Ana Luzio

Copper induced upregulation of apoptosis related genes in zebrafish (Danio rerio) gill.

Copper (Cu) is an essential micronutrient that, when present in high concentrations, becomes toxic to aquatic organisms. It is known that Cu toxicity may induce apoptotic cell death. However, the precise mechanism and the pathways that are activated, in fish, are still unclear. Thus, this study aimed to assess which apoptotic pathways are triggered by Cu, in zebrafish (Danio rerio) gill, the main target of waterborne pollutants. Fish where exposed to 12.5 and 100 μg/L of Cu during 6, 12, 24 and 48 h. Fish gills were collected to TUNEL assay and mRNA expression analysis of selected genes by real time PCR. An approach to different apoptosis pathways was done selecting p53, caspase-8, caspase-9 and apoptosis inducing factor (AIF) genes. The higher incidence of TUNEL-positive cells, in gill epithelia of the exposed fish, proved that Cu induced apoptosis. The results suggest that different apoptosis pathways are triggered by Cu at different time points of the exposure period, as the increase in transcripts was sequential, instead of simultaneous. Apoptosis seems to be initiated via intrinsic pathway (caspase-9), through p53 activation; then followed by the extrinsic pathway (caspase-8) and finally by the caspase-independent pathway (AIF). A possible model for Cu-induce apoptosis pathways is proposed.

Effects of 17α-ethinylestradiol at different water temperatures on zebrafish sex differentiation and gonad development

In the current climate change scenario, studies combining effects of water contaminants with environmental parameters, such as temperature, are essential to predict potentially harmful impacts on aquatic organisms. In zebrafish (Danio rerio), sex determination seems to have a polygenic genetic basis, which can be secondarily influenced by environmental factors, such as temperature and endocrine disrupting chemicals (EDCs). The present study aimed to evaluate the effects of the EDC 17α-ethinylestradiol (EE2), a potent synthetic estrogen, on zebrafish sex differentiation and gonad development at different water temperatures. Therefore, zebrafish raised at three distinct water temperatures (23, 28 or 33±0.5°C), were exposed to 4ng/L of EE2, from 2hours to 60days post-fertilization (dpf). Subsequently, a quantitative (stereological) assessment of zebrafish gonads was performed, at 35 and 60dpf, to identify alterations on gonadal development and differentiation. The results show that low temperature delayed general growth of zebrafish, as well as gonad differentiation and maturation, while high temperature induced an opposite effect. Moreover, sex ratio was skewed toward males when zebrafish were exposed to the high temperature. In general, EE2 exposure promoted gonad maturation in both genders, independently of the temperature. However, at the high temperature condition, exposure to EE2 induced a delay in the male gonad development, with some individuals still showing differentiating gonads at 60dpf. The findings of this study support the notion that zebrafish has a genetic sex determination mechanism highly sensitive to environmental factors and show that it is essential to study the effects of water contaminants at different climate scenarios in order to understand potential future impacts on organisms.

Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole).

Exposure of wildlife to endocrine disrupting chemicals (EDCs) is not necessarily continuous. Due to seasonal changes and variable industrial and agricultural activities it often occurs intermittently. Thus, it is possible that aquatic organisms may be more affected by periodic peak exposure than by chronic exposure. Therefore, an experimental scenario including an exposure from 2h to 90 days post-fertilization (dpf) and a subsequent recovery period until 150 dpf was chosen to assess the potential reversibility of the effects of sex steroids on sexual and gonad development of zebrafish (Danio rerio). The aim of this study was to investigate the persistence of the endocrine effects of an estrogen (EE2-17α-ethinylestradiol, 4ng/L), an inhibitor of estrogen synthesis (Fad-fadrozole, 50μg/L) or their binary mixture (Mix-EE2+ Fad, 4ng/L+50μg/L). Afterwards, a semi-quantitative histological assessment was used to investigate histopathological changes on gonad differentiation and development. The data showed that fadrozole, alone or in combination with EE2, permanently disrupts the sexual development, inducing masculinization and causing severe pathological alterations in testis, such as intersex associated to the enlargement of sperm ducts, interstitial changes, asynchronous development and detachment of basal membrane. After exposures to both EDCs and their mixture, the gonad histopathology revealed interstitial proteinaceous fluid deposits and, in ovaries, there were atretic oocytes, and presumably degenerative mineralization. On the other hand, the gonadal changes induced by EE2 alone seem to be partially reversible when the exposure regime changed to a recovery period. In addition, EE2 enhanced zebrafish growth in both genders, with male fish presenting signs of early obesity such as the presence of adipocytes in testis. Moreover, sex ratio was slightly skewed toward females, at 90 and 105 dpf, in zebrafish exposed to EE2. The data further indicate that long-term studies on impacts of single EDCs and their mixtures with recovery periods are crucial to reveal the possibility of sex reversal and pathological changes of gonads that can adversely affect breeding.

