The last frontier: Coupling technological developments with scientific challenges to improve hazard assessment of deep-sea mining.

The growing economic interest in the exploitation of mineral resources on deep-ocean beds, including those in the vicinity of sensitive-rich habitats such as hydrothermal vents, raise a mounting concern about the damage that such actions might originate to these poorly-know ecosystems, which represent millions of years of evolution and adaptations to extreme environmental conditions. It has been suggested that mining may cause a major impact on vent ecosystems and other deep-sea areas. Yet, the scale and the nature of such impacts are unknown at present. Hence, building upon currently available scientific information it is crucial to develop new cost-effective technologies embedded into rigorous operating frameworks. The forward-thinking provided here will assist in the development of new technologies and tools to address the major challenges associated with deep sea-mining; technologies for in situ and ex situ observation and data acquisition, biogeochemical processes, hazard assessment of deep-sea mining to marine organisms and development of modeling tools in support of risk assessment scenarios. These technological developments are vital to validate a responsible and sustainable exploitation of the deep-sea mineral resources, based on the precautionary principle.

The lek mating system of the worm pipefish (Nerophis lumbriciformis): a molecular maternity analysis and test of the phenotype-linked fertility hypothesis.

The origin and maintenance of mating preferences continues to be an important and controversial topic in sexual selection research. Leks and lek-like mating systems, where individuals gather in particular spots for the sole purpose of mate choice, are particularly puzzling, because the strong directional selection imposed by mate choice should erode genetic variation among competing individuals and negate any benefit for the choosing sex. Here, we take advantage of the lek-like mating system of the worm pipefish (Nerophis lumbriciformis) to test the phenotype-linked fertility hypothesis for the maintenance of mating preferences. We use microsatellite markers to perform a parentage analysis, along with a mark-recapture study, to confirm that the worm pipefish has an unusual mating system that strongly resembles a female lek, where females display and males visit the lek to choose mates. Our results show that the most highly ornamented females occupy positions near the centre of the breeding area, and males mating with these females receive fuller broods with larger eggs compared to males mating with less-ornamented females. We also conduct a laboratory experiment to show that female ornaments are condition-dependent and honestly signal reproductive potential. Overall, these results are consistent with the predictions of a sex-independent version of the phenotype-linked fertility hypothesis, as male preference for female ornaments correlates with fertility benefits.

Toxicological impact of cadmium-based quantum dots towards aquatic biota: Effect of natural sunlight exposure.

Cadmium-based quantum dots (QDs) are increasingly applied in existent and emerging technologies, especially in biological applications due to their exceptional photophysical and functionalization properties. However, they are very toxic compounds due to the high reactive and toxic cadmium core. The present study aimed to determine the toxicity of three different QDs (CdS 380, CdS 480 and CdSeS/ZnS) before and after the exposure of suspensions to sunlight, in order to assess the effect of environmentally relevant irradiation levels in their toxicity, which will act after their release to the environment. Therefore, a battery of ecotoxicological tests was performed with organisms that cover different functional and trophic levels, such as Vibrio fischeri, Raphidocelis subcapitata, Chlorella vulgaris and Daphnia magna. The results showed that core-shell type QDs showed lower toxic effects to V. fischeri in comparison to core type QDs before sunlight exposure. However, after sunlight exposure, there was a decrease of CdS 380 and CdS 480 QD toxicity to bacterium. Also, after sunlight exposure, an effective decrease of CdSeS/ZnS and CdS 480 toxicity for D. magna and R. subcapitata, and an evident increase in CdS 380 QD toxicity, at least for D. magna, were observed. The results of this study suggest that sunlight exposure has an effect in the aggregation and precipitation reactions of larger QDs, causing the degradation of functional groups and formation of larger bulks which may be less prone to photo-oxidation due to their diminished surface area. The same aggregation behaviour after sunlight exposure was observed for bare QDs. These results further emphasize that the shell of QDs seems to make them less harmful to aquatic biota, both under standard environmental conditions and after the exposure to a relevant abiotic factor like sunlight.

