Lead in the marine environment: concentrations and effects on invertebrates.

Lead (Pb) is a non-essential metal naturally present in the environment and often complexed with other elements (e.g., copper, selenium, zinc). This metal has been used since ancient Egypt and its extraction has grown in the last centuries. It has been used until recently as a fuel additive and is currently used in the production of vehicle batteries, paint, and plumbing. Marine ecosystems are sinks of terrestrial contaminations; consequently, lead is detected in oceans and seas. Furthermore, lead is not biodegradable. It remains in soil, atmosphere, and water inducing multiple negative impacts on marine invertebrates (key species in trophic chain) disturbing ecological ecosystems. This review established our knowledge on lead accumulation and its effects on marine invertebrates (Annelida, Cnidaria, Crustacea, Echinodermata, and Mollusca). Lead may affect different stages of development from fertilization to larval development and can also lead to disturbance in reproduction and mortality. Furthermore, we discussed changes in the seawater chemistry due to Ocean Acidification, which can affect the solubility, speciation, and distribution of the lead, increasing potentially its toxicity to marine invertebrates.

Overview on the European green crab Carcinus spp. (Portunidae, Decapoda), one of the most famous marine invaders and ecotoxicological models.

Green crabs (Carcinus, Portunidae) include two species native to Europe--Carcinus aestuarii (Mediterranean species) and Carcinus maenas (Atlantic species). These small shore crabs (maximal length carapace, approximately 10 cm) show rapid growth, high fecundity, and long planktonic larval stages that facilitate broad dispersion. Carcinus spp. have a high tolerance to fluctuations of environmental factors including oxygen, salinity, temperature, xenobiotic compounds, and others. Shipping of Carcinus spp. over the past centuries has resulted in its invasions of America, Asia, and Australia. Classified as one of the world's 100 worst invaders by the International Union for Conservation of Nature, Carcinus spp. are the most widely distributed intertidal crabs in the world. Their voracious predatory activity makes them strong interactors in local communities, and they are recognized as a model for invasiveness in marine systems as well as a sentinel species in ecotoxicology. This review shows an exhaustive analysis of the literature on the life cycle, diversity, physiological tolerance, genomic investigations, ecotoxicological use, historical invasion, control programs, and putative economical valorization of shore crabs.

Alternation of anthelmintic treatments: a molecular evaluation for benzimidazole resistance in nematodes.

The evolution of benzimidazoles (BZ) resistance in Teladorsagia circumcincta was investigated in a controlled trial with lambs, submitted to different treatment regimens. Four paddocks were seeded with a T. circumcincta strain constituted by 25% of BZ-resistant nematodes. Ten permanent lambs were allocated to each paddock, from April to November in order to renew the contamination of pasture. Monthly, three tracer lambs were allocated in each paddock. BZ-resistant nematode frequency was determined (PCR diagnosis). The faecal egg count reduction test (permanent lambs) and the number of nematodes in lambs were also determined (permanent and tracer lambs). Four different regimens of treatments were performed: control, levamisole (a non-BZ drug), fenbendazole (a BZ drug), and an alternation of levamisole and fenbendazole every second treatment. The same protocol was repeated on two consecutive grazing seasons, increasing the number of treatments (3 in first year and 5 in second year). The proportions of BZ-resistant nematodes did not change during all the study in both the control and the levamisole paddocks, supporting an equal global fitness of BZ-resistant and susceptible nematodes. Thus, no reversion of BZ resistance is to be expected. In the alternated drug group and in the BZ treated group, BZ-resistant nematodes increased from 25% to 47% and to 78%, respectively. BZ resistance increased proportionally to the selective pressure (number of BZ treatments). The drug alternation is not a good solution to delay importantly the evolution of resistance when more than 25% of nematodes are BZ-resistant. This study is the first evaluation of BZ-resistance evolution (using individual genotyping) in controlled conditions. It showed that when a monogenic anthelmintic resistance is established at 25% in a sexually reproducing nematode population, it seems to be impossible to prevent its increase even when using limited number of BZ treatments.

