The Pennsylvania State University
The Graduate School
The Eberly College of Science
ECOLOGICAL PHYSIOLOGY AND BIOCHEMISTRY OF SULFIDE
ACQUISITION BY TWO HYDROCARBON SEEP VESTIMENTIFERANS,
LAMELLIBRACHIA LUYMESI AND SEEPIOPHILA JONESI
A Thesis in
Biology
by
John Karl Freytag
2003 John Karl Freytag
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
December 2003 The thesis of John Karl Freytag has been reviewed and approved* by the following:
Charles R. Fisher Professor of Biology Thesis Adviser Chair of Committee
James Marden Associate Professor of Biology
Roger Koide Professor of Horticultural Ecology
Michael A. Arthur Professor of Geosciences
James J. Childress Professor Ecology, Evolution, and Marine Biology The University of California Santa Barbara Special Signatory
Douglas R. Cavenar Professor of Biology Head of the Department of Biology
*Signatures are on file in the Graduate School. iii
Abstract
Two species of vestimentiferan tubeworm, Lamellibrachia luymesi and
Seepiophila jonesi, co-occur in aggregations at northern Gulf of Mexico cold
hydrocarbon seep sites. Like all vestimentiferans, L. luymesi and S. jonesi obtain
nutrition from sulfide-oxidizing chemoautotrophic bacterial endosymbionts that must be
supplied with sulfide, oxygen, and carbon dioxide. Results from previous studies that
examined the environmental sulfide chemistry of northern Gulf of Mexico hydrocarbon
seeps suggested that the ecological physiology of seep tubeworms was not analogous to
that of the hydrothermal vent tubeworm, Riftia pachyptila, which obtains sulfide, oxygen,
and carbon dioxide across the anterior plume portion of its body. The focus of this study
was to better understand the physiological ecology of environmental sulfide acquisition
of L. luymesi and S. jonesi. Whole animal respiration studies were conducted using split-
vessel respiration chambers built specifically for this series of experiments. Methods for
sulfide equilibrium dialysis experiments were determined and utilized to estimate the
sulfide-binding characteristics of the intact fluids and component hemoglobins of L.
luymesi and S. jonesi.
L. luymesi and S. jonesi grow a posterior extension of their tube and tissue, termed
a “root,” down into sulfidic sediments below the point of original attachment.
Preliminary blood sulfide uptake experiments confirmed that sulfide uptake across the
posterior portions of live L. luymesi can occur. Sulfide was not detectable in the blood of any control animals that did not have their roots exposed to sulfide (detection limit of 3.0
µM) but was present in the blood of experimental animals that had their roots exposed to iv
500 µM sulfide (152 and 170 µM). Split-vessel respiration experiments which exposed the root portions of L. luymesi to sulfide concentrations between 51 and 561 µM demonstrated that L. luymesi can utilize their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 µmoles*g-1*h-1. Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior, root portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan L. luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake.
Vestimentiferan vascular blood and coelomic fluid contain giant extracellular hemoglobins (Hbs; a 3,500 kDa and a 400 kDa Hb in the vascular blood, and a different
400 kDa Hb in coelomic fluid) that reversibly and simultaneously bind large quantities of hydrogen sulfide and oxygen at binding sites unique for each chemical species. These
Hbs are fundamental to the uptake, accumulation, transportation, and delivery of both sulfide and oxygen to their endosymbionts. Sulfide-binding dialysis experiments were conducted in order to estimate the sulfide-binding affinity and capacity for the purified R. pachyptila 3,500 kDa Hb and a mixture of 400 kDa Hbs from vascular and coelomic fluids. Under experimental conditions, the 3,500 kDa Hb bound 2.2 moles sulfide per
mole of heme when saturated. Half-saturation occurred at 5.2µM ΣH2S (the sum of H2S,
- - HS , and S2 ) for this Hb. The 400 kDa Hbs were able to bind 0.47 moles sulfide per mole of heme when saturated, and 50% saturation occurred at 5.4µM ΣH2S. The very similar sulfide affinities of the 3,500 kDa and 400 kDa Hb fractions would allow bi- directional exchange and storage of sulfide in both fluid compartments and is consistent with the proposed role of the coelomic fluid as a temporary storage reservoir for sulfide. v
During this series of experiments, significant limitations in the methods commonly used in sulfide-binding studies were found. At equilibrium, sulfide concentrations inside small volume dialysis bags were not equal to external dialysate concentrations, and the effect varied with the bag volume. Sulfide-binding by purified 3,500 kDa and 400 kDa Riftia pachyptila Hb fractions was also positively correlated to heme concentration. Sulfide binding per heme did not increase significantly above concentrations of 0.4 mM heme in the experiments with the 400 kDa Hbs. However, sulfide bound per heme by the 3,500 kDa Hb increased significantly over the full range of heme concentrations tested (0.15 to
1.8 mM). The mechanism for increased sulfide-binding at increased heme concentrations has not been determined.
Utilizing an improved methodology resulting from the sulfide-binding experiments with R. pachyptila fluid and hemoglobins, experiments were conducted to estimate the sulfide-binding affinity and capacity for purified 3,500 kDa Hbs and mixtures of 400 kDa Hbs from the vascular and coelomic fluids of L. luymesi and S. jonesi. In addition, the heme concentrations and relative Hb abundance of intact vascular blood and coelomic fluids and the sulfide-binding characteristics of the component Hbs were determined for both L. luymesi and S. jonesi. Results from sulfide-binding experiments show that the 3,500 kDa Hb from the fluids of S. jonesi has a high affinity
for sulfide (C50 value of 8.8µM) while the 3,500 kDa Hb from the fluids of L. luymesi has only a moderate affinity for sulfide (C50 value of 96µM). S. jonesi has elevated fluid heme concentrations that may facilitate the survival of individuals in environments where they are exposed to low concentrations of oxygen and/or short periods without any oxygen. The high affinity of the predominant hemoglobin in the vascular fluid of S. vi jonesi for sulfide suggests that large S. jonesi may be capable of acquiring sulfide with the anterior plume portion of its body from low concentration pools found just above the sediment-water interface. The low affinity of the most prevalent hemoglobin in the vascular fluid of L. luymesi for sulfide suggests that large L. luymesi are not likely able to use their plumes for sulfide uptake and likely depend upon the root portion of the body and tube for sulfide acquisition. The sulfide-binding data for the L. luymesi 3,500 kDa
Hb suggests that L. luymesi may have a 3,500 kDa Hb with multiple sulfide-binding
mechanisms that have different sulfide capacity and C50 values (one lower and one higher) or express two different 3,500 kDa Hbs with different sulfide-binding characteristics.
Together these data have fundamentally changed our model for the ecological physiology of seep vestimentiferans. Although closely related to hydrothermal vent tubeworms, L. luymesi and S. jonesi do not acquire metabolites from the environment in the same way as R. pachyptila. We are now beginning to understand how sulfide, carbon dioxide, and oxygen can be acquired by L. luymesi and S. jonesi. vii
TABLE OF CONTENTS
List of Figures………………………………………………………………….……….. ix List of Tables………………………………………………………………….………... x Preface ………………………………………………………………….……………… xi Acknowledgements………………………………………………………………….….. xii
Chapter 1: Introduction……………………………………………………………..… 1 I. Communities based on chemoautotrophy………………………………..… 1 II. Vestimentiferan Biology…………………………………………………… 4 III. Gulf of Mexico seep vestimentiferans and their sulfide physiology…….… 6 IV. References……………………………………………………………….… 12
Chapter 2: A paradox resolved: Sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy…………………………………………...……………… 21 Abstract………………………………………………………………….…..…... 22
Introduction…………………………………………………………………...… 23
Methods………………………………………………………..……………….. 25
Results……………………………………………………..……………………. 31
Discussion…………………………..…………………………………………… 33
Acknowledgements…………………………………………………..………….. 40
References……………………………………………………………………….. 40
Table and Figure Legends………………………………………………………. 44
Table and Figures……………………………………………………….………. 45
Chapter 3: Sulfide binding by the giant hemoglobins from the hydrothermal vent tubeworm Riftia pachyptila …………………………………………………………… 49
Summary………………………………………………………………….…..… 49
Introduction…………………………………………………………………...… 50
Materials and Methods……………………………………………………….. 53 Animal Collection…………………………………………………….. 53 Hemoglobin purification and quantification………………………….. 54 Dialysis experiments………………………………………………….. 55 Quantification of sulfide……………………………………………… 56 Data analysis………………………………………………………….. 57 viii
Evaluation of dialysis methodology: Correction for control dialysis bag volume……………………………………………………………. 59 Evaluation of dialysis methodology: Other variables………………... 59 Effect of heme concentration…………………………………………. 60
Results………………………………………………………………………... 60 Sulfide binding by purified hemoglobins…………………………….. 60 Evaluation of dialysis methodology………………………………….. 61 Effect of heme concentration on sulfide binding……………………... 62
Discussion……………………………………………………………………. 62
References……………………………………………………………………. 67
Tables and Figures…………………………………………………………… 72
Chapter 4: Sulfide binding properties of hemoglobins isolated from two species of cold seep vestimentiferans………………………………………………………... 76
Summary……………………………………………………………………… 76
Introduction…………………………………………………………………… 77
Materials and Methods………………………………………………………... 82 Animal collection……………………………………………………… 82 Hemoglobin purification and quantification…………………………... 83 Fluid relative hemoglobin abundance…………………………………. 83 Dialysis experiments…………………………………………………... 84 Quantification of sulfide……………………………………………….. 84 Data analysis…………………………………………………………... 85
Results…………………………………………………………………………. 85 Composition vascular and coelomic fluids……………………………. 85 Sulfide binding by purified Lamellibrachia luymesi and Seepiophila jonesi hemoglobins…………………………………………………….. 86
Discussion…………………………………………………………………….. 87
References…………………………………………………………………….. 96
Tables and figures…………………………………………………………….. 104
Chapter 5: Summary……………………………………………………………….. 111 Conclusions……………………………………………………………. 113 Most significant contributions to hydrothermal vent and hydrocarbon seep biology…...……………………………………………………….. 115 ix
LIST OF FIGURES
Figure 2.1. Split-vessel respiration system………………………………………… 48
Figure 3.1. Sulfide binding by the Riftia pachyptila 3,500 kDa hemoglobin as a function of free sulfide concentration……………………………………….. 73
Figure 3.2. Sulfide binding by the Riftia pachyptila 400 kDa hemoglobins as a function of free sulfide concentration. ……………………………………… 74
Figure 3.3. Sulfide binding by the Riftia pachyptila 3,500 kDa hemoglobin, and 400 kDa hemoglobins as a function of hemoglobin concentration. ………... 75
Figure 4.1. The outer edge of a vestimentiferan aggregation on the upper Louisiana slope of the Gulf of Mexico……………………………………… 105
Figure 4.2. Sulfide binding by the Lamellibrachia luymesi 3,500 kDa hemoglobin as a function of free sulfide concentration. …………………………………. 106
Figure 4.3. Sulfide binding by the Lamellibrachia luymesi 400 kDa hemoglobins as a function of free sulfide concentration. …………………………………. 107
Figure 4.4. Sulfide binding by the Seepiophila jonesi 3,500 kDa hemoglobin as a function of free sulfide concentration.………………………………………. 108
Figure 4.5. Sulfide binding by the Seepiophila jonesi 400 kDa hemoglobins as a function of free sulfide concentration.………………………………………. 109
Figure 4.6. Sulfide binding by the Lamellibrachia luymesi and Seepiophila jonesi 3,500 kDa hemoglobins as a function of free sulfide concentrations……….. 110 x
LIST OF TABLES
Table 2.1. Seawater H2S concentrations amongst mature hydrocarbon seep tubeworm aggregations. …………………………………………………….. 45
Table 2.2. H2S, DIC, and O2 consumption rates across root and plume of Lamellibrachia cf luymesi…………………………………………………... 46
Table 2.3 Plume DIC and Oxygen flux prior to, during, and after root H2S exposure. ……………………………………………………………………. 47
Table 3.1. Percent diffusion data from sulfide and dye dialysis experiments……… 72
Table 4.1. Hemoglobin sulfide-binding characteristics and properties of intact fluids of Seepiophila jonesi, Lamellibrachia luymesi, and Riftia pachyptila……………………………………………………………………. 104 xi
PREFACE
Contributions to the research contained within this thesis:
Chapter 2. The respirometry experiments were conducted in collaboration with and utilized the mass spectrometer respiration system of the James J. Childress laboratory at the University of California Santa Barbara. Dr. Peter R. Girguis was instrumental in the operation and maintenance of this mass spectrometry system and the experimental setup. Jason Andras collected the low-level sulfide environmental data. The designs of the split-vessel incubation and respiration chambers were based upon a prototype conceived and built by Dr. Derk C. Bergquist. Dr. Charles R. Fisher provided the means for sample collection and transportation and was also instrumental during the design stage of the respirometry experiments.
Chapter 3. Ronald M. Smith assisted during all stages of hemoglobin purification and sulfide dialysis experiments. Jason F. Flores identified the dialysis bag volume effect on sulfide diffusion. Dr. Stéphane M. Hourdez assisted with some of the initial data analysis. Dr. Charles R. Fisher was involved with the overall project design and funding.
Chapter 4. Ronald M. Smith and Steven R. Breault assisted during hemoglobin purification and sulfide dialysis experiments. Dr. Stéphane M. Hourdez collected the pure fluid samples. Christopher S. Jones conducted the low-level enzymatic assay for sulfide quantification. Dr. Charles R. Fisher provided the means for sample collection and transportation and was involved with the overall project design and funding. xii
ACKNOWLEDGEMENTS
Professional Acknowledgements: I’d like to thank Dr. Chuck Fisher for providing me the support and opportunity that allowed me to work on such unique organisms. I’d also like to thank Chuck for the guidance he provided and the patience he demonstrated during my graduate career. Thanks to Jim Childress for providing me with the opportunity to work in his lab as both an undergraduate and graduate student, to participate in several mid-water and vent cruises, and for introducing me to the field of deep sea biology. I’d like to thank Dr. Stéphane Hourdez and Dr. Peter Girguis for their guidance, suggestions, and discussions. Thank you to Erik Cordes and Sue Carney for collecting invaluable samples for me at sea and to Dr. Craig Young for the opportunity to participate in a cruise and collect additional samples. Thank you to Ron Smith, Steve Breault, Wadi Gomero, Megan Merril, Janine Fisler, Stacy DiFrank, and Chris Jones for assistance in the lab. I’d like to thank Mark Van Horn for his assistance and training in lab. I’d also like to thank my committee members: Jim Marden, Michael Arthur, Roger Koide, and Jim Childress for their guidance, patience, time, discussion and comments. The work in this thesis was supported by The Minerals Management Service project RFP-6899, The Minerals Management Service, Gulf of Mexico Regional OCS Office through contract number 1435-01-96-CT30813, the NOAA National Undersea Research Program at the University of North Carolina, Wilmington, and NSF OCE 0117050. Special thanks are due to Harbor Branch Oceanographic Institution, Chris Tietz, and the captains, pilots and crews of the R/V Seward Johnson and the DSRV Johnson Sea Link.
Personal Acknowledgements: First and foremost, to my parents, Karl and Jan, and my sister, Kari: Thank you for your continuous love and support throughout my graduate career. I couldn’t have made it to and through Penn State without all of your guidance, encouragement, and help! To Breea: Thank you for loving, supporting and encouraging me, lifting me up when I needed it, helping me keep everything in perspective, and helping me stay healthy and (relatively) sane throughout my thesis writing and defense preparation months. I couldn’t have done it without you and I look forward to being there for you as you finish and defend your thesis. Special thanks to all of my friends in California and State College that have helped to make my undergraduate and graduate careers excellent and fulfilling times! Thank you fellow Fisherettes, past and present, for all of the fun and laughs inside and outside of lab: K.T., Ish, Derk, Emily, Erin, Stéphane, Jason, Breea, Erik, and Sharmishtha. Huge thanks (again) to Ron Smith for all of his time and assistance during the “Summer (and Fall) of Sulfide-Binding”. There is no way I could have conquered all those Hbs by myself. Lastly, thank you Chuck for giving me the occasional kick in the ass!
This thesis is dedicated to my family. 1
Chapter 1
Introduction
I. Communities based on chemoautotrophy
The discovery of hydrothermal vents in 1977 and hydrocarbon seeps in the mid
1980’s dramatically changed the way people thought about the biology of the deep-sea
(Jones 1985; Sibuet and Olu 1998). Previously, the deep-sea had been considered a dark, cold desert of sorts, with a low biomass and the only input of nutrients thought to come from surface waters (Grassle and Maciolek 1992). Hydrothermal vent and hydrocarbon seep environments have been referred to as deep-sea “oases” and have biomasses similar to those found in tropical rain forests (Laubier 1989; Carney 1994). However, primary production at these deep-sea environments is based upon chemosynthesis rather than photosynthesis (Jannasch 1985). Free living and endosymbiotic sulfur-oxidizing or methanotrophic bacteria utilize reduced chemicals obtained from the environment as an energy source, just as photosynthetic bacteria and the chloroplasts of green plants harvest energy from sunlight (Jannasch and Mottl 1985). Metazoans, including all vent and seep chemoautotrophic symbioses, require oxygen to survive. Sulfur and methane-oxidizing bacteria, including vestimentiferan endosymbionts, require oxygen in addition to an electron donor to survive. Thus, primary production in these lush communities is dependent upon the presence and availability of reduced chemicals such as hydrogen sulfide or methane, and oxygen (Fisher 1996).
Deep-sea hydrothermal vents are associated with tectonically active areas such as spreading centers, subduction zones, and fracture zones (Gage and Tyler 1999). 2
Hydrothermal venting results from the circulation of seawater through the earth’s crust near areas of tectonic activity. As it circulates, the water is heated by geothermal energy and becomes saturated with reduced inorganic compounds (Jannasch and Mottl 1985).
Hot, reduced fluids then rise back towards the sea floor where they emerge and mix with the surrounding bottom water. Vent environments along the East Pacific Rise are characterized by rocky, basaltic substrates, high variations in temperature, and areas where active mixing of vent fluid and ambient water occurs. End member hydrothermal fluids are rich in hydrogen sulfide and carbon dioxide, are anoxic and have pH values as low as 6.0, whereas ambient deep-sea water contains no sulfide, moderate levels of oxygen, and has a pH around 7.8 (Childress and Fisher 1992; Fisher 1995). Some vent environments are dominated by large, towering chimney-like structures from which superheated (250°C to more than 350°C), sulfide rich waters are actively expelled, while other vent areas are simply cracks in the basaltic ocean floor from which diffuse, warm fluids (up to 30°C) flow (Hessler, Lonsdale et al. 1988; Childress and Fisher 1992; Fisher
1995). Many symbiont-containing vent organisms, including vescomyid clams, bathymodiolid mussels, and the vestimentiferan tubeworm Riftia pachyptila, thrive in the areas where vent fluid actively mixes with ambient seawater (Hessler, Lonsdale et al.
1988; Scott and Fisher 1995).
