...all the diversity of [rain forests and the abyssal benthos] may be dwarfed by that of bacteria, organisms that saturate the two extreme environments and every other place on earth" (p. 141). If biodiversity and the possibility of microbial life on Mars (McKay et al. 1996) are not incentives enough to support the study of microorganisms, consider some other reasons why microbiology is perhaps one of the most important of the biological sciences. For instance, much of what is known about molecular biology today, from the discovery of DNA to the polymerase chain reaction, relies upon the use of microorganisms. Microbiology also has a direct impact on people’s lives, as it plays a critical role in areas such as medicine, biotechnology, agriculture, and industrial production (Stanier 1986). 

The microbial world is a ubiquitous one. Bacteria, known as psycrophiles, have been discovered at temperatures of 0 ^oC or lower in Anarctica (Morita 1975). At the opposite end of the spectrum, some species grow optimally at 105 ^oC (Stetter 1982)! Other extreme environments in which bacteria can exist include high salt (5.2 M salt) and high acidity (pH 1.8). 

The metabolic capacities of prokaryotes are as impressive as their abilities to thrive in practically any environment. For their source of energy, "higher" organisms such as plants rely mainly on photosynthesis, and animals feed on other living things. Microorganisms, on the other hand, possess a multitude of metabolic pathways that include: photosynthesis; fermentation, a process that does not require oxygen; oxidation of various inorganic compounds, such as hydrogen and iron; and many others (see Dworkin 1992 and references within). 

B. Symbiosis 

Such metabolic diversity, combined with their resilience in most conditions, have made microorganisms prime candidates for symbiosis. The term "symbiosis" was first coined in 1879 by de Bary and defined as the living together of differently named organisms (Smith & Douglas 1987). In most symbioses, the partners are of unequal sizes. Thus, the larger partner can be differentiated as the "host," while the smaller partner is known as the "symbiont" (Smith & Douglas 1987). Note that "host" does not imply "provider." 

Symbioses involving microorganisms are widespread throughout the world and exist on every trophic level. A brief survey begins with mycorrhiza, which are associations between fungi and plants (Harley & Harley 1986). Another example of symbiosis is that between nitrogen-fixing rhizobial bacteria and leguminous plants. Symbioses exist in animals as well as in plants. The guts of many animals and insects are habitats for flourishing microorganisms. 

In the early 1980s, the concept of a symbiosis between a larger host and its micro-symbionts was dramatically shown to exist between chemoautotrophic bacteria and certain marine invertebrates. These symbioses were originally discovered at deep-sea hydrothermal vents and have since been discovered in a variety of marine environments. 

A. Hydrothermal Vents 

Before 1977, the prevailing notion concerning the deep sea was one of low biomass. Presumably, the majority of the deep sea, with no solar energy source of its own, is thus limited by the small amount of photosynthetically-derived organic matter drifting down from the uppermost layers of the ocean (Conway et al. 1994). However, the discovery of high living biomass assembled around hydrothermal vents along the Galapagos Rift at depths greater than 2500 meters shattered any remaining doubts about the life-sustaining possibilities of certain deep-sea regions (Lonsdale, 1977; Corliss et al. 1979).

Deep-sea hydrothermal vents are known to occur worldwide at sea floor spreading centers (Juniper et al. 1990). With temperatures reaching up to 400 ^oC, hydrothermal vents are driven by tectonic and volcanic activity that convectively circulate sea water through newly formed oceanic rifts. Such circulation causes numerous hot springs to spew forth from vents along the sea floor spreading centers (Lutz and Kennish 1993). Vent water emitted from these areas is enriched in metals, methane, hydrogen, and geothermally-reduced sulfides (Roesijadi and Crecelius 1984; Tunnicliffe 1991).

