Evaluation of heavy metals contamination and pollution indices levels in surface sediments of the Bizerte coastal line, Tunisia.

Heavy metal concentrations in surface sediments collected from 17 stations samples in Bizerte coastal line were determined in order to evaluate the contamination status in the region. Heavy metal concentrations (mg/kg) of Bizerte coast line followed the decreasing trend of Fe > Pb > Zn > Mn > Cu > Cr > Ni > Co > Cd. The pollution degree of the surface sediment collected from 17 samples station in Bizerte coast line by heavy metals was evaluated on the basis of pollution indices (EF, Igeo, CF, PLI and CD). Results of Igeo, EF, CF, PLI and DC demonstrated that pollution levels of Pb and Cd were moderate, which should require more attentiveness as main pollution factors. However, no pollution was found for Zn, Cu, Ni, Cr, Co and Mn. Hierarchical cluster analysis was used to identify the influence of anthropogenic activities on the surface sediment.

Developmental exposure to the A6-pesticide causes changes in tyrosine hydroxylase gene expression, neurochemistry, and locomotors behavior in larval zebrafish.

Purpose: In recent years, the increase in the biopesticides synthesis for alternative agricultural uses has required their impacts study. Among these compounds, several of them are known to exert endocrinedisrupting (EDs) effects causing deregulation of physiological functions affecting cell signaling pathways involved in neural cell differentiation leading to developmental neurotoxicity. The objective of our study was to determine the impact of the biopesticide A6 structurally related to estrogenic EDs on zebrafish larvae, to define its toxicity, the mechanisms responsible, and to monitor the locomotors activity at nanomolar concentrations (0. 0.5, 5 and 50 nM).Materials and methods: Using imaging analysis tools, immunohistochemistry, quantitative PCR, and an automated behavior recording system (Zebrabox) we were able to assess these effects.Results: We have shown through its blue fluorescence properties that it accumulates in different parts of the body such as the intestine, adipose tissue, muscles, yolk sac and head. A6 also disrupted swimming behavior by affecting the expression of tyrosine hydroxylase (TH) in dopaminergic neurons.Conclusions: In conclusion, our study provided a mechanistic understanding of the A6 neurotoxic effect which could be the result of its binding to the estrogen receptor.

Diuron environmental levels effects on marine nematodes: Assessment of ecological indices, taxonomic diversity, and functional traits.

Coastal marine systems are the most sensitive zones to emerging pollutants. The present study aims to investigate the effect of Diuron on the meiofaunal assemblages, collected from the Bizerte channel (Tunisia). Microcosm experiments were set up using four increasing Diuron concentrations [D1 (10 ng g-1 dry weight (DW)), D2 (50 ng g-1 DW), D3 (250 ng g-1 DW) and (1250 ng g-1 DW)] compared to non-contaminated sediments (controls) and all plots were incubated for 30 days. Our results show that Diuron-supplemented sediments provoked the significant decrease of meiofaunal abundance as well as a change in nematodes' diversity and structure composition. All univariate indices, as well as the cumulative k-dominance, were lower in the Diuron than the control plot. Additionally, the ordination of treatments according to the two-dimensional nMDS plots analysis showed a clear structural separation of the Diuron treated replicates from the controls based on the functional groups lists. These current data emphasize the utility of the use of biological traits in the detection of disturbances in the aquatic biotope.

Ecotoxicity of polybrominated diphenyl ether (BDE-47) on a meiobenthic community with special emphasis on nematodes: Taxonomic and trophic diversity assessment.