Bioremediation of an iron-rich mine effluent by Lemna minor.

Contamination of water resources by mine effluents is a serious environmental problem. In a old coal mine, in the north of Portugal (São Pedro da Cova, Gondoma),forty years after the activity has ended, a neutral mine drainage, rich in iron (FE) it stills being produced and it is continuously released in local streams (Ribeiro de Murta e Rio Ferreira) and in surrounding lands. The species Lemna minor has been shown to be a good model for ecotoxicological studies and it also has the capacity to bioaccumulate metals. The work aimed test the potential of the species L. minor to remediate this mine effluent, through the bioaccumulation of Fe, under greenhouse experiments and, at the same time, evaluate the time required to the maximum removal of Fe. The results have shown that L. minor was able to grow and develop in the Fe-rich effluent and bioaccumulating this element. Throughout the 21 days of testing it was found that there was a meaningful increase in the biomass of L. minor both in the contaminated and in the non-contaminated waters. It was also found that bioaccumulation of Fe (iron) occurred mainly during the first 7 days of testing. It was found that L. minor has potential for the bioremediation of effluents rich in iron.

Validating the use of colouration patterns for individual recognition in the worm pipefish using a novel set of microsatellite markers.

In studies of behaviour, ecology and evolution, identification of individual organisms can be an invaluable tool, capable of unravelling otherwise cryptic information regarding group structure, movement patterns, population size and mating strategies. The use of natural markings is arguably the least invasive method for identification. However, to be truly useful natural markings must be sufficiently variable to allow for unique identification, while being stable enough to permit long-term studies. Non-invasive marking techniques are especially important in fishes of the Family Syngnathidae (pipefishes, seahorses and seadragons), as many of these taxa are of conservation concern or used extensively in studies of sexual selection. Here, we assessed the reliability of natural markings as a character for individual identification in a wild population of Nerophis lumbriciformis by comparing results from natural markings to individual genetic assignments based on eight novel microsatellite loci. We also established a minimally invasive method based on epithelial cell swabbing to sample DNA. All pipefish used in the validation of natural markings, independently of sex or time between recaptures, were individually recognized through facial colouration patterns. Their identities were verified by the observation of the same multilocus genotype at every sampling event for each individual that was identified on the basis of natural markings. Successful recaptures of previously swabbed pipefish indicated that this process probably did not induce an elevated rate of mortality. Also, the recapture of newly pregnant males showed that swabbing did not affect reproductive behaviour.

A real-time PCR assay for differential expression of vitellogenin I and II genes in the liver of the sentinel fish species Lipophrys pholis.

Abstract The recent advances in molecular biology techniques have prompted the use of vitellogenin (VTG) gene expression as a sensitive and reliable indicator of estrogenic chemicals (EC) exposure. However, data on the dynamic response of the different VTGs genes upon EC exposure is still poorly understood, particularly in sentinel fish species used in field monitoring studies. Hence, the present study aimed at developing a sensitive real-time PCR assay for determining the response of VTG I and II in the recently proposed marine sentinel species Lipophrys pholis upon exposure to the model EC 17α-ethinylestradiol (EE2). The findings of the laboratory study indicate that L. pholis VTG I proved to be not only more inducible but also more sensitive to EE2 exposure than VTG II, for the same range of concentrations. In fact, VTG I gene induction was 475-fold higher than VTG II at 15 ng/L EE2, and 13-fold at 5 ng/L EE2. Overall, the findings of the present study indicate that in the field, expression of VTG I in L. pholis should be preferentially used in the screening of EC exposure because of its higher sensitivity. Furthermore, the present study favors L. pholis integration in monitoring programs associated with EC's pollution within the European water policy legislation.

Body composition measures of obese adolescents by the deuterium oxide dilution method and by bioelectrical impedance.