Cold seeps are associated with active and passive margins in oceans all around the globe (Sibuet and Olu 1998). Often referred to as “cold seeps” due to the absence of superheated venting, these environments are found where organic carbon seeps upwards into surface sediments. Hydrocarbon seeps are a type of cold seep and were discovered 3 on the continental margin in the northern Gulf of Mexico (GoM) in 1984 (Brooks, Cox et al. 1986). Oil and gases are released by and migrate vertically through deep, Jurassic salt induced faults in layers of oil-bearing shale and reach the sea floor over shallow salt and along fault traces (Brooks, Kennicutt II et al. 1987; Sassen, Brooks et al. 1995). As a result of this vertical migration of compounds, methane and other hydrocarbon gasses of thermogenic or biogenic origin, crude oil, hydrogen sulfide, and supersaturated salt brine can be found in seep sediments (Behrens 1988; Roberts and Aharon 1994).
Hydrocarbon seeps, like hydrothermal vents, often support large communities based on chemoautotrophy (MacDonald, Boland et al. 1989; MacDonald, Guinasso Jr. et al. 1990; Fisher 1996). In contrast to hydrothermal vents, which are highly ephemeral and only stable for years to decades, hydrocarbon seep sites in the northern Gulf of
Mexico are able to support chemoautotrophic species for thousands of years (Powell
1995). Active venting of hydrogen sulfide rich fluids into ambient waters at hydrocarbon seeps is greatly reduced, also in stark contrast to hydrothermal vent environments. These differences are due to the underlying geologic conditions and forces that govern the formation and cessation of these environments and also are ultimately the source of any reducing chemicals present. In shallow cold seep sediments (<30cm) most sulfide is produced biogenically, along with carbonate rock, via the reduction of sulfate by free living bacteria (Sassen, Roberts et al. 1993). This process occurs just below the sediment-seawater interface, is coupled to the oxidation of methane, and is thought to be a primary source of sulfide at seep sites (Arvidson and Morse 2000; Boetius,
Ravenschlag et al. 2000). There are, however, likely to be additional deeper sources of sulfide and/or sulfate beneath cold seep environments (Carney 1994). One possible 4 source is the bacterial reduction of gypsum on limestone caprock associated with deep,
methane rich brine reservoirs according to the reaction: (CaSO4 + CH4 ! H2S (aq. or gas)
+ H2O + CaCO3) (Roberts and Carney 1997; Kirkland, Denison et al. 2000). This hydrogen sulfide could then be carried up into more shallow sediments by rising hydrocarbons and brine. Lastly, significant quantities of hydrogen sulfide in cold seep sediments may result from sulfate reduction coupled to microbial degradation of long chain hydrocarbons (Wade, Kennicutt II et al. 1989; Spormann and Widdel 2000;
Zwolinski, Harris et al. 2000).
II. Vestimentiferan Biology
Vestimentiferan tubeworms are considered community-structuring animals at both hydrothermal vent and hydrocarbon seep environments (Bergquist, Ward et al.
2003; Tsurumi and Tunnicliffe 2003). One of the most prominent organisms at East
Pacific Rise hydrothermal vents is the vestimentiferan tubeworm Riftia pachyptila (Jones
1981). Much of what is currently known about vestimentiferan biology comes from studies on this species. Like all other known vestimentiferans, R. pachyptila secretes a chitinous tube (Gaill, Herbage et al. 1988), that can reach lengths of two meters or more, and from which only the bright red anterior plume is obtruded into the environment. R. pachyptila uses this tube to anchor itself to the substrate of the vent environment, and can pull its plume completely inside of the tube for protection from occasional extreme temperature or predation. The body of this large annelid worm can reach 1.5 m in length and 40 mm in diameter (Jones 1988). R. pachyptila has also been reported to be one of the fastest growing invertebrates known, and reaches sexual maturity in just one to two 5 years (Lutz, Shank et al. 1994). All vestimentiferans have four distinct body regions: the anterior, highly vascularized plume, the muscular vestimentum, the trunk, and the posterior opisthosome (Jones 1988). The vestimentum contains a simple heart that pumps hemoglobin-rich blood through an extensive, closed vascular system. The trunk portion is basically a sac that contains a non-circulating coelomic fluid that bathes a large tissue called the trophosome and is freely able to exchange materials with the vascular system (Childress and Fisher 1992). The trophosome is highly vascularized and houses chemoautotrophic bacterial endosymbionts inside of batcteriocytes produced by the host
(Hand 1987). Adult vestimentiferans do not have a mouth, gut, or intestinal tract, and are dependent upon carbon compounds (succinate and glutamate) synthesized by their sulfide-oxidizing bacterial endosymbionts for nutrition (Jones 1981; Felbeck and
Childress 1988; Wilmot and Vetter 1990; Felbeck and Jarchow 1998).
As vestimentiferan symbionts are located deep inside the body of the tubeworm
- host, reactive gasses (HS , O2, and CO2) must be transported from the environment and supplied to the bacteria by the host (Childress, Fisher et al. 1991). The vestimentiferan vascular system is able to transport large quantities of these chemicals to the trunk of the animal where they can then be temporarily stored by the coelomic fluid (Fisher, Childress et al. 1988; Childress, Fisher et al. 1991). Both the vascular blood and coelomic fluid of
Riftia pachyptila contain giant, extracellular hemoglobins that are able to reversibly bind both oxygen and sulfide with high affinity and high capacity (Arp and Childress 1983;
Arp, Childress et al. 1987; Fisher, Childress et al. 1988). As there are separate sulfide and oxygen binding sites on vestimentiferan hemoglobins, there is no interaction between sulfide binding and oxygen binding (Childress, Arp et al. 1984). The vascular blood 6 contains two hemoglobins that have masses of ca. 3,500 kDa and 400 kDa, respectively, while the coelomic fluid contains a different 400 kDa hemoglobin (Zal, Lallier et al.
1996). For reference, human hemoglobins are intracellular and are 68 kDa in size. Both the 400 kDa and 3,500 kDa vestimentiferan hemoglobins are composed of monomer and dimer heme-containing chains. The 3,500 kDa hemoglobin also contains linker chains that appear to hold the quaternary hexagonal bilayer structure together (Toulmond 1992;
Zal, Suzuki et al. 1997). As a result of the high affinity of these hemoglobins for sulfide and their high concentrations in R. pachyptila vascular blood, concentrations of bound sulfide can be as high as two orders of magnitude greater than external sulfide concentrations, while free sulfide in the blood of R. pachyptila can simultaneously be maintained at concentrations an order of magnitude lower than environmental concentrations (Fisher, Childress et al. 1988; Childress, Fisher et al. 1991; Scott and
Fisher 1995). Thus, vestimentiferan hemoglobins provide not only mechanisms for the transportation and storage of vital metabolites, but also allow highly reactive hydrogen sulfide to move throughout the animal without ill effects for its respiration, or its symbionts (Powell and Somero 1983; Fisher, Childress et al. 1988).
III. Gulf of Mexico seep vestimentiferans and their sulfide physiology
Vestimentiferan tubeworms are very abundant at cold seep sites on the upper
Louisiana Slope in the northern Gulf of Mexico (MacDonald, Boland et al. 1989;
MacDonald, Guinasso Jr. et al. 1990). Aggregations of tubeworms at these cold seeps can consist of hundreds of thousands of individuals and cover areas as great as 1600 m2
(Brooks, Kennicutt II et al. 1989). Seep vestimentiferans are morphologically and 7 taxonomically similar to vent vestimentiferans (Nelson and Fisher 2000), however, there are some notable differences. Unlike the vent vestimentiferan R. pachyptila, seep tubeworms grow posteriorly as well as anteriorly, extending posterior portions of their tube and tissues into the sediment below the original point of attachment (Julian, Gaill et al. 1999). Seep vestimentiferans are thinner in diameter than the robust R. pachyptila, with mature seep tubeworms typically tapering from an anterior tube diameter of approximately ten mm to less than one mm at their posterior end. And also unlike R. pachyptila, seep tubeworms inhabit the entire length of their tubes (Scott and Fisher
1995).
Two vestimentiferan species, Lamellibrachia luymesi and Seepiophila jonesi, co- occur in bush-like aggregations at northern Gulf of Mexico cold seeps sites. Presently, data suggests that the planktonic larvae of L. luymesi and S. jonesi require hard substrate and active seepage for settlement to occur and areas suitable for settlement are limited
(Bergquist, Urcuyo et al. 2002). Juvenile tubeworms at Gulf of Mexico cold seeps are often found attached to hard substrate, generally authigenic carbonate rock, in areas of active seepage where bacterial mats and mussel beds are common on the surface of the sediments. Over the course of the long lives of these tubeworms, their posterior ends and original attachment points may become buried by over 0.5 m of sediment that accumulates in the bush formed by their tubes (MacDonald, Guinasso Jr. et al. 1990). L. luymesi, the more abundant species in Gulf of Mexico cold seep tubeworm communities, grows quite slowly to lengths exceeding two meters and lives in excess of 170 to 250 years (Fisher, Urcuyo et al. 1996; Bergquist, Williams et al. 2000; Bergquist, Urcuyo et al. 2002). Typically, S. jonesi does not exceed 1 m in length and grows so that its 8 anterior plume is much closer to the sediment level than L. luymesi, which grows so that its plume reaches heights as great as 1.5 meters above the sea floor (MacDonald, Boland et al. 1989). Aggregations consisting entirely of very large S. jonesi have been observed and may be an indication that S. jonesi can live even longer than L. luymesi (Bergquist,
Urcuyo et al. 2002).
As do all vestimentiferans, L. luymesi and S. jonesi depend upon their sulfide- oxidizing symbionts for nutrition and, therefore, must acquire hydrogen sulfide from the environment and supply it to their endosymbionts in order to survive (Fisher 1996).
When vestimentiferans were discovered at Gulf of Mexico seep sites scientists assumed that seep tubeworms were, like their hydrothermal vent relatives, using their anterior plumes as the primary site of gas exchange. Therefore, a paradox emerged in the understanding of vestimentiferan physiology when sulfide was found to be generally undetectable around the plumes of Lamellibrachia luymesi (MacDonald, Boland et al.
1989; Scott and Fisher 1995). If any sulfide were to reach the plumes of L. luymesi, it would have to diffuse from the sediment-water interface upward through the water column. As sulfide spontaneously reacts with oxygen (Cline and Richards 1969), the likelihood of significant amounts of sulfide existing in ambient waters at cold seep sites decreases as distance from the sediment-water interface increases.
Further examination of Gulf of Mexico cold seep environments and tubeworms produced a substantial body of circumstantial evidence that suggested mature L. luymesi may acquire sulfide from the environment using the posterior extension of their tubes and tissue which Julian and coworkers (1999) termed a ‘root’ (Julian, Gaill et al. 1999).
Detectable levels of sulfide are not only absent around the plumes of most 9 vestimentiferans on the Upper Louisiana Slope, sulfide is often undetectable within the aggregations (much closer to the sediment) and even in pore waters from 10 cm beneath the sediment under mature tubeworm aggregations (Scott and Fisher 1995). However, using a device that allowed collection of deep interstitial water samples, concentrations of hydrogen sulfide as high as 7.9 mM have been measured in water samples collected amongst the buried posterior ends of the tubeworms at greater depths in the sediments
(Bergquist, Andras et al. 2003). Julian and coworkers (1999) also reported that posterior roots extend into the sediment below the original point of attachment and that these buried root tubes are 700 times more permeable to hydrogen sulfide than the more robust anterior portions of the tubes which extend into the water column. The purpose of
Chapter Two is to determine if L. luymesi is able to use its posterior root to acquire sulfide, and if sulfide uptake by the root could sustain net chemoautotrophy.
The hydrothermal vent tubeworm Riftia pachyptila positions its plume where venting effluent actively mixes with the surrounding bottom water (Hessler, Lonsdale et al. 1988). As a result, sulfide concentrations around the plume of R. pachyptila can be highly variable, ranging from > 300 µM to 0 µM in only seconds (Johnson, Childress et al. 1988). It has been hypothesized that the high affinity for sulfide of the hemoglobins in the vascular fluids of R. pachyptila enable sulfide to be acquired from low environmental concentrations while the high capacity of these same hemoglobins allow sulfide concentrations to be as high as 10mM in the vascular fluids (Childress, Fisher et al.
1991). In an attempt to understand the processes of sulfide acquisition and physiology of
R. pachyptila the sulfide capacities of vascular and coelomic hemoglobins and the sulfide 10
affinity of mixed R. pachyptila hemoglobins have been estimated (Arp, Childress et al.
1987; Fisher, Childress et al. 1988; Zal, Suzuki et al. 1997). The objectives of Chapter
Three are twofold; to describe the most appropriate methods for determining the sulfide-
binding properties of vestimentiferan vascular and coelomic hemoglobins, and to provide
estimates of the sulfide-binding affinity and capacity for purified R. pachyptila
hemoglobins.
By definition, niche differentiation takes place when co-occurring species
decrease interspecific competition by dividing limited resources (Schoener 1974). The
noticeably different growth morphologies of L. luymesi and S. jonesi have led to the hypothesis that, though these two cold seep tubeworm species live in extremely close association with one another, they may exploit different sources of sulfide in their environment. The growth morphology of S. jonesi may be an adaptation for obtaining hydrogen sulfide across its plume from seep fluids near the sediment-seawater interface.
If S. jonesi is able to obtain sulfide from the low concentrations that exist above the
sediment and L. luymesi preferentially acquires sulfide from the much higher
concentrations that are found sub-sediment, then there are likely differences in the
sulfide-binding properties of the vascular and coelomic hemoglobins and intact fluids of
each species. The purpose of Chapter Four is to determine the relative hemoglobin
abundance of intact L. luymesi and S. jonesi vascular and coelomic fluids, the sulfide-
binding affinity and capacity of the isolated component hemoglobins, and to compare
these estimates to those of R. pachyptila hemoglobins. The implications of these results 11 as they pertain to the whole-animal sulfide physiology of L. luymesi and S. jonesi is discussed.
Though chemoautotrophic communities were discovered in the Northern Gulf of
Mexico almost twenty years ago, there is still much to learn about the sulfide physiology of the vestimentiferan tubeworm species that are community-structuring organisms in these environments (Bergquist, Ward et al. 2003). It is known that these animals are often located in areas where hydrocarbons are actively released from the seafloor and that active seepage is an indication that hydrocarbon reservoirs lay beneath (Kennicutt II,
Brooks et al. 1988). At present, it can only be hypothesized that the survival of
Lamellibrachia luymesi and/or Seepiophila jonesi is associated with hydrocarbon availability. With a more extensive understanding of the ecological physiology of hydrocarbon seep vestimentiferan species there is an increased likelihood that the exploitation of natural resources in Gulf of Mexico seep environments may take place in a fashion that ensures the survival of these deep-sea communities. This thesis increases the understanding of autotrophic symbioses in general and vestimentiferan physiological ecology in particular, while focusing on the physiology of Lamellibrachia luymesi and
Seepiophila jonesi. The purpose of Chapter Five, the final chapter, is to summarize the results and conclusions and discuss the relevance of this work. 12
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Chapter 2
(As published in The Proceedings of the National Academy of Science)
Manuscript Classification: Biological Sciences: Physiology
A paradox resolved: Sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy
John K. Freytag1, Peter R. Girguis2, Derk C. Bergquist1, Jason P. Andras1, James J. Childress2, and Charles R. Fisher1
1 - Department of Biology, The Pennsylvania State University, University Park, PA 16802
2 - Department of Ecology, Evolution and Marine Biology, University of California Santa Barbara, CA 93106
Corresponding Author: John K. Freytag, Department of Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, PA 16802 Phone: (814) 863-8360, Fax: (814) 865-9131, Email: [email protected]
Manuscript Information:
Twenty four text pages. One figure on one page. Three tables on three pages.
Word and character counts:
219 words in Abstract 46,880 characters in paper 22
Abstract:
Vestimentiferan tubeworms, symbiotic with sulfur oxidizing chemoautotrophic bacteria, dominate many cold seep sites in the Gulf of Mexico. The most abundant vestimentiferan species at these sites, Lamellibrachia cf luymesi, grows quite slowly to lengths exceeding two meters and lives in excess of 170 to 250 years. L. cf luymesi can grow a posterior extension of its tube and tissue, termed a “root,” down into sulfidic sediments below its point of original attachment. This extension can be longer than the anterior portion of the animal. Here we show, using methods optimized for detection of hydrogen sulfide down to 0.1 µM in seawater, that hydrogen sulfide was never detected around the plumes of large cold-seep vestimentiferans and only rarely detectable around the bases of mature aggregations. Respiration experiments which exposed the root portions of L. cf luymesi to sulfide concentrations between 51 and 561 µM demonstrate that L. cf luymesi utilize their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 µmoles*g-1*h-1. Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior, root, portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan L. cf luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake. 23
Introduction:
Hydrothermal vent and hydrocarbon seep environments support large communities
sustained by chemoautotrophy, which is in turn based on local sources of reduced chemicals
(1). Vestimentiferan tubeworms are the most conspicuous organisms at many of the
hydrothermal vent sites in the eastern Pacific as well as many of the cold seep sites in the
Gulf of Mexico (2, 3). All vestimentiferans harbor chemoautotrophic bacterial
endosymbionts and live autotrophically with hydrogen sulfide as their energy source (4, 5).
The vestimentiferan Riftia pachyptila inhabits ephemeral hydrothermal vent habitats and is
reported to be among the fastest growing of invertebrates, reaching tube lengths of 1.5 m
and sexual maturity in two years (6). R. pachyptila thrives in areas of the vent field where
warm, sulfide-rich hydrothermal fluids rise and actively mix with the overlying oxic bottom
water, and it obtains both the hydrogen sulfide and oxygen required by the symbiosis
directly across its plume (4, 7-9).
Lamellibrachia cf luymesi is the dominant species of vestimentiferan found at most of the known cold seep sites at less than 1000 m depth on the upper Gulf of Mexico continental slope (10-12). Single aggregations of tubeworms at these cold seep sites normally contain between 500 and 2000 individuals, however some aggregations include hundreds of thousands of individuals and cover areas as great as 1600 m2 (11). In comparison to its vent relative R. pachyptila, L. cf luymesi grows quite slowly, averaging about one cm per year, and lives in excess of 170 to 250 years (13, 14).
Based on studies of R. pachyptila, it was expected that other vestimentiferans would exclusively utilize their plumes for gas exchange, including acquisition of sulfide from the environment (4). A paradox emerged in the understanding of seep vestimentiferan 24 physiology when investigators consistently discovered that sulfide was generally undetectable (with detection limits between two and ten µmol l-1) around the plumes of
Gulf of Mexico vestimentiferan tubeworms (15-17). In retrospect, this is not surprising as the sulfide that supports tubeworm communities at cold seeps in the Gulf of Mexico is produced biogenically in shallow sediments (18), and seeping pore fluids are not warm and buoyant like those that bathe the plumes of hydrothermal vent vestimentiferans. There now exists a substantial body of circumstantial evidence suggesting that mature L. luymesi take up sulfide from the environment using the posterior extension of their tubes and tissue which Julian and coworkers have termed a ‘root’ (17). These workers report that posterior roots extend into the sediment below the original point of attachment and that these buried root tubes are 700 times more permeable to hydrogen sulfide than the more robust anterior portions of the tubes, which extend upwards into the water column. They also report that although sulfide was often undetectable in pore waters 5-10 cm beneath mature tubeworm aggregations (16), concentrations as high as 2,700 µM were present in water samples collected amongst the buried posterior ends of the tubeworms at greater depths in the sediments (17).
