Alteration in molecular markers of oocyte development and intersex condition in mullets impacted by wastewater treatment plant effluents.

Wastewater Treatment Plant (WWTP) discharges are an important source of endocrine disrupting chemicals (EDCs) into the aquatic environment. Fish populations inhabiting downstream of WWTP effluents show alterations in gonad and gamete development such as intersex condition, together with xenoestrogenic effects such as vitellogenin up-regulation. However, the molecular mechanisms participating in the development of intersex condition in fish are not elucidated. The aim of this study was to assess the impact of two WWTPs effluents (Gernika and Bilbao-Galindo situated in the South East Bay of Biscay) with different contaminant loads, in thicklip grey mullet (Chelon labrosus) populations inhabiting downstream, examining the presence and severity of intersex condition, during two seasons. Molecular markers of xenoestrogenicity and oocyte differentiation and development (vtgAa, cyp19a1a, cyp19a1b, cyp11b, foxl2, dmrt1 and gtf3a) were also studied. Intersex mullets were identified downstream of both WWTPs and vtgAa was upregulated in intersex and non intersex males. Sex dependent differential transcription levels of target genes were detected in mullets from Galindo. However, no such pattern was observed in mullets from Gernika, suggesting an attenuating effect over studied genes caused by a higher presence of EDCs in this site, as indicated by the elevated prevalence of intersex mullets in this population. In conclusion, no direct association between xenoestrogenic responses and intersex condition was established. Mullets from Gernika showed signs of severe EDC exposure compared to those from Galindo, as demonstrated by the higher prevalence of intersex males and the reduction in transcription profile differences between sexes of gametogenic gene markers.

The white mullet (Mugil curema) as biological indicator to assess environmental stress in tropical coastal lagoons.

Several coastal lagoons and estuaries in Mexico receive untreated domestic and industrial discharges which contain complex mixtures of contaminants. In order to assess the effects of chemical contamination, we used the White mullet (Mugil curema) as biological indicator. We worked in two estuaries located in Northwest Mexico: Urias (highly polluted) and Teacapan (less polluted, therefore used as reference site). We measured several endpoints at different levels of biological organization: vitellogenin transcription in males as biomarker of estrogenic contamination, as well as reproductive, morphological (deformities), morphometric, and meristic parameters. Total RNA was isolated from the liver, and a partial sequence of the mullet vitellogenin gene was obtained; gene expression was analyzed by quantitative PCR. At the same time, gonad samples were analyzed by histologic techniques to determine sex ratios and the reproductive cycle stage. The reproductive season was detected from February to June in both sites, but the gonadosomatic index was consistently higher in Teacapan. Sex ratios were female-biased in both estuaries, and one intersex gonad and several malformations were found in fish from Urias. Vitellogenin gene transcription in males was detected in both sites, although gene expression was slightly higher in Urias. The results obtained in this study indicate that biological effects of contamination are evident in fish, environmental estrogens may be present in both estuaries, and the white mullet is useful as biological indicator to identify and characterize environmental stressors in tropical coastal ecosystems.

Diseases of captive yellow seahorse Hippocampus kuda Bleeker, pot-bellied seahorse Hippocampus abdominalis Lesson and weedy seadragon Phyllopteryx taeniolatus (Lacépède).

Seahorses, pipefish and seadragons are fish of the Family Syngnathidae. From 1998 to 2010, 172 syngnathid cases from the Toronto Zoo were submitted for post-mortem diagnostics and retrospectively examined. Among the submitted species were yellow seahorses Hippocampus kuda Bleeker (n=133), pot-bellied seahorses Hippocampus abdominalis Lesson (n=35) and weedy seadragons Phyllopteryx taeniolatus (Lacépède; n=4). The three most common causes of morbidity and mortality in this population were bacterial dermatitis, bilaterally symmetrical myopathy and mycobacteriosis, accounting for 24%, 17% and 15% of cases, respectively. Inflammatory processes were the most common diagnoses, present in 117 cases. Seven neoplasms were diagnosed, environmental aetiologies were identified in 46 cases, and two congenital defects were identified.

Frequency of micronuclei and of other nuclear abnormalities in erythrocytes of the grey mullet from the Mondego, Douro and Ave estuaries--Portugal.