The objectives of the present study were to describe and compare the body composition variables determined by bioelectrical impedance (BIA) and the deuterium dilution method (DDM), to identify possible correlations and agreement between the two methods, and to construct a linear regression model including anthropometric measures. Obese adolescents were evaluated by anthropometric measures, and body composition was assessed by BIA and DDM. Forty obese adolescents were included in the study. Comparison of the mean values for the following variables: fat body mass (FM; kg), fat-free mass (FFM; kg), and total body water (TBW; %) determined by DDM and by BIA revealed significant differences. BIA overestimated FFM and TBW and underestimated FM. When compared with data provided by DDM, the BIA data presented a significant correlation with FFM (r = 0.89; P < 0.001), FM (r = 0.93; P < 0.001) and TBW (r = 0.62; P < 0.001). The Bland-Altman plot showed no agreement for FFM, FM or TBW between data provided by BIA and DDM. The linear regression models proposed in our study with respect to FFM, FM, and TBW were well adjusted. FFM obtained by DDM = 0.842 x FFM obtained by BIA. FM obtained by DDM = 0.855 x FM obtained by BIA + 0.152 x weight (kg). TBW obtained by DDM = 0.813 x TBW obtained by BIA. The body composition results of obese adolescents determined by DDM can be predicted by using the measures provided by BIA through a regression equation.

Body composition measures of obese adolescents by the deuterium oxide dilution method and by bioelectrical impedance

The objectives of the present study were to describe and compare the body composition variables determined by bioelectrical impedance (BIA) and the deuterium dilution method (DDM), to identify possible correlations and agreement between the two methods, and to construct a linear regression model including anthropometric measures. Obese adolescents were evaluated by anthropometric measures, and body composition was assessed by BIA and DDM. Forty obese adolescents were included in the study. Comparison of the mean values for the following variables: fat body mass (FM; kg), fat-free mass (FFM; kg), and total body water (TBW; percent) determined by DDM and by BIA revealed significant differences. BIA overestimated FFM and TBW and underestimated FM. When compared with data provided by DDM, the BIA data presented a significant correlation with FFM (r = 0.89; P < 0.001), FM (r = 0.93; P < 0.001) and TBW (r = 0.62; P < 0.001). The Bland-Altman plot showed no agreement for FFM, FM or TBW between data provided by BIA and DDM. The linear regression models proposed in our study with respect to FFM, FM, and TBW were well adjusted. FFM obtained by DDM = 0.842 x FFM obtained by BIA. FM obtained by DDM = 0.855 x FM obtained by BIA + 0.152 x weight (kg). TBW obtained by DDM = 0.813 x TBW obtained by BIA. The body composition results of obese adolescents determined by DDM can be predicted by using the measures provided by BIA through a regression equation.

Rapid-behaviour responses as a reliable indicator of estrogenic chemical toxicity in zebrafish juveniles.

Whereas biochemical and molecular parameters have been well recognised as important "signposts" of individual disturbance to endocrine disrupting chemical's (EDCs) exposure, behavioural endpoints are yet greatly overlooked as a routine tool in environmental risk assessment of EDCs. However, life histories are intimately associated with numerous inter- and intra-specific interactions, which invariably depend on the performance of effective behaviours. Within fish species, one of the most important factors influencing energy turnover earlier in the development is locomotor activity. This essential trait reflects the organism's ability to generate and coordinate the metabolic energy required for both reproductive and non-reproductive behaviours. Inappropriate movement responses due to toxic effects of contaminants may ultimately impact important ecological variables. Therefore, in the present study, the swimming bursts of zebrafish juveniles exposed for 40 d to the synthetic estrogen ethinylestradiol (EE(2)), tested at environmentally relevant concentrations (nominal concentrations of 0.5, 1 and 2 ng L(-1)), were investigated in order to address the potential of rapid-behaviour patterns as an effective response indicator of estrogenic endocrine disrupting chemical's exposure. This synthetic estrogen was selected due to its high prevalence in aquatic ecosystems, ability to mimic natural estrogens and proven record of causing negative effects in fish reproduction. The behavioural responses were compared with established endpoints used in the screening of EE(2) effects at adulthood. Results indicate that zebrafish juveniles' swimming activity was significantly decreased upon EE(2) exposure. Since reduced locomotion of zebrafish may impact foraging, predator avoidance, drift and transport, and even interfere with social and reproductive behaviours, a fitness decline of wild fish populations can ultimately be hypothesized. Furthermore, behavioural endpoints were found to display higher sensitivity to EE(2) than either vitellogenin gene induction or reproductive parameters determined at adulthood. Overall, the findings of this work not only demonstrate the power of high-throughput behavioural responses, able to act as sensitive early warning signals of EDC exposure, but also highlight the potential of behavioural endpoints in providing a more comprehensive and non-invasive measure of EDC's exposure.

