Reviews in Environmental Science and Bio/Technology

, Volume 5, Issue 2, pp 193–201

Post-capture investigations of hydrothermal vent macro-invertebrates to study adaptations to extreme environments

Authors

    • Department of Oceanography and FisheriesIMAR Centre of the University of Azores
  • Jonathan J. Powell
    • MRC Human Nutrition ResearchElsie Widdowson Laboratory
Review Paper

DOI: 10.1007/s11157-006-0006-z

Cite this article as:
Kadar, E. & Powell, J.J. Rev Environ Sci Biotechnol (2006) 5: 193. doi:10.1007/s11157-006-0006-z

Abstract

Typical survival strategies, developed by macro-invertebrates at a variety of reducing marine habitats including deep-sea hydrothermal vents, have been the subject of the laboratory experimentation over the past three decades. This review provides an insight into the international efforts that have converged on the area of laboratory maintenance of such species whose nutritional requirements are outside the usual scope of metazoan life. We emphasise the methodology used in post-capture manipulations that are designed to identify the physiological limits of adaptation to the harsh conditions known at various vent sites worldwide, and to understand the mechanisms involved. The concepts behind appropriately designed experiments and the choice of suitable model organisms for such physiological studies are also discussed.

Keywords

Deep-sea hydrothermal ventPost-capture experimentsHydrostatic pressureAdaptation to the extreme environment

“When we look at our own planet’s most challenging environments, we are really looking for clues to what may be the normal conditions on other planets. We want a hint of what we may be searching for when we investigate those other worlds for signs of life. We will be better prepared to recognize and study them because our minds have been expanded by our knowledge of our own terrestrial biology....”(Penelope Boston)

Introduction

Definition of “extreme environment” per se is a significant challenge to ecologists, since physicochemical factors that actually prevent life in some organisms may correspond to living requirements for others. However, the textbook definition simplifies the issue by: “conditions that are far outside the boundaries in which most organisms live comfortably. Conditions include: pH, air pressure, temperature, salinity, radiation, dryness (desiccation), and oxygen level”. Deep-sea hydrothermal vents certainly fall under this definition with respect to many of their attributes, including: low pH, high hydrostatic pressure (more that 10-fold the pressures tolerated by humans), reduced conditions, presence of toxicants (heavy metals and radio nucleides), total absence of sun-light, hyper-dynamic conditions and geographical limits to species dispersal. Consequently, studying species that are endemic to such challenging conditions is widely accepted as one of the best approaches of studying adaptation to the “extreme”. However, there is an increasing body of evidence that avoidance response in hydrothermal vent metazoans is in fact a more important adaptation strategy as compared to the previously predicted biochemical adaptation/tolerance to the harsh conditions (Shillito et al. 2001, 2004, 2006; Company et al. 2005; Kadar et al. in press a & b), which suggests that further experimentation and in situ investigations are required. Such acquired adaptation is in contrast to previous speculations on novel, genetically imprinted mechanisms of tolerance in hydrothermal organisms, and points to life on this planet surviving under a wide range of conditions with a restricted biochemical tool kit (Dixon 2005) that deserves more attention.

Over the past 20 years deep sea vent research, especially that involving experimental approaches, was limited by sample availability. Biological studies on live specimens were, and largely still are, restricted to shipboard studies which were mainly carried out during periods that allowed access to underwater locations (usually summer months in the Atlantic). Thus a complete assessment of the life cycle and general physiology of these species was not possible. Laboratory experimentation with these species was, and still remains, hampered by the extreme stress imposed during recovery from depths around 2,000 m (even the shallowest site along the Mid-Atlantic Ridge, Menez Gwen, is 850 m). However, development of pressurized flow-through systems (Quetin and Childress, 1980) enabled on-board or even laboratory experiments, but these were still limited to short, post-capture, episodic studies following scientific cruises to vent locations. However, recent technologies that involve the use of acoustically retrievable mussel cages to supply permanent land-based laboratories (http://www.horta.uac.pt/projectos/fisiovent/cagefilling.htm) have considerably extended access to live animal samples and, as a consequence have increased duration of experimentation and garnered the involvement of a larger scientific community (Dixon et al. 2004; Company et al. 2004; Kadar et al. 2005a; Kadar et al. 2005b). The main improvement provided by this technology is that it enables sampling at times when submersible operation is impossible, and thus it allows investigation of seasonal patterns. The cages (Sonardyne International Ltd., UK), of 1.25 m2 size consist of a frame constructed of glass-reinforced plastic, covered in 2 cm wide plastic mesh, and surrounded by a weighted rubber skirt around the cage base to divert sulphide- and methane-laden fluid through the bottom. They are fitted with floats with transponders to signal their exact position and also with acoustic release mechanisms for later recovery. Using the robot arm of Victor 6,000, the cages can be filled with approximately 500 mussels (2–3 h/cage to fill), and oriented to allow fluid penetration (as indicated by a temperature probe).

