Bisphenol A and 17α-ethinylestradiol-induced transgenerational differences in expression of osmoregulatory genes in the gill of medaka (Oryzias latipes).

Embryonic bisphenol A (BPA) and 17α-ethinylestradiol (EE2) exposure can have far reaching health effects in fish, including adult onset transgenerational reproductive abnormalities, anxiety, and cardiac disorders. It is unknown whether these two environmental estrogens can induce transgenerational abnormalities in the gill. The present study examined transgenerational effects of BPA or EE2 exposure on genes that are critical for osmoregulation in fish. Medaka (Oryzias latipes) embryos were exposed to either BPA (100 µg/L) or EE2 (0.05 µg/L) for the first 7 days of embryonic development and never thereafter for the remainder of that generation (F0) and in subsequent generations of this study (F1, F2, and F3). Expression of osmoregulatory genes (NKAα1a, NKAα1b, NKAα1c, NKAα3a, NKAα3b, NKCC1a, and CFTR) were examined in gills of the first-generation (F0) adults which were directly exposed as embryo and in the fourth-generation adults (F3), which were never exposed to either of these environmental estrogens. Significant alterations in expression of osmoregulatory genes were observed in both F0 and F3 generations. Within the F0 generation, a sex-specific expression pattern was observed with a downregulation of osmoregulatory genes in males and an upregulation of osmoregulatory genes in females. At the F3 generation, this pattern reversed with the majority of the osmoregulatory genes upregulated in males and downregulated in females, suggesting that exposure to BPA and EE2 during embryonic development induced transgenerational impairment in molecular events associated with osmoregulatory functions in subsequent generations. These adverse outcomes may have impacts on physiological functions related to osmoregulation of fish inhabiting contaminated aquatic environments.

The Enhancer Trap in Ciona.

Enhancer trap is a famous application of transposons. This method is useful for the creation of marker transgenic lines that express a reporter gene in tissue- or organ-specific manner, characterization of enhancers in the genome, finding novel patterns of gene expression, and mutagenesis. In Ciona intestinalis, efficient enhancer traps with Minos and Sleeping beauty transposons have been reported. With the enhancer trap lines, the intronic enhancers regulating the expression of the Musashi gene, the compartment in the digestive tube, the presence of enhancers sensitive to the orientation of the gene that they regulate, and the functions of the Hox1 gene have been revealed. The enhancer trap lines generated with the transposon vectors are valuable resources for use as visual markers.

Ontogenesis of opercular deformities in gilthead sea bream Sparus aurata: a histological description.

The aim of this study was to characterize histological changes during opercular osteogenesis in farmed gilthead sea bream Sparus aurata larvae from 7 to 69 days post hatching (dph) and compare normal osteogenesis with that of deformed opercles. Mild opercular deformities were first detected in 19 dph larvae by folding of the opercle's distal edge into the gill chamber. Here, the variation in the phenotype and the irregular bone structure at the curled part of the opercles is described and compared with the histology of normal opercles. Results indicated that deformed opercles still undergo bone growth with the addition of new matrix by osteoblasts at the opercular surface, especially at its edges. No significant difference was found in bone thickness between deformed and normal opercles. In addition to differences in bone architecture, differences in collagen fibre thickness between normal and deformed opercles were also found.

Habitat quality affects the incidence of morphological abnormalities in the endangered salamander Ambystoma ordinarium.