Disruption of zebrafish (Danio rerio) embryonic development after full life-cycle parental exposure to low levels of ethinylestradiol.

Exposure of fish to the synthetic estrogen ethinylestradiol (EE2) has been shown to induce a large set of deleterious effects. In addition to the negative impact of EE2 in reproductive endpoints, concern has recently increased on the potential effects of EE2 in fish embryonic development. Therefore, the present study aimed at examining the effects of EE2 on the full embryonic development of zebrafish in order to identify the actual phases where EE2 disrupts this process. Hence, zebrafish were exposed to environmentally relevant low levels of EE2, 0.5, 1 and 2ng/L (actual concentrations of 0.19, 0.24 and 1ng/L, respectively) from egg up to eight months of age (F(1)), and the survival as well as the occurrence of abnormalities in their offsprings (F(2)), per stage of embryonic development, was investigated. A thorough evaluation of reproductive endpoints and transcription of vtg1 gene in the parental generation (F(1)) at adulthood, was performed. No significant differences could be observed for the two lowest EE2 treatments, in comparison with controls, whereas vtg1 transcripts were significantly elevated (40-fold) in the 2ng/L EE2 treatment. In contrast to the findings in the F(1) generation,a significant concentration-dependent increase in egg mortality between 8 and 24hours post-fertilization (hpf) was observed for all EE2 treatments, when compared with controls. The screening of egg and embryo development showed a significant increase in the percentage of abnormalities at 8 hpf for the highest EE2 concentration, a fact that might explain the increased embryo mortality at the 24 hpf time-point observation. Taken together, these findings indicate that the two lowest tested EE2 concentations impact late gastrulation and/or early organogenesis, whereas exposure to 2ng/L EE2 also disrupts development in the blastula phase. After early organogenesis has been completed (24 hpf), no further mortality was observed. These results show that increased embryo mortality occurs at EE2 levels below those inducing reproductive impairment and vtg1 gene induction in the male parental generation, thus suggesting that EE2 may impact some fish populations at levels below those inducing an increase in vtg1 transcripts. Hence, these findings have important implications for environmental risk assessment, strongly supporting the inclusion of embryonic development studies in the screening of endocrine disruption in wild fish populations.

Organotin levels in seafood from Portuguese markets and the risk for consumers.

Because of their ubiquity in the aquatic environment, the antifouling agent tributyltin (TBT) and other organotins (OTs) accumulate through the food chain, resulting in the occurrence of OTs in seafood products. Despite a high number of studies on the negative impact of TBT in female prosobranch gastropods, few works exist in Europe reporting the levels of these compounds in edible parts of marine organisms used in Human diet. Therefore, within the scope of an EU project OT-SAFE the levels of several OTs were evaluated in the most relevant seafood products for Portuguese consumers. Butyltins (BTs) have been detected in all analysed groups (fish, crustaceans, bivalves, cephalopods), whereas triphenyltin, tricyclohexyltin, monooctyltin and dioctyltin could not be detected and tetrabutyltin was present above detection limits in a single sample. In general, levels of BTs in edible parts of fish, crustaceans and cephalopods collected in Portuguese markets during this study are in the lower range of that reported for these animal groups from other locations (i.e. below 30ngg(-1) wet weight). In contrast, moderate to high concentrations have been observed in bivalves (up to 275ng TBTg(-1) wet weight). While most samples showed TBT plus DBT levels below the tolerable average residue levels (TARL), which may indicate low risk for consumer, four bivalve samples displayed BT levels above TARL, thus indicating that higher bivalve consumer groups may be at risk. The results found are discussed in relation to the potential risk for consumers and integrated with recent finds on the molecular targets of OTs in mammals.