At lower, more life-friendly temperatures (2-40 ^oC), diluted hydrothermal fluids were observed to support high densities of large invertebrates (Corliss and Ballard 1977). One such invertebrate, from the Galapagos Rift site near South America, is the vestimentiferan tube worm, Riftia pachyptila. This organism can grow to lengths of 2 meters or more (a little shy of a tall basketball player) (Jones 1981). Other hydrothermal vent communities have been found at areas such as the East Pacific Rise near Mexico, the Juan de Fuca Ridge off the coast of the American Northwest, and the Mid-Atlantic Ridge in the middle of the Atlantic Ocean (see Figure 3). Soon after the discovery of hydrothermal vents, it was found that high biomass with vent-like taxa can also thrive at non-vent, deep-sea locations. These occur at cold, hypersaline and hydrocarbon seeps found in places such as the Florida Escarpment and Louisiana Slope (see Figure 3) (Paull et al 1984; Kennicutt et al. 1985). Fissures and faults along these seeps emit large amounts of hypersaline waters, oils, and/or gas that have been implicated in maintaining the high biomass of deep-sea invertebrates (Paull et al. 1985; Brooks et al. 1987). Like the hydrothermal vents, these seeps also appear to contain reduced inorganic compounds, such as sulfides, at the sediments of the sea floor (Cary et al. 1989). 

B. Chemolithoautotrophic Symbioses  

Because photosynthesis can be ruled out as the driving force behind primary production in the deep-sea, a hypothesis proposes that the highly enriched vent waters, containing reduced compounds such as H₂S, H₂, and CH₄, serve as a "nutrient broth" for chemosynthetic bacteria (Rau 1981; Ruby et al. 1981). This hypothesis places chemosynthetic bacteria as the basis of primary production for hydrothermal vent communities. Chemosynthetic bacteria have been interchangeably referred to as chemoautotrophs, chemolithotrophs, or chemolithoautotrophs (Cavanaugh 1985). The term "chemolithoautotrophic" refers to metabolic capabilities of the bacteria. "Chemo" means that the bacteria utilize chemical compounds as their primary energy source, while "litho" indicates that the chemicals are inorganic, such as reduced sulfides, hydrogen gas, and iron. Finally, "auto" applies to organisms that acquire their carbon from inorganic sources (i.e., carbon dioxide). Thus, chemolithoautotrophic bacteria oxidize reduced inorganic compounds to produce energy; the energy is utilized to fix carbon dioxide and synthesize organic compounds (Madigan et al. 1996). 

Subsequent research has indeed shown that chemoautotrophs are responsible for primary production at certain thriving deep-sea locations. More than 250 free-living chemoautotrophs have been isolated from these deep-sea environments (Lutz and Kennish 1993), and their existence provide sustenance for huge populations of invertebrates near the vents (Jannasch and Mottl 1985). Furthermore, some of these microbes exist as symbionts in several of the vent invertebrates, including vesicomyid clams, mussels, and vestimentiferan tube worms (Fisher 1990; Cavanaugh 1994). This scenario certainly explains the lack of a digestive system in R. pachyptila because it contains intracellular bacterial symbionts (within a highly vascularized trophosome) for nourishment (Cavanaugh et al. 1981). 

The presence of chemoautotrophic symbionts within marine metazoans is now known to be widespread across the world, occurring in coastal sediments, hypersaline and hydrocarbon seeps, hydrothermal vents, and corralline sands (Cavanaugh 1994). Symbiotic relationships between chemoautotrophs and invertebrate hosts have been suggested for over 100 species in five different phyla (Childress and Fisher 1992; Cavanaugh 1994).  

C. Model and Examples of Chemoautotrophy  

The following model of simultaneous access to oxic and anoxic environment is applicable to aerobic sulfur-oxidizing microbes (called thioautotrophic bacteria), which exist in a large majority of chemoautotrophic symbioses. These bacteria, whether free-living or symbiotic, require both an oxygen source and a sulfide source (Smith and Strohl 1991). When reduced sulfide compounds and oxygen co-occur, though, the sulfides are spontaneously oxidized with a half life of less than 20 minutes (Chen and Morris 1972; Millero 1986). In sulfide-rich environments (e.g., certain areas of sediment), there is a narrow interface between reduced sulfide compounds and oxygen where they can co-exist; but, these regions are very thin and are compressed to only a few millimeters (Kepkay et al. 1979). Invertebrate animals offer a solution to the seemingly Catch-22 situation of certain thioautotrophs. Because of their mobility, size, physiology and organ development, invertebrate hosts can simultaneously access anoxic and oxic environments to obtain sulfides and oxygen respectively for their symbionts (Cavanaugh 1994). 