Identification of early warning signals previous to the occurrence of population decline or extinction is a major challenge for the conservation of animal species. Prevalence of morphological abnormalities in a population can be one of these signals. We registered morphological abnormalities in the salamander Ambystoma ordinarium. We also evaluated the relation between habitat quality and the prevalence of abnormalities in this species. We used scores from rapid bioassessment protocols (RBPs) to assess the habitat quality of streams inhabited by A. ordinarium. A preliminary survey indicated that of 29 streams where this species has been historically registered, 13 might have few or no A. ordinarium. The association between habitat quality and the incidence of morphological abnormalities was evaluated in these 16 streams. Of 502 sampled individuals, 224 (44.62%) had at least one body abnormality. Of the 224 individuals with body abnormalities, 84 (37.5%) presented more than one abnormality. Of a total of 5,522 evaluated morphological characters, 344 (6.74%) were abnormal. Partial loss of gills and missing digits were the most frequent abnormalities. Results of a binomial logistic regression indicated that the probability of a character of an individual to be abnormal was significantly associated with habitat quality; as the levels of the quality of the habitat increased, the prevalence of morphological abnormalities decreased. These results suggest that RBPs are a quick and useful method for assessing the habitat quality of streams inhabited by A. ordinarium. Given that RBPs provide rapid and cost-effective assessments of the ecological health of aquatic ecosystems, it will be important to test if the RBPs protocols can be used to rapidly assess habitat quality for other species of stream amphibians. The negative association between habitat quality and the prevalence of morpohological abnormalities that we found indicates that habitat condition plays an important role in the high number of abnormalities registered in A. ordinarium. Therefore, our results suggest that one of the several negative effects of habitat degradation on amphibians is an increase in the frequency of morphological abnormalities with marked consequences for the survival and general fitness of aquatic amphibians.

Malformations of the gill filaments of the ruffe Gymnocephalus cernuus (L.) (Pisces) caused by echinostomatid metacercariae.

In parasite surveys of fishes from Lake Balaton and its tributaries in Hungary, infections with metacercariae of a species of the digenean genus Echinochasmus (Trematoda: Echinostomatidae) were found in seven species of fish. In ruffe, Gymnocephalus cernuus, malformations of the gill filaments apparently caused by these infections were observed. These malformations were in the form of bifurcations of the filaments at about their mid-length. At the point where the filaments bifurcate, an Echinochasmus metacercaria was always embedded in the cartilaginous ray of the gill filament. All specimens of the ruffe were found to be infected by these metacercariae, and each ruffe specimen was infected by 30-300 metacercariae. Such a bifurcation was found in all of the ruffe specimens, but, apart from these gill malformations, the metacercariae produced only local changes in the cartilage. In the other six infected fish species, only local signs were observed in the cartilage. Experimental infections of chicks with metacercariae resulted in the finding of the sexual adult (marita) of an unidentified species of Echinochasmus. ITS sequences of the adult and metacercaria corresponded with each other, and also with a cercaria isolated from a gravel snail (Lithoglyphus naticoides), with a 99.5-100% similarity.

Toxicity of endosulfan on embryo-larval development of the South American toad Rhinella arenarum.

Endosulfan is a widely used pesticide despite its extreme toxicity to a variety of taxa and its worldwide ban. The aim of the present study was to evaluate the acute and chronic toxicity of endosulfan on the embryonic-larval development of the common South American toad Rhinella arenarum. The results showed that lethal and sublethal effects increased with concentration and exposure time. The sensitivity to endosulfan increased during the larval period, the complete operculum stage (S.25) being the most sensitive (504-h median lethal concentration [LC50] = 0.01 mg endosulfan/L; 10% lethal concentration [LC10] = 0.004 mg endosulfan/L). Endosulfan exposure caused morphological abnormalities such as general underdevelopment, edema, gill malformations, and cellular dissociation as well as neurotoxicity. Our results also showed that larvae exposed to concentrations of 0.005 mg endosulfan/L and 0.01 mg endosulfan/L completed metamorphosis earlier than controls, but with underdevelopment. The 240-h teratogenic index was 6.13, implying a high risk for embryos to be malformed in the absence of significant embryonic lethality. Because the hazard quotients for chronic exposure were over 1, the level of concern value and toxicity endpoints obtained in the present study for R. arenarum occurred at concentrations lower than the levels of endosulfan reported in the environment, this pesticide should be considered a potential risk for this species.

Stage-dependent toxicity of bisphenol a on Rhinella arenarum (anura, bufonidae) embryos and larvae.