The purpose of this review is to present a synthesis of the available data concerning laboratory experimentation on vent macro-invertebrates while specific physiological processes that are involved in adaptation to their challenging environment are highlighted. Thus, rather than simply showcasing some well known examples, emphasis is on the validation of suitable post-capture studies to indicate adaptation to the vent environment in the hope of encouraging further work in the area.

Post-capture experimentation on vent macro-invertebrates

Laboratory experimentation performed at atmospheric pressure

Laboratory investigations of specific adaptations to reducing ecosystems were commenced in the early 1980s on molluscs inhabiting shallow environments that survived well in the laboratory under atmospheric pressure. Thus experiments such as those aimed at C pathways in symbiont-bearing organisms were possible without specialized pressure apparatus (Distel and Felbeck 1988a; 1988b). These experiments were the first to establish a nutritional reliance of the host macro-invertebrates on symbiont bacteria that actively fix carbon from methane. Additionally, symbiont transmission mode was examined that resulted in the development of molecular tools for the determination of bacterial–host specificity, again in species inhabiting shallow reducing environments (sea grass beds and mangrove swamps) in laboratory manipulations conducted for over a decade by Gros and his co-workers (Gros et al. 1996a; 1996b; Gros et al. 1997; Gros et al. 1998a; 1998b; Gros et al. 1999; Gros et al. 2000; Gros et al. 2003a; 2003b). These works stimulated current hydrothermal vent experimentation that is feasible at atmospheric pressure, which uses species from depths where the pressure does not exceed 100 bar as this is the threshold for barofilic adaptations (Dixon et al. 2004). Thus, the hydrothermal bivalve of the genus Bathymodiolus (e.g. B. azoricus), became an important model organism for post-capture investigations, being euribarofilic and thus adapted to different depths at hydrothermal vents along the Mid-Atlantic Ridge (MAR) ranging from 850 m at Menez Gwen (37°35′N–38°N) to 1,700 m at Lucky Strike (37°00′N–37°35′N) and 2,300 m at Rainbow (36°14′N) or even below 3,000 m depth at Broken Spur (29°10′N). Consequently, this organism, when taken from environments <100 bar (i.e. Menez Gwen), not only survives at atmospheric pressure over the time span term (Kadar et al. 2005a; Kadar et al., in press d), but it can also be subjected to high-pressure simulation studies (Company et al., 2004). In addition, it is the biomass-dominant species at many MAR vent sites, and thus is an appropriate choice for the study of distinct exposure conditions within these “natural pollution laboratories” (Kadar et al., in press a & b). Furthermore, its nutritional flexibility to survive on a whole range of nutrient supplies from dual endosymbiosis to filter-feeding (Fiala-Medioni et al. 2002; Kadar et al. 2005a), enables laboratory studies on selective nutritional reliance. Mytilus edulis, from which B. azoricus have evolved (Von Cosel et al., 1999), is a widely accepted pollution biomonitor, and thus results from metal uptake and detoxification studies can be compared with abundant data available on its shore analogues from polluted sites (Kadar et al., in press a, b, & c). Finally and most importantly, this species has been naturally co-exposed to a variety of toxic compounds (Sarradin et al. 1999; Kadar et al. 2005), on a geological time scale, which may have determined the development of genetically-enforced, highly efficient detoxification mechanisms (Kadar, et al. 2005b, Kadar et al, in press a, c), the study of which deserves closer scrutiny. Consequently, B. azoricus has been subjected to studies of both basic and more elaborate physiological processes such as: symbiont transmission mode (Kadar, et al. 2005a), metal toxicology (Kadar, et al. 2005b), reproduction studies (Colaco et al. in press; Kadar et al. in press d), parasitological assessments (Kadar et al. in press e), immunological response studies (Bettencourt et al. in preparation) and ongoing work on selective nutritional reliance. All these experiments were conducted in a laboratory set-up that provided a variety of feeding regimes under controlled conditions, and have been successful in maintaining the vent mussel for over 12 months in captivity (Kadar et al. in press d). These feeding regimes required the supply of aquaria with methane and/or sulphide thus supporting endosymbiosis in B. azoricus for prolonged periods. For a detailed description of the laboratory set up, water parameters and other technological details readers are referred to the work of Kadar et al. (e.g. 2005a, b, and in press d) and http://www.horta.uac.pt/projectos/fisiovent/labhorta.htm. Decompression stress associated with sampling is, however, still an unresolved problem because of the significant gene damage caused by hydrostatic pressure variations. The damage, however, appears to be reversible over a relatively short period of time when animals are allowed to recover, as reported by Dixon et al. (2004). From these data it was concluded that for mussels from locations where pressure did not exceed 100 bar (Menez Gwen vent site for instance) certain investigations would be appropriate following careful sampling and with a recovery period of at least 15 days prior to experimentation. Nonetheless, extrapolation of results obtained on organisms from relatively shallow sites to true deep-sea vents must be made with caution. It is thus worth mentioning that mussels from Lucky Strike vent site (1,700 m depth) have so far resisted every attempt to be maintained in the laboratory for periods longer than 1 month. Experimentation at atmospheric pressure, therefore, only enables long-term studies on species from relatively shallow vents (one only known to date, Menez Gwen), and this remains the main limitation of this approach in spite of it facilitating a whole range of studies that would not be possible under pressurized conditions. An accurate experimental design and investigation of physiological adaptation to hydrostatic pressure requires hyperbaric simulation.