Here we present two independent data sets that support the hypothesis that L.cf luymesi utilize the posterior portions of their tubes to acquire sulfide in situ. First, because the very high affinity of vestimentiferan hemoglobin for sulfide would allow uptake from very low environmental levels (19), we use a very sensitive sampling and analysis technique to confirm that at most, very low (<0.1 µM) levels of sulfide are present around the plumes of mature cold seep vestimentiferans. Second, we demonstrate that in the laboratory, L. cf luymesi take up sufficient sulfide across posterior portions of its tube and body to support net autotrophic carbon 25 uptake by the intact symbiosis. Although these results do not indicate to what extent seep vestimentiferans rely on their “roots” for sulfide acquisition in situ, they do demonstrate a need for a subsurface sulfide uptake mechanism and that a viable mechanism exists.
Methods:
Water samples were taken through small diameter polyetheretherketone (PEEK) tubing directly into a syringe in the rear chamber of the DSRV Johnson Sea Link (20), during a July, 1998 research expedition. The samples were taken from among four young aggregations (largest animals < 0.5m in length) and eight mature aggregations (largest animals >1.0m in length). At each aggregation, water samples were taken from around the plumes of animals, near the sediment-water interface at the base of the aggregations, and approximately halfway between plume level and sediment level among the tubes of the animals. Total sulfide was quantified using a modification of the methods of Singh et. al. (21). 700 µl of the sample was immediately combined with 150 µl of papain-
SSCH3 solution (2.0 mg/ml papain-SSCH3 (Molecular Probes), 8.0 mM ethylenediaminetetraacetic acid (EDTA), 60 mM NaH2PO4, 1.0 mM sodium acetate) under a nitrogen head and stored on ice until the end of the dive. Upon recovery of the submersible the samples were incubated at room temperature for one hour and then combined with 150 µL of a solution of chromogenic substrate [7.2 mg/ml N-benzoyl-L- arginine, p-nitroanilide (L-BAPNA) (Molecular Probes), 3 mM EDTA, 150 mM bis-Tris] for an additional one hour, at which point the enzymatic digestion was stopped by addition of 5.0 µl of 1.0 mM phenylmethylsulfonyl fluoride (PMSF) (Boehringer
Mannheim) and absorbance of the samples at 410 nm was noted. Sulfide stock solutions
(about 1 mM) were made up at sea from pre-weighed and washed sodium sulfide crystals 26 stored in sealed vials under nitrogen and their concentrations determined by gas chromatography (22). The sulfide stock solutions were diluted to concentrations of 20 and 100 µM with degassed distilled water in a glove bag under a nitrogen atmosphere and used immediately to prepare the sulfide standards (0.1-5.0µM) that were processed in parallel with the water samples. This relationship between sulfide concentration and sample absorbance was linear between 0.3 µM and 5.0 µM, with concentrations between
0.1 and 0.3 µM detectable as consistent non-zero absorbance values. Samples taken between 100 and 400 m above the bottom at the beginning and end of each dive served as experimental sulfide-free controls.
Aggregations containing Lamellibrachia cf luymesi and an escarpid-like species of tubeworm were collected in July, 1998 from the outskirts of the Brine Pool NR1 hydrocarbon seep site in the Gulf of Mexico (27°43’24” N, 91°16’30” W) from a depth of 640 meters. Tubeworms were collected with the DSRV Johnson Sea Link using a hydraulically actuated net (“Bushmaster Jr.”) that collects intact aggregations of vestimentiferans along with the carbonate rock to which the animals are attached (14).
The animals were brought to the surface in a temperature-insulated box mounted on the front of the submersible and, upon reaching the surface, were immediately transferred to aerated, chilled (7 °C) seawater and held at atmospheric pressure. The blood sulfide uptake experiments were initiated at sea 24 hours after collection of the animals used in these experiments. Upon returning to port, two intact aggregations of tubeworms were shipped in cold seawater to Santa Barbara, CA. At U.C.S.B. the animals were maintained in a flow-through aquarium at 5°C. Only apparently healthy, active, and undamaged animals with intact tubes were used in this study. The first set of split-vessel respiration 27 experiments and the balance of the blood sulfide uptake experiments were conducted within two weeks of collection of the animals. The second set of split vessel respiration experiments and the undivided vessel respiration measurements were made on animals held in the laboratory for ten months.
Blood sulfide uptake experiments were conducted in split vessel incubation chambers that allowed isolation of the posterior and anterior halves of the tubeworms into different pools of flowing water (3-4 ml/min). Vessel halves were constructed of polycarbonate tubing (7.6 cm inner diameter) that were separated by two layers of latex sheeting sandwiching 1.25 cm of lard, resulting in a watertight seal around the animals and between the two halves of the vessel.
Single individuals or pairs of L.cf luymesi were first maintained in sulfide free seawater for a minimum of 24 hours and then were placed into the system for periods of time ranging from 24 to 120 hours. Their roots were exposed to anoxic seawater containing dye (to confirm the integrity of the seal between chamber halves) and 500 µM sulfide in the posterior half of the vessel, while the plumes of the animals were exposed to ambient Gulf of Mexico surface
seawater ([O2] = 200µM) in the anterior half of the vessel. Control animals were run in parallel and had their roots exposed to dyed deoxygenated seawater that did not contain sulfide. All experiments were conducted at 7°C. Upon termination of the experiment, animals were removed from the incubation chambers, and immediately sacrificed. Samples of mixed coelomic and vascular fluids were collected and total sulfide (free and bound) concentration was determined by gas chromatography (22).
Respiration experiments were conducted at 5 °C at ambient pressure in split vessel flow- through respiration chambers that were similar in design to the vessels used for the sulfide uptake experiments, except that the vessel halves had smaller interior diameters (2.6 cm) and the 28 lengths were customized to accommodate the anterior or posterior of the animals in a minimum volume of water (105 ml in the top half and 160 ml in the bottom half of the chamber). To further assure that no sulfide leaked into the upper half of the vessel (around the animals’ plumes) during the respiration experiments, a slight positive pressure (0.2-0.4 atm) was maintained between the anterior and posterior halves of the vessels when both were flowing, using back-pressure valves (Circle Seal, Inc.) on the respirometer chamber exit streams (Fig 2.1).
For each experiment, groups of animals were placed into the split-vessel flow-through respirometry chamber while a chamber without animals was simultaneously run in parallel as a control. The desired gas concentrations in the bottom stream of inlet water were achieved by bubbling the filtered seawater with appropriate mixtures of H2S, O2 and N2 gases in a gas equilibrium column (23). Gas flow into the equilibration column was regulated with mass flow controllers (Sierra Instruments, Inc.) and the resultant concentrations of gases in the inlet water determined by gas chromatography (22). Flow (between 1 and 3 ml/min depending on the experiment) was maintained with low pressure metering pumps (Prominent Industries), and effluent streams were directed to analysis via stream selection valves (VICI, Inc.). Flow through the chambers served as the only means of mixing inside of the chambers. The relative concentrations of dissolved inorganic carbon (DIC), O2 and H2S in the effluent streams
(experimental and control) were measured by membrane inlet mass spectrometry (23, 24), and absolute concentrations determined periodically by gas chromatography (22) in samples removed from the in-line sampling ports (effluent samples for calibration were taken immediately before effluent analysis by the mass spectrometer; Fig 2.1). The pH of the seawater in the lower half of the experimental vessels ranged from 6.9 in the presence of 800 µM sulfide to 8.0 in the absence of sulfide. All calibration correlations were linear within our operating range, and had r2 values 29 in excess of 0.90. This system allowed the independent monitoring of oxygen, DIC and hydrogen sulfide flux by anterior (plume) and posterior (root) portions of the worms under experimental conditions of our choosing. Each of the effluent streams (experimental and control) was analyzed for periods of 13.6 minutes with continuous rotation from one sample stream to another. Only data collected a minimum of 5 hours after the animals had been introduced into this system was used for calculation of the flux rates, as this allowed the animals sufficient time to acclimate to the chamber and for the levels of gases in the effluent streams to stabilize.
Mass specific flux rates were determined by calculating the differences in dissolved gas concentrations between the experimental and control chambers, while taking into consideration flow rates and the mass of animals in the experimental chamber. During the experiments, each effluent stream was sequentially analyzed by the mass spectrometer for 13.6 minutes per stream
(the time required for the mass spectrometer to complete 25 scans). The data acquisition software eliminated the first 5 scans (to allow for flushing of our membrane inlet system) and the remaining data were averaged and logged as partial pressures. These data points were converted to concentrations via our calibration curves. Each rate (“n” in Tables 2.2 and 2.3) used to determine the average respiration rate for a given set of conditions was calculated from the difference between gas concentration values measured in control and experimental chambers in two subsequent time periods of 13.6 minutes, with a break of 13.6 minutes between each paired analysis. When conditions were not altered for several hours, these rates were averaged to yield a mean rate for a given set of conditions (Tables 2.2 and 2.3). Therefore, the values for “n” given in tables 2.2 and 2.3 are not independent experiments, but rather independent measurements made during a single extended experiment. 30
In a first set of respiration experiments the apparatus was configured to alternate between
analyzing gas flux in the anterior and posterior compartments of the split experimental and
control vessels. The posterior ends of groups of three L. cf luymesi were exposed to oxygen-free seawater containing approximately 220, 310, or 430 µM sulfide for at least 24 hours while the anterior ends of the same animals were exposed to seawater containing 200 µM oxygen and no sulfide. The first group of three animals was exposed to 220µM sulfide and then removed to sulfide free water for a period of 66 hours while a different group of three animals was exposed to 430 µM sulfide. The first group was then returned to the system and exposed to 310 µM sulfide.
For the second set of experiments the apparatus was configured to monitor only changes in the concentrations of gases around the anterior ends of a group of three L. cf luymesi before, during, and after sulfide was included in the water bathing their roots. This improved our power to detect differences in dissolved inorganic carbon flux, as twice the number of measurements could be made during each set of conditions. For this experiment a different group of three L. cf luymesi was maintained in the system for 8 days with the posterior ends of the animals exposed sequentially to anoxic sulfide-free water, anoxic sulfidic water, and anoxic sulfide-free water.
To change conditions in the posterior half of the vessel, the anterior vessel half was sealed to prevent leakage from the posterior portion and the posterior half was flushed at a rate of about 20 ml/min. with the new solution for 2 - 5 hours. Flow rates of 1.6 to 2.0 ml/min were then reestablished in the upper half of the system. Rates were calculated after a minimum of an additional 5 hours had passed to assure the system had reached equilibrium. In these experiments, sulfide levels in the posterior compartments were monitored only by gas chromatography of discrete samples (22). As an additional control, the animals were sacrificed 31 and removed from their tubes after the last set of experiments, and the empty tubes were returned to the respiration system and monitored for 15.5 hours.
Determining the lengths of the “root” portion of most individuals collected to date has proven to be very difficult as the posterior portions of all animals in most aggregations are tightly intertwined and virtually impossible to untangle. This is especially true of larger animals in larger aggregations. However, 59 L. cf luymesi with intact roots were successfully separated from a single aggregation of young individuals and their root and anterior tubes measured using twine to trace the lengths.
Statistical analyses and comparisons (two sample t-tests, ANOVA, regression analysis, and descriptive statistics) were calculated using Minitab 12 (Minitab Inc.).
Results
Sulfide was detected in four (25%) of the sixteen water samples taken among the tubes of vestimentiferans in young aggregations and in four (66%) of the six samples taken at the sediment water interface at the bases of these aggregations. These young aggregations occurred in areas of active seepage as evidenced by the co-occurrence of bacterial mats, partially exposed authigenic carbonates, and/or mussel beds. The highest level of sulfide in any of these water samples was 2.70 µM (avg. = 0.53 µM, SD = 0.91, n = 22). However, sulfide was never detected in water samples taken around the plumes of older (partially buried) tubeworm aggregations and was only rarely detected in samples taken half-way between the plumes and the sediment surface or even at the sediment surface in these aggregations (Table 2.1). In fact, four of the five water samples from mature aggregations that contained detectable levels of sulfide 32 were taken from among one aggregation that was immediately adjacent to an aggregation of young tubeworms.
The initial blood sulfide uptake experiments confirmed that sulfide uptake across the posterior portions of live L. cf luymesi could occur. Sulfide was not detectable in the blood of any of the three control animals (detection limit of 3.0 µM) but was present in the blood of both experimental animals (152 and 170 µM). Even with the low level of replication, the difference between experimental and control animals was significant
(T=17.67, DF=1, P = 0.036).
Sulfide uptake by posterior portions of L. cf luymesi occurred at all sulfide concentrations tested in the respiration experiments and was accompanied by DIC and oxygen uptake across the plumes (anterior ends) of the animals (Table 2.2). No DIC flux was detectable across the root portion of the worms. Before exposure of their roots to sulfide, DIC was produced and oxygen was consumed by the worms (Table 2.3), as is typical of heterotrophic animals. Shortly after sulfide was added to the water bathing the animals’ roots DIC flux reversed direction to net consumption (Table 2.3). When sulfide-free water was again supplied to the posterior chamber, oxygen consumption rates decreased sharply and DIC flux again reversed direction to heterotrophic production over a period of about 17 hours (Table 2.3).
The root portion of the tubes of the 59 small L. cf luymesi (2.0 – 50.2 cm total length) from an aggregation of young animals ranged from 26% to 91% (avg. = 61%) of the total tube length. 33
Discussion:
Vestimentiferan tubeworms have no mouth or gut and obtain all of their nutrition from their symbionts. Their symbionts are sulfide oxidizing chemoautotrophs and require sulfide to fuel autotrophic carbon fixation (and ultimately support their host) (4). Any sulfide that reaches the plumes of the vestimentiferan tubeworm Lamellibrachia cf luymesi, must travel from the sediment upward through the water column. However, cold seep fluid is not as buoyant as hydrothermal fluid and the plumes of L. cf luymesi are often located well over a meter above the sediment. As a result of spontaneous reactions between sulfide and oxygen (25), the likelihood of significant amounts of sulfide existing in bottom waters at cold seep sites decreases as distance from the sediment-water interface increases. However, hydrothermal vent vestimentiferan blood has a high affinity for sulfide (19), and preliminary data (J.K.F. unpublished) indicates that seep vestimentiferan hemoglobins have a similarly high affinity for sulfide. Thus, although earlier workers had found sulfide levels to be below their detection limits of 2 – 10 µM around the plumes of seep vestimentiferans (15-17), these observations did not rule out the presence of sufficient sulfide around the plumes to support significant sulfide uptake rates. Using a much more sensitive sampling and analysis protocol, the highest concentration of sulfide found in water samples taken from the sediment-water interface around mature tubeworm aggregations was 3.7 µM, with 81% of water samples taken from this locale containing no detectable sulfide (< 0.1µM). Not surprisingly, detection of sulfide in water samples taken halfway between the sediment-water interface and the plumes of tubeworms in mature aggregations (0.5-0.75m above the sediment-water interface) was even more rare, and the maximum concentration of sulfide detected was 0.5 µM. No sulfide was detected in any water sample taken from around the plumes of tubeworms in mature aggregations (Table 2.1). Taken 34 together, these data offer strong support for the hypothesis that, as previously suggested (15-17), adult seep tubeworms must acquire sulfide from interstitial sources.
The results of the three sets of experiments in the split experimental vessels clearly demonstrate that live L. cf luymesi are capable of sulfide acquisition via their roots.
Furthermore, these experiments demonstrate that in the laboratory L. cf luymesi can acquire sufficient sulfide across its roots to elicit and sustain net DIC uptake (net autotrophy). The blood uptake experiments demonstrated that blood sulfide levels similar to those of freshly collected animals could result from root exposure to sulfide alone. In the split vessel respiration experiments, all animals demonstrated net DIC uptake across their plume when exposed to sulfide at their posterior end only (Tables 2.2 and 2.3). When not exposed to sulfide, the animals’ metabolism is in heterotrophic poise, releasing DIC into the water. However, net DIC flux changed from positive to negative (production to consumption) when sulfide was added to the water around their posterior end (Table 2.3). None of the gas fluxes measured during sulfide exposure were significantly correlated with posterior chamber sulfide concentrations, which is not unexpected as gas flux was measured with three different sets of animals and only 4 independent experiments were conducted.
Acquisition of sulfide by L. cf luymesi across the root portion of its body results in the spatial separation of hydrogen sulfide uptake from oxygen uptake and may allow this species to survive in a habitat where sulfide and oxygen do not co-exist in the water column. The ability to spatially separate the acquisition of sulfide and oxygen has been described in bivalves endemic to vent and seep habitats (26-28), and thus is not unique to L. cf luymesi. However, adult L. cf luymesi are much longer animals, and can potentially separate sulfide and oxygen acquisition by distances measured in meters. 35
Julian and co-workers modeled root sulfide uptake by a 10 gram L. cf luymesi with a 40 cm root and concluded that sufficient sulfide could be taken up across the root by diffusion to meet the animal’s metabolic requirements. This was based on the rates of sulfide diffusion across the root tube measured by the authors, an assumed metabolic rate approximately one tenth that of R. pachyptila, and a variable sulfide concentration along the length of the root tube that averaged 155µM (17). The ratio of animal biomass to vessel volume necessitated by the size and shape of the experimental animals resulted in a relatively small signal in the respirometer system, and as a result the absolute rates reported here should be considered estimates. However, the sulfide and oxygen consumption rates, and heterotrophic DIC production rates reported here are about 40% of those most recently reported for R. pachyptila (29). These lower metabolic rates are quite reasonable considering the large differences in the growth rates and environments of these two species and, may be a factor in the extreme longevity of L. cf luymesi (14, 30, 31).
Nevertheless, Julian and co-workers may have underestimated the metabolic rate of L. cf luymesi in their calculations. On the other hand, measurements of the roots of very small animals and estimates of root length for larger animals indicate that even animals as small as one gram can have a 40 cm root, and larger animals have much larger roots. These two refinements to the model of Julian et al. have opposite effects of similar magnitude that offset each other and their general conclusions stand: root sulfide uptake can be sufficient to support the sulfide demands of the association if sufficient sulfide is present (and replenished) around the animals’ roots.
The animals selected for use in the respirometers were chosen because the aggregation was collected completely intact and several of the animals could be disentangled from the rest without damaging them. The clean collection and separation of individuals from this aggregation were facilitated because the roots of the animals in this aggregation were not nearly 36 as extensive as on most other intact aggregations successfully collected with the bushmaster collection devices. The animals used in this study averaged about 50 cm in total length, approximately one half of which was root tube. Repeated attempts to collect larger aggregations with completely intact roots have been unsuccessful, and have anchored the submersible to the seafloor with the bushmaster collection nets during the attempts. We suggest that the roots of adult L. cf luymesi are not only longer than that of the hypothetical animal modeled by Julian et al., but are also considerably more extensive than those of the animals used in this study, and thus are capable of even higher rates of sulfide uptake.