D. Evidence for Chemoautotrophy 

chemoautotrophic symbionts in host invertebrates, including electron microscopy to actually visualize the bacterial symbionts (Fiala-Medioni and Metivier 1986) and certain enzyme activity experiments to ascertain chemoautotrophy. When photoautotrophy has been ruled out (i.e., at the deep sea), the detection of carbon fixation enzymes, such as ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), is strong evidence for chemoautotrophy (Felbeck 1981; Cavanaugh et al. 1988). Rubisco is an enzyme of the Calvin-Benson cycle, a carbon fixation pathway.  

The Calvin-Benson cycle, known only in autotrophic organisms, is an essential metabolic pathway upon which all of life depends (Tabita 1988). Through this cycle of reactions, the energy harnessed from photosynthesis or chemosynthesis is used to convert carbon dioxide into carbohydrate, i.e., food. The catalyst which forms the nexus between energy-gathering and energy-storage is Rubisco, the most abundant protein in the world (Ellis 1979). Rubisco catalyzes carbon fixation by carboxylating (attaching on a CO₂) ribulose-1,5-bisphosphate (RuBP), which breaks down to form two C₃ compounds called 3-phosphoglycerate (Pierce et al. 1986; Hartman and Harpel 1993). The chemical equation for carbon fixation is as follows:_Rubisco

The compound 3-phosphoglycerate serves as a building block of carbohydrates such as sucrose (Gutteridge 1995). Rubisco is thus directly involved in the annual fixation of 10¹¹ tons of CO₂ per year. In addition, all organic carbon in the biosphere derives ultimately from the catalysis of this very important enzyme (Schneider et al. 1990b). 

E. Stable Carbon Isotopes  

Lastly, another method widely utilized to identify a possible chemoautotrophic symbiosis is the use of stable carbon isotopes (Rau 1979; Rau 1985). Before discussing its relevance to chemoautotrophic symbioses, a few basics and definitions in isotope chemistry are required. The basis of isotopic analysis is that isotopes of the same element have different physical and chemical properties due to quantum mechanical effects related to their different interatomic distances (Hoefs 1987). These different physical and chemical properties are called isotope effects. The following words are used throughout this presentation, and it is necessary to properly define them here. "Fractionation" is similar to "discrimination," and it simply means the change in isotopic content that occurs due to a physical, chemical, or biological processes (O’Leary 1988).  

When the rate of a chemical reaction is affected by atomic mass in a reacting species, a "kinetic isotope effect" occurs (Hoefs 1987). A kinetic isotope effect is defined as k₁/k₂, where k₁ and k₂ are the rate constants for the reaction of light and heavy isotopes (Hoefs 1987). In general, molecules with the lighter isotope react slightly more easily than molecules with the heavier isotope. Thus, there is a discrimination against the heavier isotope. For example, in carbon fixation, the enzyme Rubisco tend to fix ¹²CO₂ more readily than it does ¹³CO₂.  

Stable carbon analysis relies on the observation that carbon exists as two stable isotopes (¹²C and ¹³C) and that organisms differ in their isotopic ratio of ¹²C to ¹³C (Hoefs 1987). These differences are the result of mainly two factors. The first factor is the isotopic composition of the source carbon. Source carbon for heterotrophs is mainly derived from matter that they consume, while source carbon for autotrophs is principally derived from CO₂. The second factor concerns the isotopic discrimination during uptake and metabolism of the carbon, such as the fractionation by Rubisco (Goericke et al. 1994). Stable carbon analysis is so useful because, in any ecosystem and at any trophic level, carbon is an essential element. Differences in carbon isotopic composition, caused by processes such as photosynthesis and chemosynthesis (both of which are catalyzed by Rubisco), are isotopic signatures that can be traced through various trophic levels, including the primary production level and the consumer levels above it. Thus, stable carbon isotopic analysis may help to elucidate the source of a carbon supply and the various biological processes in an ecosystem that affect the carbon input (Goericke et al. 1994).

Isotopic values are shown as d¹³C values, where the isotopic composition of a sample is given with respect to a known standard: 

d¹³C = {(Rsample/Rstandard) - 1} x 1000 with R = ¹³C/¹²C (Hoefs 1987). 