Laboratory experimentation at in situ pressures

Conveniently, a flow-through system was developed in 1980 to re-create hydrostatic pressure characteristics of the environment for two bathypelagic mysids to enable the assessment of behavioral responses to various stress factors, in the laboratory (Quetin and Childress, 1980). This system inspired deep-sea vent researchers to adopt the methodology and thus instigated a new era in deep-sea vent research (Mickel and Childress 1980; 1982a; 1982b; 1982c; Arndt et al. 1998; Arndt et al. 2001; Page et al. 2001; Shillito et al. 2001, 2003; Ravaux et al. 2003; Pradillon et al. 2004 and 2005; Chausson et al. 2004). These laboratory-based pressure culture systems provided settings for specific experiments that have, above all, resolved several misconceptions about hydrothermal vent organisms, such as their relatively slow metabolism and growth (as we knew in non-vent deep-sea fauna) or their extreme thermo-tolerance, based on in-situ observations (reviewed in Van Dover and Lutz 2004).

Inventive experiments have been carried out on the hydrothermal tubeworm, Riftia pachyptila, the species that is arguably at the highest level of adaptation to vent-conditions (Van Dower and Lutz 2004). Tubeworms were kept in pressurized aquaria and exposed to variable H2S concentrations by simply controlling the pH, which provided valuable insights not only into their sulphide metabolism, but also into their laboratory maintenance requirements (Goffredi et al. 1997a, 1997b). Another high-pressure aquaria operating at the Scripps Institute enabled a series of stress response studies on the same species (Arndt et al. 1998; 2001), which demonstrated the fundamental role of “sulphur respiration” in anoxia-tolerant symbioses. Carbon fixation and its transfer from symbionts to the host cells were described following laboratory exposure of the tube worm to radio-labeled CO2 (Bright et al. 2000), and evidenced the importance of direct digestion of symbionts in its diet.

Extensive studies on the adaptive strategies of another hydrothermal species, Bythograea thermydron, experimentally exposed to sulfide, reported its ability to regulate oxygen consumption via thiosulphate regulation in the haemolymph, and demonstrated its increased hemocyanin-oxygen (Hc-O-2) affinity (Childress and Mickel 1980; Sanders and Childress 1985; 1992; Gorodezky and Childress 1994).

Using a high-pressure recirculation aquarium and radiolabeled bacteria, Page and co-workers established the nutritional flexibility of vent bivalves by providing experimental evidence of filter-feeding in Bathymodiolus thermophilus on particulate organic matter and thus supplementing nutrients provided by endosymbiotic chemoautotrophic bacteria (Page et al., 2001).

