Nuno Miguel Monteiro

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.

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.

Controlling for false positives: interpreting MBL Etest and MBL combined disc test for the detection of metallo-β-lactamases

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Stronger Sexual Selection in Warmer Waters: The Case of a Sex Role Reversed Pipefish

In order to answer broader questions about sexual selection, one needs to measure selection on a wide array of phenotypic traits, simultaneously through space and time. Nevertheless, studies that simultaneously address temporal and spatial variation in reproduction are scarce. Here, we aimed to investigate the reproductive dynamics of a cold-water pipefish simultaneously through time (encompassing variation within each breeding cycle and as individuals grow) and space (by contrasting populations experiencing distinct water temperature regimes) in order to test hypothesized differences in sexual selection. Even though the sampled populations inhabited locations with very different water temperature regimes, they exhibited considerable similarities in reproductive parameters. The most striking was the existence of a well-defined substructure in reproductive activity, where larger individuals reproduce for longer periods, which seemed dependent on a high temperature threshold for breeding rather than on the low temperatures that vary heavily according to latitude. Furthermore, the perceived disparities among populations, such as size at first reproduction, female reproductive investment, or degree of sexual size dimorphism, seemed dependent on the interplay between seawater temperature and the operational sex ratio (OSR). Contrary to our expectations of an enhanced opportunity for sexual selection in the north, we found the opposite: higher female reproductive investment coupled with increased sexual size dimorphism in warmer waters, implying that a prolonged breeding season does not necessarily translate into reduced sexual selection pressure. In fact, if the limited sex has the ability to reproduce either continuously or recurrently during the entire breeding season, an increased opportunity for sexual selection might arise from the need to compete for available partners under strongly biased OSRs across protracted breeding seasons. A more general discussion on the effects of climate change in the pressure of sexual selection is also presented.

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.

Microsatellite markers for identification and parentage analysis in the European wild boar (Sus scrofa)

BackgroundThe wild boar (Sus scrofa) is among the most widespread mammal species throughout the old world. Presently, studies concerning microsatellites in domestic pigs and wild boars have been carried out in order to investigate domestication, social behavior and general diversity patterns among either populations or breeds. The purpose of the current study is to develop a robust set of microsatellites markers for parentage analyses and individual identification.FindingsA set of 14 previously reported microsatellites markers have been optimized and tested in three populations from Hungary, Portugal and Spain, in a total of 167 samples. The results indicate high probabilities of exclusion (0.99999), low probability of identity (2.0E-13 – 2.5E-9) and a parentage assignment of 100%.ConclusionsOur results demonstrate that this set of markers is a useful and efficient tool for the individual identification and parentage assignment in wild boars.

Drifting towards the surface: A shift in newborn pipefish's vertical distribution when exposed to the synthetic steroid ethinylestradiol

Endocrine disrupting chemical (EDC) effects during early life have the potential to modulate population structure, either directly through increased mortality or by causing inappropriate aggregation events, thus affecting the number of young that will reach adulthood. An alteration in the dispersal and recruitment patterns can also impair the connectivity among geographically distant populations. However, the detection of EDC-induced effects occurring after egg hatch, when newborns increase their chances of contacting with environmentally dispersed contaminants, is not a simple process as effects might be masked by the large natural mortality rates that usually occur during fish early life. Since there is a lack of information regarding the impact of EDCs on fish early life dispersal patterns, particularly on vertical migrations patterns, the effects of environmentally relevant concentrations of EE(2) on the vertical distribution of newborn fish was assessed through an ex situ exposure experiment. Syngnathus abaster newborns were exposed to EE(2) (nominal concentrations of 8, 12 and 36 ng L(-1)) and the dynamics of their vertical distribution was monitored for up to 40 d. No significant differences in overall mortality were observed between treatments or in the dynamics of the registered death curves. Nevertheless, an alteration in the distribution patterns was observed. The commonly benthic newborn tended to shift their vertical distribution towards the surface, in a dose-dependent manner. Curiously, a follow up of the exposed pipefish confirmed that EE(2) effects were also noticeable upon sexual maturity, namely by the alteration of several primary and secondary sexual characters. The observation that vertical distributional patterns, at least in pipefish, are clearly altered at environmentally relevant EE(2) concentrations indicates that EDCs impact in fish larvae behaviour should be considered when addressing the effects of contaminants, given the obvious implications on population connectivity, stability and persistence.