Françoise Gaill

Structure and rheology of gelatin and collagen gels

This paper undertakes a parallel analysis of the gelation mechanisms, structure and rheological properties of gelatin and collagen gels. Although the molecular compositions of collagen and gelatin are almost identical, gelation proceeds from distinct mechanisms and leads to different types of molecular assemblies. First are presented the properties of the solutions, based on their structural and rheological characterization; then the mechanisms of gelation in the networks, observed by Transmission Electron Microscopy, of three types of gels: gelatin gels, Type I collagen gels and gels made of cuticle collagen extracted from annelid worms. The rheological investigation of the sol-gel transition of gelatin is described within the context of the theories of percolation and scaling laws. Different experimental approaches to the kinetics of gelation are presented, combining dynamic light scattering and rheology in respect to gelatin gels.

Biology and ecology of the “Pompeii worm” (Alvinella pompejana Desbruyères and Laubier), a normal dweller of an extreme deep-sea environment: A synthesis of current knowledge and recent developments

Abstract Alvinella pompejana, the “Pompeii worm” lives on active hydrothermal edifices at deep-sea vents of the East Pacific Rise. The physical and chemical patterns of its microhabitat were determined from temperature probe measurements, temperature time series, and on-board and shore-based chemical analyses based on discrete sampling (pH, H2S, CO2, CH4, S2O2-3, Ca, Mg, Cu, Cd, Zn). The microhabitat is characterised by high temporal and microscale spatial variability, with temperature values in the range of 20°–45°C at the immediate periphery of tubes but reaching higher, still undetermined, values inside the tubes. The difference observed between in vitro temperature limits for the stability of biomolecules and metabolic rates, and suggested in situ conditions seems to indicate a significant protective role of biological interfaces (tubes and cuticle). Temporal instability possibly also plays an important role in the ability for these worms to colonise such an extreme habitat. The functional role of dominant epibiotic bacteria is discussed in the light of recent biochemical and molecular data: the tube-worm–bacteria system can be considered as a symbiotic entity where carbon is probably metabolised and recycled. Sulphide detoxification occurs by oxidation at the gill level and possibly at the intracellular haemoglobin level. Heavy metals, ingested or absorbed, are trapped in spherocrystals and bound to metallothionein-like proteins. Anatomical, physiological and molecular adaptations to hypoxia allow the worm to successfully colonise the chimneys. A. pompejana lives in an ephemeral environment and must reproduce and disperse accordingly. It is a gonochoric species that displays a pseucopulatory behaviour allowing transfer of sperm to female spermathecae, thus avoiding dispersion of the gametes. The size of the oocytes suggests a lecithotrophic or benthic development. The population size structure is polymodal, indicating discontinuous recruitment. Population genetics data indicate the occurrence of a microscale level of population differentiation that does not increase with increasing geographical distances, thus suggesting the occurrence of a metapopulation-like system and/or the possibility that enzyme loci evolve under stabilising selective driving forces inherent to the vents’ highly variable conditions.

Deep-sea ecology: Developmental arrest in vent worm embryos

Temperature is a key factor in controlling the distribution of marine organisms and is particularly important at hydrothermal vents, where steep thermal gradients are present over a scale of centimetres. The thermophilic worm Alvinella pompejana, which is found at the vents of the East Pacific Rise (2,500-m depth), has an unusually broad thermotolerance (20–80 °C) as an adult, but we show here that the temperature range required by the developing embryo is very different from that tolerated by adults. Our results indicate that early embryos may disperse through cold abyssal water in a state of developmental arrest, completing their development only when they encounter water that is warm enough for their growth and survival.

Heat-shock response and temperature resistance in the deep-sea vent shrimp Rimicaris exoculata.

SUMMARY The shrimp Rimicaris exoculata swarms around hydrothermal black smoker chimneys at most vent sites along the Mid-Atlantic Ridge. This species maintains close proximity to the hydrothermal fluid, where temperatures can reach 350°C and steep thermal and chemical gradients are expected. We performed in vivo experiments in pressurized aquaria to determine the upper thermal limit [critical thermal maximum (CTmax)] of R. exoculata and to investigate some characteristics of the shrimp stress response to heat exposure. These experiments showed that the shrimp does not tolerate sustained exposure to temperatures in the 33-37°C range (CTmax). A heat-inducible stress protein belonging to the hsp70 family was identified in R. exoculata, and its synthesis threshold induction temperature is below 25°C. The R. exoculata optimal thermal habitat may thus be restricted to values lower than previously expected (<25°C).