The response taxonomic and trophic of meiobenthic organisms, especially marine nematodes to polybrominated diphenyl ether (BDE-47) was studied using a community from Bizerte lagoon (Nord-East Tunisia). Four concentrations of BDE-47 [D1 (2.5 µg/kg dw), D2 (25 µg/kg dw), D3 (50 µg/kg dw), and D4 (100 µg/kg dw)] were applied, and responses were determined 30 days after exposure. Species abundance and all univariate indices were significantly affected in all treated microcosms compared to the control. The non-parametric cluster based on species abundance separated the nematode population into two groups: control + all treated microcosms. After grouping nematode species according to their trophic diversity, their abundance showed differential responses. The non-metric multi-dimensional scaling analysis and cumulative k-dominance based on the abundance of trophic groups abundances reflected significant separation between the control microcosm and each treatment condition. The correspondence analysis 2D plot generated from nematode species and trophic groups abundance showed the control microcosm was dominated by microvores, represented by two species of Terschellingia. However, when treated with the highest concentration of BDE-47, the community was occupied by the resistant trophic groups of facultative predators and epigrowth feeders represented by Metoncholaimus pristiurus and Paracomesoma dubium, respectively.

Meiobenthic nematode Oncholaimus campylocercoides as a model in laboratory studies: selection, culture, and fluorescence microscopy after exposure to phenanthrene and chrysene.

Numerous studies have focused on the response of meiofauna after exposure to polycyclic aromatic hydrocarbons (PAHs), but none has been devoted to their uptake into nematode body compartments. The present study monitored PAH uptake by Oncholaimus campylocercoides which was selected after 40 days in the laboratory through original protocols from natural sediments collected in the Old Harbor of Bizerte, Tunisia. To achieve the mono-species level, a grain size magnification was applied by gradually adding a biosubstrate made from either the crushed shells of Mytilus galloprovincialis or minced leaves of Posidonia oceanica. After selection, O. campylocercoides was cultured and fed with earthworm powder (560 mg.l-1). Thereafter, it was exposed for 3 weeks to phenanthrene and chrysene (38, 116, and 348 ppb). Fluorescence microscopy revealed higher intensities of PAHs at the spicules, mouths, and pharynges compared with the other organs considered. Moreover, the buccal fluorescence showed a significant correlation with that measured in the biosubstrate made with shells of M. galloprovincialis.

Ethinylestradiol (EE2) residues from birth control pills impair nervous system development and swimming behavior of zebrafish larvae.

The ubiquitous use of ethinylestradiol (EE2), an active constituent of birth control preparations, results in continuous release of this synthetic estrogen to surface waters. Many studies document the untoward effects of EE2 on the endocrine system of aquatic organisms. Effects of environmental EE2 on the nervous system are still poorly documented. We studied effects of pico- to nanomolar concentrations of EE2 on early nervous system development of zebrafish larvae. EE2 disrupted axonal nerve regeneration and hair cell regeneration up to 50%. Gene expression in larval brain tissues showed significantly upregulated expression of target genes, such as estrogen and progesterone receptors, and aromatase B. In contrast, downregulation of the tyrosine hydroxylase, involved in the synthesis of neurotransmitters, occurred concomitant with diminution of proliferating cells. Overall, the size of exposed fish larvae decreased by 25% and their swimming behavior was modified compared to non-treated larvae. EE2 interferes with nervous system development, both centrally and peripherally, with negative effects on regeneration and swimming behavior. Survival of fish and other aquatic species may be at risk in chronically EE2-contaminated environments.

Using meiobenthic taxa, nematofauna biological traits, and bacterial abundance to assess the effects of the polybrominated diphenyl ethers compound: Case study of tetrabromo diphenyl ether BDE-47.