The acute and chronic toxicity of bisphenol A (BPA) was evaluated on the common South American toad Rhinella arenarum embryos and larvae by means of continuous and pulse exposure treatments. Embryos were treated continuously from early blastula (S.4) up to complete operculum (S.25), during early larval stages and by means of 24 h pulse exposures of BPA in concentrations ranging between 1.25 and 40 mg L(-1) , in order to evaluate the susceptibility to this compound in different developmental stages. For lethal effects, S.25 was the most sensitive and gastrula was the most resistant to BPA. The Teratogenic Index for neurula, the most sensitive embryonic stage for sublethal effects was 4.7. The main morphological alterations during early stages were: delayed or arrested development, reduced body size, persistent yolk plug, microcephaly, axial/tail flexures, edemas, blisters, waving fin, underdeveloped gills, mouth malformations, and cellular dissociation. BPA caused a remarkable narcotic effect from gill circulation stage (S.20) onwards in all the organisms exposed after 3 h of treatment with 10 mg L(-1) BPA. After recovering, the embryos exhibited scarce response to stimuli, erratic or circular swimming, and spasmodic contractions from 5 mg L(-1) onwards. Our results highlight the lethal and sublethal effectsof BPA on R. arenarum embryos and larvae, in the last case both at structural and functional levels.

Deformación de branquias en salmónidos: análisis macroscópico, histológico, ultraestructural y de elementos / Gill deformation in salmonids: macroscopic, histological, ultrastructural and elements analysis

Las branquias son los órganos destinados al proceso de respiración en los peces, estas están consideradas, entre las estructuras más delicadas del cuerpo de los teleósteos ya que están expuestas a sufrir daños de cualquier agente irritante, los cuales constituyen la causa más frecuente y significativa de sus cambios patológicos. Los objetivos del presente estudio son caracterizar las alteraciones branquiales desde los puntos de vista anatómico, histológico, además, determinar niveles de Ca, P y Zn del tejido óseo de la columna vertebral y cráneo de salmones afectados. Se utilizaron 47 salmones, divididos en dos grupos: control y con deformaciones, a los cuales se les realizaron análisis macroscópico, histológico, ultraestructural y de elementos. Se observaron alteraciones tanto en el contexto anatómico como histológico, así como gran presencia de bacterias y hongos que afectaban principalmente al primer arco, pudiendo concluir que los peces tendrían una susceptibilidad génica a la deformación branquial.

Gills act as the respiratory organ in the respiration process in fish. They are considered among the most delicate body structures of teleost fish as they are subject to damage from any irritant, which are the most frequent and significant causes of their pathological changes. The objective of this study is to characterize gill alterations from the anatomical and histological standpoint, and to determine levels of Ca, P and Zn in bone tissue of the spine and skull of affected salmon. For purposes of this study 47 salmon were used in two groups: control and deformation, subjected to complete histological, ultrastructural and element inspection. Abnormalities were observed in both the anatomical and histological context and extensive presence of bacteria and fungi affecting mainly the first arc were found. Therefore, we can conclude that these fish have a genetic susceptibility to gill deformation.

Recovery of opercular anomalies in gilthead sea bream, Sparus aurata L.: morphological and morphometric analysis.

Opercular anomalies are very frequent in reared gilthead sea bream and these can negatively influence the product value. Field observations have suggested that opercular malformations can recover over time. In order to verify this hypothesis, 140-day-old gilthead sea bream with monolateral opercular anomalies were divided into three groups, according to the type and increasing seriousness of the opercular malformations, and another group was composed of fish with bilateral opercular anomalies. All groups were monitored for 16 months. In the group with monolateral anomalies, the opercular recovery process was documented by morphological (stereomicroscope) and morphometric analysis. For the latter analysis, two relevant areas, A and T, were identified in the cephalic region. The ratio (T - A)/T, tending to 1, represents the recovery index (RI) of anatomical integrity and quantifies the recovery level of opercular complex anomalies. Results suggested that the recovery process was considerable over the 16 months of investigation but should not be considered complete. At the end of the study, 61% of the gilthead sea bream population with monolateral opercular defects recovered external integrity, whereas the population with bilateral defects showed a poorer recovery capability.