At this point we have insufficient data to draw conclusions concerning the relative importance of root versus plume sulfide uptake by seep vestimentiferans in situ. It is likely that it varies considerably between aggregations and over the life of the vestimentiferans. The vestimentiferan plume is a very efficient gas exchange organ due to its large surface area and short diffusion distances into blood spaces, and if sulfide is present around the plumes of animals in an aggregation this is likely to be the main route of sulfide uptake. Unlike in larger aggregations, sulfide is often detectable around the plumes of animals in younger aggregations, where the common co-occurrence of mussels with methanotrophic symbionts also argues for a surface expression of seepage. It is likely that substantial sulfide uptake across the plumes occurs in these young aggregations. As the tubeworms grow and the aggregations age over several centuries, surface expression of seepage diminishes and the animals’ growth places their plumes as high as 1.5 meters above the sediment. At this point, the relative importance of sulfide uptake by buried portions of their body should increase. Because we do not know the extent of the buried roots of adult vestimentiferans, the density of these roots, the levels of sulfide in the 37 midst of the “rhizosphere”, or the rate of sulfide replenishment in this zone, we cannot calculate in situ rates of sulfide uptake across buried roots at this time.
As noted previously, the pH of the seawater in the posterior compartments of the vessels ranged from 6.9 during the experiment that exposed animals to the highest levels of sulfide tested
(800 µM) to that of ambient seawater (ca. 8.0) when no sulfide was present. Differences in
- seawater pH shift the proportion of the sulfide species present from ca. 40%H2S and 60%HS at
- pH 6.9, to 5%H2S and 95% HS at pH 8 (32), and further preclude using this data set to investigate the relation between sulfide exposure level and gas flux rates as we do not know
- which species of sulfide (H2S or HS ) is preferentially acquired by L. cf luymesi. This variation in the pH of the water surrounding the posterior portions of the animals during the experiments described here may have affected the rate of sulfide uptake by the roots, but would have had no effect on the DIC species present around the plume and therefore no direct effect on DIC uptake.
The fact that L. cf luymesi maintained in the laboratory for ten months were still capable of net autotrophy was not unexpected (Table 2.3). We have maintained L. cf luymesi for over two years in recirculating aquaria at PSU partially buried in sand, with sulfate reducing bacteria producing sulfide in the sediment and active aeration depleting sulfide that diffuses into the water column. The fact that many of these animals have grown at both their anterior and posterior ends (J.K.F. and D.C.B. unpublished observations) is further circumstantial evidence that sulfide uptake across their roots can support net autotrophy.
This study was conducted to confirm that, at most, extremely low levels of sulfide are available to L. cf. luymesi at plume level in situ and to test the hypotheses that L. cf.
Luymesi is capable of using the root portion of its body to acquire sulfide at rates sufficient to sustain net autotrophic DIC uptake. The next step is to collect the data 38 necessary to model sulfide requirements and acquisition by intact aggregations in order to constrain the magnitude of the importance of root sulfide uptake in situ. The morphology and extent of the buried portion (the “rootball”) of larger aggregations is still almost completely unknown, and are important components of a model because they will constrain the area available for sulfide uptake and intraspecific competition for buried sulfide among the individuals in an aggregation. Another key unknown at this time is the source of the deep interstitial sulfide and rates of replenishment of the rhizosphere sulfide. The primary source of sulfide at these seeps was thought to be anaerobic oxidation of seawater sulfate, but this process occurs almost exclusively in the upper 10 –
20 cm of sediment (33), and diffusion alone would be insufficient to supply significant amounts of sulfide to roots at greater depth. Other possible sources of sulfide include sulfide produced as a by-product of bacterial reduction of Castile gypsum on limestone caprock, which is often found in association with methane rich brine reservoirs (34, 35), and production of sulfide from the degradation (decomposition) of buried detritus (18).
An intriguing possibility suggested by Julian and co-workers is that L. cf luymesi releases sulfate into sediments around their roots. This could occur either through the pumping of seawater down the length of the tube by a peristaltic motion of the worm, or through the directed release of the sulfate produced as a waste product of chemoautotrophic sulfide oxidation (36).
Free-living chemoautotrophic sulfide-oxidizing bacteria require access to both sulfide and oxygen and most commonly occur in interface environments. These environments are normally only a few mm thick, although in areas where sulfide rich waters are actively expelled into oxic waters (as at hydrothermal vents and actively 39 venting cold seeps), both sulfide and oxygen co-occur in a much larger volume of water.
At cold seeps in the Gulf of Mexico active expulsion of seep fluid from the substrate, driven by rising methane gas and hydrocarbons, is very patchy and limited to relatively small areas within much more extensive seep communities (12). In areas of most active seepage, exposed authigenic carbonates, thick bacterial mats, mussel beds, and/or young vestimentiferan aggregations commonly occur. Existing data suggest that young seep vestimentiferans require active seepage of sulfidic fluids and acquire the sulfide required to support chemoautotrophy across their anterior plumes until posterior roots can be grown that allow interstitial sulfide sources to be tapped. Though expression of seepage at the sediment/seawater interface slows down over decadal time scales, vestimentiferan aggregations can persist for centuries and the ability to tap deep interstitial pools of sulfide may be a key factor contributing to their remarkable longevity. With the ability to draw sulfide from well below the sulfide/oxygen interface, seep tubeworms may limit the expression of sulfide at the sediment surface. They thereby exclude, via competion for this required resource, both mats of free-living aerobic chemoautotrophic bacteria (that are restricted to habitats where oxygen and sulfide co-occur) as well as other symbiont- containing species that do not have the ability to acquire sulfide from as deep in the sediment. Furthermore, because the pool of sulfidic water is tapped before it mixes with oxygen-containing waters, the tubeworms can utilize most of what would otherwise be lost through spontaneous reaction with oxygen in the bottom water before reaching the tubeworm’s plume.
Since the adult tubeworms are obtaining sulfide before it is released from the sediment, massive aggregations of mature vestimentiferans at cold seep sites form 40 thickets of tubes in a relatively non-toxic environment (compared to aggregations of hydrothermal vent vestimentiferans). Thus, they provide a nontoxic area of refuge for many transient and endemic fauna. The autotrophic life style, longevity, and role of cold seep vestimentiferans in creating and providing habitat for other species suggests they are in many ways analogous to ecosystem engineering plants (sensu (37)). This analogy is further strengthened by the demonstration that L. cf luymesi grow posterior extensions of their bodies into the sediment and use these root-like structures to acquire essential dissolved compounds.
Acknowledgements:
This work was supported by The Mineral Management Service Gulf of Mexico Regional OCS
Office through contract number 1435-01-96-CT30813 to CRF, NSF OCE 9632861 and 0002464 to JJC, and the NOAA National Undersea Research Program at University of North Carolina,
Wilmington. Special thanks are due to Mark Delacruz, Sean Murphy, Mark Van Horn, Chris
Tietze, Dr. Istvan Urcuyo, Dr. Michael Arthur, Dr. Stéphane Hourdez, The Harbor Branch
Oceanographic Institution, and the captains, pilots and crew of the RV Seward Johnson and the
DSRV Johnson Sea Link.
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Figure 2.11 Legend: Split vessel respiration system. Arrows indicate positions of manual valves for stream selection and calibrations. “X” indicates the position of an in- line sampling port.
Table 2.2 Legend: All data are given as mean ± one S.D. Posterior (root containing) chamber H2S concentration was determined periodically (”n” times) throughout each experiment via analysis of a discrete sample by gas chromatography (22). The n listed for each flux rate represents the number of independent calculations of flux rate for that condition as described in the text.
Table 2.3 Legend: All data are given as mean ± one S.D. Positive numbers indicate consumption and negative numbers indicate production. Each n represents the number of independent calculations of flux rate for that condition as described in the text. 45
Table 2.1 Seawater H2S concentrations amongst mature hydrocarbon seep tubeworm
aggregations.
Sample Location Range of [H2S] Samples Taken Samples with No % ND
(µM) Detectable H2S
Water Column ND 24 24 100
Plume Level ND 17 17 100
Mid-Aggregation ND – 0.5 15 13 87
Sediment Level ND – 3.7 16 13 81 46
Table 2.2 H2S, DIC, and O2 consumption rates across root and plume of
Lamellibrachia cf luymesi
Posterior Root H2S Plume DIC Plume O2
chamber [H2S] Uptake Uptake Uptake
(µM) (µmoles*g-1*h-1) (µmoles*g-1*h-1) (µmoles*g-1*h-1)
218±82 (n=7) 2.9 ± 0.3 (n=19) 1.1 ± 0.3 (n=28) 7.2 ±2.3 (n=28)
307±124 (n=11) 4.2 ± 0.6 (n=18) 2.3 ± 0.2 (n=18) 5.3 ±1.8 (n=18)
431±103 (n=7) 4.7 ± 1.1 (n=39) 2.7 ± 0.1 (n=24) 10.4 ±3.4 (n=24) 47
Table 2.3 Plume DIC and Oxygen flux prior to, during, and after root H2S exposure.
Experimental Conditions DIC Flux O2 Flux
(µmoles*g-1*h-1) (µmoles*g-1*h-1)
Pre-H2S Exposure - 2.4 ± 0.8 (n=73) 6.2 ± 0.9 (n=73)
800µM H2S in posterior chamber 2.1 ± 1 (n=78) 8.8 ± 0.9 (n=78)
Post-H2S Exposure (17 hrs) - 4.9 ± 1.8 (n=38) 4.7 ± 2.3 (n=38) 48
Figure 2.1 Split-vessel respiration system 49
Chapter 3
SULFIDE BINDING BY THE GIANT HEMOGLOBINS FROM THE
HYDROTHERMAL VENT TUBEWORM RIFTIA PACHYPTILA
JOHN K. FREYTAG*, RONALD M. SMITH1, JASON F. FLORES, STÉPHANE M.
HOURDEZ, and CHARLES R. FISHER
Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA
*e-mail: [email protected] 1 Present Address: Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104, USA
Summary
The hydrothermal vent tubeworm, Riftia pachyptila, like all vestimentiferans, obtains nutrition from sulfide-oxidizing chemoautotrophic bacterial endosymbionts.
Vestimentiferan vascular blood and coelomic fluid contain giant extracellular hemoglobins (Hbs; a 3,500 kDa and a 400 kDa Hb in the vascular blood, and a different
400 kDa Hb in coelomic fluid) that reversibly and simultaneously bind large quantities of hydrogen sulfide and oxygen at binding sites unique for each chemical species. These
Hbs are fundamental to the uptake, accumulation, transportation, and delivery of both sulfide and oxygen to their endosymbionts. In this study, we estimate the sulfide-binding affinity and capacity for the purified R. pachyptila 3,500 kDa Hb and a mixture of 400 kDa Hbs from vascular and coelomic fluids. Under our experimental conditions, the 50
3,500 kDa Hb bound 2.2 moles sulfide per mole of heme (95% confidence interval (CI) =
- 2.0 – 2.3 HS /Heme) when saturated. Half-saturation occurred at 5.2µM ΣH2S (95% CI =
3.9 – 7.1µM) for this Hb. The 400 kDa Hbs were able to bind 0.47 moles sulfide per mole of heme (95% CI = 0.42 – 0.51 HS-/Heme) when saturated, and 50% saturation
occurred at 5.4µM ΣH2S (95% CI= 3.0 – 9.8 µM). In addition, we found significant limitations in the methods commonly used in sulfide-binding studies, and discuss these in the context of previous studies.
Key words: sulfide-binding, Riftia pachyptila, vestimentiferan, tubeworm, hemoglobin, affinity, capacity, extracellular, chemoautotrophy, sulfide, hydrothermal vent, diffusion, dialysis
Introduction
The vestimentiferan tubeworm Riftia pachyptila visually dominates many hydrothermal vent sites along the East Pacific Rise, thriving where warm vent effluent mixes with cold, ambient seawater (Hessler et al., 1988). This giant annelid worm can grow to lengths of 1 m or more and reach sexual maturity in less than two years (Lutz et al., 1994). R. pachyptila, like all mature vestimentiferans, has no mouth or digestive system and relies upon chemolithoautotrophic bacterial endosymbionts for nutrition
(Cavanaugh et al., 1981; Felbeck, 1981; Jones, 1981). Symbionts are contained in the trunk of the tubeworm in a vascularized organ known as the trophosome and are dependent upon their host for all required metabolites. Thus, for the intact symbiosis to 51 survive the host animal must acquire and transport chemicals from the vent environment to the endosymbionts so that they can in turn provide food to the host (Fisher, 1996).
Vestimentiferan bacterial endosymbionts utilize hydrogen sulfide as an electron donor to fuel carbon fixation, a process that also requires carbon dioxide and oxygen
(Childress et al., 1991; Wilmot and Vetter, 1990). These essential chemicals are taken up by the tubeworm from the environment directly across its plume, a highly vascularized organ that is exposed to both sulfide and carbon dioxide rich vent effluent, and oxygenated bottom waters. Riftia pachyptila acquires hydrogen sulfide from the
- environment as HS rather than H2S (Goffredi et al., 1997a). Upon diffusion into the
- animal, carbon dioxide is converted into HCO3 and remains in this form as it, sulfide and oxygen are transported to the symbionts by the vascular blood of the tubeworm (Goffredi et al., 1997b). Vestimentiferan blood is hemoglobin (Hb) rich and is pumped throughout the body in a closed vascular system (Jones, 1988; Terwilliger et al., 1980). In the trunk of the worm, capillaries are interspersed throughout the trophosome allowing metabolites to be transferred from the vascular blood to the symbionts (Jones, 1988).
Both the coelomic and vascular fluids of vestimentiferans contain large amounts of giant, extracellular hemoglobins (Hbs) that are able to reversibly bind large quantities of both oxygen and hydrogen sulfide with high affinity (Arp and Childress, 1981; Arp et al., 1987; Terwilliger et al., 1980). The binding sites on vestimentiferan hemoglobins for sulfide and oxygen are separate and there is no interaction between sulfide binding and oxygen binding (Childress et al., 1984). The vascular blood contains two Hbs that are approximately 3500kDa and 400 kDa, while the coelomic fluid contains a different
400kDa Hb (Arp et al., 1987; Childress et al., 1991; Zal et al., 1996a). As a result of the 52 high sulfide affinity of these hemoglobins and their abundance in the blood, concentrations of bound sulfide in vascular blood of R. pachyptila can be as high as two orders of magnitude greater than external sulfide concentrations, while free sulfide in the vascular blood can simultaneously be maintained at concentrations an order of magnitude lower than environmental concentrations (Childress et al., 1991; Scott and Fisher, 1995).
Thus, vestimentiferan hemoglobins provide not only a transportation mechanism, but also allow the normally toxic sulfide to move throughout the body with no ill effects to the animal or its symbionts (Fisher et al., 1988; Powell and Somero, 1983).
Early studies revealed much about the physiological roles of the vascular and coelomic fluids, and their hemoglobins, in Riftia pachyptila (Arp and Childress, 1981;
Arp and Childress, 1983; Arp et al., 1987; Childress et al., 1984; Childress et al., 1991;
Fisher et al., 1988; Powell and Somero, 1983). The polypeptide composition of the hemoglobins and mechanisms of sulfide-binding, however, were determined relatively recently. The 3,500 kDa Hb is made up of monomer and dimer heme-containing chains
(globins) as well as linker chains that help to maintain the quaternary hexagonal bilayer structure (Zal et al., 1996a). Additional work by Zal et al (1998) demonstrated two mechanisms of sulfide binding for the 3,500 kDa Hb; sulfide is bound by free cysteine
(Cys) residues on the globin chains to form S-sulfohemoglobin and by reduction of disulfide bridges on the linker chains. Though distinctly different proteins, the 400 kDa coelomic and vascular Hbs are both composed entirely of heme-containing chains, and therefore all sulfide-binding occurs through S-sulfohemoglobin formation (Zal et al.,
1998). Structural data indicates that the two 400 kDa Hbs contain the same number of 53
heme-containing chains and free Cys residues, and therefore likely have the same sulfide-
binding capacities (Zal et al., 1996b; Zal et al., 1998).
The only data currently available on the sulfide affinities of R. pachyptila fluids are from
a binding curve generated from a mixture of vascular and coelomic fluids, and all three Hbs
(Fisher et al., 1988). The present study examines the sulfide binding characteristics (affinity and
capacity) of purified R. pachyptila 3,500 kDa Hb and a mixture of 400 kDa hemoglobins from
vascular and coelomic fluids. In addition, we discuss limitations of materials and methods used
in sulfide binding studies, and the implications for previous sulfide binding data and these types
of dialysis experiments in general.
Materials and methods
Animal collection
Riftia pachyptila Jones were collected by submersible (D.S.R.V Alvin) during a
research expedition to 13° N (12˚48.7’N, 103˚56.7’W) along the East Pacific Rise in
1990. Animals were brought to the surface in a temperature-insulated sampling box.
Upon recovery of the submersible animals were transferred to a cooler containing cold
seawater (5˚C). Dissection and fluid collection occurred within three hours of recovery of the submersible following the methodology of Childress et al. (1991) (Childress et al.,
1991). Mixed fluid samples (vascular blood and coelomic fluid) were frozen at –20˚C in
15 ml. aliquots onboard ship, transferred to the laboratory on dry ice and stored at -70˚C until use. 54
Hemoglobin purification and quantification
Fifteen ml. aliquots of mixed R. pachyptila fluids were thawed, centrifuged at
4,000g for 5 minutes in order to remove particulate impurities, divided into 1.5 ml
aliquots and refrozen at –70˚C. Later, each 1.5 ml aliquot was thawed and separated by gel filtration on a 1 X 30 cm Superose 6-C column (Pharmacia LKB Biotechnology) using a low-pressure fast protein liquid chromatography system (ISCO). Riftia saline
buffer (50 mM HEPES/400 mM NaCl/3mM KCl/32 mM MgSO4/11 mM CaCl2, pH 7.5
at 23ºC) (Fisher et al., 1988; Zal et al., 1996b) was used as the eluent. After separation,
Hb-containing solutions were adjusted to heme concentrations between 0.2 and 0.6 mM
heme using 4 ml and 25 ml Ultrafree Centrifugation Filters (10,000 MWCO; Millipore)
and were then refrozen. All Hb-containing solutions were kept on ice during purification
and collection, and were refrozen within 24 hours of being thawed to minimize protein
degradation. When 20-30 ml of each purified Hb solution had been accumulated, the
samples were thawed and combined and adjusted to a final working heme concentration
of 0.6 mM. The composition of each Hb solution was confirmed to be at least 98% the
desired Hb (by area on the elution profile) by gel filtration separation of a small aliquot of
the solution. The two solutions were aliquoted into replicate 0.95 ml experimental
samples that were then refrozen at -70˚C until use.
