Philippe Pernet

Structure of Sediment-Associated Microbial Communities along a Heavy-Metal Contamination Gradient in the Marine Environment

ABSTRACT Microbial community composition and structure were characterized in marine sediments contaminated for >80 years with cadmium, copper, lead, and zinc. Four sampling sites that encompass a wide range of sediment metal loads were compared in a Norwegian fjord (Sørfjord). HCl-extractable metals and organic matter constantly decreased from the most contaminated site (S1) to the control site (S4). All sampling sites presented low polychlorinated biphenyl (PCB) concentrations (Σ7PCB < 7.0 ng g [dry weight]−1). The biomass ranged from 4.3 × 108 to 13.4 × 108 cells g (dry weight) of sediments−1 and was not correlated to metal levels. Denaturing gradient gel electrophoresis indicated that diversity was not affected by the contamination. The majority of the partial 16S rRNA sequences obtained were classified in the γ- and δ-Proteobacteria and in the Cytophaga-Flexibacter-Bacteroides (CFB) bacteria. Some sequences were closely related to other sequences from polluted marine sediments. The abundances of seven phylogenetic groups were determined by using fluorescent in situ hybridization (FISH). FISH was impaired in S1 by high levels of autofluorescing particles. For S2 to S4, the results indicated that the HCl-extractable Cu, Pb, and Zn were negatively correlated with the abundance of γ-Proteobacteria and CFB bacteria. δ-Proteobacteria were not correlated with HCl-extractable metals. Bacteria of the Desulfosarcina-Desulfococcus group were detected in every site and represented 6 to 14% of the DAPI (4′,6′-diamidino-2-phenylindole) counts. Although factors other than metals may explain the distribution observed, the information presented here may be useful in predicting long-term effects of heavy-metal contamination in the marine environment.

Adherent bacteria in heavy metal contaminated marine sediments

Abstract The eubacterial communities adherent to sediment particles were studied in heavy metal contaminated coastal sediments. Six sampling sites on the Belgian continental plate and presenting various metal loads, granulometries, and organic matter content, were compared. The results indicated that the total microbial biomass (attached + free-living bacteria) was negatively correlated to HCl-extractable metal levels (p < 0.05) and that the percentage of cells adherent to sediment particles was close to 100% in every site even in highly contaminated sediments. Consequently, it seems that heavy metal contamination does affect total bacterial biomass in marine sediments but that the ratio between attached and free living microorganisms is not affected. The composition of the eubacterial communities adherent to the fine fraction of the sediments (<150 µm) was determined using fluorescent in situ hybridisation (FISH). The FISH results indicated that the proportion of γ- and δ-Proteobacteria, and Cytophaga-Flexibacter-Bacteroides (CFB) bacteria, was not related to the HCl extractable metal levels. Most of the 79 complete 16S rRNA sequences obtained from the attached microbial communities were classified in the γ- and δ-Proteobacteria and in the CFB bacteria. A large proportion of the attached γ-Proteobacterial sequences found in this study (56%) was included in the uncultivated GMS clades that are indigenous to marine sediments.

Field contamination of the starfish Asterias rubens by metals. Part 2: Effects on cellular immunity.

To study the effects of metals on starfish in field conditions, immune responses were measured in starfish from natural populations along a metal pollution gradient (long-term contamination) and in starfish that were transferred up the gradient (short-term contamination). Coelomic amoebocyte concentration (CAC) and production of reactive oxygen species (ROS) by amoebocytes were measured in two varieties of Asterias rubens occurring in the fjord: The black variety which lives only in the low salinity upper waters (22-26/1000) and the red variety which live both in the upper layer and in the deeper layer characterized by a salinity close to that of seawater (30/1000). The studied immune responses were stimulated in starfish living along the metal pollution gradient according to the contamination of these starfish by cadmium. However, the sensitivity of these responses toward metals appeared to be strongly modulated by the salinity stress. In red starfish living at 30/1000 and transferred up the contamination gradient, the immune responses were inhibited and closely followed the short-term accumulation of metals in the animal organs. Starfish transferred down the gradient did not recover normal immune responses in the short-term and appeared highly sensitive to caging stress. It is suggested that the impact of metals on the immune responses of A. rubens in field conditions occurs in three phases. Short-term inhibitory effects are exerted by a direct action of metals on the immune cells and are followed by a recovery due to the induction of protective mechanisms. Eventually, when these mechanisms are overwhelmed by a long-term contamination, indirect and durable stimulatory effects on the immune responses appear due to a global disruption of the animal physiology.

Field contamination of the starfish Asterias rubens by metals. Part 1: Short‐ and long‐term accumulation along a pollution gradient

The accumulation of Cd, Pb, Zn, and Cu in the starfish Asterias rubens was studied in a Norwegian fjord characterized by a gradient of metal pollution in the sediments, ranging from very high metal concentrations at its head to much lower levels at its opening. The concentrations of metals in starfish from natural populations along the gradient (long-term accumulation) and in starfish that were transferred up the gradient (short-term accumulation) were compared. At long-term, Cd and Pb accumulations by starfish living at normal salinity (30/1000) were related to the level of contamination of the environment while Cu and, to a lesser extent, Zn accumulations appeared strictly controlled. At short-term, Pb was accumulated steadily, Cd and Zn were accumulated transiently in the pyloric caeca (fast compartment), and Cu was not accumulated at all. Depuration experiments (transfer down the gradient) showed that Cd and Pb were efficiently eliminated from the pyloric caeca but not from the body wall (slow compartment). It is concluded that Pb is chronically accumulated, without apparent control, Cd is subjected to a regulating mechanism in the pyloric caeca which is overwhelmed over the long-term; Zn is tightly controlled in the pyloric caeca and Cu in both pyloric caeca and body wall. A distinct color variety of starfish is restricted to the low salinity (22-26/1000) superficial water layer. This variety showed a different pattern of metal accumulation over the long-term. This pattern is attributed to the particular hydrological conditions prevailing in this upper layer.