Gill histopathological alterations in Nile tilapia, Oreochromis niloticus exposed to treated sewage Water

Adult Nile tilapia, Oreochromis niloticus, of both sexes were exposed in wastewater from a sewage treatment plant for a period of 4 days. Gill samples were collected after 24, 48, 72 and 96 h and histopathological changes were analyzed by light and scanning electronic microscopy. Gill epithelium of control O. niloticus (freshwater group) was similar to that of other teleosts, while histopathological lesions were observed in exposed fishes. The main histopathological changes were edema, lifting of lamellar and filamentar epithelia and lamellar fusion. Cell proliferation with consequent thickening of the filament epithelium was also found in fishes exposed to the treated sewage water. The severity of the lesions increased with the time of exposure, namely the hyperplasia of the epithelial cells with proliferation of filamentar epithelium and fusion of lamellae observed at 96 h. Additionally, several histopathological results obtained by light microscopy were confirmed through scanning microscopy.

Screening and identification of potential sex‐associated sequences in Danio rerio

Current knowledge on zebrafish (Danio rerio) suggests that sex determination has a polygenic genetic basis in this species, although environmental factors may also be involved. This study aimed to identify sex‐associated genomic regions using two different marker systems: inter‐simple sequence repeats (ISSRs) and random‐amplified polymorphic DNA (RAPDs). Two bulks were constructed: one with DNA from zebrafish females and the other from males; then, a total of 100 ISSR and 280 RAPD primers were tested. Three DNA fragments presenting sexual dimorphism (female‐linked: OPA17436 and OPQ191027; male‐linked: OPQ19951) were determined from sequential analysis of the bulks followed by assessment in individuals. These fragments were cloned and convert into the following sequenced characterized amplified regions (SCAR): DrSM_F1, DrSM_F2, and DrSM_M, which share identities with sequences located in chromosomes 2, 3, and 11 (Zv9), respectively. Using these potential markers in zebrafish samples it was possible to correctly identify 80% of the males (DrSM_M) and 100% of the females (DrSM_F1 + DrSM_F2) in the analyzed population. Mol. Reprod. Dev. 82: 756–764, 2015.

Zebrafish sex differentiation and gonad development: A review on the impact of environmental factors

Zebrafish (Danio rerio) is extensively used in research; however the mechanisms that control this species sex determination are still poorly understood. In the latest decades, it has been established that zebrafish sex is determined by genetic factors on a polygenic basis, as various candidate genes with sex dimorphic expression, as well sex-linked loci have been identified in different zebrafish strains. However, it has been evidenced that sex determination in this species is also influenced by environmental factors. For instance, temperature can have a crucial role in zebrafish sex determination. Likewise, the exposure to endocrine disrupting chemicals (EDCs), the most studied zebrafish sex changing factor, can strongly influence the course of sex differentiation and unbalance the sex ratio of zebrafish populations. Despite this, so far the influence of environmental factors is still less understood and only few studies have addressed this topic. Therefore, this review intends to gather current knowledge on the environmental factors involved in sex determination of zebrafish and identify important gaps in this research area. Briefly, the current understanding on zebrafish sex related genetics is also addressed.

MS-222 short exposure induces developmental and behavioural alterations in zebrafish embryos

MS-222 has been widely used as an anaesthetic in fish, thus, raising the need to infer about its toxicological safety during development. In this study, MS-222 toxicity in zebrafish embryos was evaluated after a 20-min exposure at different stages of development. Embryos exposed during the 256-cell stage displayed an increase in mortality, associated with defective early developmental pathways. Following exposure during the 50% epiboly stage, an increase in mortality and abnormal cartilage development, as well as changes in noggin expression were observed. Locomotor deficits were detected and associated with changes in early signalling pathways through the involvement of noggin. When exposed at the 1-4 somites stage, zebrafish were phenotypically normal, although presenting changes in the expression pattern of developmental genes. These findings indicate a teratogenic impact, independent of sodium channels that should be taken into consideration when MS-222 toxicity is discussed.

General Whole-Mount Immunohistochemistry of Zebrafish (Danio rerio) Embryos and Larvae Protocol

Immunohistochemistry (IHC) is a powerful research tool to localize specific antigens in whole-mount or tissue sections of embryos with labeled antibodies based on antigen-antibody interactions. In whole-mount IHC, the distribution of an antigen can be mapped rapidly and reliably in the embryos. Immunofluorescence microscopy is one of the numerous IHC methods that may be used to assess both the localization and endogenous levels of proteins of interest. In this chapter, a general protocol for whole-mount immunofluorescent labeling of zebrafish embryos and larvae is described.