Heme concentrations of intact vestimentiferan fluids and experimental Hb- containing solutions were quantified using methods adapted from Van Assendelft, 1970 and Toulmond et al. 1990 (Toulmond et al., 1990; Van Assendelft, 1970). Five µl of
heme containing solution was diluted in1000 µl of double distilled H20 and mixed with
10 µl potassium ferricyanide solution (50 mmol l-1). After a 5 min incubation at room 55 temperature to allow for oxidation of the hemoglobin to methhemoglobin, 10µl potassium cyanide (500 mmol l-1) was added to obtain cyan-methemoglobin which was then quantified by measuring the absorbance of the solution at 540 nm (extinction coefficient of 11.01 mmol-1cm-1) with a Beckman DU-64 spectrophotometer.
Dialysis experiments
Lengths of 5-6 cm of Spectra/Por dialysis membrane material (MWCO 6,000-
8,000 kDa; Spectrum Laboratories) were cut, soaked in dionized H2O for at least 15 minutes, rinsed, placed into chilled Riftia saline solution where they were allowed to soak for an additional 15-30 minutes, and then rinsed with Riftia saline. Dialysis bags 1.5-2.5 cm in length were made with a length of soaked membrane tubing and two SpectraPor closures (one weighted, one unweighted). Each bag was filled with 300 µl Hb solution
(0.6 mM heme) and dialyzed against 0.2µm-filtered, deoxygenated Riftia saline buffer containing sulfide (pH 7.5 at 5ºC) for a minimum of 18 hours. At each dialysate sulfide concentration three replicate dialysis bags containing Hbs were run in parallel with three replicate dialysis bags (300µl or 1000µl) containing deoxygenated Riftia saline as controls. All dialysis experiments were conducted at 5˚C in a sealed 2.56 l acrylic chamber that was constantly stirred and secondarily contained within a 15 l AtmosBag
(Aldrich Chemical Company, Inc.) filled with nitrogen. At the end of each experiment, control and experimental bags were removed from the chamber individually and samples were drawn into a 500 µl gas tight syringe (Hamilton, Co.) and analyzed. All manipulations were conducted in the AtmosBag and the dialysis chamber was resealed between the removal of each dialysis bag. 56
Quantification of sulfide
- - ΣH2S (the sum of H2S, HS , and S2 ) of dialysate solutions with ΣH2S concentrations greater than 5µM and of all Hb solutions was quantified by gas chromatography (GC; (Childress et al., 1984; Childress et al., 1991)). Each Hb or dialysate sample was injected into an in-line heated extractor, acidified with phosphoric acid, and the stripped gasses were then analyzed by a Hewlett Packard 5880A series gas chromatograph. This methodology removes bound sulfide from Hbs and volatizes free sulfide in solution for quantification. The lower detection limit of the GC was approximately 3 µM, and quantification below 10µM approximate.
The ΣH2S of dialysate samples containing less than 5µM ΣH2S was quantified by using a modification of the methods of Singh et al. 1990, and Freytag et al, 2001 (Freytag et al., 2001; Singh et al., 1993). For quantification in the range of 0.5-5.0 µM total
sulfide, 700 µl of the dialysate sample was combined with 150 µl of papain-SSCH3
solution (0.6 mg/ml papain-SSCH3 (Molecular Probes), 2.0 mM ethylenediaminetetraacetic acid (EDTA), 40 mM NaH2PO4, 5.0 mM sodium acetate, pH
7.4) under a nitrogen head and incubated at room temperature for one hour. Samples were then combined with 150 µL of a solution of chromogenic substrate [2.1 mg/ml N- benzoyl-L-arginine, p-nitroanilide (L-BAPNA) (Molecular Probes), 1 mM EDTA, 50 mM bis-Tris, pH 6.3] and incubated at room temperature for an additional hour, at which point 5.0 µl of 1.0 mM phenylmethylsulfonyl fluoride (PMSF) (Boehringer Mannheim) was added to stop the enzymatic reaction and the absorbance of the samples at 410 nm
was noted. For higher precision in the range of 0.2-2.0 µM ΣH2S, the solutions were 57
modified: the concentrations of the components of the papain-SSCH3 were increased to
2.0 mg/ml papain-SSCH3, 8.0 mM (EDTA), 60 mM NaH2PO4, and 10 mM sodium acetate). The concentrations of the components of the chromogenic substrate solution were increased to 7.2 mg/ml L-BAPNA, 3 mM EDTA, and 150 mM bis-Tris. The pH of
each component solution was the same as described for the 0.5-5.0 µM ΣH2S assay.
Under these conditions the addition of PMSF did not stop the enzymatic reaction and sample absorbance continued to increase for 20 hours at which point the r2-value of the standard curve was above 0.98. The incubation period used for this modified assay was
20 hours.
Data analysis
Since the GC analysis methodology measures the total (bound and unbound) sulfide in samples of vestimentiferan Hb, bound sulfide was calculated by subtracting the
concentration of ΣH2S in the control bags (free ΣH2S) from the concentration of ΣH2S
measured in the Hb solutions (bound and free ΣH2S). The bound sulfide was then standardized to the amount of heme in the sample to allow comparisons between Hbs and to other studies.
To estimate the sulfide-binding capacity of each type of Hb the data was plotted
- to show the relationship between bound sulfide (HS /Heme) and free ΣH2S (ΣH2S concentration in control dialysis bags). When plotted in this manner, Hb sulfide-binding
data forms a sigmoid shaped curve as ΣH2S increases from zero, and behaves in a manner similar to Hb oxygen-binding data with Hb sulfide-binding approaching a point of 58 saturation. A non-linear function was fitted to the data (Hourdez et al., 2002) using the computer program JMP 5:
aHSc([∑− ] ) HS− / Heme = 2 1+ bHSc([∑−2 ] )
where ΣH2S is in mM, c is the value of ΣH2S where bound sulfide is zero, and a and b are constants. As the sulfide concentration tends to infinity, HS-/Heme tends to a/b, which is the theoretical maximum amount of sulfide that each Hb is capable of binding (capacity).
χ2 values were used to determine the goodness of fit for each regression; both sulfide- binding curves were highly significant having P values <0.001. Regression 95%
confidence intervals (CIs) were calculated as x ±tDF,0.025 * Standard Error (SE) of
ˆ ∧ Regression. 95% CIs were calculated for capacity estimates asθσ± Ζαθ2 , where θ is the
estimated parameter value, α = 0.05, Ζα 2=1.96, and σθ is Var[]θ .
C50 values (the ΣH2S concentration at which the Hb is half-saturated with sulfide and a parameter used to describe the sulfide affinity of a Hb), and cooperativity of
binding were estimated for each Hb. A plot of log[Y/(1-Y)] versus log[ΣH2S], where Y is the fraction of Hb molecules that are saturated with ligand, was generated for each Hb for saturation values between 25 and 75% (Antonini and Brunori, 1971). The intercept at
log[Y/(1-Y)]=0 gives C50, and the slope of the regression line provides an indication of the degree of cooperativity of binding. CIs at 95% were calculated for affinity estimates and linear regression parameters as described by Sokal and Rohlf (Sokal and Rohlf,
1998). 59
Evaluation of dialysis methodology: Correction for control dialysis bag volume
Two different control bag volumes (300µl and 1000µl) were used in approximately the first 1/3 of the sulfide concentrations analyzed. Unexpectedly, this resulted in two distinct sets of data. An additional series of experiments were conducted at that point to confirm the apparent effect of bag volume on the equilibrium concentration of sulfide inside the bags. These experiments were conducted using the same methodology as all dialysis experiments with the addition of consistent direct measurement of external dialysate concentration. These experiments were conducted at both “High” sulfide concentrations (between 255 and 330 µM) and “Low” sulfide concentrations (between 5 and 20 µM), however, no effect of sulfide concentration was detected and the data were combined for further analysis. To confirm that the internal and external concentrations of sulfide were in equilibrium after the 24 hour experimental incubation period, a five-day experiment was conducted with both volumes of bags.
There was no significant increase in the internal concentration of sulfide with time after
24 hours (Table 1). A consistent effect of bag volume on the equilibrium between internal and external sulfide concentration was confirmed and a numerical correction factor was applied to all early experiments that used 1000 µl control bags and 300 µl experimental bags before inclusion and further analysis of these data.
Evaluation of dialysis methodology: Other variables
To investigate the cause of bag volume effect on the equilibrium between internal and external sulfide concentrations additional experiments were conducted in which the size of the bag or volume of the samples were varied, and several membrane pre- 60 treatments were tested. Additional dialysis experiments utilizing the chemical dyes
Allura Red AC (0.1 mg/ml; Becker-Underwood, Inc.) and Acid Blue #158 (0.08 mg/ml;
J.S. Vila Corp.) were conducted to explore the generality of the effect noted with sulfide.
In these experiments the concentration of the dyes were measured spectrophotometrically.
Effect of heme concentration
Additional sulfide-binding experiments were conducted to investigate the effect of hemoglobin concentration on in vitro sulfide-binding of the isolated hemoglobins.
These experiments employed the same basic dialysis methodology as the other experiments, with sulfide concentrations between 150 and 330 µM, and heme concentrations from 0.15 to 2.4 mM.
Results:
Sulfide binding by purified hemoglobins
The relationships between free sulfide (the ΣH2S concentration in control dialysis bags) and sulfide bound by the hemoglobins (mM HS-/mM Heme) for the purified hemoglobin fractions are shown in Figures 3.1 and 3.2. At saturation the 3,500 kDa Hb bound 2.2 moles HS- per mole of heme (parameter 95% confidence interval (CI) = 2.0 –
- 2.3 HS /Heme) with a C50 of 5.2 µM sulfide (parameter 95% CI = 3.9 – 7.1 µM). The
400 kDa Hbs have a lower capacity for sulfide and bind 0.47 moles HS- per mole of heme
- (parameter 95% CI = 0.42 - 0.52 HS /Heme) at saturation (See Fig. 2). The C50 of the 400 kDa Hbs of 5.4 µM sulfide (parameter 95% CI = 3.0 – 9.8 µM) is similar to that of the 61
3,500 kDa Hb. The Hill plots of sulfide-binding for the 3,500 and 400 kDa Hbs were linear with slopes of 0.74 (parameter 95% CI = 0.47-1.01) and 0.90 (parameter 95% CI =
0.54-1.26), respectively.
Evaluation of dialysis methodology
A consistent and significant effect of bag volume on the equilibrium between internal and external sulfide concentration was confirmed, with the smaller volume bags reaching an average of only 68.2% (95% CI = 65.0-71.4%) of the external dialysate sulfide concentration and the larger volume bags reaching an average of 86.0% (95% CI
= 84.2-87.7%) of the external sulfide concentration (Table 1). There was no significant increase in the internal concentration of sulfide with time between 24 and 120 hours of incubation suggesting that equilibrium in sulfide concentration had been reached with the dialysate after 24 hours (Table 1). Larger volume dialysis bags (2000 µl, and 3000µl) reached sulfide concentrations that were 93.0 and 89.4% of dialysate sulfide concentrations, respectively (Table 1). Membrane pretreatments designed to clean and neutralize the dialysis membranes did not have a significant effect on the sulfide equilibrium concentrations (Table 1). A significant bag volume effect was evident in the
24 hour dialysis experiments conducted with two species of chemical dye (Allura Red
AC which carried a net negative charge, and Acid Blue #158 which carried no net charge) in Riftia saline at pH 7.5. However, the effect was in the opposite direction as with sulfide (i.e. – larger volume bags were farther from equilibrium) which suggested that the internal and external dye concentrations had not reached equilibrium. When the experiments were repeated with the apparently most slowly diffusing dye (Allura Red 62
AC) for 72 hours, the internal concentration in all bags was the same as the external concentration.
Effect of heme concentration on sulfide binding
Sulfide-binding by purified 3,500 kDa and 400 kDa Riftia pachyptila Hb fractions was positively correlated to heme concentration (Figures 3). Sulfide binding per heme did not increase significantly above concentrations of 0.4 mM heme in the experiments with the 400 kDa Hbs. Sulfide bound per heme by the 3,500 kDa Hb increased significantly over the full range of heme concentrations tested (0.15 to 1.8 mM; Fig. 3A).
Discussion:
Results from sulfide-binding experiments presented here demonstrate that both the 3,500 kDa Hb and 400 kDa Hbs purified from the fluids of R. pachyptila have a high
affinity for sulfide. The estimated sulfide C50 value (the ΣH2S concentration at which
Hbs are 50% saturated) of the 3,500 kDa Hb and the 400 kDa Hbs are both quite low, 5.2
and 5.4µM, respectively, and are not significantly different from each other. These C50 values estimated by the present study are similar to the value of 11.2µM estimated for mixed fluids of R. pachyptila by Fisher and others in 1988 which suggests the affinity of these Hbs for sulfide was not affected by the purification process. Based on the reported
1.7:1 ratio of the 3,500 kDa to 400 kDa Hbs in R. pachyptila vascular fluid (Arp et al.,
1987; Childress et al., 1991), we estimate that the mixed fluids the Hbs were purified from contained approximately equal amounts of vascular and coelomic fluids. The purified 400kDA Hb fraction therefore consisted of approximately 70% coelomic fluid 63
400kDa Hb and 30% vascular 400 kDa Hb. This and the very similar sulfide affinities of the 3,500 kDa and 400 kDa Hb fractions suggest that the intact vascular and coelomic fluids also have very similar affinities for sulfide. This would allow bi-directional exchange and storage of sulfide in both fluid compartments and is consistent with the proposed role of the coelomic fluid as a temporary storage reservoir for sulfide (Arp et al., 1987; Childress et al., 1991; Fisher et al., 1988). The storage of sulfide by coelomic fluid assures a regular supply of sulfide to the symbionts even though the host tubeworm lives in an environment where sulfide exposure fluctuates dramatically.
The binding capacity of purified R. pachyptila 3,500 kDa Hb of 2.2 HS-/Heme reported here is less than the capacities calculated from previous binding experiments
(approx. 3.0 HS-/Heme; (Arp et al., 1987; Zal et al., 1997)), and estimates based upon structural data (3.5 HS-/Heme; (Zal et al., 1998)). This is most likely due to the lower concentrations of Hb used in this study (Fig. 3). Since the intact Riftia pachyptila vascular fluid has a heme concentration of approximately 3.5 mM (Arp et al., 1987;
Childress et al., 1984) a capacity of 3.0 HS-/Heme is the most appropriate value to consider with respect to the in vivo activity of the 3,500 kDa Hb. In contrast to the 3,500 kDa Hb, there was no significant effect of concentration on sulfide binding capacity of the 400 kDa Hbs above heme concentrations of 0.6 mM (See Fig. 3). At this time, a mechanism to explain the observed increase in the sulfide-binding capacity of the 3,500 kDa Hb at increased heme concentrations has not been determined. Riggs (1998) suggests that heme concentration affects the level of association between subunits of some vertebrate Hbs as well as the oxygen binding properties of these Hbs {Riggs, 1998
#495}. The level of Hb subunit assembly has also been shown to play a role in the 64 binding properties of invertebrate Hbs {Weber, 2001 #410}. This, or a mechanism that similarly increases the level of interaction between multiple subunits or Hbs at increased heme concentrations, may prove to be affecting sulfide-binding by the R. pachyptila
3,500 kDa Hb. The capacity of the R. pachyptila 400 kDa Hbs of 0.47 HS-/Heme reported here corresponds well with the most recent estimate from binding studies (0.5
HS-/Heme; (Zal et al., 1997)) and structural data, 0.58 HS-/Heme (Zal et al., 1998).
However, it is about half of the capacities estimated by earlier binding studies (1.0 HS-
/Heme; (Arp et al., 1987; Childress et al., 1991)). It is unlikely that the differences are due to differing amounts of the vascular and coelomic Hbs in the different studies because Zal and coworkers also have shown that the two 400 kDa Hbs contain identical numbers of globin chains and free cysteine residues, and thus should have identical sulfide-binding capacities (Zal et al., 1998).
While conducting these studies an unexpected discovery was made: At equilibrium, sulfide concentrations inside small volume dialysis bags were not equal to external concentrations, and the effect varied with the bag volume. The fact that the
300µl bags accumulated sulfide to lower concentrations (68% of external sulfide, n=22) than the 1000µl bags (86% of external sulfide, n=28) suggested that this effect was not because of insufficient time to reach equilibrium. This was confirmed in a series of experiments that extended the dialysis time up to 5 days (Table 1). In a second set of experiments, extending the range of volume of fluid in bags of uniform size was tested.
The concentration of sulfide in the larger bags was closer to that of the external dialysate, however the internal concentrations never equaled the internal concentrations. In a third set of experiments the sample volumes were kept constant and bag length was varied. 65
This depressed the sulfide levels inside the bags, although the difference was only significant in the case of the 1000 µl sample volume (Table 1, t-test: t=2.71, P=0.024).
Taken together, all of the data above suggest that this effect is related to the relationship between the surface area of the bag and the volume of the sample in the dialysis bag, with the largest discrimination against sulfide occurring when the surface area/volume ratio is highest.
Two additional sets of experiments were conducted to provide further insights into this phenomenon. Three different membrane pre-treatments designed to neutralize the net negative charges on the membrane at the experimental pH were tested, and none had any effect on the sulfide equilibrium. To determine if this effect was limited to sulfide, equilibrium experiments with two dyes were conducted. In the case of both dyes, the time necessary for the internal concentrations to come to equilibrium with the external concentrations was a function of the volume of the bags, but when equilibrium was reached, internal and external dye concentrations were equal.
At this time, we are unable to explain the fact that at equilibrium, sulfide concentrations inside small volume dialysis bags do not equal external sulfide concentrations. Dialysis membrane material is typically used for the bulk separation of salts or other impurities from macromolecular solutions placed inside the dialysis tubing.
This application does not depend upon the establishment of equal concentrations inside and outside of the bags, and thus it should be noted that using the dialysis bags as described here falls outside of the purpose the bags were designed for. Nonetheless, if appropriate controls are conducted that measure the actual concentrations of the 66 compound of interest inside the bags, as was done here, this methodology can continue to be used successfully in studies of this sort.
Previous sulfide-binding studies on Riftia pachyptila fluids and Hbs (Arp and
Childress, 1983; Arp et al., 1987; Childress et al., 1984; Fisher et al., 1988; Zal et al.,
1997) also utilized regenerated cellulose or cellulose esther dialysis membrane material.
With the exception of the study by Childress and others (1984) which estimated bound sulfide by reference to a control bag containing distilled water, these studies incorrectly
assumed that the concentration of ΣH2S in the dialysate equaled the free ΣH2S concentration in the dialysis bags and used this value to calculate bound sulfide. Most of the experiments in these studies used larger volumes of intact fluids (1-2 ml) at relatively high heme concentrations compared to those used by the present study (J.J. Childress pers. comm) so the bag volume and heme concentration effects would be small. None the less, the sulfide-binding capacities and affinities estimated by these studies were very likely underestimated by 10-15%.
The ability of the giant Hbs of vestimentiferans to bind and transport hydrogen sulfide has long been known to be fundamental to the survival of these unique animals.
We now confirm that the 3,500 kDa and 400 kDa Hbs of Riftia pachyptila are able bind sulfide with similarly high affinity, a finding that supports the previously proposed role of the coelomic fluid as a short-term store for sulfide. Sulfide-binding affinity and capacity estimates for isolated R. pachyptila Hbs have lead to an increased understanding of the sulfide physiology of this species and its Hb-containing fluids. The methodology presented here will allow future sulfide-binding studies to be conducted in a manner that 67 will maximize the usefulness of the data and will allow comparisons to be made within and between vestimentiferan species.
