Maria Natividade Vieira

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.

Implications of different brood pouch structures in syngnathid reproduction

The evolutionary radiation of the family Syngnathidae was accompanied by a diversi¢cation of structures involved in male parental care whose anatomical variations may signal diierences in reproductive strategies, with increasing egg protection possibly aiecting female investment in oispring or larvae quality at the end of the embryonic development phase. An analysis of egg numbers showed signi¢cant diierences between syngnathids with and without marsupium, suggesting that the brood pouch, besides increasing protection to the male and its eggs, also introduced the ability to carry an additional number of oispring per pregnancy, from one or more females. Curiously, even though larvae sizes do not signi¢cantly diier between the considered brooding structures (controlling for male length), seahorse larvae were smaller than expected, given the large volume of the pear-shaped eggs. These observations suggest that the sealed seahorse pouch, although allowing the transport of a great number of oispring and capable of multiple consecutive pregnancies, may impose serious functional constraints, namely those related with oxygenation of the developing larvae, thus explaining the increased egg surface together with the presence of speci¢c salinity control mechanisms inside the marsupium.

Reversing sex role reversal: compete only when you must

The operational sex ratio (OSR) is thought to be a major factor influencing the intensity of mating competition and sexual selection. Even though many studies on species with conventional sex roles have shown that alterations in the OSR can either intensify male–male competition or promote female–female competitive interactions, sometimes resulting in a reversal of sex roles, it is not known how, and how quickly, individuals with reversed sex roles respond to fluctuations in this ratio. We tested for a direct influence of adult sex ratios (as a direct estimation of the OSR) on the reproductive behaviour of the sex role-reversed black striped pipefish, Syngnathus abaster. Although imbalances in the OSR effectively modulated the expression of sex roles, with males and females varying in the degree of choosiness and competitive displays, alterations in the sex ratios did not promote a similar response pattern in both sexes. A surplus of males resulted in a reversion to conventional sex roles observed when both sexes coexist in similar numbers, with males competing intensely and exhibiting a conspicuous ornament towards other males. An excess of females, in contrast, did not result in an overall increase in female competitive interactions. Only small, less attractive, females were more prone to compete as the proportion of males decreased. Large females, however, seemed to rely on their greater mating prospects, thereby avoiding the hypothetical costs of intrasexual competition.

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.

The courtship behaviour of the pipefish Nerophis lumbriciformis: reflections of an adaptation to intertidal life

Abstract. The courtship behaviour of Nerophis lumbriciformis (Pisces: Syngnathidae) consists of three distinct phases (initial courtship, spawning, and embrace) marked by prominent behavioural changes. The first courtship phase is characterised by female quivering. Courtship activity increases from low to high levels of quivering, causing undulatory head movements in the female. In the second phase, the female transfers her eggs onto the male incubating ventral surface. During the final phase, the male wraps his body around the female (embrace). Females actively initiate courtship indicating that these pipefishes are courtship role reversed. The complete lack of swimming and vertical movements in the courtship behaviour of N. lumbriciformis, unlike in the behaviour of other syngnathid species, suggests an adaptation to intertidal conditions.

The breeding ecology of the pipefish Nerophis lumbriciformis and its relation to latitude and water temperature

The breeding season of Nerophis lumbriciformis (Pisces: Syngnathidae), has not yet been determined for the southernmost part of its range. A total of 863 individuals was examined between March 1997 and November 1999. In Portugal, the breeding season of this species occurs throughout the year, with a marked reduction during summer and autumn, whilst in Britain it occurs from May to September. Despite these temporal differences, the water temperature at which breeding takes place is similar in the two areas (13–16°C). Together with preliminary laboratory observations with animals kept at different temperatures, these data support the hypothesis that the decrease in breeding activity during summer and autumn in Portugal is due to an inhibitory effect of high water temperature.

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 males 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.

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, Gondomar), 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.

Activity rhythms and cyclical changes of microhabitat preferences in the intertidal pipefish Nerophis lumbriciformis (Pisces: Syngnathidae)

Abstract. As an inhabitant of the intertidal zone, Nerophis lumbriciformis predictably should show a clear rhythmic activity, with its maximum closely correlated with the high tide period, as observed in several other rocky intertidal fish species. We investigated the rhythmic activity patterns of N. lumbriciformis and analysed to what extent specific substratum preferences may be linked to water level and, ultimately, to the particular activity rhythm patterns of the fish. To determine N. lumbriciformis substratum preferences, two different experiments were conducted. The first evaluated substratum preferences from among a group of the four most common substrates found in the sample area. The second experiment evaluated the fishs maintenance of that substratum preference, after alteration of the water level in a simulated ebb tide. From the available substrata, N. lumbriciformis displayed a clear preference for algae until the water level began to decrease. Then, the preference radically shifted from algae to boulders. N. lumbriciformis showed a clear rhythmic activity, affected by both tidal and circadian periodicity, producing asymmetrical activity peaks. Unlike the rhythms of other rocky intertidal fish species, the maximum activity peaks were not centred at the expected high tide period. The adaptive explanation for the apparently peculiar activity peaks appears to be related to the particular substratum preferences of N. lumbriciformis. The detected circatidal period seems, consequently, closely related to small migrations between substrata, whilst the observed increase in diurnal activity, independent of the tidal cycle, may be related to predatory activity, since N. lumbriciformis is a visual feeder.