The first in-vivo experiment on behavioral response to heat shock on the barofilic vent polychaete, Hesiolyra bergi, implemented a novel pressurized incubator namely, IPOCAMP, which was able to simulate pressures as high as 260 bar, while nutrient supply could be controlled and simultaneous video observations were recorded (Shillito et al. 2001). The relatively normal physiological state of experimental specimens, indicated by the 100% survival rate of control animals as well as their high oxygen uptake rate, suggested suitable maintenance conditions. This, together with data from another recent thermal resistance study (Shillito et al. 2006) on a hydrothermal shrimp (Mirocaris fortunate) that exists across the hydrothermal gradient of 2–25°C and at depths from 850 to 2,300 m, have concluded that thermal physiology should be studied at each population’s native pressure. However, collection stress still imposed limitations to this experimental design (i.e. this could have affected thermal resistance). Seeing that heat-shock proteins are induced by various non-temperature related factors, and these may directly influence thermo tolerance, the same authors recommended, for the first time, the use of isobaric collection cells for deep-sea biota sampling. Unfortunately, this technology awaits ongoing field testing until there is wider availability to the research community. Depressurization remains a major problem during experiments; for example whenever water is renewed or dead specimens are removed. However, this problem has been solved, at least at the micro-scale, in a study conducted by Pradillon et al. (2004) that reports on a micro-volume device for pressure simulation. This system has improved features, that allow automatic adjustment of gas (H2S, O and CH4) and/or the supply of other compounds as well as water renewal and interim specimen sampling, without depressurization of the reactor during long-term experiments, for embryonic development studies and/or the study of small organisms, larvae, cell cultures or bacteria.

With the lack of such technology on the macro-scale only short term experiments, such as heat-shock response were possible for various deep-sea mcro-invertebrates: namely, Rimicaris exoculata (Ravaux et al. 2003), Alvinella pompejana (Shillito et al. 2004), and various paralvinnelids (Lee 2003). Surprisingly, these studies collectively established that temperature tolerance of these species is well below values expected for “extremofiles” and based on field observations. An “avoidance response” was proposed as a preferred mechanism (Dixon et al. 2002) rather than the previously stipulated extreme thermo-tolerance (reviewed in Van Dover and Lutz 2004).

Concomitant studies on other physiological parameters have contributed to our present knowledge of various mechanisms involved in adaptation to deep sea hydrothermalism, which would not have been possible without the advantages of such an experimental tool that enables independent control of parameters. A remarkable study, carried out by Chausson and co-workers, not only established that respiratory adaptations of the vent crab Segonzacia mesatlantica are derived from specific functional properties of its haemocyanin (Hc), but also correlated the structure plasticity of this Hc with local environmental factors (Chausson et al. 2004). Ecotoxicological investigations of Company and co-workers, also conducted using HIPOCAMP, on the enzymatic defence of the vent bivalve B. azoricus were the first to document increased resistance of this species, from exposure to heavy metals such as Cd, Cu and Hg, as compared to non-hydrothermal bivalves from polluted areas (Company et al. 2004). However, these experiments were limited to a relatively short time span of exposure conditions (up to 1 month) due to high mortality and the problems associated with bio-corrosion on the surface of the pressure-chamber (discussed in Beech and Gaylarde 1999). Nonetheless in a recent developmental study of the early life stage of Alvinella pompejana in a pressure vessel (Pradillon et al. 2005), the authors have recognized the limitations of the technique (pressure vessels do not simulate the complex and dynamic in situ thermal and chemical conditions typical of hydrothermal vents). Consequently, they have incubated embryos directly at the vent site along a fluid gradient with conditions similar to those re-created in the laboratory. Beyond the scientific finding of this work, the authors have accomplished an accurately designed experiment that was calibrated with a reference field experiment, which normally is not possible due to the prohibitive costs involved. From the above experimental investigations it may be presumed that baseline evaluation of specimen condition (such as oxygen consumption rate, water-, lipid-, and glycogen-content, as well as tissue structure), indicative of animal health, should be determined in situ for each species before undergoing experimental investigation.

While we have learned much over this past 25-years of experimentation, many of the limitations of this pressurized system approach have still not been fully overcome. The most important problems are related to pressure-acclimatization, such as isobaric sampling and maintenance of constant pressure during water changes in the laboratory. It should be noted that the latter has been solved for small size pressure chambers composed of two reactors fed by a common pressure line which can be manipulated separately using a set of valves (for further details see Pradillon et al. 2004). Furthermore, long-term maintenance requires provision of specimens with the typical hydrothermal nutrients (methane and sulphide) which bring about a series of challenges for engineers (development of novel inert materials resistant to both pressure and bio-corrosion; prevention of complexation reactions causing clogging of the tubing system in metal exposure experiments; development of the pressure-resistant version of the classical instrumentation used in behavioral response studies; etc) and for physiologists trying to tackle complex questions relevant to dynamic systems such as those typical of deep-sea hydrothermal vent conditions.