Trophic web structuring in aquatic sediments is dependent on the biological interactions between metazoans and microbial communities. The presence of pollutants in these biotas can therefore impact the meiofauna structure via the modification of the microbial communities. The current study examined in a laboratory bioassay the response of meiobenthic communities, particularly marine nematode taxa from the Bizerte Lagoon to the effect of the most detected polybrominated diphenyl ether in this aquatic environment, BDE-47. Four doses [D1 (2.5 ppb Dry weight (DW)), D2 (25 ppb DW), D3 (50 ppb DW), and D4 (100 ppb DW)] were gradually applied and sediment microcosms were incubated for 30 days in the presence or absence of meiofauna. Our results show that BDE-47-enriched sediments decreased the meiofaunal taxa and bacterial abundance. A lower taxonomic diversity of the nematodes' general structure was observed with all doses used. The numerical analysis of the two dimensional (2D) non-metric multidimensional scaling (nMDS) plots and the evolution of the relative abundances of each functional group of nematode genus assemblages revealed that the abundance of all biological traits was modified. Nevertheless, only three of the functional traits, adult length, feeding group, and amphid shape, showed a clear difference between the control and the treated microcosms. The similarity percentage analysis (SIMPER) revealed that the average dissimilarity between nematode genera communities and biological traits increased with BDE-47-enriched sediments. The nMDS second-stage ordination of inter-matrix rank correlations for matrices including genera and biological traits showed that the amphid shape was the functional trait closest to the generic distribution. Finally, the Principal Component Analysis (PCA) for the nematode biological traits and bacteria indicated a positive correlation of these microbes with the functional groups [1A, Cr, and ef], and a negative correlation only with the "cla"-type tail shape.

Do presence of gray shrimp Crangon crangon larvae influence meiobenthic features? Assessment with a focus on traits of nematodes.

In this study, a microcosm experiment was conducted for 30 days to assess the impact of the presence of juvenile gray shrimp Crangon crangon on meiofauna. The results suggested that juvenile shrimp had a significant negative impact on the abundance of nematodes and copepods, but no effect on polychaetes. Moreover, nematodes showed a significant decline in individual weight. The collected nematodes were taxonomically identified and assigned to five functional traits: shapes of the tail and amphid, life history, feeding types, and adult length. The nematode traits were affected by the number of shrimp introduced, and descriptors followed normal or inversed bell-shaped curves. When no shrimp were present, the nematofauna had a higher species richness compared with treatments of 4, 8, and 12 shrimp. Bell-shaped curve patterns were common in relation to the two phases of feeding for C. crangon. During the first phase, C. crangon consumed the nematode species Oncholaimus campylocercoides; thereafter, shrimp fed mostly on the nematode Anticoma eberthi and copepods.

Experimental selection of Marylynnia puncticaudata (Cyatholaimidae, Nematoda) and effects of organic enrichment.

Meiobenthic nematodes are well-known bioindicators in aquatic ecosystem health programs. However, the explored taxa are still limited and practically devoted to the community level. The present study provided a new method of experimental isolation of a species from a pristine nematofauna. In our method, the nematofauna faced two types of sediment, namely, the leaves of Posidonia oceanica and shells of Mytilus galloprovincialis, under controlled laboratory conditions, and several changes in species composition occurred through gradual selection of the most adaptable nematode taxa to the new environments, which were previously defaunated. We used the selected nematode taxon, Marylynnia puncticaudata (Cyatholaimidae), to examine the possible effects of organic enrichment, and the results clearly showed that the body size of the nematodes significantly increased and they became fat when after enrichment using a powder made of marine agar (1200 mg l-1) and cuticles of Crangon crangon (900 mg l-1), but their relative body growth showed no discernible changes.

Nematodes trophic groups changing via reducing of bacterial population density after sediment enrichment to ciprofloxacin antibiotic: Case study of Marine Mediterranean community.