Metal concentrations, sperm motility, and RNA/DNA ratio in two echinoderm species from a highly contaminated fjord (the Sørfjord, Norway)

The present study evaluated the effects of field metal contamination on sperm motility and the RNA/DNA ratio in echinoderms. Populations of Asterias rubens and Echinus acutus that occur naturally along a contamination gradient of sediments by cadmium, copper, lead, and zinc in a Norwegian fjord (the Sorfjord) were studied. Sperm motility, a measure of sperm quality, was quantified using a computer-assisted sperm analysis system. The RNA/DNA ratio, a measure of protein synthesis, was assessed by a one-dye (ethidium bromide)/one-enzyme (RNase), 96-well microplate fluorometric assay. Although both species accumulate metals at high concentrations, neither sperm motility parameters in A. rubens nor the RNA/DNA ratio in both species were affected. The Sorfjord is still one of the most metal-contaminated marine sites in Europe, but even so, populations of A. rubens and E. acutus are able to endure under these conditions.

Factors controlling juvenile growth and population structure of the starfish Asterias rubens in intertidal habitats: field and experimental approaches

The dynamics of intertidal populations of the starfish Asterias rubens, living in contrasted habitats and over a broad geographical range, were studied from March 2000 to November 2002 using modal analysis. As only 1 juvenile (first year after recruitment) and 1 adult (subsequent years) modes could be distinguished; only juvenile growth was quantified. Concomitantly, experiments were carried out to test several factors assumed to influence juvenile growth: food quantity and quality, emersion, salinity variations and temperature. Three different juvenile growth patterns were evidenced: (1) a fast and protracted growth linked to high food availability and lack of disturbance; (2) a winter cessation of growth likely due to a seasonal increase of emersion-related stress and salinity variations; and (3) disrupted juvenile dynamics, which was encountered in 2 populations. In the first one, estuarine salinity conditions limited growth and, combined with food depletion, led to the extinction of the population. In the second one, wave action confined most of the population to a restricted area with low food levels. In the third scenario, intraspecific competition for food was probably at the source of an unusual growth pattern in which most juveniles did not grow while a small proportion achieved a medium growth rate.

Effects of field contamination by metals (Cd, Cu, Pb, Zn) on biometry and mechanics of echinoderm ossicles

Echinoderms are known to readily incorporate metals in their calcified endoskeleton. It is currently unclear if this has an impact on the skeleton function or if this can be considered as a detoxification mechanism. In the present work, populations of the sea urchin Echinus acutus and the starfish Asterias rubens were studied in stations distributed along a metal contamination gradient in a Norwegian fjord (Sørfjord). Ossicles involved in major mechanical functions - sea urchin spine and starfish ambulacral plate - were analyzed for their metal concentration (Cd, Cu, Pb and Zn) and their biometric and mechanical properties. Starfish plates were more contaminated by Cd, Pb and Zn than sea urchin spines. Cu concentrations were at background levels. In E. acutus, metals principally affected size. In A. rubens, material stiffness and toughness were decreased in the most contaminated station. This reduction is attributed either to the direct incorporation of metals in the calcite lattice and/or to deleterious effects of metals during skeleton ontogenesis. The contrasting incorporation of metals in the skeleton of the two investigated species accounts for the different impact of the metals, including in terms of fitness. The present results clearly indicate that, at least in A. rubens, incorporation of metals in the skeleton cannot be considered as a detoxification mechanism.

Contrasting relationships between pyloric caecum and gonad growth in the starfish Asterias rubens: Combined field and experimental approaches

The common starfish, Asterias rubens, occurs in fluctuating environments in the North Atlantic. To better understand energy allocation dynamics, we recorded gonad, body wall, and pyloric caeca (storage organ) indices between 2000 and 2004 from three different habitats. We applied a Fourier transform to the data to evaluate and compare the seasonal variation in these indices. Specific effects of emersion and salinity variation were examined in two laboratory studies. Differences in energy allocation were found between sites and temporally within sites. Food availability appeared to be the most important factor controlling allocation dynamics while fluctuating salinity and/or emersion had a significant but smaller impact. Only severe food shortage reduced reproductive investment indicating a preferential energy allocation to gonads. This study is the first to encompass a broad range of populations over several reproduction cycles and emphasizes the ability of A. rubens to adapt to a fluctuating environment. Copyright

Marking bark beetle parasitoids within the host plant with rubidium for dispersal studies

A technique using rubidium chloride (RbCl), a trace element, as internal label is proposed for marking hymenopteran parasitoids attacking a concealed host, Ips typographus L. (Coleoptera: Scolytidae). RbCl was introduced directly into spruce via the vascular system using glass tubing. RbCl passed through the food chain and was detected at the parasitoid level by electrothermal atomic absorption spectroscopy (EAAS) or flame emission spectroscopy (FES). Mark persistency until day 8 after emergence was tested in labelled Rhopalicus tutela (Walker) (Hymenoptera: Pteromalidae). The mark decreased with time only in fed females, probably due to excretion and/or egg resorption. The proportion of marked males did not vary with time or treatment.