The standard most commonly used is a limestone fossil from the Cretacious Pee Dee formation in South Carolina, and values are expressed on a per mil (^o/_oo) range (Boutton 1991). When d¹³C has a more negative value for a certain sample, this means that the sample is either enriched in ¹²C or depleted in ¹³C. Conversely, when d¹³C has a more positive value, a sample is either depleted in ¹²C or enriched in ¹³C. 

Stable carbon analysis has been a useful tool in many diverse fields. It has been espoused as a means to protect endangered species such as ivory-tusked elephants and rhinoceros (van der Merwe et al. 1990; Martin et al. 1991); to study the dynamics of greenhouse gases (Francey et al. 1995); and to gauge the effects of deforestation (Veldkamp 1994).  

Stable isotopic analysis was one of the first methods used to indicate that hydrothermal vent organisms are obtaining their nutritional carbon from a local chemoautotrophic source (Rau and Hedges 1979; Rau 1981). The d¹³C values for most vent organisms were not similar to those of typical marine organisms that depend on organically-derived matter (Rau and Hedges 1979; Rau 1981). Thus, researchers (Rau and Hedges 1979) concluded that chemosynthetic bacteria were probably the primary producers at vents because their carbon fixation pathways distinctly fractionate against ¹³C and thus leave isotopic signatures of chemoautotrophy. Though researchers were correct about chemosynthetic bacteria, the relationship between chemoautotrophy and stable carbon isotopes is actually more complicated than first thought. The elucidation of this relationship forms the basis of the research discussed in this presentation. 

A. Disparity in d¹³C Values  

During the course of using stable carbon isotopes to determine nutritional sources and metabolic pathways of diverse invertebrate-chemoautotroph symbioses, a disparity in d¹³C values developed. Certain organisms, including vestimentiferan tube worms, alvinellid polychaetes, and a Mid-Atlantic Ridge shrimp, fell into the range of -9 to -15 ^o/_oo (called the -11 ^o/_oo group) (Childress & Fisher 1992). Others, including symbiotic bivalves and a gastropod, had d¹³C values ranging from -27 to -35 ^o/_oo (called the -30 ^o/_oo group) (Childress & Fisher 1992).  

The -30 ^o/_oo group members have d¹³C values that suggest their chemoautotrophic symbionts are producing organic carbon that is about 30 ^o/_oo more depleted than the source carbon. This reflects the "normal" situation because free-living chemoautotrophic bacteria have similar fractionation against ¹³C (Ruby et al. 1987). The -11 ^o/_oo group, though, presents a challenging problem. It is clear, from our discussion above on evidence for chemoautotrophy, that members of the -11 ^o/_oo group rely on chemoautotrophic symbionts. This is especially true for the vestimentiferan tube worms which do not even possess digestive systems. Yet, it would appear that these symbionts have practically no fractionation against ¹³C when producing their organic carbon (as interpreted from their host tissues). Host tissues are different from the source carbon by only about 10 ^o/_oo or less. Several hypotheses have been proposed to explain the difference in isotopic signatures between these two groups. All of them focus on explaining the "abnormal" situation of the -11 ^o/_oo group. These hypotheses include: varying sources of dissolved inorganic carbon, carbon limitation, and C₃ vs. C₄ carbon fixation pathways.  

B. Hypotheses for Disparity 

The first hypothesis suggests that the -11 ^o/_oo group members are utilizing a heavy carbon source that is enriched in ¹³C (Childress and Fisher 1992). This explanation is probably not correct because some members of both the -11^o/_oo and -30 ^o/_oo groups live in similar microhabitats; they can also sometimes co-exist and compete in the same area (Hessler 1985). 

The second hypothesis is related to problems with supplying carbon to bacterial symbionts, which rely on the host circulatory system to supply it with metabolites (Childress and Fisher 1992). The carbon limitation hypothesis deals with carbon substrate availability after diffusion or active intake through at least two cellular membranes (Rau 1985; Fisher et al. 1990). The third hypothesis proposes a carbon fixation pathway for -11 ^o/_oo group members that is similar to the one possessed by C₄ plants (Felbeck 1985; Distel and Felbeck 1988). The C₄ fixation pathway is a variation on the Calvin-Benson cycle, but there is little discriminatory effect against ¹³C during carbon uptake. The evidence supporting these two hypotheses are limited.  

