Bruce Shillito

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).

Microbial diversity associated with the hydrothermal shrimp Rimicaris exoculata gut and occurrence of a resident microbial community

Rimicaris exoculata dominates the megafauna of several Mid-Atlantic Ridge hydrothermal sites. Its gut is full of sulphides and iron-oxide particles and harbours microbial communities. Although a trophic symbiosis has been suggested, their role remains unclear. In vivo starvation experiments in pressurized vessels were performed on shrimps from Rainbow and Trans-Atlantic Geotraverse sites in order to expel the transient gut contents. Microbial communities associated with the gut of starved and reference shrimps were compared using 16S rRNA gene libraries and microscopic observations (light, transmission and scanning electron microscopy and FISH analyses). We show that the gut microbiota of shrimps from both sites included mainly Deferribacteres, Mollicutes, Epsilon- and Gammaproteobacteria. For the first time, we have observed filamentous bacteria, inserted between microvilli of gut epithelial cells. They remained after starvation periods in empty guts, suggesting the occurrence of a resident microbial community. The bacterial community composition was the same regardless of the site, except for Gammaproteobacteria retrieved only in Rainbow specimens. We observed a shift in the composition of the microbiota of long-starved specimens, from the dominance of Deferribacteres to the dominance of Gammaproteobacteria. These results reinforce the hypothesis of a symbiotic relationship between R. exoculata and its gut epibionts.

Inorganic carbon fixation by chemosynthetic ectosymbionts and nutritional transfers to the hydrothermal vent host-shrimp Rimicaris exoculata

The shrimp Rimicaris exoculata dominates several hydrothermal vent ecosystems of the Mid-Atlantic Ridge and is thought to be a primary consumer harbouring a chemoautotrophic bacterial community in its gill chamber. The aim of the present study was to test current hypotheses concerning the epibiont’s chemoautotrophy, and the mutualistic character of this association. In-vivo experiments were carried out in a pressurised aquarium with isotope-labelled inorganic carbon (NaH13CO3 and NaH14CO3) in the presence of two different electron donors (Na2S2O3 and Fe2+) and with radiolabelled organic compounds (14C-acetate and 3H-lysine) chosen as potential bacterial substrates and/or metabolic by-products in experiments mimicking transfer of small biomolecules from epibionts to host. The bacterial epibionts were found to assimilate inorganic carbon by chemoautotrophy, but many of them (thick filaments of epsilonproteobacteria) appeared versatile and able to switch between electron donors, including organic compounds (heterotrophic acetate and lysine uptake). At least some of them (thin filamentous gammaproteobacteria) also seem capable of internal energy storage that could supply chemosynthetic metabolism for hours under conditions of electron donor deprivation. As direct nutritional transfer from bacteria to host was detected, the association appears as true mutualism. Import of soluble bacterial products occurs by permeation across the gill chamber integument, rather than via the digestive tract. This first demonstration of such capabilities in a decapod crustacean supports the previously discarded hypothesis of transtegumental absorption of dissolved organic matter or carbon as a common nutritional pathway.

Pressure tolerance of the shallow-water caridean shrimp Palaemonetes varians across its thermal tolerance window

SUMMARY To date, no published study has assessed the full physiological scope of a marine invertebrate species with respect to both temperature and hydrostatic pressure. In this study, adult specimens of the shallow-water shrimp species Palaemonetes varians were subjected to a temperature/pressure regime from 5 to 30°C and from 0.1 to 30 MPa. The rate of oxygen consumption and behaviour in response to varying temperature/pressure combinations were assessed. Rates of oxygen consumption were primarily affected by temperature. Low rates of oxygen consumption were observed at 5 and 10°C across all pressures and were not statistically distinct (P=0.639). From 10 to 30°C, the rate of oxygen consumption increased with temperature; this increase was statistically significant (P<0.001). Palaemonetes varians showed an increasing sensitivity to pressure with decreasing temperature; however, shrimp were capable of tolerating hydrostatic pressures found outside their normal bathymetric distribution at all temperatures. ‘Loss of equilibrium’ (LOE) in ≥50% of individuals was observed at 11 MPa at 5°C, 15 MPa at 10°C, 20 MPa at 20°C and 21 MPa at 30°C. From 5 to 20°C, mean levels of LOE decreased with temperature; this was significant (P<0.001). Low mean levels of LOE were observed at 20 and 30°C and were not distinct (P=0.985). The physiological capability of P. varians to tolerate a wide range of temperatures and significant hydrostatic pressure is discussed.

Structure and phylogeny of the crustacean hyperglycemic hormone and its precursor from a hydrothermal vent crustacean: the crab Bythograea thermydron.