The chitin system in the tubes of deep sea hydrothermal vent worms

Abstract The ultrastructure of the protective tubes secreted by the vestimentiferan Tevnia jerichonana has been investigated. The tube wall is made of a hierarchy of structures and substructure which are visualized by scanning and transmission electron microscopy (SEM and TEM). When the tubes are observed at a low magnification, a layering of the wall is observed. The layers consist of a crisscrossed arrangement of flat straight ribbons, each of them having widths of 10–50 μm and thicknesses ranging from 0.1 to 1 μm. When viewed at higher magnification, the ribbons themselves are seen to consist of a substructure of endless parallel chitin microfibrils embedded in a protein matrix (estimated weight ratio: 55% protein and 45% chitin). The structure of the chitin microfibrils was investigated by diffraction contrast electron microscopy (DCTEM) together with electron diffraction analysis. These techniques, applied to either tube wall cross sections or microfibrils that were unhinged after protein removal, reveal that the chitin microfibrils have unusually large widths, reaching up to 50 nm in some cases. Each microfibril is a β chitin whisker-like single crystal having a four-sidedpolygonal cross section. Within a given ribbon, all crystals have the same polarity. The Tevnia chitin microfibrils are susceptible to intracrystalline hydration: this is denoted by an increase in one of the lattice parameters when they are subjected to hydration under temperature and pressure conditions. Upon dehydration, the anhydrous β chitin unit cell is restored, but a cracking of the crystalline microfibrils observable in their cross sections seems to be connected with this dehydration. The overall organization of the microfibrillar chitin network within the tube wall is reminiscent of the continuous twisted arrangement found in many cholesteric arthropod cuticles. However in the Tevnia tubes, the microfibrillar twist is discrete and irregular as there is an aperiodic variation of the fibrillar orientation within the tubes. Their overall organization which is still of the “plywood type” cannot therefore be described as cholesteric but rather as “random twisted nematic.”

Symbioses between deep-sea mussels (Mytilidae: Bathymodiolinae) and chemosynthetic bacteria: diversity, function and evolution.

Mussels of the subfamily Bathymodiolinae thrive around chimneys emitting hot fluids at deep sea hydrothermal vents, as well as at cold seeps and on sunken organic debris (sunken wood, whale falls). Despite the absence of light-driven primary production in these deep-sea ecosystems, mussels succeed reaching high biomasses in these harsh conditions thanks to chemosynthetic, carbon-fixing bacterial symbionts located in their gill tissue. Since the discovery of mussel symbioses about three decades ago our knowledge has increased, yet new findings are published regularly regarding their diversity, role and evolution. This article attempts to summarize current knowledge about symbiosis in Bathymodiolinae, focusing on mussel species for which information is available regarding both hosts and symbionts. Moreover, new data obtained from small mussels inhabiting sunken woods around the Philippines are provided. Indeed, mussel species from organic falls remain poorly studied compared to their vent and seep relatives despite their importance for the understanding of the evolution of symbiosis in the subfamily Bathymodiolinae.

Unusual nutrition of the “Pompeii worm” Alvinella pompejana (polychaetous annelid) from a hydrothermal vent environment: SEM, TEM, 13C and 15N evidence

Morphological and histological studies of Alvinella pompejana (a polychaete living in the vicinity of hydrothermal vents of the East Pacific Ocean) were performed using light, scanning and transmission electron microscopy. The worms were collected in April–May 1979, during the “Rise” cruise by the submersible “Alvin” on the crest of East Pacific Rise at 21°N. The digestive tracts contained many sulfide particles (as determined by microprobe analysis) associated with organic matter and bacteria. Bacterial communities of different morphological types (cocci and filaments) were also observed at different levels of the worms outer teguments. An atypical (possibly bacteria-derived) nutritional source of carbon and nitrogen for A. pompejana is indicated by the natural abundances of 13C:12C and 15N:14N in its tissues.