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Toulmond, A. (1997). Primary structure of the common polypeptide chain b from the multi-hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila: An insight on the sulfide binding site. Proteins: Structure, Function, and Genetics 29, 562-
574. 72
Table 3.1. Percent diffusion data from sulfide and dye dialysis experiments Treatment 300 µl 1000 µl 2000 µl 3000 µl Sulfide Diffusion Experiments Regular membrane treatment, 20-24 hour expts., 6-8000 kDa MWCO Spectra/Por 1 RC membrane, bag length 1.5-2.5 cm 68.2 (65.0-71.4) (22) 86.0 (84.2-87.7) (28) Duration of Experiment 120 Hour Dialysis Experiment 24 hour Incubation 69.4 (59.3-74.6) (2) 85.1 (84.3-85.9) (2) 48 hour Incubation 76.3 (1) 83.1 (78.7-87.1) (2) 96 hour Incubation 73.3 (71.0-75.5) (2) 90.9 (90.2-91.5) (2) 120 hour Incubation 75.4 (72.8-77.9) (2) 87.4 (1) Bag Size and Volume Larger Bag Volumes 68.2 (66.2-70.2) (3) 83.2 (79.4-86.8) (2) 93.0 (88.4-96.5) (3) 89.4 (87.0-91.6) (3) Longer Bag Lengths (2.6-4.6 cm) 63.5 (56.4-70.4) (2) 82.1 (79.8-84.3) (4) Membrane Treatments
Soaked 30 min. in DI H2O, 30 min. in Riftia Saline, 30 min. in 1% BSA/DI H2O. 70.2 (63.0-76.9) (2) 88.7 (81.9-94.0) (2) Soaked 30 min. in DI H2O, 30 min. in Riftia Saline, 30 min. in 5% Tween 20/DI H2O. 72.3 (66.6-77.6) (2) 90.1 (88.8-91.3) (2) Soaked 15 min. in DI H2O, 30 min in 2% Sodium Bicarb, 1 mM EDTA @ 60º. 65.5 (63.8-67.1) (3) 82.5 (76.4-87.8) (3) Dye Diffusion Experiments
Acid Blue #158 99.9 (99.7-100.0) (6) 94.8 (88.0-98.9) (6) 86.5 (83.7-89.2) (7) Allura Red AC 40.1 (1) 21.1 (20.2-22.0) (3) 15.5 (15.0-16.0) (3) 72 hour incubation 100* (3) 100* (3) 100* (3)
Values are means, (95% Confidence Interval for mean), (N). N is the number of replicates (measurements) used to determine mean values. Unless otherwise noted, experiments were conducted at 5ºC, in Riftia saline (pH 7.5) as dialysate, for 20-24 hours. “100*” - indicates that internal concentration had reached 100% of the external concentration. 73
4
3.5
3
2.5 S/Heme) 2
2
1.5
Bound Sulfide (H 1
0.5
0 0.0001 0.001 0.01 0.1 1 10
[H2S] mM
Fig. 3.1. - Sulfide binding by the Riftia pachyptila 3,500 kDa hemoglobin as a function of free sulfide 322.([ 5∑−HS ] 0 . 0002 ) 2 concentration. The solid line fits the equation: HS− / Heme = 2 , χ =4.98, N=66, 1+148.([ 8∑−HS2 ] 0 . 0002 ) P=<0.001. The thin dashed lines represent the 95% confidence bands for the regression line. At the estimated point of saturation, this hemoglobin binds 2.2 moles sulfide per mole of heme (parameter
95% confidence interval = 2.0 – 2.3 H2S/Heme). The predicted 50% saturation occurs at 5.2µM ΣH2S (parameter 95% confidence interval = 3.9 – 7.1µM). 74
1.2
1
0.8 S/Heme) 2
0.6
0.4
Bound Sulfide (H 0.2
0 0.0001 0.001 0.01 0.1 1 10
[H2S] mM
Fig. 3.2. Sulfide binding by Riftia 400 kDa hemoglobins as a function of free sulfide concentration. The 82.([ 06∑−HS ] 0 . 0002 ) 2 solid line fits the equation: HS− / Heme = 2 ,χ =3.55, N=82, P=<0.001. The thin 1+175.([ 34∑−HS2 ] 0 . 0002 ) dashed lines represent the 95% confidence bands for the regression line. At the estimated point of saturation, these hemoglobins bind 0.47 moles sulfide per mole of heme (parameter 95% confidence interval = 0.42 – 0.51 H2S/Heme). The predicted 50% saturation point of the 400 kDa Hbs occurs at
5.4µM ΣH2S (parameter 95% confidence interval = 3.0 – 9.8 µM). 75
3
2.5 3,500 kDa Hb
2 400 kDa Hb /Heme) -
1.5
1 Bound Sulfide (HS
0.5
0 0 0.5 1 1.5 2 2.5 3 3.5 [Heme] mM
Fig. 3.3. Sulfide-binding by the Riftia pachyptila 3,500 kDa Hb, and 400 kDa Hbs as a function of hemoglobin concentration. Each point represents mean bound sulfide ± 1 S.D. In most cases, sample size (N) was three. The solid line fits the equation: y = 0.654Ln(x) + 1.955, R2 = 0.800, P=<0.001. The dashed line fits the equation: y = 0.190Ln(x) + 0.521, R2 = 0.602, P=<0.001. Regressions were fitted to entire data set and not to averages. 76
Chapter 4
SULFIDE BINDING PROPERTIES OF HEMOGLOBINS ISOLATED FROM
TWO SPECIES OF COLD SEEP VESTIMENTIFERANS
JOHN K. FREYTAG*, RONALD M. SMITH1, STEVEN R. BREAULT, STÉPHANE
M. HOURDEZ, CHRISTOPHER S. JONES, and CHARLES R. FISHER
Department of Biology, The Pennsylvania State University, University Park, PA
16802, USA
*e-mail: [email protected]
1 Present Address: Department of Bioscience and Biotechnology, Drexel University,
Philadelphia, PA 19104
Summary:
Two species of vestimentiferan tubeworm, Lamellibrachia luymesi and
Seepiophila jonesi, co-occur in aggregations at northern Gulf of Mexico cold hydrocarbon seep sites. Like all vestimentiferans, L. luymesi and S. jonesi obtain nutrition from sulfide-oxidizing chemoautotrophic bacterial endosymbionts, which must be supplied with sulfide, oxygen, and carbon dioxide. Vestimentiferan vascular blood and coelomic fluid contain giant extracellular hemoglobins that reversibly bind large quantities of hydrogen sulfide and oxygen at binding sites unique for each compound. 77
These Hbs are fundamental to the uptake, accumulation, transportation, and delivery of
both sulfide and oxygen. Based upon the patterns of hydrogen sulfide presence in their
environment and the growth habits of L. luymesi and S. jonesi, we hypothesized that these
co-occurring seep vestimentiferans may be able to obtain sulfide from different points in
the habitats where they co-occur. Here we provide estimates of the sulfide-binding
affinity and capacity for purified 3,500 kDa Hbs and mixtures of 400 kDa Hbs from the
vascular and coelomic fluids of L. luymesi and S. jonesi. We discuss how differences in
functional properties of L. luymesi and S. jonesi Hbs and fluids support the hypothesis
that these tubeworm species are acquiring sulfide from different environmental pools.
Key words: sulfide, sulfide-binding, vestimentiferan, tubeworm, Lamellibrachia luymesi,
Seepiophila jonesi, hemoglobin, affinity, capacity, chemoautotrophy, hydrocarbon seep, resource partitioning
Introduction
Like hydrothermal vents, cold hydrocarbon seeps are environments characterized by high productivity and animal biomass (Bergquist et al., 2003b; Carney, 1994; Sibuet and Olu, 1998). Primary production at vents and seeps is based upon chemoautotrophy, which is dependent upon the availability of reduced chemicals such as hydrogen sulfide
(Brooks et al., 1987; Jannasch, 1985). The majority of hydrogen sulfide in cold seep sediments is produced biogenically in the top 30 cm of sediment via the reduction of seawater sulfate that is coupled with the oxidation of methane and other organics 78
(Arvidson and Morse, 2000; Boetius et al., 2000; Sassen et al., 1993). There are, however, thought to be additional deeper sources of biogenic and thermogenic sulfide beneath cold seep environments (Carney, 1994). Hydrocarbons are released in deep sediment layers when Jurassic salt induces faults in oil-bearing shale (Brooks et al., 1987;
Sassen et al., 1995). Released hydrocarbons then migrate vertically towards the sea floor along fault traces and, as a result, hydrogen sulfide, methane and other hydrocarbon gasses, crude oil, and supersaturated salt brine can be present in surface sediments and bottom water (Behrens, 1988; Roberts and Aharon, 1994). Surface expression of seepage in northern Gulf of Mexico seep environments is patchy, with vast areas of bare sediment separating areas of active seepage and the associated chemoautotrophic communities
(Kennicutt II et al., 1988; MacDonald et al., 1989; MacDonald et al., 1990).
Vestimentiferan tubeworms can dominate the biomass at hydrothermal vents in the eastern Pacific Ocean and at hydrocarbon seeps in the northern Gulf of Mexico
(Bergquist et al., 2003b; Hessler et al., 1988). Vestimentiferans contribute to an increase in species richness at vent and seep environments as they frequently live in large groups or bush-like aggregations that generate and provide habitat for endemic species and also, at seeps, opportunistic and transient species (Bergquist et al., 2003b; Sarrazin and
Juniper, 1999). These large, sessile, annelid worms have no mouth or gut, and are dependent upon sulfide-oxidizing chemoautotrophic bacterial endosymbionts for nutrition (Jones, 1981; Wilmot and Vetter, 1990). Most of what is known about vestimentiferan physiology comes from studies of the hydrothermal vent tubeworm,
Riftia pachyptila. Previous studies have demonstrated that the tubeworm host must acquire sulfide, oxygen, and carbon dioxide from the environment and supply these 79 chemicals to its bacterial endosymbionts housed deep in its body (Arp et al., 1985;
Childress and Fisher, 1992; Felbeck and Childress, 1988). R. pachyptila acquires each of these chemicals across the anterior plume portion of its body, which is the only portion of the animal that is extended into the environment from a chitinous tube (Childress et al.,
1984; Gaill et al., 1988; Goffredi et al., 1997a; Goffredi et al., 1997b). Hemoglobin-rich vascular and coelomic fluids have been shown to be key to the acquisition, transport, and storage of oxygen and sulfide in R. pachyptila (Arp et al., 1987; Childress et al., 1984;
Childress et al., 1991; Fisher et al., 1988). Both oxygen and sulfide are bound reversibly with high affinity and capacity, and are transported within the closed vascular system by two giant (3,500 kDa and 400 kDa), extracellular hemoglobins (Hbs) (Arp and Childress,
1981; Arp and Childress, 1983; Arp et al., 1987; Fisher et al., 1988; Jones, 1988;
Terwilliger et al., 1980; Zal et al., 1996). The coelomic fluid is non-circulating and contains a 400 kDa hemoglobin (Hb) that is distinct from the vascular 400 kDa Hb
(Childress et al., 1991; Jones, 1988). As a result of the high sulfide-binding capacity and affinity of the vascular and coelomic Hbs, concentrations of sulfide in the vascular blood of R. pachyptila can be two orders of magnitude greater than in the environment, while concentrations of free (unbound) sulfide are maintained at low, non-toxic levels (Fisher et al., 1988).
Two species of vestimentiferan tubeworm, Seepiophila jonesi and Lamellibrachia luymesi, co-occur in aggregations at hydrocarbon seep sites on the upper Louisiana slope of the Gulf of Mexico (Bergquist et al., 2002; MacDonald et al., 1989). Single, bush-like aggregations normally contain between 100 to 2000 individual tubeworms, though
“fields” of tubeworms can cover areas as great as 1600 m2 and contain hundreds of 80 thousands of individuals (Bergquist et al., 2003b; Brooks et al., 1989). In most aggregations, L. luymesi is more abundant than S. jonesi (Bergquist et al., 2002).
Settlement of both L. luymesi and S. jonesi larvae takes place during a limited recruitment period (between 8 and 25 years), with both the larval settlement and the survival of young tubeworms dependent upon the availability of hard substrate (exposed authigenic carbonate rock) and active seepage (Bergquist et al., 2002). Both L. luymesi and S. jonesi grow very slowly and are believed to be extremely long-lived (Bergquist et al., 2000).
The plumes of large L. luymesi can be more than 2 m above the sediment, whereas the plumes of large S. jonesi are usually only a few centimeters above the sediment (See Fig.
4.1; Gardiner et al., 2001; MacDonald et al., 1989; JKF personal observation).
Unlike their vent relative R. pachyptila, both cold seep tubeworm species have posterior extensions of their body (“roots”) present deep in the sediment (Julian et al.,
1999). The apparent absence of sulfide around the plumes of L. luymesi prompted studies that sought to determine how this species was obtaining sulfide from the hydrocarbon seep environment. An initial study by Julian and coworkers (1999) measured concentrations of hydrogen sulfide as high as 2.7 mM present in water samples collected among the buried posterior “root” portions of the tubeworms, and found that the posterior root tube of L. luymesi is 700 times more permeable to sulfide than anterior tube (Julian et al., 1999). Later studies have measured even higher concentrations of sulfide in sediments beneath tubeworm aggregations, determined that concentrations of hydrogen sulfide around the plumes of large L. luymesi are not sufficient for the sulfide requirements of this species and demonstrated that L. luymesi could use the root portion 81 of its tube and tissue to acquire sulfide at rates sufficient to sustain net chemoautotrophy
(Bergquist et al., 2003a; Freytag et al., 2001).
Although both L. luymesi and S. jonesi have roots, it has proven difficult to collect and characterize intact roots or to determine to what extent each species relies on its root for environmental sulfide uptake. Any sulfide available to the plumes of either seep tubeworm species would have to move from the sediment upward through the water column and, since sulfide and oxygen react spontaneously (Cline et al, 1969), the probability of sufficient quantities of sulfide existing in the water column decreases as distance above the sediment-seawater interface increases. Considering this and observed differences in the growth habits of L. luymesi and S. jonesi (see Fig. 4.1), it has been suggested that large S. jonesi may rely more on the anterior plume portions of their bodies for sulfide acquisition than large L. luymesi.
We hypothesize that the functional properties of the vascular and coelomic Hbs of each species will reflect any differences in method of environmental sulfide acquisition.
The present study examines the sulfide binding characteristics (affinity and capacity) of purified 3,500 kDa Hb and a mixture of 400 kDa hemoglobins from vascular and coelomic fluids from L. luymesi and S. jonesi. In addition, we estimate the sulfide- binding capacities of intact vascular and coelomic fluid from each species and compare these results to the sulfide-binding capacities of Riftia pachyptila Hbs and fluids. Lastly, we discuss how differences in functional properties of L. luymesi and S. jonesi Hbs and fluids may facilitate acquisition of sulfide from different environmental sulfide sources. 82
Materials and methods
Animal collection
Animals were collected in July 1998, July 2000, August 2000, and February 2003 during research expeditions to three hydrocarbon seep sites on the upper Louisiana slope in the Gulf of Mexico: Bush Hill (27º 46’96”N, 91º 30’46”W), a site in Green Canyon leasing block 234 (27º 44’7”N, 91º 13’3”W), and Brine Pool NR-1 (27º 43’24” N,
91º16’30”). L. luymesi and S. jonesi were collected using the DSRV Johnson Sea-Link I and were brought to the surface in a temperature-insulated sampling box. Upon recovery of the submersible animals were transferred to a cooler containing aerated, chilled (7˚C) seawater where they stayed until dissection. Fluid samples were only taken from animals that appeared healthy, active and undamaged. Dissection and fluid collection occurred within 72 hours of recovery of the submersible following the methodology of Childress et al. (1991) (Childress et al., 1991). Vascular blood was collected prior to coelomic fluid in an attempt to prevent cross-contamination of each fluid type. As only small volumes
(5 -100µl) of pure vascular blood could be collected from an individual tubeworm, collections from 8-10 individuals were pooled together upon collection to produce a larger, analyzable volume. Larger volumes (200-1000µl) of pure coelomic fluid could often be collected and, in most cases, were not pooled. Only vascular and coelomic fluid samples that were believed to be uncontaminated and pure were used when determining the relative Hb abundance the fluid type. Mixed fluid samples (vascular blood and coelomic fluid) were much easier to obtain and were collected in larger volumes (0.5 - 3 ml) from individual tubeworms. The Hbs used in sulfide-binding dialysis experiments were purified from mixed fluid samples. After collection, each sample of pure or mixed 83 fluids was mixed with 1µl of 1.0 mM PMSF (Roche Molecular Biochemicals) to prevent
Hb degradation and was frozen at –20˚C or in liquid nitrogen onboard ship. Samples were transferred to the laboratory on dry ice or in liquid nitrogen and stored at -70˚C until use.
Fluid relative hemoglobin abundance
Subsamples of pure vascular and coelomic fluid sample were separated by gel filtration into component Hbs as described in Chapter 3. The eluent was monitored at
280 nm. After separation, the relative protein abundance (in cm2/µl) was determined for each component Hb by measuring the area of each peak on the chromatographic tracing and standardizing the area for the volume of fluid that was injected. As the 3,500 kDa
Hb and the 400 kDa Hbs do not have the same protein/heme ratios (24,300:1 and
16,670:1, respectively) and relative Hb abundance values were calculated based on the relative protein abundance and these ratios.
Hemoglobin purification and quantification
Samples of L. luymesi and S. jonesi fluids were thawed, centrifuged at 4,000g for
5 minutes in order to remove impurities, and separated by gel filtration on a 1 X 30 cm
Superose 6-C column (Pharmacia LKB Biotechnology) using methods described in
Chapter 3.
Heme concentrations of intact vestimentiferan fluids and experimental heme containing solutions were quantified as described in Chapter 3, based upon methods adapted from Van Assendelft, (1970) and Toulmond et al. (1990). 84
Dialysis experiments
Hemoglobin sulfide-binding dialysis experiments were conducted as described in
Chapter 3. At each dialysate sulfide concentration, three replicate dialysis bags containing 300µl of 3,500 kDa or 400 kDa Hbs were run in parallel with three replicate dialysis bags containing 300µl of deoxygenated Riftia saline as controls.
Quantification of sulfide
- - ΣH2S (the sum of H2S, HS , and S2 ) of dialysate solutions with ΣH2S concentrations greater than 5µM, and of all Hb solutions, was quantified by gas chromatography (GC; (Childress et al., 1984; Childress et al., 1991)).