An experiment was carried out using microcosms to evaluate the impact of the fluoroquinolone antibiotic on nematode trophic groups structure and bacterial abundance. Sediment samples were experimentally enriched with four increasing doses of ciprofloxacin [D1 (50 ppm), D2 (100 ppm), D3 (200 ppm) and D4 (500 ppm)] and compared to non-enriched sediments (used as control). Ciprofloxacin changed the trophic composition of nematodes taxa where the relative abundance of microvores (M), epigrowth feeders (EF) and ciliate consumers (CF), raised in a control microcosm, was highly affected and significantly decreased in response to the increasing doses. Nevertheless, the abundance of deposit feeders (DF), optional predators (FP) and exclusive predators (Pr) showed a significant increase. Results from the multivariate analysis showed a clear impact of this antibiotic on nematode trophic assemblages. Microcosms treated with the three highest doses [D2, D3 and D4] were different from the control. The exceptions were those treated with the lowest dose, D1, and which were grouped with the control. The SIMPER analysis results showed that the average dissimilarity continuously increased in the treated microcosms compared to the control. Furthermore, our results have shown that ciprofloxacin also leads to a significant decrease in bacterial density with the highest dose, which could explain the results obtained for nematode trophic groups distribution. Thus, the bacteriophages nematodes only use bacteria as a nutrition source and the lack or presence in small quantity of this food could induce a decrease in their abundance as well as changing of nematodes groups repartition. Our work demonstrates that the nematode responses were dependent on sediment enrichment with ciprofloxacin and opens new perspectives on the potential impact of antibiotics on functional nematode diversity.

Restructuring of a meiobenthic assemblage after sediment contamination with an antibacterial compound: Case study of ciprofloxacin.

A microcosm experiment was conducted to evaluate the impacts of the fluoroquinolone antibiotic ciprofloxacin on meiobenthic taxa abundance, nematode genus structure, and functional trait parameters. Sediment samples were experimentally enriched with four different doses of ciprofloxacin [D1 (50 ppm Dry weight 'DW'), D2 (100 ppm DW), D3 (200 ppm DW), and D4 (500 ppm DW)] and were then compared with non-enriched sediments (controls). After one month of exposure, the data showed that ciprofloxacin had altered the meiofaunal taxa abundance. A change in the structure of nematofaunal genera was observed, particularly with the highest dose (D4), which was characterized by the lowest taxonomic diversity. The SIMPER analysis revealed that the average dissimilarity between nematode communities increased with increasing doses of ciprofloxacin. Two dimensional (2D) non-metric multidimensional scaling (nMDS) plots and relative abundances of functional groups of nematode genus assemblages revealed that all functional trait abundances were affected, particularly with the highest dose. However, only the amphid shape and feeding group functions showed a clear distribution separation between the control and ciprofloxacin treatments. The nMDS second-stage ordination of inter-matrix rank correlations for matrices including genus and functional traits showed that the tail shape was the closest functional trait to the generic distribution. Thus, only the curves of cumulative dominance related to the tail shape mirrored discernibly the sedimentary concentrations in ciprofloxacin.

New protocols for the selection and rearing of Metoncholaimus pristiurus and the first evaluation of oxidative stress biomarkers in meiobenthic nematodes.

Meiobenthic nematodes have been designated as sensitive global models in the development of biomonitoring and ecotoxicology monitoring programs howbeit the sensitivity of these organisms against oxidative stress biomarkers have never been addressed. The present study aimed to decipher this research axis after selecting and culturing a single nematode species from an entire community through original laboratory protocols. The purpose of this investigation was to change the grain size of the sediment into the immediate environment of nematodes by progressively adding a biosubstrate made from Sepia officinalis endoskeletton. At the end of the experiment, Metoncholaimus pristiurus became the unique component of the nematode species when the sediment was enriched with 80% of S. officinalis powder. After the mono-species level had been achieved, the selected species was fed on an another biosubstrate made from bodies of Porcellio scaber under the identical laboratory controlled conditions of light and temperature adopted during the selection process. Accordingly, the bioassay protocol this study layed new foundations for the study of meiobenthic nematodes in the biomarker field. Our results revealed that, in case of M. pritiurus, discernible oxidative stress responses are valid for catalase and gluthatione S-transferase. Indeed, for both enzymes, a clear increase in the activity was recorded, and the response was more reinforced when zinc and permethrin were administrated in combination. The relevance of the protocols proposed in this work parallels their global applicability to reach and maintain the monospecific level in laboratory by using biosubstrates made from animals widely distributed. It is true also that our data provided the first results in terms of biochemical biomarkers for meiobenthic nematodes and showed that the selected taxa, M. pristiurus, could be one of the first marine taxa responding early to the tested stressors, zinc and permethrin, even at very low concentrations.