Fish are bioindicators of water pollution, and an increased rate of their erythrocyte nuclear morphological abnormalities (ENMAs)-and particularly of erythrocyte micronuclei (EMN)-is used as a genotoxicity biomarker. Despite the potential value of ENMAs and MN, there is scarce information about fish captured in Iberian estuaries. This is the case of the Portuguese estuaries of the Mondego, Douro and Ave, suffering from different levels of environmental stress and where chemical surveys have been disclosing significant amounts of certain pollutants. So, the aim of this study was to evaluate genotoxicants impacts and infer about the exposure at those ecosystems, using the grey mullet (Mugil cephalus) as bioindicator and considering the type and frequency of nuclear abnormalities of erythrocytes as proxies of genotoxicity. Sampling of mullets was done throughout the year in the important Mondego, Douro and Ave River estuaries (centre and north-western Portugal). The fish (total n = 242) were caught in campaigns made in spring-summer and autumn-winter, using nets or fishing rods. The sampled mullets were comparable between locations in terms of the basic biometric parameters. Blood smears were stained with Diff-Quik to assess the frequencies of six types of ENMAs and MN (given per 1,000 erythrocytes). Some basic water physicochemical parameters were recorded to search for fluctuations matching the ENMAs. Overall, the most frequent nucleus abnormality was the polymorphic type, sequentially followed by the blebbed/lobed/notched, segmented, kidney shaped, vacuolated, MN and binucleated. The total average frequency of the ENMAs ranged from 73 ‰ in the Mondego to 108 ‰ in the Ave. The polymorphic type was typically ≥50 % of the total ENMAs, averaging about 51 ‰, when considering all three estuaries. The most serious lesion-the MN-in fish from Mondego and Douro had a similar frequency (≈0.38 ‰), which was significantly lower than that in the Ave (0.75 ‰). No significant seasonal differences existed as to the MN rates and seasonal differences existed almost only in the Douro, with the higher values in AW. In general, the pattern of ENMAs frequencies was unrelated with the water physicochemical parameters. Considering the data for both the total ENMAs and for each specific abnormality, and bearing in mind that values of MN in fish erythrocytes >0.3 ‰ usually reflect pollution by genotoxicants, it is suggested that mullets were likely being chronically exposed to such compounds, even in the allegedly less polluted ecosystem (Mondego). Moreover, data supported the following pollution exposure gradient: Mondego < Douro < Ave. The scenario and inferences nicely agree with the published data from chemical monitoring.

Relationship between DNA damage in sperm after ex vivo exposure and abnormal embryo development in the progeny of the three-spined stickleback.

Many xenobiotics released in the aquatic environment exhibit a genotoxic potential toward organisms. Long term exposure to such compounds is expected to lead to multigenerational reproductive defects, further influencing the recruitment rate and hence, the population dynamics. Paternal exposure to genotoxicants was previously shown to increase abnormal development in the progeny of mammalian or aquatic species. The aim of this study was to evaluate the relationship between DNA damage in sperm of the fish three-spined stickleback and progeny developmental defects. Spermatozoa were exposed ex vivo to an alkylating agent (methyl methanesulfonate) before in vitro fertilization and DNA damage was assessed by the alkaline comet assay. A significant relationship between abnormal development and sperm DNA damage was underlined. This study illustrates the interest to use germ cell DNA damage after ex vivo exposure to evaluate the impact of genotoxic compounds on progeny fitness in aquatic organisms.

Chronic perchlorate exposure causes morphological abnormalities in developing stickleback.

Few studies have examined the effects of chronic perchlorate exposure during growth and development, and fewer still have analyzed the effects of perchlorate over multiple generations. We describe morphological and developmental characteristics for threespine stickleback (Gasterosteus aculeatus) that were spawned and raised to sexual maturity in perchlorate-treated water (G(1,2003)) and for their offspring (G(2,2004)) that were not directly treated with perchlorate. The G(1,2003) displayed a variety of abnormalities, including impaired formation of calcified traits, slower growth rates, aberrant sexual development, poor survivorship, and reduced pigmentation that allowed internal organs to be visible. Yet these conditions were absent when the offspring of contaminated fish (G(2,2004)) were raised in untreated water, suggesting a lack of transgenerational effects and that surviving populations may be able to recover following remediation of perchlorate-contaminated sites.

Spinal deformities in the black-striped pipefish Syngnathus abaster (Pisces, Syngnathidae) from the Tunis North Lake, Tunisia.

Spinal deformities are seen in fishes. Deformities in the family Syngnathidae is rarely reported. Spine curvatures in natural population of pipefish, Syngnathus abaster are reported from the Tunis North Lake. The species is euryhaline and occurs along the Mediterranean coasts. Four hundred specimens of S. abaster were collected from January 2006 to December 2007 from the Tunis North Lake. Two specimens with skeletal abnormalities were described for the first time. X-rays radiographies revealed several vertebral deformities for each deformed specimen, especially at five curvation places of the caudal part. Several factors are believed to be the cause of spinal deformities of S. abaster in the Tunis North Lake, such as environmental disturbances, parasitism infection and nutritional factors.

Survey of contamination of estrogenic chemicals in Japanese and Korean coastal waters using the wild grey mullet (Mugil cephalus).

We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan. Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0 microg/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka were also greater than 1.0 microg/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullets living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms.