In situ monitoring of thin alumina passive film growth by surface plasmon resonance (SPR) during an electrochemical process.

This article presents a sensing technique to characterize the growth of an alumina passive film on an aluminum micro structured layer in situ. The technique uses surface plasmon resonance (SPR) on aluminum coated gratings with spectroscopic measurements during electrochemical polarization in 0.02M Na2SO4. The structure of the sensor was first simulated and then fabricated by photolithography. The grating was then replicated by nanoimprint (NIL) in Sol-Gel before pure aluminum layer was deposited by RF magnetron sputtering to produce the samples used in this study. Coupled plasmonic and electrochemical measurements confirmed the feasibility of in situ characterization (thickness) of alumina passive film on aluminum-based gratings in neutral aqueous media. Combining both measurements with an appropriated SPR spectrum fitting lead to alumina thickness monitoring within a few nanometers' accuracy. The objectives and challenges of this study are to better characterize the alumina growth during electrochemical process combining in situ electrochemical process and SPR spectra in order to determine thin passive layer characteristics.

Replication of natural surface topographies to generate advanced cell culture substrates.

Surface topographies of cell culture substrates can be used to generate in vitro cell culture environments similar to the in vivo cell niches. In vivo, the physical properties of the extracellular matrix (ECM), such as its topography, provide physical cues that play an important role in modulating cell function. Mimicking these properties remains a challenge to provide in vitro realistic environments for cells. Artificially generated substrates' topographies were used extensively to explore this important surface cue. More recently, the replication of natural surface topographies has been enabling to exploration of characteristics such as hierarchy and size scales relevant for cells as advanced biomimetic substrates. These substrates offer more realistic and mimetic environments regarding the topographies found in vivo. This review will highlight the use of natural surface topographies as a template to generate substrates for in-vitro cell culture. This review starts with an analysis of the main cell functions that can be regulated by the substrate's surface topography through cell-substrate interactions. Then, we will discuss research works wherein substrates for cell biology decorated with natural surface topographies were used and investigated regarding their influence on cellular performance. At the end of this review, we will highlight the advantages and challenges of the use of natural surface topographies as a template for the generation of advanced substrates for cell culture.

The biomimetic surface topography ofRubus fruticosusleaves stimulate the induction of osteogenic differentiation of rBMSCs.

The interaction between cells and biomaterials is essential for the success of biomedical applications in which the implantation of biomaterials in the human body is necessary. It has been demonstrated that material's chemical, mechanical, and structural properties can influence cell behaviour. The surface topography of biomaterials is a physical property that can have a major role in mediating cell-material interactions. This interaction can lead to different cell responses regarding cell motility, proliferation, migration, and even differentiation. The combination of biomaterials with mesenchymal stem cells (MSCs) for bone regeneration is a promising strategy to avoid the need for autologous transplant of bone. Surface topography was also associated with the capacity to control MSCs differentiation. Most of the topographies studied so far involve machine-generated surface topographies. Herein, our strategy differentiates from the above mentioned since we selected natural surface topographies that can modulate cell functions for regenerative medicine strategies.Rubus fruticosusleaf was the selected topography to be replicated in polycaprolactone (PCL) membranes through polydimethylsiloxane moulding and using soft lithography. Afterwards, rat bone marrow stem cells (rBMSCs) were seeded at the surface of the imprinted PCL membranes to characterize the bioactive potential of our biomimetic surface topography to drive rBMSCs differentiation into the osteogenic lineage. The selected surface topography in combination with the osteogenic inductive medium reveals having a synergistic effect promoting osteogenic differentiation.

Biomimetic surface topography as a potential modulator of macrophages inflammatory response to biomaterials.