Laboratory bioassay exploring the effects of anti-aging skincare products on free-living marine nematodes: a case study of collagen.

Organic enrichment due to human impact is one of the major threats that affect benthic communities in semi-enclosed marine ecosystems, such as the Mediterranean Sea. However, many emerging sources of organic pollutants, such as those released to nature through human practices related to esthetics and cosmetics, remain underestimated, despite being an increasingly important source of organic matter input following a decade of expansion by the cosmetic industry. Therefore, an experiment was designed to explore the influence of collagen, the main component of commercialized skin anti-aging products, on a Mediterranean community of free-living marine nematodes from a beach in Rimel, northeast Tunisia. The effects of exposure for 30 days to a control treatment and three test treatments, corresponding to three levels of sedimentary enrichment with collagen (3, 6, and 12 ppm dry weight (DW)), were examined using a microcosm approach. Reductions in abundance and diversity were noted with an increase in collagen enrichment, together with a slight increase in individual weight. The presence of three species characteristic of control microcosms, Ptycholaimellus ponticus, Theristus modicus, and Kraspedonema reflectans, was clearly affected at the lowest dose; these were therefore classified as "collagen-sensitive." In contrast, the numbers of "collagen-tolerant" species, including Sigmophoranema rufum, Lauratonema hospitum, Enoploides spiculohamatus, and Trichotheristus mirabilis, increased significantly in the treated microcosms.

Neurotoxicity of a Biopesticide Analog on Zebrafish Larvae at Nanomolar Concentrations.

Despite the ever-increasing role of pesticides in modern agriculture, their deleterious effects are still underexplored. Here we examine the effect of A6, a pesticide derived from the naturally-occurring α-terthienyl, and structurally related to the endocrine disrupting pesticides anilinopyrimidines, on living zebrafish larvae. We show that both A6 and an anilinopyrimidine, cyprodinyl, decrease larval survival and affect central neurons at micromolar concentrations. Focusing on a superficial and easily observable sensory system, the lateral line system, we found that defects in axonal and sensory cell regeneration can be observed at much lower doses, in the nanomolar range. We also show that A6 accumulates preferentially in lateral line neurons and hair cells. We examined whether A6 affects the expression of putative target genes, and found that genes involved in apoptosis/cell proliferation are down-regulated, as well as genes reflecting estrogen receptor activation, consistent with previous reports that anilinopyrimidines act as endocrine disruptors. On the other hand, canonical targets of endocrine signaling are not affected, suggesting that the neurotoxic effect of A6 may be due to the binding of this compound to a recently identified, neuron-specific estrogen receptor.

Trophic restructuring (Wieser 1953) of free-living nematode in marine sediment experimentally enriched to increasing doses of pharmaceutical penicillin G.

Trophic structure of free living nematode from Bizerte lagoon was tested by a microcosmic study after 30 days of exposure with 5 increasing doses of pharmaceutical penicillin G (D1: 3 mg L(-1), D2: 30 mg L(-1), D3: 300 mg L(-1), D4: 600 mg L(-1), D5: 700 mg L(-1)). Results showed significant differences between nematode assemblages from undisturbed controls and those from penicillin G treatments. Selective deposit-feeders (1A) or nonselective deposit-feeders (1B), very abundant in the control microcosm, were significantly affected and their dominance declined significantly. Epistrate feeders (2A) were significantly gradual increase for all microcosms treated with penicillin G, appeared to be more tolerant to the antibiotic and to take advantage of the growing scarcity of other trophic groups. Compared to the control microcosms, omnivorous-carnivorous (2B) was found to be higher in all treated microcosms, with the exception of those treated with D5. Trophic index (Σθ(2)) was significantly reduced in all microcosms treated whereas trophic ratio 1B/2A appears to be insignificant.

Steroid modulation of neurogenesis: Focus on radial glial cells in zebrafish.