Toxic effect of the harmful dinoflagellate Cochlodinium polykrikoides on the spotted rose snapper Lutjanus guttatus.

The dinoflagellate Cochlodinium polykrikoides isolated from Bahía de La Paz, Gulf of California, showed an important short-term toxic effect on the spotted rose snapper Lutjanus guttatus. This microalga was able to decrease fish liver catalase activity and lipid peroxidation. Fish exposed to live dinoflagellates developed an abnormal mucus secretion on the gills that was directly related to algal cell concentration. Hepatic catalase inhibition and an increase in mucus secretion on the gills occurred when fish were exposed to 2 x 10(6) cells L(-1) of C. polykrikoides. Lipid peroxidation was significantly different at 4 x 10(6) cells L(-1) and the hepatosomatic index decreased at 3 x 10(6) cells L(-1). Our results suggest that oxidative stress contributes, at least in part, to the ichthyotoxic effect of C. polykrikoides from the Gulf of California.

Abnormal features of Macoma balthica (Bivalvia) in the Baltic Sea: alerting symptoms of environmental adversity?

Recent studies of the Baltic clam Macoma balthica (L.) from the southern Baltic (the Gulf of Gdansk) have revealed striking morphological, histological and cytogenetic features. Strong deformation of the shell, including elongation of the posterior end and the appearance of an easily visible flexure in this part, has been recorded. The population contribution of the deformed blunt shelled ("irregular") clams ranged from 0% to 65% and tended to increase with depth. The morphologically "irregular" clams had higher accumulated tissue concentrations of trace metals (As, Ag, Cd, Pb, Cu and Zn), indicating a different metal handling ability. Adverse conditions in deeper water regions of the Gulf (e.g. hypoxia, hydrogen sulphide, elevated bioavailability of contaminants) have been suggested as inducers of the phenotypical changes (morphological deformation) in part of the population and, in parallel, of the specific physiological adaptations that result in higher metal accumulation in the "irregular" clams. Cytogenetic and histological analyses showed the presence of tumours in gill cells and digestive system of the affected clams, the prevalence of disseminated neoplasia ranging from 0% to 94% depending on the site. The disease was manifested by a modified karyotype (i.e. an abnormal number and morphology of chromosomes), a higher activity of nucleolar organizer regions (AgNORs), and tissue lesions (enlarged cells, actively proliferative with pleomorphic nuclei). Bottom sediments showed acute toxicity and have been proposed as a source of an initialising carcinogenic factor. However, none of the ecotoxicological studies provided was successful in the clear demonstration of a single (or multifactorial) agent that can account for the disseminated neoplasia.

Fístula de segundo arco branquial

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Zinc protection against delayed development produced by cadmium.

The protective effect of zinc against slight teratogenical action, exerted by low cadmium concentrations, was evaluated in Bufo arenarum embryos treated simultaneously with both cations or preincubated with Zn before Cd treatment. Data on survival, malformations, and delay in development pointed out that Zn could prevent the deleterious effects of Cd in previous and simultaneous treatments with that heavy metal.

Experimental studies on a mutant gene (p) causing premature death of Ambystoma mexicanum embryos.

The premature death (p) mutation is a recessive lethal, which, in the homozygous condition, gives rise to a complex of abnormalities. The mutant embryos develop only to stage 37, at which time disintegration of superficial tissue begins. Many of the abnormalities observed in sections of the stage-37 mutant embryo are related to its failure to establish a functioning circulatory system, or to the resulting edema and/or ascites that distend the abdomen and flanks. There are, however, abnormalities of heart, liver, gill and muscle development which cannot be attributed to lack of circulation and edema. All of these abnormalities can be indirectly related to the endoderm, particularly the anterior and dorsal endoderm. The findings, therefore, suggest that the mutation leads to a fairly general defect of the endoderm.