The implantation of biomaterial devices can negatively impact the local microenvironment through several processes including the injury incurred during the implantation process and the associated host inflammatory response. Immune cell responses to implantable biomaterial devices mediate host-material interactions. Indeed, the immune system plays a central role in several biological processes required for the integration of biomaterials such as wound healing, tissue integration, inflammation, and foreign body reactions. The implant physicochemical properties such as size, shape, surface area, topography, and chemistry have been shown to provide cues to the immune system. Its induced immune-modulatory responses towards inflammatory or wound healing phenotypes can determine the success of the implant. In this work, we aim to evaluate the impact of some biomimetic surface topographies on macrophages' acute inflammatory response. For that, we selected 4 different biological surfaces to replicate through soft lithography on spin casting PCL membranes. Those topographies were: the surface of E. coli, S.eppidermidis and L929 cells cultured in polystyrene tissue culture disks, and an Eggshell membrane. We selected a model based on THP-1-derived macrophages to study the analysis of the expression of both pro-inflammatory and anti-inflammatory markers. Our results revealed that depending on the surface where these cells are seeded, they present different phenotypes. Macrophages present a M1-like phenotype when they are cultured on top of PCL membranes with the surface topography of E. coli and S. epidermidis. When cultured on membranes with L929 monolayers or Eggshell membrane surface topography, the macrophages present a M2-like phenotype. These results can be a significant advance in the development of new implantable biomaterial devices since they can help to modulate the inflammatory responses to implanted biomaterials by controlling their surface topography.

Fabrication of biomimetic patterned PCL membranes mimicking the complexity of Rubus fruticosus leaves surface.

The development of bioresponsive interfaces that can induce a beneficial impact on cell mechanisms, such as adhesion, proliferation, migration and differentiation are of utmost relevance in Tissue engineering (TE) approaches. The surface topography is a captivating property that contribute to interesting cell responses, being inspired by several cues found in nature. Therefore, the study herein presented reports the fabrication of a surface topography using the Rubus fruticosus leaf on spin casting polycaprolactone (PCL) membranes. The topography was replicated by replica molding rapid fabrication technique and nanoimprint lithography (NIL). The biomimetic patterned PCL membranes (bpM) were successfully produced revealing high detail due to the complexity of the leaf's surface ranging from the stroma structures to nerves structures. The thermal evaluation revealed a slight increase of crystallinity of the bpM compared with the other tested conditions. However, did not induce significant effects on the melting and recrystallization temperatures. The mechanical properties revealed that the young modulus increase from 3.2 MPa to 4.4 MPa during the imprinting process. However, bpM presents a lowest elongation capacity than bare membrane (bM) (1076 to 444 %, respectively) due to the heterogeneous thickness induced by the topography. The selected topography revealed to promote a positive bioresponse, depicted by the improvement of the cellular behaviour and different organization. This promising strategy revealed that circumventing the traditional topographies by nature mimetic topographies is fundamental for the development of innovative bioresponsive substrates that can tune cellular behaviour in TE strategies.

Anestesia intravenosa contínua com cetamina racêmica ou dextrocetamina e detomidina em cadelas / [Continuous intravenous anesthesia with racemic ketamine or dextroketamine and detomidine in female dogs]

Objetivou-se com este estudo comparar a associação de detomidina e cetamina ou dextrocetamina, por via intravenosa contínua, em oito cadelas submetidas a dois protocolos: GCD - indução anestésica com 5mg/kg e infusão intravenosa contínua de 20mg/kg/h de cetamina; e GDD - indução com 3,5mg/kg e infusão de 14mg/kg/h de dextrocetamina. Associou-se detomidina, 30µg/kg/h, em ambos os grupos. Registraram-se frequência cardíaca (FC), pressão arterial (PA), frequência respiratória (f), temperatura (TC), miorrelaxamento, analgesia, hemogasometria e eletrocardiograma, antes e 15 minutos após a MPA (Mbasal e Mmpa); após o início da infusão (Mic); a cada 10 minutos até 90 minutos (M10, M20, M30, M40, M50, M60, M70, M80 e M90); e 30 minutos após o fim da infusão (M120). Foi observada bradicardia em Mmpa no GCD e de Mmpa a M10 no GDD. Ocorreu hipotensão em Mmpa e hipertensão a partir de Mic. A f diminuiu de M10 a M30. Foram observados: onda T de alta amplitude, bloqueios atrioventriculares e parada sinusal. Ocorreu acidose respiratória. O período de recuperação foi de 219,6±72,3 minutos no GCD e de 234,1±96,8 minutos no GDD. A cetamina e a dextrocetamina, associadas à detomidina por infusão contínua, causam efeitos cardiorrespiratórios e anestésicos similares.(AU)