Characterization and sequence analysis of manganese superoxide dismutases from Brachyura (Crustacea: Decapoda): hydrothermal Bythograeidae versus littoral crabs.

Hydrothermal vent conditions are particular and organisms living in these environments may have developed detoxification mechanisms and/or genetic adaptations. In particular, physico-chemical conditions are thought to generate reactive oxygen species, highly toxic for organisms. The enzyme superoxide dismutase constitutes the first line of defense against oxidative damage. To improve our understanding of the environmental impacts exerted on the vent organisms, we have characterized the two manganese superoxide dismutase cDNAs (mitochondrial: mMnSOD and cytoplasmic: cMnSOD) of three members of the Bythograeidae (Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica), the only endemic crab family living in hydrothermal vents. In comparison, the isolation of manganese superoxide dismutase cDNAs was also carried out in several littoral crab families. MnSOD signatures were found in both sequences from each species studied, as well as different residues involved in metal coordination and protein activity. The phylogenetic analysis performed confirms the probable ancient duplication that gave rise to the two MnSODs (cMnSOD and mMnSOD). This study describes two potential distinct mMnSOD isoforms presenting particular peptide signals. Nevertheless, no sequence particularity that could support the hypothesis of a genetic adaptation was found in Bythograeidae's MnSODs compared to the other sequences. The mRNA expression analysis performed by real-time PCR on B. thermydron and S. mesatlantica compared to Cancer pagurus and Necora puber revealed a higher cMnSOD and mMnSOD mRNA expression in hydrothermal crabs compared to littoral crabs.

Widespread occurence of mariner transposons in coastal crabs.

Mariner-like elements (MLEs) are ubiquitous DNA mobile elements found in almost all eukaryote genomes. Nevertheless most of the known copies are inactive and the question of the genome invasion by MLEs remains largely hypothetical. We have previously reported the presence of highly homologous copies of MLEs in the genome of phylogenetically distant crustacea living in the same hydrothermal environment suggesting the possibility of horizontal transfer. In order to further support the hypothesis that horizontal transmission of MLEs might occur between crustacean sympatric species, we described here 85 MLE sequences found in the genome of a large spectrum of coastal crab species. The number of the MLEs copies in genomes was variable. Half of these MLEs fit with the irritans subfamily of MLEs whereas the second half grouped in a new subfamily called marmoratus. In addition, a molecular phylogeny of crabs was established by using the 16S information. The comparison between 16S and MLEs based trees reveals their incongruence, and suggests either the existence of horizontal transfer events between phylogenetically distant species, or an ancestral MLE polymorphism followed by different evolution and stochastic loss.

Small metallothionein MT-10 genes in coastal and hydrothermal mussels.

Metallothioneins (MTs) are important proteins in the intracellular regulation of metals. In the Mytilidae family, which includes many economically important species, 2 major forms of MTs have been reported: MT-10 (10 kDa) and MT-20 (20 kDa). Many different MT-10 proteins have been isolated from the common species Mytilus edulis, which suggests that distinct MT-10 genes may occur in a single specimen. Some MT genes, involving 3 exons and 2 large introns, have been isolated in Mytilidae. Our aim was to determine whether intron-free forms of the MT-10 genes can exist, which could allow rapid transcription in response to exposure to metals. Our study focused on 2 species living under very different environmental conditions: Mytilus edulis (a coastal mussel) and Bathymodiolus thermophilus (a hydrothermal mussel). We report here the first description of small, intron-free MT-10 genes, possessing a correct open reading frame in these 2 species.

Molecular approaches to studying benzimidazole resistance in trichostrongylid nematode parasites of small ruminants.