Future advances

This review of the major experimental approaches to understand adaptation to vent conditions highlights that laboratory maintenance allows for some specific investigations to be conducted with great advantages over the costly and resource-consuming in situ observations. However, by no means can current technologies address all of the issues needed in mimicking vent site conditions, and the need for continued improvements are widely emphasized. Critical issues in all the above are, for example, sampling limitations that preclude continuous research, or the poor animal condition at- and post-capture. Additionally, empirical data on natural “health-condition” is lacking for almost all experimental species. New approaches, therefore, must focus on these deficiencies to ensure viability of experimentation in the long-term.

Choosing the most suitable specimens

In considering comprehensive experimental design, aimed at studying adaptation mechanisms to “extreme” environments, the model organisms should (1) be adapted to and/or respond to a range of environmental changes relevant to the question to be answered; (2) be representative of typical conditions to allow organism-habitat inferences; (3) be abundant enough in their natural environment to enable long-term study without endangering the population or interfering with management of protected areas; (4) be studied both in the laboratory and in in situ experiments for validation of results; (5) have non-vent analogues with well studied physiology for comparison and, finally; (6) be able to resist the stress associated with laboratory handling. Certainly the list could be extended and recommendations are welcome.

Continuity in sample supply

Relatively frequent (even monthly) animal supply has been achieved using the acoustically retrievable cage technique that operates at significantly reduced costs with simultaneously increased flexibility/frequency of sampling, since it is only dependent upon the ROV for deployment and filling of cages, white recovery can be pursued using less expensive vessels at any time of the year. Moreover, our experience also shows that cage-recovery dramatically improves animal condition as compared to those collected using the classical slurp gun technique.

Alternatively, continuous laboratory-cultures of vent organisms would be an option, although so far this has been limited to experiments on vent microorganisms (Postec et al. 2005; Daughney et al. 2004; Mergeay 2000). Current advances in laboratory reproduction of vent bivalves (Colaco et al. in press) are encouraging attempts.

Animal condition during sampling and post-capture

Possible tools helping to solve the problems related to sampling-stress include isobaric sampling cells that are connectable to hyperbaric chambers on-board without pressure loss, as first recommended by Shillito et al. (2001). It is only a matter of time before development and testing will make such tools available for field-use (the equipment-testing cruise, EXOCET/D, is scheduled for summer 2006).

Regarding specimen condition during experiments, re-creation of their natural environment in the laboratory has proved to be successful for various species. Arguably one of the best examples is the functional experimental set-up for the long-term maintenance of B. azoricus that enables preservation and manipulation of endosymbiosis (Kadar et al. 2005a), with the potential to develop groups with distinct nutritional reliance, and thus creating the basis for more elaborate ecophysiological research. Although improvements will be necessary in the future in terms of pressure simulations and control of water parameters (to which infra-structural conditions are currently being developed), this system has provided evidence for the good condition of mussels as shown not only by long-term survival (over 12 months), but also by gametogenesis (Kadar et al. in press d) and larval spawning in captivity (Colaco et al. in press). In addition, by maintaining endosymbiosis (both types: methane and/or sulphide oxidizers) within the host, these bacteria, otherwise uncultivable under laboratory conditions, provide a new experimental tool in vent research (Kadar et al. 2005a).

Conclusions

In this review we have shown how recent studies have taken advantage of various experimental tools to address specific issues related to adaptation to extreme environments that are typical of deep-sea hydrothermal vents. The greatest understanding will come, however, when inferences are made in conjunction with physiological data collected in the field. In the future, rapid evolution of new technologies will enable the development of dedicated in situ experimental strategies to calibrate laboratory set-ups and provide baselines for animal condition. Instrumental platforms are particularly needed to monitor real-time chemical and temperature gradients at scales relevant to individual organism, which may only be addressed through integrated multi-disciplinary studies.

Acknowledgments

The research was undertaken under the scope of the research project FISIOVENT (Physiological adaptations to extreme conditions at deep sea hydrothermal vents) funded by FCT (POCTI/MAR/55547/2004). We acknowledge the postdoctoral fellowship (SFRH/BPD/19625/2004) to EK. The kind efforts of the two anonymous reviewers and of David Dixon to improve this paper are gratefully acknowledged.

Copyright information

© Springer Science+Business Media B.V. 2006