Estrogens are known as steroid hormones affecting the brain in many different ways and a wealth of data now document effects on neurogenesis. Estrogens are provided by the periphery but can also be locally produced within the brain itself due to local aromatization of circulating androgens. Adult neurogenesis is described in all vertebrate species examined so far, but comparative investigations have brought to light differences between vertebrate groups. In teleost fishes, the neurogenic activity is spectacular and adult stem cells maintain their mitogenic activity in many proliferative areas within the brain. Fish are also quite unique because brain aromatase expression is limited to radial glia cells, the progenitor cells of adult fish brain. The zebrafish has emerged as an interesting vertebrate model to elucidate the cellular and molecular mechanisms of adult neurogenesis, and notably its modulation by steroids. The main objective of this review is to summarize data related to the functional link between estrogens production in the brain and neurogenesis in fish. First, we will demonstrate that the brain of zebrafish is an endogenous source of steroids and is directly targeted by local and/or peripheral steroids. Then, we will present data demonstrating the progenitor nature of radial glial cells in the brain of adult fish. Next, we will emphasize the role of estrogens in constitutive neurogenesis and its potential contribution to the regenerative neurogenesis. Finally, the negative impacts on neurogenesis of synthetic hormones used in contraceptive pills production and released in the aquatic environment will be discussed.

Effects of increasing levels of pharmaceutical penicillin G contamination on structure of free living nematode communities in experimental microcosms.

A microcosm experiment was conducted to examine the effects of the pharmaceutical (penicillin G) on free living nematode communities of a Tunisian coastal zone (South-Western Mediterranean Sea). Sediments were contaminated with five penicillin G dose [D1 (3 mgL(-1)), D2 (30 mgL(-1)), D3 (300 mgL(-1)), D4 (600 mgL(-1)), D5 (700 mgL(-1))], and effects were examined after 30 days. Results showed significant differences between nematode assemblages from undisturbed controls and those from penicillin G treatments. Most univariate measures, including diversity (H'), species richness (d), equitability (J) and number of species (S) decreased significantly with increasing level of the antibiotic contamination. Results from multivariate analyses of the species abundance data demonstrated that responses of nematode species to the penicillin treatments were varied: Kraspedonema octogoniata and Paracomesoma dubium were eliminated at all the antibiotic doses tested and seemed to be intolerant species to penicillin G contamination; Oncholaimus campylocercoides although survived even the highest dose D5, showed definite reduction in its abundance and may be classified as "opportunistic" species at this dose, whereas, Nannolaimoides decoratus which showed a positive response with an increase in density even at highest concentration of contaminant, seems to be "penicillin G resistant" species.

Aromatase, estrogen receptors and brain development in fish and amphibians.

Estrogens affect brain development of vertebrates, not only by impacting activity and morphology of existing circuits, but also by modulating embryonic and adult neurogenesis. The issue is complex as estrogens can not only originate from peripheral tissues, but also be locally produced within the brain itself due to local aromatization of androgens. In this respect, teleost fishes are quite unique because aromatase is expressed exclusively in radial glial cells, which represent pluripotent cells in the brain of all vertebrates. Expression of aromatase in the brain of fish is also strongly stimulated by estrogens and some androgens. This creates a very intriguing positive auto-regulatory loop leading to dramatic aromatase expression in sexually mature fish with elevated levels of circulating steroids. Looking at the effects of estrogens or anti-estrogens in the brain of adult zebrafish showed that estrogens inhibit rather than stimulate cell proliferation and newborn cell migration. The functional meaning of these observations is still unclear, but these data suggest that the brain of fish is experiencing constant remodeling under the influence of circulating steroids and brain-derived neurosteroids, possibly permitting a diversification of sexual strategies, notably hermaphroditism. Recent data in frogs indicate that aromatase expression is limited to neurons and do not concern radial glial cells. Thus, until now, there is no other example of vertebrates in which radial progenitors express aromatase. This raises the question of when and why these new features were gained and what are their adaptive benefits. This article is part of a Special Issue entitled: Nuclear receptors in animal development.