Vitellogenin gene expression in the intertidal blenny Lipophrys pholis: a new sentinel species for estrogenic chemical pollution monitoring in the European Atlantic coast?

The presence of estrogenic chemicals (ECs) in the aquatic environment is a growing problem. While most attention was initially given to fresh water and estuarine ecosystems, it is now evident that coastal marine areas are also vulnerable to these pollutants. The use of vitellogenin induction in male fish, a specific biomarker of EC exposure, has been the most widely applied methodology. However, in some occasions, the high mobility and migratory behaviour of common sentinel fish species makes data interpretation difficult. Hence, there is the need to validate new sentinel marine fish species which should display, among other features, a strong homing behaviour. The shanny, Lipophrys pholis, is an intertidal fish that combines many of the required characteristics for a sentinel species: abundance and easy of catch, wide geographical distribution and restricted home range. Thus, in order to evaluate, in the field, the species sensitivity to ECs, L. pholis males were collected at two sites reflecting different degrees of anthropogenic contamination. The vitellogenin II gene (VTGII) was isolated and its liver expression evaluated by RT-PCR in the field samples. A significant induction of gene expression was observed in the specimens collected in the urban area, if compared to the reference site, which suggests exposure to ECs. Moreover, a 21-days laboratory exposure to environmental relevant concentrations of ethinylestradiol (EE2) was also performed. A significant induction of L. pholis VTGII gene in EE2 exposed males was observed suggesting similar sensitivity to that of other marine/estuarine fishes. Even though further validation is currently in progress, the available data indicates that L. pholis is responsive to ECs, thus favouring its future integration in monitoring programmes designed to evaluate the presence of ECs in European marine ecosystems.

Can the limited marsupium space be a limiting factor for Syngnathus abaster females? Insights from a population with size-assortative mating.

1. Some syngnathid species show varying degrees of sex role reversal aside from male pregnancy, with females competing for access to mates and sometimes presenting conspicuous secondary sexual characters. Among other variables, brooding space constraints are usually considered a key element in female reproductive success, contributing strongly to the observed morphological and behavioural sexual differences. Nevertheless, a close relationship between sex role reversal and male brooding space limitation has not yet been accurately demonstrated in field studies. 2. The present work, conducted over two consecutive breeding seasons in a wild population of the sex role-reversed pipefish Syngnathus abaster, simultaneously analysed egg number and occupied space, as well as the free area in the male's marsupium. The number of eggs that would fit in the observed unoccupied space was estimated. 3. Contrary to what would be expected, given the marked sexual dimorphism observed in the population studied, where females were larger and more colourful, male brooding space did not appear to limit female reproduction as neither large nor small individuals presented a fully occupied pouch. Interestingly, the largest unoccupied areas of marsupium were found in the larger individuals, although they received more and larger eggs. Laboratory data also showed that larger females lay larger eggs. 4. Together, these results suggest the existence of assortative mating, which may result from: (i) the reluctance of larger males (which tend not to receive small eggs usually laid by small females) to mate with lower quality females, even at the expense of a smaller number of offspring; or (ii) female-female competition, which might strongly reduce the hypothesis of a small female mating with a large male. The potential impact of temperature on reproduction and population dynamics is also discussed in the light of ongoing climatic changes.

New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus.