The combination of detomidine and ketamine or dextrocetamine for continuous intravenous infusion was compared in eight female dogs submitted to two protocols: GCD - 5mg/kg of anesthetic induction and continuous intravenous infusion of ketamine 20mg/kg/h; and GDD - induction with 3.5mg/kg and infusion of 14mg/kg/h of dextrocetamine. Detomidine, 30µg/kg/h was associated in both groups. Heart rate (HR), blood pressure (BP), respiratory rate (RR), temperature (CT), myorelaxation, analgesia, blood gas analysis and electrocardiogram were recorded before and 15 minutes after MPA (Mbasal and Mmpa); after the start of infusion (Mic); every 10 minutes to 90 minutes (M10, M20, M30, M40, M50, M60, M70, M80 and M90); and 30 minutes after the end of infusion (M120). Bradycardia was observed in Mmpa in GCD and from Mmpa to M10 in GDD. There was hypotension in Mmpa and hypertension from Mic. The RR decreased from M10 to M30. High amplitude T wave, atrioventricular blocks and sinus arrest were observed. Respiratory acidosis occurred. The recovery period was 219.6±72.3 minutes in GCD and 234.1±96.8 minutes in GDD. Ketamine and S+ ketamine associated with detomidine for continuous infusion cause cardiorespiratory and similar anesthetic effects.(AU)

The 'Woman in Red' effect: pipefish males curb pregnancies at the sight of an attractive female.

In an old Gene Wilder movie, an attractive woman dressed in red devastated a man's current relationship. We have found a similar 'Woman in Red' effect in pipefish, a group of fish where pregnancy occurs in males. We tested for the existence of pregnancy blocks in pregnant male black-striped pipefish (Syngnathus abaster). We allowed pregnant males to see females that were larger and even more attractive than their original high-quality mates and monitored the survival and growth of developing offspring. After exposure to these extremely attractive females, males produced smaller offspring in more heterogeneous broods and showed a higher rate of spontaneous offspring abortion. Although we did not observe a full pregnancy block, our results show that males are able to reduce investment in current broods when faced with prospects of a more successful future reproduction with a potentially better mate. This 'Woman in Red' life-history trade-off between present and future reproduction has similarities to the Bruce effect, and our study represents, to our knowledge, the first documentation of such a phenomenon outside mammals.

Bioengineered Tooth Buds Exhibit Features of Natural Tooth Buds.

Tooth loss is a significant health issue currently affecting millions of people worldwide. Artificial dental implants, the current gold standard tooth replacement therapy, do not exhibit many properties of natural teeth and can be associated with complications leading to implant failure. Here we propose bioengineered tooth buds as a superior alternative tooth replacement therapy. We describe improved methods to create highly cellularized bioengineered tooth bud constructs that formed hallmark features that resemble natural tooth buds such as the dental epithelial stem cell niche, enamel knot signaling centers, transient amplifying cells, and mineralized dental tissue formation. These constructs were composed of postnatal dental cells encapsulated within a hydrogel material that were implanted subcutaneously into immunocompromised rats. To our knowledge, this is the first report describing the use of postnatal dental cells to create bioengineered tooth buds that exhibit evidence of these features of natural tooth development. We propose future bioengineered tooth buds as a promising, clinically relevant tooth replacement therapy.

GelMA-Encapsulated hDPSCs and HUVECs for Dental Pulp Regeneration.