After analyzing three different hypotheses, the problem presented by -11 ^o/_oo symbioses is more complex than first assumed. What then could be accounting for the difference in d¹³C values between the two chemoautotrophic groups, a difference that can be as large as 20 ^o/_oo? This disparity is all the more baffling considering several common aspects that some members of these two groups share: (1) they live in similar microhabitats, sometimes coexisting and competing in the same environment (Hessler et al. 1985); (2) they have closely related bacterial symbionts (Cavanaugh 1994); (3) their symbionts utilize the same carbon source species, CO₂, for carbon fixation (Fisher 1990); and (4) their symbionts have the same or very similar carbon fixation cycle (Robinson and Cavanaugh 1995).  

A recent and more plausible hypothesis capitalizes on the fact that the Rubisco enzyme in the Calvin Benson cycle exists in two different forms: form I and form II (Gibson and Tabita 1977a). Before discussing this hypothesis, a general overview of the two forms is presented. 

While both forms of Rubisco catalyze the same reaction involving the same substrates (that is, CO₂ and RuBP) in the Calvin Benson cycle (Gutteridge 1990), they also differ in several ways. One of the most obvious difference is in their quaternary structure (Gibson and Tabita 1985). Rubisco form I consists of eight large subunits and eight small subunits arranged in a hexadecameric structure (L8S8) (Torres-Ruiz and McFadden 1987). Rubisco form II possesses only large subunits situated as dimers (Lx), ranging from L₂ to aggregates of at least L₈ (Gutteridge 1990; Schneider et al. 1990a). Even with such structural differences, there are conserved regions in the two forms’ tertiary arrangement. Although the form II amino acid sequence from Rhodospirillum rubrum is only 25% identical to the large subunit of Rubisco form I in plants (Nargang et al. 1984), they both possess highly conserved peptide regions that have been identified as activation and active sites (Schneider et al. 1990b and references within). Furthermore, x-ray crystallographic studies show that form II in R. rubrum and the form I enzymes in spinach and tobacco have closely similar folding of the large subunit polypeptide chain (Schneider et al. 1990a and references within). Nevertheless, besides structure, the two forms differ in several other aspects. They differ in their Michaelis-Menton kinetics (Roeske and O’Leary 1985) and are immunologically diverse (i.e., antibodies raised against one form will not react with the other form) (Gibson and Tabita 1977a,b; Robinson and Cavanaugh 1995). They also differ in their respective ratios of carboxylation rate to oxygenase reaction rate (both CO₂ and O₂ compete for the same active site on Rubisco to react with RuBP) (Gutteridge 1990). Finally, the two forms differ in their kinetic isotope effect when utilizing substrate CO₂ (Roeske and O’Leary 1984, 1985; Guy et al. 1993; Robinson and Cavanaugh 1995).

It has been known for quite some time that there are two different forms of Rubisco. Form I is found in most autotrophs including plants while form II has been described for a few autotrophic bacteria and some dinoflagellates (Morse et al. 1995; Whitney et al. 1995). Although most organisms possess either form I or form II, it is interesting to note that some bacterial organisms do encode both forms. These organisms include Rhodobacter sphaeroides (Gibson and Tabita 1977a), Rhodopseudomonas capsulata (Gibson and Tabita 1977b), and Thiobacillus denitrificans (English et al. 1992).  

D. Varying Fractionations for Form I and Form II 

For the few Rubisco enzymes that have had their kinetic isotope effects determined, it is observed that form I has a larger fractionation factor than that of form II. For example, form I Rubisco from Spinacia oleracea (spinach) has a fractionation factor of 29 ^o/_oo (Roeske & O’Leary 1984). Form II Rubisco from the photosynthetic bacteria Rhodospirillum rubrum can have a fractionation factor as small as 17.8 ^o/_oo (Roeske & O’Leary 1985), an almost 12 ^o/_oo difference from spinach. The Rubisco form I from the cyanobacterium Anacystis nidulans, though, has a fractionation factor of 22 ^o/_oo (Guy et al. 1993), which is more similar to that of form II.  