Structural data on the intra-crystalline swelling of β-chitin

The intra-crystalline swelling of the highly crystalline β-chitin from Tevnia jerichonana was investigated by X-ray crystallography and Fourier transform infrared (FTIR) spectroscopy, using hydrogenated and deuterated hydrochloric acids as swelling agents. Three levels of swelling were identified that could be defined as inter- and intra-sheet swelling. A moderate and reversible swelling in water and methanol gave crystalline β-chitin cystallosolvates, namely dihydrate and methanolate, respectively. In these, an inter-sheet swelling was observed, corresponding to an expansion of only the b parameter of the unit cell of β-chitin. Under these swelling conditions, the use of deuterated reagents had no effect on the amide NH⋯OC hydrogen bonds that hold the structure of β-chitin together, but only induced a partial and reversible deuteration of the chitin hydroxymethyl groups. A more severe swelling — but still reversible — occurred with 6 N HCl or DCl, which converted the crystals of β-chitin into a paracrystalline gel-like product resulting from inter-sheet+intra-sheet swelling. With this acid strength, the deuteration pattern indicated that a fraction of the amide hydrogen bonds was broken and became susceptible to an irreversible deuteration. A very severe and irreversible swelling occurred with 8 N HCl or DCl. In that case, the inter- and intra-sheet swelling was extensive to the point where all memory of the parallel-chain β-chitin was lost. In addition, this swelling was accompanied by a drastic and rapid depolymerization. The treatment with 8 N HCl led invariably to crystalline α-chitin when the samples were neutralized.

Colonization of organic substrates deployed in deep-sea reducing habitats by symbiotic species and associated fauna.

In this study, our goal was to test whether typical vent/seep organisms harbouring symbionts or not, would be able to settle on organic substrates deployed in the vicinity of chemosynthetic ecosystems. Since 2006, a series of novel standardized colonization devices (CHEMECOLI: CHEMosynthetic Ecosystem COlonization by Larval Invertebrates) filled with three types of substrates (wood, alfalfa and carbonate) have been deployed in different types of reducing habitats including cold seeps in the eastern Mediterranean, a mud volcano in the Norwegian Sea, and hydrothermal vents on the Mid-Atlantic Ridge for durations of 2 weeks to 1 year. For all deployments, highest species diversities were recovered from CHEMECOLIs filled with organic substrates. Larvae from species associated with thiotrophic symbionts such as thyasirid, vesicomyid and mytilid bivalves, were recovered in the eastern Mediterranean and at the Mid-Atlantic Ridge. At the Haakon Mosby Mud Volcano, larvae of symbiotic siboglinids settled on both organic and carbonate substrates. Overall, novel colonization devices (CHEMECOLI) filled with organic substrates attracted both fauna relying on chemosynthesis-derived carbon as well as fauna relying on heterotrophy the latter being opportunistic and tolerant to sulphide.

Molecular characterization of cuticle and interstitial collagens from worms collected at deep sea hydrothermal vents

Two different collagens were isolated and characterized from the body walls of the vestimentiferan tube worm Riftia pachyptila and the annelid Alvinella pompejana, both living around hydrothermal vents at a depth of 2600 m. The acid-soluble cuticle collagens consisted of a long triple helix (2.4 microns for Alvinella, 1.5 microns for Riftia) terminating into a globular domain. Molecular masses of 2600 and 1700 kDa, respectively, were estimated from their dimensions. The two cuticle collagens were also quite different in amino acid composition, in agreement with their different supramolecular organizations within tissues. Interstitial collagens corresponding to cross-striated fibrils underneath the epidermal cells could be solubilized by digestion with pepsin and consisted of a single alpha-chain. They were similar in molecular mass (340 kDa) and length (280 nm) but differed in composition and banding patterns of segment-long-spacing fibrils. This implicates significant sequence differences also in comparison to fibril-forming vertebrate collagens, although all form typical quarter-staggered fibrils. The thermal stability of the worm collagens was, with one exception (interstitial collagen of Riftia), in the range of mammalian and bird collagens (37 to 46 degrees C), and thus distinctly above that of shallow sea water annelids. Yet, their 4-hydroxyproline contents were not directly correlated to this stability. About 20% of Riftia collagen alpha-chain sequence was elucidated by Edman degradation and showed typical Gly-X-Y repeats but only a limited homology (45 to 58% identity) to fibril-forming vertebrate collagens. A single triplet imperfection and the variable hydroxylation of proline in the X position were additional unique features. It suggests that this collagen represents an ancestral form of fibril-forming collagens not directly corresponding to an individual fibril-forming collagen type of vertebrates.