In an attempt to clarify the mechanism(s) of tributyltin-mediated imposex induction in females of the neogastropod Nucella lapillus, dogwhelks collected in an almost imposex free population were exposed to several treatments for a 3 month-period, and the effects on imposex induction and testosterone/estradiol levels were evaluated. As a positive control, tributyltin (50 ng TBT Sn/L) clearly induced imposex and led to a significant increase in the severity of the phenomenon. In contrast, although a selective P450 aromatase inhibitor (formestane at 0.3 mg/L) was capable of imposex induction, it failed to increase its severity. A vertebrate androgen receptor (AR) antagonist (cyproterone acetate at 1.25 mg/L) in combination with TBT completely blocked the imposex induction capacity of TBT. On the other hand, an estrogen receptor antagonist (tamoxifen at 0.3 mg/L) rendered no effect. The determination of steroid levels in female specimens revealed that TBT induces an elevation of free testosterone (but not the total amount, free+esterified), while the co-administration of the anti-androgen and TBT was able to rescue the increase of free testosterone levels. Despite a minor decrease in the amount of testosterone-fatty acid esters in the TBT group, significant differences in esterified testosterone were not found among treatments. On the contrary, free estradiol levels were elevated in the TBT, anti-androgens and TBT plus anti-androgens groups. These results indicate that free estradiol biosynthesis in TBT-exposed females does not seem to be affected. Overall, our results demonstrate that a selective aromatase inhibitor can induce imposex in N. lapillus but not to a similar extent of TBT, which may suggest the involvement of other mechanism in imposex induction, besides aromatase inhibition. Additionally, the study points to the involvement of AR receptors in imposex induction.

Comparative assessment of electroglottographic and acoustic measures of jitter in pathological voices.

Jitter, or the amount of cycle-to-cycle variation in the fundamental frequency, is a characteristic of the vocal folds' vibration that may affect electroglottographic (EGG) and acoustic signals in similar ways, because the translaryngeal conductance and the oral pressure are modulated by the same physiological mechanism. Despite the apparent simplicity in jitter computation and the relative facility in recording and analyzing EGG signals, only a few studies comparing EGG and acoustic jitter have been reported. This can be attributed to the remarkable sensitivity of measures of acoustic jitter to such features as the type of sound being analyzed, the equipment used for data acquisition, or the algorithms used to identify glottal cycle boundaries. To assess the agreement achievable by measures from these signals in pathological voices, acoustic and EGG waveforms of sustained vowels (/i/, /a/, /u/) produced by 15 dysphonic patients were analyzed by jitter extraction methods on the basis of peaks, zero crossings, and a waveform matching technique. The agreement, expressed as normalized absolute differences between acoustic- and EGG-derived jitter, was poorer for /i/ and /u/ than for /a/ vowels. For /a/ vowels, a method of acoustic jitter estimation is also proposed that combines peaks and zero crossings and resulted in increased consistency with the zero crossing-based EGG measures (mean normalized absolute difference: 10.95%, SD: 6.44%; range: 23.81%). The proposed method, which has a built-in-test intended to reject unreliable estimates, may provide more confidence in acoustic measures in dysphonic speakers and lead to a better understanding of the relationships between acoustic and EGG signals.

Pesquisa de aglutininas antileptospira em diferentes grupos profissionais na cidae de Londrina, Paraná / Anti-Leptospira agglutinins research in different professional groups in the city of Londrina, Paraná

Realizou-se um levantamento soroepidemiológico na cidade de Londrina - Paraná, para leptospirose, em 49 indivíduos näo expostos a risco, 75 trabalhadores da limpeza pública, 55 indivíduos com atividade em ambiente hospitalar e 29 trabalhadores do departamento de Agua e Esgoto. Dos 208 soros analisados pela reaçäo de soroaglutinaçäo microscópica, 28,4 por cento apresentaram aglutininas antileptospira. A maior positividade foi encontrada nos soros dos trabalhadores da limpeza pública (46,7 por cento)

[Anti-Leptospira agglutinins in different professional groups in the city of Londrina, Paraná]. / Pesquisa de aglutininas antileptospira em diferentes grupos profissionais na cidade de Londrina, Paraná.

Serum samples were obtained from 208 individuals in Londrina, Paraná, Brazil. The serum were analysed for leptospiral agglutinins by agglutination microscopic tests and 28.4% were positive. The highest positivity was found for the serum of garbage collector (46.7%).