The ΣH2S of dialysate samples containing less than 5µM ΣH2S was quantified by using a modification of the methods of Singh et al. (1990), and Freytag et al, (2001). For quantification of total sulfide between 0.2-2.0 µM or 0.5-5.0 µM, 700 µl of the dialysate
sample was combined with 150 µl of papain-SSCH3 solution (3.4 mg/ml papain-SSCH3
(Molecular Probes), 8.0 mM ethylenediaminetetraacetic acid (EDTA), 60 mM NaH2PO4,
10.0 mM sodium acetate, pH 7.4) under a nitrogen head and incubated on ice for one hour. Samples were then combined with 150 µL of a solution of chromogenic substrate
[7.2 mg/ml N-benzoyl-L-arginine, p-nitroanilide (L-BAPNA) (Molecular Probes), 3 mM
EDTA, 150 mM bis-Tris, pH 6.3] and incubated in a 28ºC water bath an additional 16 to
20 hours. A series of sulfide standards (0.2-2.0 µM or 0.5-5.0µM) were run in parallel with dialysate samples. The duration of the final, 28ºC incubation was between 18 and 20 hours for the 0.2-2.0 µM sulfide series and between 16 an 18 hours for the 0.5-5.0µM sulfide series. The absorbance of the samples at 410 nm was noted after the final incubation. 85
Data analysis
Since the GC analysis methodology measures the total (bound and unbound)
sulfide in samples of vestimentiferan Hb, bound sulfide was calculated by subtracting the
concentration of ΣH2S in the control dialysis bags (free ΣH2S) from the concentration of
ΣH2S measured in the Hb solutions (bound and free ΣH2S). The bound sulfide was then
standardized to the amount of heme in the sample to allow comparisons between Hbs and
to other studies.
The capacity and affinity of each hemoglobin for sulfide was estimated as
described in Chapter 3.
Results
Composition of vascular and coelomic fluids
Fluid heme concentrations and the relative abundance of each size Hb in
Lamellibrachia luymesi vascular blood and coelomic fluid are shown in Table 4.1. Heme
concentrations of pooled L. luymesi vascular blood ranged from 3.2 to 4.4 mM heme and
had a mean value of 3.9±0.45 mM heme (S.D., N=7 pooled samples). All L. luymesi vascular blood samples contained both 3,500 kDa and 400 kDa Hbs, and the 3,500 kDa
Hb constituted, on average, 90.4% (range= 82%-94.5%, N=7 pooled samples) of the Hbs present. Heme concentrations of L. luymesi coelomic fluid ranged from 0.78 to 2.1 mM and had a mean value of 1.3±0.3 mM heme (S.D., n=39). Most L. luymesi coelomic fluid samples contained both 3,500 kDa and 400 kDa Hbs. However, one sample contained no 3,500 kDa Hb. The 3,500 kDa Hb constituted, on average, 39%
(range=0%-57%, n=18) of the Hbs present in L. luymesi coelomic fluid. 86
Fluid heme concentrations and the relative abundance of each size Hb in
Seepiophila jonesi vascular blood and coelomic fluid are shown in Table 4.1. Heme
concentrations in pooled samples of S. jonesi vascular blood ranged from 4.3 to 7.1 mM
heme and had a mean value of 5.5±0.94 mM heme (S.D., N=4 pooled samples). All S. jonesi vascular blood samples contained both 3,500 kDa and 400 kDa Hbs, and the 3,500 kDa Hb constituted, on average, 80% (range= 69%-91%, N=4 pooled samples) of the
Hbs present. Heme concentrations of S. jonesi coelomic fluid ranged from 1.2 to 3.3 mM and had a mean value of 2.2±0.54 mM heme (S.D., n=11). No 3,500 kDa Hb was present in two of seven analyzed samples. The 3,500 kDa Hb constituted, on average, 23%
(range=0%-45%, n=7) of the Hbs present in S. jonesi coelomic fluids.
Sulfide binding by purified Lamellibrachia luymesi and Seepiophila jonesi
hemoglobins
The relationships between free sulfide (the ΣH2S concentration in control dialysis
bags) and sulfide bound by the hemoglobins (mM HS-/mM Heme) for the purified
hemoglobin fractions from Lamellibrachia luymesi are shown in Figures 4.2 and 4.3. At
saturation the 3,500 kDa Hb bound 1.85 moles HS- per mole of heme (parameter 95%
- confidence interval (CI) = 1.1 – 2.6 HS /Heme) with a C50 of 96 µM sulfide (parameter
95% CI = 70 – 132 µM). The 400 kDa Hbs have a lower capacity for sulfide and bind
0.58 moles HS- per mole of heme (parameter 95% CI = 0.50 - 0.66 HS-/Heme) at
saturation (See Fig. 4.3). The C50 of the 400 kDa Hbs of 100 µM sulfide (parameter 95%
CI = 46 – 218 µM) is similar to that of the 3,500 kDa Hb. The Hill plots of sulfide- 87 binding for the 3,500 and 400 kDa Hbs were linear with slopes of 1.0 (parameter 95% CI
= 0.65-1.45) and 0.7 (parameter 95% CI = 0.11-1.2), respectively.
The relationships between free sulfide (the ΣH2S concentration in control dialysis bags) and sulfide bound by the hemoglobins (mM HS-/mM Heme) for the purified hemoglobin fractions from Seepiophila jonesi are shown in Figures 4.4 and 4.5. At saturation the 3,500 kDa Hb bound 1.33 moles HS- per mole of heme (parameter 95%
- confidence interval (CI) = 1.25 – 1.41 HS /Heme) with a C50 of 8.8 µM sulfide (parameter
95% CI = 5.3 – 14.7 µM). The 400 kDa Hbs have a lower capacity for sulfide and bind
0.35 moles HS- per mole of heme (parameter 95% CI = 0.21 - 0.49 HS-/Heme) at
saturation (See Fig. 4.5). In contrast to the 3,500 kDa Hb, the C50 of the 400 kDa Hbs is quite high at 246 µM sulfide (parameter 95% CI = 105 – 576 µM). The Hill plots of sulfide-binding for the 3,500 and 400 kDa Hbs were linear with slopes of 1.1 (parameter
95% CI = 0.73-1.50) and 0.6 (parameter 95% CI = 0.20-1.0), respectively.
Discussion
Three limitations of the collection and experimental methodology described here must be taken into account when interpreting the relative Hb abundance data, and the Hb and fluid sulfide-binding estimates presented here. First, the collection of pure vascular and coelomic fluids from Lamellibrachia luymesi and Seepiophila jonesi remains difficult and, as a result, it is impossible to know if the presence of the 3,500 kDa Hb in the majority of the coelomic fluid samples is real or is the result of contamination of the coelomic fluid by vascular blood. The absence of the 3,500 kDa Hb from the elution profiles of several coelomic fluid samples from both L. luymesi and S. jonesi suggests 88 that this Hb may not be a component of the coelomic fluids of these species (as suggested for the hydrothermal vent tubeworm, Riftia pachyptila; (Arp et al., 1987)). Another possibility is that the relative abundance of the 3,500 kDa Hb in the coelomic fluids of L. luymesi and S. jonesi actually does range from 0% to ca. 50% in vivo. Thus, coelomic fluid sulfide capacities have been estimated in two ways in Table 4.1: with the measured relative abundance of the 3,500 kDa Hb, and assuming 0% 3,500 kDa Hb in the coelomic fluid. Estimates of fluid sulfide capacity that include the 3,500 kDa Hb in the coelomic fluids of both species are higher than the estimates that exclude the 3,500 kDa Hb.
Second, the inability to separate vascular 400 kDa Hbs from coelomic 400 kDa
Hbs in mixed fluid samples must also be considered when interpreting the sulfide-binding results for the 400 kDa Hbs of each species. As fluids from many different individual tubeworms were required to produce enough purified Hbs to conduct the sulfide-binding experiments, we are unable to estimate the percentage of vascular 400 kDa Hb versus coelomic 400 kDa Hb in our purified 400 kDa Hb fraction. Thus, we are not able to
attribute the sulfide capacity or C50 estimates of the 400 kDa Hbs to either the coelomic or vascular 400 kDa Hb in either species. Structural studies by Zal and others (1996,
1998) on the R. pachyptila vascular and coelomic 400 kDa Hbs suggest that these Hbs contain the same number of heme-containing chains and free Cys residues, and therefore likely have the same sulfide-binding capacities (Zal et al., 1996; Zal et al., 1998). In addition, the relatively steep slope in the sulfide-binding curve for L. luymesi 400 kDa
Hbs around the C50 (Fig. 4.2) indicates that the vascular and coelomic 400 kDa Hbs have very similar C50 values. The resolution of the respective curve for S. jonesi 400 kDa Hbs 89
(Fig. 4.4) is not sufficiently high to determine if the vascular and coelomic 400 kDa Hbs
have similar C50 values.
Third, sulfide-binding experiments with purified R. pachyptila 3,500 kDa Hb demonstrated that in vitro this Hb bound more sulfide per heme at heme concentrations greater than 0.6 mM (See Chapter 3), the experimental heme concentration used in this study. Therefore, the sulfide-binding capacities of purified L. luymesi and S. jonesi 3,500 kDa Hbs are also likely higher at the in vivo heme concentrations (≥3.5 mM heme) of the
3,500 kDa Hb in the vascular blood of each species. Sulfide-binding by R. pachyptila
400 kDa Hbs did not increase at heme concentrations greater than 0.6 mM, and, therefore, the sulfide-binding capacity estimates for 400 kDa Hbs presented here should represent in vivo capacities. In Table 4.1, we estimate fluid sulfide capacity for the vascular blood of L. luymesi and S. jonesi with the 3,500 kDa Hb sulfide-binding capacity estimates reported here, and using the value of 3.0 HS-/Heme that has been reported for the R. pachyptila 3,500 kDa Hb (Arp et al, 1987; Zal et al, 1997) to permit comparison to other studies. L. luymesi and S. jonesi vascular blood sulfide capacity estimates are greater than that of R. pachyptila using either calculation method.
By definition, niche differentiation takes place when multiple, ecologically similar species that coexist in a stable environment are able to decrease interspecific competition by dividing a limiting resource (Morin, 1999; Schoener, 1974). Niche differentiation, or resource partitioning, is well documented in co-occurring plant and animal species and is likely occurring between Lamellibrachia luymesi and Seepiophila jonesi in aggregations at northern Gulf of Mexico cold seep sites. S. jonesi normally grow so that the plumes of large individuals are only centimeters above the sediment- 90
water interface. Co-occurring L. luymesi are much longer than S. jonesi (Bergquist, et al
2002), a growth habit that results in the plumes of large L. luymesi positioned one meter
or more above the sediment-water interface, and gives the aggregations their bush-like
appearance. These observations led us to hypothesize that large S. jonesi may
supplement whatever sulfide they acquire through their roots with sulfide from low
concentration pools just above the sediment-water interface while large L. luymesi
acquire most, if not all, sulfide directly from pools in seep sediments.
The Hb abundance and sulfide-binding data indicate that the 3,500 kDa Hb is
responsible for ∼95% of the sulfide binding by the vascular blood of both L. luymesi and
S. jonesi. The sulfide affinity of the 3,500 kDa Hb in the vascular blood of a
vestimentiferan is physiologically significant because a high sulfide affinity (low C50
value) facilitates uptake of sulfide from low environmental concentrations (Childress et
al., 1991). Results from sulfide-binding experiments show that the 3,500 kDa Hb of S.
jonesi has a high affinity for sulfide while the 3,500 kDa Hb of L. luymesi has a much
lower affinity for sulfide. Figure 4.6 illustrates the differences between the sulfide-
binding properties of the S. jonesi and L. luymesi 3,500 kDa Hbs and the environmental sulfide concentrations of functional relevance to these Hbs. At sulfide concentrations of
3.7 µM the S. jonesi 3,500 kDa Hb can accumulate sulfide to about 30% saturation. This suggests that S. jonesi could efficiently take up sulfide from the low concentration
environmental pools documented at the sediment-water interface. In contrast, the L.
luymesi 3,500 kDa can only increase saturation by 2.5% (from 10% to 12.5%) at these
sulfide concentrations (Fig. 4.6) suggesting that the L. luymesi vascular fluid cannot
efficiently acquire sulfide from very low concentrations. At sulfide concentrations above 91
50 µM both species can bind significant amounts of sulfide. Sulfide concentrations of the extracellular space into which sulfide is offloaded from the Hbs to the bacteriocytes have not been measured. It is known, however, that the bacteria have a very high affinity for sulfide and likely create a large sink for sulfide around the bacteriocytes (Arp et al.,
1985). Fisher and others have demonstrated that sulfide can be acquired by vestimentiferan endosymbionts from Hb-containing vascular and coelomic fluids (Fisher et al., 1988; Fisher et al., 1989). These workers have also shown that vestimentiferan vascular fluids (and Hbs) make high concentrations of sulfide available to the symbionts while maintaining very low environmental free sulfide concentrations, conditions that elicited maximal carbon fixation rates by the bacteria (Fisher et al., 1988). This is additional evidence that the free sulfide concentration in the extracellular space is very low (< 0.5 µM). These concentrations would facilitate offloading of sulfide from the
3,500 kDa Hbs of both S. jonesi and L. luymesi (See Fig. 4.6).
As sulfide concentrations measured in seawater just above the sediment-water interface around the bases of mature tubeworm aggregations (and around S. jonesi plumes) are both low and variable (0-3.7µM; (Bergquist et al., 2003a; Freytag et al.,
2001)), we believe that the high sulfide affinity of the 3,500 kDa Hb in the vascular blood of S. jonesi could allow this species to exploit this environmental sulfide pool. We do not know to what extent S. jonesi utilizes its plume for sulfide uptake in situ. We suggest only that this species is likely able to acquire sulfide across its plume and cannot rule out that S. jonesi obtains some or most of the sulfide it requires across its buried root. S. jonesi also likely utilizes its plume for carbon dioxide and oxygen uptake. With a low affinity for sulfide, the L. luymesi 3,500 kDa Hb is very unlikely able to facilitate sulfide 92 uptake from the environmental concentrations found around the plumes of large L. luymesi (< 0.1µM; (Freytag et al., 2001)). However, the low binding affinity of the L. luymesi 3,500 kDa Hb for sulfide is still likely sufficient to facilitate sulfide uptake from interstitial pools that have sulfide concentrations as high as 7.9 mM (Bergquist et al.,
2003a). We suggest that large L. luymesi do not utilize the plume portion of their bodies for sulfide uptake in situ and, therefore, rely predominantly on the buried root portion of its tube and tissue for sulfide acquisition. Whole-animal respiration experiments demonstrated that L. luymesi could acquire sulfide in this manner in vitro while simultaneously taking up carbon dioxide and oxygen across its anterior plume (Freytag et al., 2001).
Though we cannot attribute the C50 values of the 400 kDa Hbs from L. luymesi fluids to either the vascular or coelomic 400 kDa Hb, the very similar sulfide affinities of the L. luymesi 3,500 kDa and 400 kDa Hbs suggest that the intact vascular and coelomic fluids of this species have similar affinities for sulfide. Similar vascular and coelomic fluid affinities would allow bi-directional exchange and storage of sulfide in both fluid compartments. The ability to transfer sulfide between coelomic fluid and vascular blood would also facilitate root sulfide uptake by L. luymesi. Sulfide diffusing from interstitial pools across the root tube and into the tissues of L. luymesi that was first bound by coelomic Hbs could be passed to vascular Hbs and bacterial endosymbionts. Our estimate of the sulfide affinity of the 400 kDa Hbs from S. jonesi fluids is extremely low and is not at all similar to that of the S. jonesi 3,500 kDa Hb. This suggests that exchange between the two fluids is not bi-directional and that the coelomic fluid of S. jonesi is unlikely to serve as a temporary store of sulfide. This is not consistent with previous 93 hypotheses regarding role of vestimentiferan coelomic fluid in vent species (Childress et al., 1991; Fisher et al., 1988). Discrete water samples have been used to determine the sulfide concentrations in the bottom waters around and within vestimentiferan aggregations. These data only provide the sulfide concentrations present at the time that the samples were taken and do not represent the temporal variability of sulfide concentrations that might be available to the plumes of L. luymesi and S. jonesi. Plants that live on forest floors are able to survive with less than 1% of the light available to canopy plants, 40-80% of which arrives as sunflecks, occasional short bursts of sunlight that reach the forest floor when small gaps in the overlying canopy are opened by winds
(Pfitsch and Pearcy, 1989; Björkman and Ludlow, 1972). There are lines of argument however, that suggest that large L. luymesi do not subsist by occasionally taking up small amounts of sulfide that might reach their plumes. Based on binding data present here, the plumes of L. luymesi would need to be exposed occasionally and regularly to a minimum of 50µM sulfide for individuals to survive in a manner analogous to understory plants.
Concentrations less 50µM would not be able to be concentrated by the vascular 3,500 kDa Hb of L. luymesi (See Fig. 4.6) and thus would be useless to the animal. It is hard to conceive of a mechanism where sulfide moves vertically from seep sediments to tubeworm plumes in a way similar to how a ray of light reaches the forest floor through a canopy of trees. Sulfide is highly unstable, reacting spontaneously with oxygen, and would have to rely on diffusion to slowly transport it through 0.5 to 2 m of oxygenated seawater to the plumes. Additionally, sulfide concentrations that have been measured just above the seafloor sediment are very low (≤ 3.7 µM). As a result we consider it 94 likely that large L. luymesi, with their plumes high above the sediment-seawater interface, rely heavily if not entirely upon their roots for sulfide acquisition.
The previous discussion of the sulfide affinities of the L. luymesi and S. jonesi
3,500 kDa Hbs assumes that there is one 3,500 kDa Hb with one sulfide affinity in the vascular blood of each tubeworm species. However, the sulfide-binding data for the L. luymesi 3,500 kDa Hb data suggests that this 3,500 kDa Hb may have multiple sulfide- binding mechanisms with different sulfide affinities or that there may be actually two different 3,500 kDa Hbs in the fluids of L. luymesi with different sulfide-binding characteristics (See the thick dashed line in Fig 4.2). Work by Zal and others has demonstrated two separate mechanisms of sulfide-binding by the 3,500 kDa Hb of R. pachyptila (Zal et al., 1998). Analysis of the first “step” of the L. luymesi 3,500 kDa Hb
- data provides a capacity estimate of approximately 0.7 HS /Heme and a C50 of 2.2µM sulfide (and would have a high sulfide affinity). Analysis of the second “step” gives a
- capacity estimate of approximately 1.5 HS /Heme and a C50 of 174µM sulfide (a very low affinity for sulfide). The planktonic larvae of both S. jonesi and L. luymesi settle out of the water column onto hard substrate in areas of active seepage that has been shown by
Bergquist and coworkers (2002) to be both temporally and spatially limited (Bergquist et al., 2002). New recruits of both seep species must acquire sulfide across their anterior plumes until they can grow posterior roots down into interstitial sulfide pools. Thus young L. luymesi may require a higher affinity 3,500 kDa Hb during their first few years of life. The presence of either two 3,500 kDa Hbs or one with a range of affinities for sulfide would be very advantageous to new L. luymesi recruits. 95
Although this study focuses on Hb-sulfide binding, vestimentiferan Hbs also
function as oxygen carriers (Arp et al., 1985). Based upon studies of the hydrothermal
vent worm R. pachyptila it has been suggested that the high Hb concentrations of
vestimentiferan fluids are also required to transport and store the oxygen required by the
symbiosis (Childress and Fisher, 1992). The heme concentrations of the vascular blood
and coelomic fluid of S. jonesi are significantly greater than the heme concentrations of
the respective fluids of L. luymesi (See Table 4.1; vascular blood t-test: t = 2.36, df = 7, P
= 0.005; coelomic fluid t-test: t = 2.18, df = 12, P = 0.0002). Accordingly, the high fluid heme concentrations of S. jonesi may facilitate the survival of this species in environments where they may be exposed to low concentrations of oxygen and/or short periods without any oxygen, such as in the middle of an aggregation with the plume near the sediment-water interface. As the plumes of large L. luymesi are usually well over a meter above the sediment-water interface and on the external surface of the aggregations, large individuals of this species are exposed to a relatively constant supply of oxygen and may not require elevated fluid Hb concentrations for the temporary storage of oxygen.