The unresolved issue presented at the beginning of this section was the disparity in d¹³C values between -11 ^o/_oo and -30 ^o/_oo symbioses. Three previous hypotheses (carbon source, carbon limitation, and C₄ pathway) have failed to provide a satisfactory explanation for this disparity. The most recent hypothesis suggests that the difference in discrimination against ¹³C isotopes by the two different forms of Rubisco can explain the disproportion in d¹³C values of chemoautotrophic symbioses (Robinson & Cavanaugh 1995). Because discrimination by form II is much less than that by form I, it is hypothesized that the CO2-fixing enzyme in members of the -30 ^o/_oo group is a form I Rubisco, while in the -11 ^o/_oo symbioses, a form II Rubisco is expressed (Robinson & Cavanaugh 1995). Preliminary evidence (both immunological and enzyme inhibition experiments) exist for this hypothesis. Members of the -30 ^o/_oo group, the vent mussel Bathymodiolus thermophilus and Atlantic coast clam Solemya velum, were shown to express Rubisco form I. Furthermore, Rubisco form II expression was detected in certain representatives from the -11 ^o/_oo group, such as the vent vestimentiferans Tevnia jerichonana and Riftia pachyptila (Robinson & Cavanaugh, 1995). 

Correlation between Rubisco Form and Stable Carbon Isotope Values 

A. Question and Hypothesis Concerning Rubisco Form and d¹³C Values 

Is there truly a correlation between the Rubisco form and the stable carbon isotopic composition of a chemoautotrophic symbiosis? That is, can the Rubisco form be a predictor of the d¹³C values of a chemoautotrophic symbiosis, and vice versa? More specifically, do -30 ^o/_oo symbioses express a Rubisco form I and do -11 ^o/_oo group members express a Rubisco form II? 

My research seeks to confirm the hypothesis that, although other environmental and physiological factors must still be considered, there is a strong correlation between the Rubisco form and the d¹³C values of a chemoautotrophic symbiosis. Thus, it is proposed that -30 ^o/_oo symbioses will have a Rubisco form I, while -11 ^o/_oo group members will have a Rubisco form II.  

For most chemoautotrophic symbioses that do not depend on heterotrophy, the organic carbon they possess (i.e., their tissues) are mainly derived from the fixation of inorganic carbon by Rubisco. Thus, if the Rubisco enzyme discriminates against ¹³C isotope, this effect will be noticeable downstream of the carbon fixation and will show up in the invertebrate tissues. It is therefore very reasonable and probable to hypothesize that, if there is varying discrimination by the two different forms of Rubisco, there will then be varying isotopic signatures among the organisms whose symbionts express those particular Rubisco forms.  

B. Objectives to Demonstrate Relationship between Rubisco and d¹³C Values 

In order to show a correlation between the Rubisco enzymes of chemoautotrophic symbioses and their d¹³C values, a survey was conducted using diverse chemoautotrophic systems obtained from across the world. To test the above hypothesis, the following were performed: (1) d¹³C values were obtained for each particular chemoautotrophic system by analyzing the tissues of organisms and/or by referencing previously published d¹³C values; (2) through immunological detection of the actual protein, the type of Rubisco expressed was determined for a particular symbiosis; (3) via DNA hybridization experiments, the Rubisco enzyme gene(s) possessed by the chemoautotrophic symbiont was determined; and (4) the data was analyzed to ascertain a correlation between d¹³C values and Rubisco form expression. 

To briefly explain, immunological detection and DNA hybridization experiments are commonly known as Western and Southern blotting respectively. In Western blotting, antisera are raised against known proteins, such as Rubisco form I from spinach plants or Rubisco form II from a cyanobacterium. The antisera are then utilized to screen for form I or form II in the various chemoautotrophic systems. Animal tissues from these systems are subjected to electrophoresis and transferred to a membrane that is incubated with the desired form I or form II antisera. Positive signals are compared to known standards and to controls to confirm the presence of form I and/or form II proteins. In Southern blotting, similar principles apply, although the procedure screens for the presence of genes rather than of proteins. DNA probes for the Rubisco form I and form II genes are produced using the polymerase chain reaction. They are then radioactively labeled with ³²P and incubated with membranes containing genetic material from various chemoautotrophic symbioses. After washing, any remaining radioactive signals detected on film are possible positive confirmations of form I and/or form II genes.  