Pulpal revascularization is commonly used in the dental clinic to obtain apical closure of immature permanent teeth with thin dentinal walls. Although sometimes successful, stimulating bleeding from the periapical area of the tooth can be challenging and in turn may deleteriously affect tooth root maturation. Our objective here was to define reliable methods to regenerate pulp-like tissues in tooth root segments (RSs). G1 RSs were injected with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) encapsulated in 5% gelatin methacrylate (GelMA) hydrogel. G2 RSs injected with acellular GelMA alone, and G3 empty RSs were used as controls. White mineral trioxide aggregate was used to seal one end of the tooth root segment, while the other was left open. Samples were cultured in vitro in osteogenic media (OM) for 13 d and then implanted subcutaneously in nude rats for 4 and 8 wk. At least 5 sample replicates were used for each experimental group. Analyses of harvested samples found that robust pulp-like tissues formed in G1, GelMA encapsulated hDPSC/HUVEC-filled RSs, and less cellularized host cell-derived pulp-like tissue was observed in the G2 acellular GelMA and G3 empty RS groups. Of importance, only the G1, hDPSC/HUVEC-encapsulated GelMA constructs formed pulp cells that attached to the inner dentin surface of the RS and infiltrated into the dentin tubules. Immunofluorescent (IF) histochemical analysis showed that GelMA supported hDPSC/HUVEC cell attachment and proliferation and also provided attachment for infiltrating host cells. Human cell-seeded GelMA hydrogels promoted the establishment of well-organized neovasculature formation. In contrast, acellular GelMA and empty RS constructs supported the formation of less organized host-derived vasculature formation. Together, these results identify GelMA hydrogel combined with hDPSC/HUVECs as a promising new clinically relevant pulpal revascularization treatment to regenerate human dental pulp tissues.

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.

Oblique Retro-Aortic Left Renal Vein and its Clinical Importance.

Kidneys are the organs that remove the waste products of the metabolic activities. A smooth blood flow to the kidneys is essential to maintain their function. Abnormalities of the renal vasculature may result not only in impairing the renal function but can lead to conditions like varicocele. During an autopsy of an adult male, we observed renal vascular variations. The left renal vein had a retro-aortic course before its termination into the inferior vena cava. It was joined with the inferior vena cava at the level of inferior mesenteric artery with an acute angle. The left testicular vein joined the left renal vein with an acute angle. The right kidney was supplied by two renal arteries. The knowledge about retro-aortic course of the left renal vein may be important during renal transplantation. The oblique course of left renal vein and the termination of left testicular vein into it with an acute angle may increase the chances of left sided varicocele.

Degradation of tetracycline at a boron-doped diamond anode: influence of initial pH, applied current intensity and electrolyte.

The anodic oxidation of tetracycline was performed in an up-flow reactor, operating in batch mode with recirculation, using as anode a boron-doped diamond electrode. The influence on the degradation rate of solution initial pH (2 to 12), applied current intensity (25 to 300 A m(-2)) and type of electrolyte (sodium sulphate or sodium chloride) were investigated. For the assays run at equal current density, with sodium sulphate as electrolyte, the solution's initial pH of 2 presented the highest absorbance and chemical oxygen demand removals. Regarding the influence of current density, for equal charge passed, the organic load removal rate decreased with the increase in applied current. When sodium sulphate was used as an electrolyte, high-performance liquid chromatography (HPLC) results have shown an almost complete removal of tetracycline after a 2-h assay. HPLC results have also shown the presence of oxamic acid as one of the intermediates of tetracycline anodic oxidation. The complete removal of tetracycline was much faster in the presence of chloride ions that promoted the complete degradation of this antibiotic in 30 min. However, in the presence of chloride ions, the tetracycline mineralization is slower, as observed by the lower organic carbon removal rate when compared to that of the tetracycline degradation in the presence of sulphate.

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.

Zebrafish (Danio rerio) life-cycle exposure to chronic low doses of ethinylestradiol modulates p53 gene transcription within the gonads, but not NER pathways.

Parental full life-cycle exposure to ethinylestradiol (EE2) significantly affects embryo development and survival. One of the possible mechanisms of action of EE2 may involve the impairment of an organism's ability to repair DNA damage. DNA repair mechanisms have sophistically evolved to overcome DNA damaging hazards that threaten the integrity of the genome. In the present study, changes in the transcription levels of key genes involved in two of the most thoroughly studied DNA repair systems in mammals were evaluated in adult zebrafish (Danio rerio) gonad upon full life-cycle exposure to chronic environmentally low levels of EE2 (i.e., 0.5, 1 and 2 ng/L EE2). Real time PCR was used to analyse the expression levels of nucleotide excision repair genes (NER) as well as the tumor suppressor p53 and downstream selected effectors, i.e., p21 (cyclin-dependent kinase inhibitor), GADD45α (growth arrest and DNA damage induced 45, alpha), bax (bcl2-associated X protein) and p53 key regulator MDM2 (murine double minute 2 protein). NER genes transcription levels in gonads did not differ significantly among treatments. In contrast, the number of transcripts of p53 gene was significantly increased in male gonads at all EE2 exposure concentrations and in females at 1 ng/L EE2. Despite the increase in p53 transcripts, transcription levels of p21, GADD45α and bax genes were not affected upon EE2 treatment, whereas MDM2 gene expression significantly increased in females at the intermediate EE2 dose (1 ng/L). Overall, the present study indicate that chronic low levels of EE2 significantly modulates the transcription of p53, a key gene involved in DNA repair, particularly in male zebrafish gonads, which supports the hypothesis of an impact of EE2 in male gonad DNA repair pathways.

Influence of reduction processes on the colour and photochromism of amorphous mesoporous TiO(2) thin films loaded with a silver salt.

Silver nanoparticles were created inside mesoporous titania thin films by different reduction processes. We investigated the influence of the reduction method on the colour and photochromism of these amorphous TiO(2) films. The results highlight brown films by optical reduction, gray films by thermal reduction, and red, purple or orange films by chemical reduction. The different size distributions and localizations of the nanoparticles, characterized by UV-visible spectroscopy and electron microscopy, give various photochromic behaviours when exposed to visible laser light. We especially report the bleaching of different film colours under laser exposure.

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.

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 EDC's impact in fish larvae behaviour should be considered when addressing the effects of contaminants, given the obvious implications on population connectivity, stability and persistence.

The unpredictable effects of mixtures of androgenic and estrogenic chemicals on fish early life.

Estuarine shallow areas and coastal lagoons are known to receive and concentrate multiple inputs, either from land, rivers or coastal areas, being intensively impacted by chemical contamination, namely endocrine disrupting chemicals (EDCs). Despite the ubiquitous coexistence of several classes of EDCs in most of these aquatic ecosystems, there is still limited information regarding their combined effects. Furthermore, given the immediate implications for population dynamics, the available laboratory studies almost invariably focus on very specific life history stages, such as embryonic development or reproduction, thus creating a gap on our knowledge of what happens in between. During this 'intermediate phase', the newborn larvae and juveniles face numerous challenges whose outcome may impair reproduction or even survival. The black-striped pipefish, Syngnathus abaster, member of the Syngnathidae family (comprising pipefish, seahorses and seadragons), usually breeds in coastal areas such as estuaries, where its newborns are immediately exposed to EDCs. Given the ongoing decline of pipefish populations, together with the observed shrinkage and fragmentation of seagrass meadows, known to be impacted by EDCs, a first reasonable question to address is if pipefish newborns respond to environmentally relevant concentrations of ubiquitous EDCs, either single or in combination. Hence, a seven days exposure experiment to the estrogenic chemical ethinylestradiol (EE(2)) and the androgenic chemical tributyltin (TBT), single and in binary mixtures, was conducted. Selected behavioural (e.g. predator avoidance) and developmental variables (e.g. growth) were monitored in pipefish juveniles after EDCs insult. The obtained results indicate that EE(2), TBT, or their combined exposure, do impact pipefish early life. However, the pattern of results emerging from the measured variables clearly indicates that mixtures significantly modulate newborn responses in distinct ways when compared to individual chemical's exposure. These findings further demonstrate the importance of addressing the issue of chemical mixtures of pollutants acting through dissimilar mode of action. Independently of all the observed response variations, an ultimate conclusion seems certain: EE(2) and TBT, single or in combination, induce disruption patterns able to imbalance pipefish survival. Since these (as well as other) contaminants are present in estuarine areas, profound implications in population structure could be expected, ranging from a decrease in recruitment to a disruption of sexual selection. Inexorably, these stressors simultaneously operate in already declining populations.

Sexing blennies using genital papilla morphology or ano-genital distance.

Information on the genital morphology of male and female Lipophrys pholis is provided, as well as for two other sympatric blenniid species, Coryphoblennius galerita and Lipophrys trigloides. The use of non-invasive sex determination procedures described may be extremely useful not only for ecological studies but also as a proxy for the detection of environmental exposure to endocrine disruptive chemicals, given that blennies have been proposed as potential sentinel species for chemical contamination.