Molecular techniques are of growing importance in the study of anthelmintic resistance in trichostrongylid worm populations. A knowledge of the genetic determinants of benzimidazole (BZ) resistance has made it possible to construct a molecular tool for genotyping individual worms, in respect of mutation of the beta-tubulin gene responsible for BZ resistance. This tool offers new possibilities in the diagnosis of BZ resistance, and also in the study of anthelmintic use and other breeding management factors that can affect the selection of BZ-resistant alleles in worm populations. New molecular methods have also made it possible to study the origin and diversity of BZ-resistant alleles in trichostrongylid populations. The results demonstrate the value of a multidisciplinary approach to the study of anthelmintic resistance, combining molecular, ecological and epidemiological techniques.

Are Teladorsagia circumcincta (Nematoda) morphs equally able to survive under anthelmintic treatment in sheep on pastures?

The nematode Teladorsagia circumcincta is a polymorphic species, morphs T. c. trifurcata and T. c. circumcincta being the most frequent. Among other traits, T. c. trifurcata has lower infectivity than T. c. circumcincta. Anthelmintic treatments that regularly remove populations should favour the more infective T. c. circumcincta. The present investigation was intended to test the respective capabilities of the two morphs to withstand anthelmintic pressure. The nematode strain was susceptible to imidazothiazole and partly resistant to benzimidazole at the onset of experiment. A 2-year grazing season study was undertaken under four levels of anthelmintic pressure [from untreated controls to frequently treated with a poorly efficient benzimidazole (fenbendazole) or an efficient imidazothiazole (levamisole)]. The strain previously maintained in laboratory conditions regained its natural, original proportion of T. c. trifurcata during the first grazing season. The two morphs were equally resistant to fenbendazole; and the proportion of resistant nematodes did not vary between the two morphs. During the second grazing season, the treatments indirectly depleted the proportions of T. c. trifurcata, probably due to the poor infectivity of this morph.

Mitochondrial DNA variation in benzimidazole-resistant and -susceptible populations of the small ruminant parasite Teladorsagia circumcincta.

The genetic diversity of the mtDNA ND4 gene in 11 Teladorsagia circumcincta populations from France and Morocco was assessed by sequencing. Some of these nematode populations were resistant to benzimidazole (BZ) anthelmintics, while others were susceptible. The nucleotide diversity in all populations studied was very high, probably due to a high mutation rate in nematodes, but there was no significant difference between them. This suggests that no strong, recurrent bottlenecks occur during the acquisition of BZ resistance by a worm population. The conservation of genetic variations during the acquisition of BZ resistance is probably due to the fact that anthelmintic treatments do not kill all the susceptible adult worms and to the presence of numerous free-living larvae that are not submitted to this anthelmintic pressure. There was no genetic subdivision between worm populations on a small geographical scale (less than 200 km), but significant F(ST)s were found on a larger geographical scale. This kind of subdivision cannot be explained by different genetic flows between populations because all these populations were isolated from each other. This subdivision is probably due to the breeding management practices and the large size of these worm populations, which limit genetic drift.

Study by ribosomal DNA ITS 2 sequencing and RAPD analysis on the systematics of four Metastrongylus species (Nematoda:Metastrongyloidea).

Four species of lungworms of the Metastrongylus genus are found sympatrically in the lungs of the wild boars (Sus scrofa L.) on the Chambord game reserve (France): Metastrongylus asymmetricus, M. confusus, M. pudendotectus, and M. salmi. These species are difficult to identify using morphological characters alone, and epidemiological evidence raises doubt about the existence of 4 distinct species. Two molecular approaches (sequencing of ribosomal DNA Internal Transcribed Spacer 2 [ITS 2] and Random Amplified Polymorphic DNA assay [RAPD]) were used to evaluate the genetic similarities between the 4 taxa. The ITS 2 sequences of M. salmi and M. confusus were identical, whereas the sequences of the other species were distinct. On the other hand, RAPD analysis indicated unambiguously that the 4 species are genetically different. These results reveal the limitation of using only 1 molecular approach for taxonomic studies and indicate the need to examine different regions (ribosomal, mitochondrial DNA) in the genome of a species in complement with morphological and epidemiological data.