Finally, the chemoautotrophic symbioses surveyed were chosen for several reasons, including: (1) the availability of specimens; (2) the different types of symbioses represented: some microbes are epibiotic (living on the surfaces of the host) while others are endosymbiotic; (3) the breadth of the environmental diversity: organisms were selected from hypersaline seeps, three different hydrothermal vents, and sulfide-rich coastal sediments; (4) there exists evidence to indicate that chemoautotrophy is the main contributor of nutrition for these symbioses.  

C. Results and Discussions 

The hypothesis that there exists a correlation between the Rubisco form and d¹³C values of a chemoautotrophic symbiosis has been substantiated by my research on diverse chemoautotroph-invertebrate symbioses: -11^o/_oo symbioses have a Rubisco form II while -30 ^o/_oo group members have a Rubisco form I. Immunological studies and, to a certain extent, DNA hybridization data indicate that -11^o/_oo symbioses express a Rubisco form II while -30 ^o/_oo group members express a Rubisco form I. Examples of -11^o/_oo symbioses that express form II include: the Juan de Fuca vestimentiferan Ridgea piscesae, the Mid-Atlantic shrimp Rimicaris exoculata, the Juan de Fuca hairy polychaetes, the Juan de Fuca "Ridgea" bacterial mats, and the Juan de Fuca "clambed" bacterial mats. Examples of -30 ^o/_oo organisms that express form I include Lucina floridana from coastal sediments and Solemya reidi from sewage waters. 

Although the two different forms of Rubisco are shown to contribute to the varying stable carbon isotope values of chemoautotrophic bacteria, other factors, both environmental and physiological, must still be taken into account. A secondary research project that I conducted was to study these various factors for two organisms, the bivalves Calyptogena magnifica and Calyptogena pacifica, that are exceptions to the hypothesis just discussed. They both possess form II enzymes but have d¹³C values that are below -30^o/_oo (Southward et al. 1994; Fisher 1995). This anomalous result poses a very interesting question as to what may be the cause of the greater ¹³C fractionation. That is, if the form II enzymes in C. magnifica and C. pacifica are responsible only for a portion of the ¹³C discrimination, what else is contributing to the carbon fractionation such that their d¹³C values are so negative? The research and possible explanations to this question will have to be addressed in another presentation. However, the question raised by these two exceptions demonstrates a well-tested notion of science: in attempting to answer a question, research tends to result in more questions that continue to challenge our curiosity and intellect.  

Symbioses between invertebrates and chemoautotrophic symbionts are widespread and occur in numerous marine habitats. Stable carbon analysis has identified two main groups of chemoautotrophic symbioses: the -11 ^o/_oo group with d¹³C values from -9 to -16 ^o/_oo and the -30 ^o/_oo group with d¹³C values from -27 to -35 ^o/_oo. Several hypotheses, including source carbon, C₄ pathway, and carbon limitation, have been proposed for this disparity in d¹³C values, but they are unsatisfactory. A new hypothesis was proposed: that two different forms of Rubisco are responsible for the distinction in d¹³C values. 

Stable carbon isotope values, immunological detections, and DNA hybridization experiments are shown to support this hypothesis, although exceptions can exist due to various other factors that affect carbon isotopic signatures. Thus, overall, this study has found that, while environmental and physiological conditions may play significant roles in certain cases, there is definitely a clear relationship between the Rubisco form and the d¹³C values of a chemoautotrophic symbiosis. Because form I tends to have a larger fractionation factor than that of form II, form I is more likely to be in those symbioses that are more depleted in ¹³C. The research presented here has shown this to be the case: -30 ^o/_oo group members tend to express form I while -11 ^o/_oo symbioses possess form II.  

The findings from this research have several implications. They demonstrate reasons for caution in the use of stable carbon isotopes for determining nutritional sources among trophic levels. They also provide a new interpretation for situations in which chemosynthesis is a known nutritional source. Finally, they show that Rubisco form II is more prevalent than first assumed.  

The sciences offer enormous opportunities in terms of intellectual and personal rewards. Independent research allows one to develop a sense of accomplishment and a belief in the search for knowledge. Biology is truly an interdisciplinary field, as one may have noticed from the above presentation. During the relatively short time spent on the above research project, I was exposed to marine biology, geochemistry, animal physiology, biochemistry, molecular biology, isotope chemistry, and others. There is no better field to enter if one wishes to contribute to the canon of scientific knowledge and to humankind in general. I wish all much luck!  

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