The structure of a well-known neurohormone involved in homeostasis regulation and stress response, the crustacean hyperglycemic hormone, was investigated in the deep-sea hydrothermal vent crab Bythograea thermydron. The neuropeptide was isolated from neurohemal organs (sinus glands) and its biological activity checked using an homologous bioassay. Partial amino acid sequence was established by a combination of Edman chemistry and mass spectrometry. Then, the sequence of the cDNA encoding the hormone precursor was determined. The preprohormone is composed of a 29 amino acid signal peptide, followed by a 41 amino acid associated peptide flanking the 72 amino acid hyperglycemic hormone. Comparison of these data with other known crab hyperglycemic hormone and prohormone sequences was performed using phylogenetic analysis methods.

Acquisition of epibiotic bacteria along the life cycle of the hydrothermal shrimp Rimicaris exoculata

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first ‘epibiotic’ association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle.

Thermal Limit for Metazoan Life in Question: In Vivo Heat Tolerance of the Pompeii Worm

The thermal limit for metazoan life, expected to be around 50°C, has been debated since the discovery of the Pompeii worm Alvinella pompejana, which colonizes black smoker chimney walls at deep-sea vents. While indirect evidence predicts body temperatures lower than 50°C, repeated in situ temperature measurements depict an animal thriving at temperatures of 60°C and more. This controversy was to remain as long as this species escaped in vivo investigations, due to irremediable mortalities upon non-isobaric sampling. Here we report from the first heat-exposure experiments with live A. pompejana, following isobaric sampling and subsequent transfer in a laboratory pressurized aquarium. A prolonged (2 hours) exposure in the 50–55°C range was lethal, inducing severe tissue damages, cell mortalities and triggering a heat stress response, therefore showing that Alvinella’s upper thermal limit clearly is below 55°C. A comparison with hsp70 stress gene expressions of individuals analysed directly after sampling in situ confirms that Alvinella pompejana does not experience long-term exposures to temperature above 50°C in its natural environment. The thermal optimum is nevertheless beyond 42°C, which confirms that the Pompeii worm ranks among the most thermotolerant metazoans.

First access to live Alvinellas

The deep-sea vent worm Alvinella pompejana is a biological enigma, regarding the adaptative features that allow it to prosper in the harsh environment of hydrothermal chimney walls (East Pacific Rise). Moreover, attempts to maintain this invertebrate alive following recovery (2600 m depth) have so far failed, thus forbidding in vivo laboratory studies. For the first time, it is shown that most of the specimens reach the surface alive, as witnessed by video monitoring of these animals after native pressure conditions have been restored. Furthermore, some individuals may show active behaviour at least 20 h after recovery, under controlled temperature and oxygen level conditions. These promising results open up a new field of investigation, and call for a major breakthrough towards our understanding of A. pompejanas biology, in the near future.

Influence of environmental conditions on early development of the hydrothermal vent polychaete Alvinella pompejana

SUMMARY Dispersal and colonisation processes at deep-sea vents are still not fully understood, essentially because early life stages of vent species remain unknown. The polychaete worm Alvinella pompejana forms colonies on chimney walls at East Pacific Rise vent sites where the temperature can frequently exceed 20°C. In vitro studies in pressure vessels showed that the early embryos tolerate temperatures in a lower range (10–14°C), suggesting that they would have to escape the colony to develop. Pressure vessels offer the advantage that each parameter can be independently controlled, but they do not simulate the more complex and dynamic conditions naturally encountered at vent sites. Accordingly, in addition to incubations in pressure vessels, we incubated embryos directly at a vent site, in different habitats along a gradient of hydrothermal influence. Embryos incubated on an adult A. pompejana colony where temperature and H2S concentrations were relatively high showed a very low survival rate and did not develop, whereas embryos incubated in a Riftia pachyptila clump environment with a lower hydrothermal signature, or at the base of the chimney where the influence of the hydrothermal activity was very weak, survived well and developed. Although the average temperature recorded in the A. pompejana colony was within the range tolerated by embryos (13°C), frequent peaks above 20°C were recorded. Estimated sulphide concentration at this site reached 200 μmol l–1. Punctuated exposure to both high temperature and elevated sulphide levels probably explain the low survival of embryos within the A. pompejana colony. The in situ experiments further support the idea that embryos require conditions with moderate hydrothermal influence not generally found within an adult colony. However, as much more benign physicochemical conditions can be found within a few tens of cm of adult colonies, embryos do not necessarily have to leave their vent of origin to develop. Further analyses are needed to pinpoint the specific factors that affect the survival and development of embryos at vents.