Our proposed models of sulfide acquisition for L. luymesi and S. jonesi suggest some additional testable hypotheses. First, if large L. luymesi are getting most of their sulfide across their roots while large S. jonesi are getting most of their sulfide from above-sediment pools, L. luymesi would then have more extensive roots than S. jonesi.
Second, the different pools of sulfide utilized by the two species may have different stable sulfur isotope signatures (δ34S values), and this should be reflected in different
tissue δ34S values between species in the same aggregation. Determining the δ34S values
of the environmental sulfide pools and the tissue δ34S values of L. luymesi and S. jonesi 96 could resolve whether these two co-occurring seep tubeworm species are indeed partitioning resources. Third, the potentially different strategies for sulfide acquisition employed by very young and large L. luymesi could require expression of different 3,500 kDa Hbs over their life cycle. Sampling fluids and generating sulfide-binding curves for
3,500 kDa Hbs from young individuals of L. luymesi could test this hypothesis.
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Table 4.1. Hemoglobin sulfide-binding characteristics and properties of intact fluids of Seepiophila jonesi, Lamellibrachia luymesi, and Riftia pachyptila.
Hemoglobin Sulfide Fluid Heme Concentrations Relative Hb Abundance of Intact Fluids Fluid Sulfide Capacities (mM Heme) Capacities - (HS /Heme) (mM H2S) Tubeworm 3,500 400 Vascular Coelomic Vascular Coelomic Vascular Coelomic Species kDa Hb kDa Hbs 3,500 kDa 400 kDa 3,500 400 kDa Hb Hb kDa Hb Hb Seepiophila 1.33 0.35 5.5 2.2 80% 20% 23% 77% 6.2 1.26 jonesi (1.25-1.4) (0.2-0.5) (4.3-7.1),0.94 (1.2-3.3),0.54 (69-91%) (9-31%) (0-45%) (55-100%) (13.6)1 (0.77) 2 Lamellibrachia 1.85 0.58 3.9 1.3 90.5% 9.5% 39% 61% 6.7 1.40 luymesi (1.1-2.6) (0.5-0.66) (3.2-4.4),0.45 (0.78-2.1),0.3 (82-94.5%) (5.5-18%) (0-57%) (43-100%) (10.8) 1 (0.75) 2 Riftia 2.2 0.47 3.5 1.9 62% 38% 14% 86% 5.4 1.35 pachyptila (2.0-2.3) (0.42-0.51) (2-4.8), 0.19 (0.9-3.6),0.21 (34.5-81%) (19-65.5%) (0-36%) (64-100%) (7.1) 1 (0.89) 2
Hemoglobin sulfide capacities are reported as: mean, (95% C.I.). Fluid heme concentrations are reported as: mean, (Range), S.D. Relative Hb Abundance values are reported as: mean, (Range). 1 - Vascular fluid sulfide capacity estimate assuming 3.0 HS-/Heme sulfide-binding capacity of 3,500 kDa Hbs (See text). 2 – Coelomic fluid sulfide capacity estimate assuming 0% 3,500 kDa Hb in coelomic fluids (See text). Riftia pachyptila Hb sulfide capacities taken from Freytag et al, 2003. Riftia pachyptila fluid heme concentrations and relative Hb abundance values taken from Childress et al, 1984, and Arp et al., 1987. 105
Figure 4.1. The outer edge of a vestimentiferan aggregation on the upper Louisiana slope of the Gulf of Mexico. The anterior plumes of both L. luymesi and S. jonesi are visible. Most S. jonesi plumes are centimeters from the sediment-seawater interface while the plumes of L. luymesi can be more than 0.5 to 1.5 meters above the sediment. 106
Fig. 4.2. - Sulfide binding by the Lamellibrachia luymesi 3,500 kDa hemoglobin as a function of free sulfide concentration. The solid line fits the equation: 20.([ 34∑−HS ] 0 . 0095 ) 2 HS− / Heme = 2 , χ =4.62, N=45, P=<0.001. The thin dashed lines 11+ 10.([20∑−HS2 ] .0095 ) represent the 95% confidence bands for the regression line. At the estimated point of saturation, this hemoglobin binds 1.85 moles sulfide per mole of heme (parameter 95% confidence interval = 1.06 – 2.64 H2S/Heme). The predicted 50% saturation occurs at
95.8µM ΣH2S (parameter 95% confidence interval = 69.4 – 132.2µM). The thick dashed line represents sulfide binding assuming multiple 3,500 kDa Hbs or sulfide-binding mechanisms (See text). 107
0.8
0.7
0.6
0.5
0.4
0.3
0.2 Bound Sulfide (HS-/Heme)
0.1
0 0.0001 0.001 0.01 0.1 1 10 [H2S] mM
Fig. 4.3. - Sulfide binding by the Lamellibrachia luymesi 400 kDa hemoglobins as a function of free sulfide concentration. The solid line fits the equation: 67.([∑−HS ]0 .0023 ) 2 HS− / Heme = 2 , χ =1.03, N=41, P=<0.001. The thin dashed lines 11+ 16.([∑−HS2 ]0 .0023 ) represent the 95% confidence bands for the regression line. At the estimated point of saturation, this hemoglobin binds 0.57 moles sulfide per mole of heme (parameter 95% confidence interval = 0.50 – 0.66 H2S/Heme). The predicted 50% saturation occurs at 100
µM ΣH2S (parameter 95% confidence interval = 46 – 218 µM). 108
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4 Bound Sulfide(HS-/Heme) 0.2
0 0.0001 0.001 0.01 0.1 1 10 [H2S] mM
Fig. 4.4. - Sulfide binding by the Seepiophila jonesi 3,500 kDa hemoglobin as a function of free sulfide concentration. The solid line fits the equation: 151.([ 8∑−HS ] 0 . 0002 ) 2 HS− / Heme = 2 , χ =2.66, N=51, P=<0.001. The thin dashed lines 1+114.([ 5∑−HS2 ] 0 . 0002 ) represent the 95% confidence bands for the regression line. At the estimated point of saturation, this hemoglobin binds 1.33 moles sulfide per mole of heme (parameter 95% confidence interval = 1.25 – 1.41 H2S/Heme). The predicted 50% saturation occurs at
8.8µM ΣH2S (parameter 95% confidence interval = 5.3 – 14.7µM). 109
0.8
0.7
0.6
0.5
0.4
0.3
0.2 Bound Sulfide (HS-/Heme) 0.1
0 0.0001 0.001 0.01 0.1 1 10 [H2S] mM
Fig. 4.5. - Sulfide binding by the Seepiophila jonesi 400 kDa hemoglobins as a function of free sulfide concentration. The solid line fits the equation: 22.([00∑−HS ] .0051 ) 2 HS− / Heme = 2 , χ =2.26, N=40, P=<0.001. The thin dashed lines 16+ .([31∑−HS2 ] 0 . 0051 ) represent the 95% confidence bands for the regression line. At the estimated point of saturation, this hemoglobin binds 0.35 moles sulfide per mole of heme (parameter 95% confidence interval = 0.21 – 0.49 H2S/Heme). The predicted 50% saturation occurs at
246µM ΣH2S (parameter 95% confidence interval = 105 – 576µM). 110
Figure 4.6. - Sulfide binding by the Lamellibrachia luymesi (dashed line) and Seepiophila jonesi (solid line) 3,500 kDa Hbs as a function of free sulfide concentration (from Figures 4.2 and 4.4). Relevant environmental and internal sulfide concentrations are indicated in order to illustrate the potential functional significance of the sulfide binding properties of the Hbs at these sulfide concentrations. The darkest gray area represents an estimated range of sulfide concentrations within the vestimentiferan trophosome where sulfide would be offloaded from the Hbs. The lightest gray area represents the range of sulfide concentrations found near the plumes of S. jonesi (<0.1- 3.7µM). The intermediate gray area represents the range of sulfide concentrations found in sediments beneath tubeworm aggregations. 111
Chapter 5
Summary
The majority of this work was conducted to increase our understanding of the sulfide physiology of Lamellibrachia luymesi and Seepiophila jonesi, two vestimentiferan tubeworm species that live together in aggregations at hydrocarbon seep sites in the northern Gulf of Mexico. The hydrothermal vent tubeworm, Riftia pachyptila, has been well studied and takes up sulfide, carbon dioxide, and oxygen from the environment across the anterior, plume portion of its body. Preliminary measurements made around the plumes of northern Gulf of Mexico hydrocarbon seep tubeworms suggested that the physiological ecology of sulfide uptake by seep tubeworms might be different from that of R. pachyptila. Broadly, the goal of this study was to provide insight into how the hydrocarbon seep vestimentiferans, L. luymesi and S. jonesi, acquire sulfide from their environments. Initial work that is presented in the second chapter of this thesis tested the following hypotheses:
- Sulfide is not present around the plumes of large L. luymesi (> 0.5 m).
- L. luymesi can use the posterior “root” portion of its tissue and tube to acquire
sulfide.
- Sulfide uptake by the root of L. luymesi is sufficient to elicit and sustain net
chemoautotrophy by the symbiosis. 112
Soon after the discovery of the hydrothermal vent tubeworm, Riftia pachyptila, researchers realized the importance of the vascular blood, coelomic fluid, and the hemoglobins suspended in these fluids to the survival of the animal and its symbionts.
The functions and structures of R. pachyptila hemoglobins and fluids have been heavily studied and resulting data have been instrumental to increasing the understanding of the sulfide physiology of this robust vent worm. The work presented in the third chapter of this thesis began with sulfide-binding experiments conducted with R. pachyptila fluids and isolated hemoglobins. This series of experiments was conducted to determine the sulfide-binding affinities of isolated vestimentiferan 3,500 kDa and 400 kDa Hbs and to test methodologies on fluids from a large vestimentiferan with abundant and easy to collect vascular and coelomic fluids. While the sulfide capacities of isolated R. pachyptila Hbs and the sulfide affinity of mixed R. pachyptila hemoglobins had previously been determined, the affinities of the isolated Hbs had not. During these experiments several limitations of materials and methods that had been used in previous sulfide binding studies were discovered and examined.
Having determined the most appropriate methods for conducting sulfide-binding experiments on isolated vestimentiferan Hbs, studies with L. luymesi and S. jonesi Hbs were started. Based upon observed differences in the growth habits of L. luymesi and S. jonesi, we hypothesized that large S. jonesi and L. luymesi exploit different environmental sulfide sources, and that the functional properties of the vascular and coelomic Hbs of each species would reflect differences in the mechanisms of environmental sulfide acquisition. Heme concentrations and relative Hb abundance of intact vascular blood and coelomic fluids, and the sulfide-binding characteristics of the 113 component Hbs, were determined for both L. luymesi and S. jonesi and are presented in the fourth chapter of this thesis. Results from these studies facilitated the modeling of the whole animal acquisition of environmental sulfide, carbon dioxide, and oxygen for both L. luymesi and S. jonesi.
Conclusions:
Lamellibrachia luymesi can use the root portion of its tube and body to take up sulfide from an environmental pool. When only the roots of individual L. luymesi were exposed to sulfide, the concentrations of sulfide in the blood of the animals were similar to concentrations in the blood of freshly collected individuals. In contrast, control animals that did not have their roots exposed to sulfide did not have any sulfide in their blood (detection limit of 3.0 µM). Live-animal respiration experiments demonstrated that
L. luymesi could use their roots to acquire sulfide at an average rate of 4.1 µmol/g*h and that root sulfide uptake was sufficient to elicit and sustain a net chemoautotrophic response from the animal (indicated by net dissolved inorganic carbon uptake across the plumes of the animals). From these laboratory experiments it can be concluded that L. luymesi has a mechanism that could be used to take up sulfide from interstitial pools. It, however, is not known at this time to what extent L. luymesi is dependent upon its root for sulfide uptake in situ.
There are limitations of materials and methods used in sulfide binding studies.
These limitations have implications for past, present, and future sulfide-binding data and dialysis experiments in general. During sulfide dialysis experiments conducted with regenerated cellulose dialysis membrane material, the concentration of sulfide inside of 114 experimental control dialysis bags was not equal to the sulfide concentration of the external dialysate. Therefore, when determining the amount of sulfide bound by vestimentiferan hemoglobins, it is most appropriate to use the sulfide concentration inside of control bags that are the same size and volume of the experimental hemoglobin containing bags as the free or unbound sulfide concentration. It was also determined that the ability of R. pachyptila hemoglobins to bind sulfide increased with the heme concentration of experimental hemoglobin containing solutions. The 3,500 kDa Hb from
R. pachyptila fluids showed a marked increase in sulfide-binding at heme concentrations above 0.6 mM heme whereas the 400 kDa Hbs from R. pachyptila fluids did not. This suggests that to most accurately determine the sulfide-binding characteristics of vestimentiferan hemoglobins, sulfide-binding dialysis experiments should be conducted at or near in vivo heme concentrations. The mechanism for increased sulfide-binding at increased heme concentrations has not been determined. R. pachyptila 3,500 kDa and
400 kDa hemoglobins have similarly high affinities (low sulfide C50 values) for binding sulfide indicating that bi-directional exchange and storage of sulfide by the vascular and coelomic fluids is possible.
Co-occurring Lamellibrachia luymesi and Seepiophila jonesi have characteristics that may allow them to acquire sulfide from different environmental pools at northern
Gulf of Mexico hydrocarbon seeps. The growth habits of these species suggested that sulfide acquisition mechanisms of L. luymesi and S. jonesi might be different and the sulfide-binding properties of the hemoglobins of each species are consistent with this hypothesis. Results from sulfide-binding experiments indicate that the sulfide-binding characteristics of the hemoglobins and fluids from L. luymesi and S. jonesi are very 115
different. Large S. jonesi may be capable of acquiring sulfide with the anterior plume
portion of its body from low concentration pools found just above the sediment-water
interface. At this time, we cannot rule out that S. jonesi obtains some or most of the
sulfide it requires across its buried root. S. jonesi has elevated fluid heme concentrations
that may facilitate the survival of individuals in environments where they may be
exposed to low concentrations of oxygen and/or short periods without any oxygen. In
situ, large L. luymesi likely do not use their plume for sulfide uptake and are probably dependant upon the root portion of their body and tube for sulfide acquisition. L. luymesi may have a 3,500 kDa Hb with multiple sulfide-binding mechanisms that have different
sulfide capacity and C50 values (one lower and one higher) or two different 3,500 kDa
Hbs with different sulfide-binding characteristics.
Most significant contributions to hydrothermal vent and hydrocarbon seep biology:
This study has fundamentally changed our model for the physiological ecology of
seep vestimentiferans. Previously, all vestimentiferans were believed to function like the
hydrothermal vent tubeworm, R. pachyptila. We know now that, as adults, not all
vestimentiferan tubeworm species rely entirely upon the anterior plume portion of their
bodies for the uptake of metabolites from the environment. Lamellibrachia luymesi can
utilize the root portion of its body and tube to acquire sulfide at rates sufficient to elicit
and sustain net chemoautotrophy. The ability to tap deep interstitial pools of sulfide may
contribute to the longevity of this species.
Results from this work suggest that niche differentiation, or resource partitioning,
may be occurring between Lamellibrachia luymesi and Seepiophila jonesi in aggregations 116 at northern Gulf of Mexico cold seep sites. In situ, large L. luymesi is likely heavily reliant upon its root for sulfide uptake from buried pools while large S. jonesi may acquire sulfide across its plume. Thus, L. luymesi and S. jonesi may be able to acquire sulfide from different environmental pools. At this time, however, it cannot be ruled out that both species are heavily reliant upon their roots for sulfide acquisition. Our proposed models of sulfide acquisition for L. luymesi and S. jonesi suggest the following additional testable hypotheses: 1) that L. luymesi would have more extensive roots than S. jonesi; 2) the different pools of sulfide utilized by the two species may have different δ34S values and this should be reflected in different tissue δ34S values between species in the same aggregation; and 3) juvenile and large L. luymesi may express different 3,500 kDa Hbs over their life cycle.
This study has improved experimental methods used to determine the sulfide- binding characteristics of vestimentiferan vascular and coelomic fluids and Hbs.
Utilization of the methodology described here will increase the usefulness and accuracy of results from future sulfide-binding studies. These methods will be particularly valuable to researchers working with small or delicate species. Vita: JOHN KARL FREYTAG
Department of Biology P.O. Box 338 The Pennsylvania State University Pine Grove Mills, PA 208 Mueller Laboratory 16868 University Park, PA 16802 814-880-9203 814-863-8360 [email protected]
EDUCATION The Pennsylvania State University, University Park, Pennsylvania, USA Ph.D., Biology Dec. ‘03 Dissertation: “ECOLOGICAL PHYSIOLOGY AND BIOCHEMISTRY OF SULFIDE ACQUISITION BY TWO HYDROCARBON SEEP VESTIMENTIFERANS, LAMELLIBRACHIA LUYMESI AND SEEPIOPHILA JONESI.” Dissertation advisor: Dr. Charles R. Fisher
University of California at Santa Barbara, Santa Barbara, California, USA B.S., Aquatic Biology, with Honors June ‘97
PROFESSIONAL EXPERIENCE Research, The Pennsylvania State University, University Park, PA, Aug. 97-Dec.‘02 - Participated in two oceanographic research expeditions to the Gulf of Mexico utilizing manned submersibles (Johnson Sea-Link). - Completed the design and built split-vessel respiration chambers in order to conduct live- animal respiration experiments with hydrocarbon seep vestimentiferan tubeworms utilizing gas chromatography and mass spectrometry.
Research, University of California at Santa Barbara, Santa Barbara, CA, Aug. 95-June‘97 - Participated in three oceanographic research expeditions in the Pacific Ocean studying the biomass and metabolic characteristics of midwater organisms and the physiology of the giant hydrothermal vent tubeworm, Riftia pachyptila.
SELECTED PUBLICATIONS Freytag, J.K., Girguis, P.R., Bergquist, D.C., Andras, J.P., Childress, J.J., & Fisher, C.R. (2001). A paradox resolved: Sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy. Proceedings of the National Academy of Science, 98, no. 23, 13408- 13413.
Girguis, P.R., Childress, J.J., Freytag, J.K., Klose, K., & Stuber, H.R. (2002). Effects of metabolite uptake on proton-equivalent elimination by two species of deep-sea vestimentiferan tubeworm, Riftia pachyptila and Lamellibrachia cf luymesi: proton elimination is a necessary adaptation to sulfide-oxidizing chemoautotrophic symbionts. The Journal of Experimental Biology. 205: 3055-3066.