Impact of exposure to glyphosate-based herbicide on morphological and physiological parameters in embryonic and larval development of zebrafish.

Glyphosate-based herbicides (GBH) have been commonly used in agriculture to inhibit weed growth and increase yields. However, due to the high solubility of these herbicides in water, they can reach aquatic environments, by infiltration, erosion, and/or lixiviation, affecting non target organisms. Thus, this study aimed to characterize the toxicity of GBH Roundup WG® (RWG®) during the embryonic and larval development of Danio rerio. Embryos (3 hours post fertilization, hpf-until hatching) and larvae (3 days post fertilization, dpf to 6 dpf) were exposed to concentrations of 0.065 and 6.5 mg L-1 . They were evaluated for survival, hatching, spontaneous movements, heartbeat, morphology, and morphometry by in vivo photographs in microscope, cell proliferation and apoptosis by immunohistochemistry, and exploratory behavior and phototropism by video recording. Our results showed an increase in embryo and larvae mortality in those exposed to 0.065 mg L-1 , as well as a reduction in spontaneous embryo movements. The larval heartbeats showed a decrease at 4 dpf in the group exposed to 0.065 mg L-1 and an increase at 5 and 6 dpf in both exposed groups. Cell proliferation was reduced in both groups exposed in embryos and only in the 0.065 mg L-1 group in larvae, while cell death increased in embryos exposed to 6.5 mg L-1 . These results demonstrated the toxic effect of low concentrations of the herbicide RWG® during embryonic and larval development of non target organisms, as well as the importance of constantly reviewing acceptable limits for exposure in natural environments.

Cell proliferation in the central nervous system of an adult semiterrestrial crab.

Neurogenesis occurs in adults of most organisms, both vertebrates and invertebrates. In semiterrestrial crabs of the infraorder Brachyura, the deutocerebrum, where neurogenesis occurs, processes the olfactory sensory information from the antennae. The deutocerebrum is composed of a pair of olfactory lobes associated with cell clusters 9 and 10 (Cl 9 and Cl 10), containing proliferating cells. Because the location of the neurogenic niche in brachyuran semiterrestrial crabs has not been defined, here we describe a neurogenic niche in the central olfactory system of the crab Ucides cordatus and report two types of glial cells in the deutocerebrum, based on different markers. Serotonin (5-hydroxytryptamine) labeling was used to reveal neuroanatomical aspects of the central olfactory system and the neurogenic niche. The results showed a zone of proliferating neural cells within Cl 10, which also contains III beta-tubulin (Tuj1)+ immature neurons, associated with a structure that has characteristics of the neurogenic niche. For the first time, using two glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), we identified two types of astrocyte-like cells in different regions of the deutocerebrum. This study adds to the understanding of neurogenesis in a brachyuran semiterrestrial crustacean and encourages comparative studies between crustaceans and vertebrates, including mammals, based on shared aspects of both mechanisms of neurogenesis and regenerative potentials.

Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean.

Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis by the p53 pathway. This study evaluated some molecular markers of the apoptosis pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in environmental doses, during five consecutive days of exposure, in the brain of the crab Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblotting the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and SIM increased the protein content of p53, increasing the content of AKT and, somehow, blocking p21, increasing the content of activated caspase-3, which led the cells to apoptosis. The signs of death affected the increase in neurotrophins, such as BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical assays and immunoblotting showed that apoptosis was present in the brains of all UV groups, while the number of mitotic cells in the same groups decreased. In conclusion, environmental doses of UV can cause apoptosis by increasing p53 and decreasing p21, revealing an UV-damage pathway for U. cordatus.

Antioxidant activity stimulated by ultraviolet radiation in the nervous system of a crustacean.

Ultraviolet (UV) radiation can produce biological damage, principally oxidative stress, by increasing the production of reactive oxygen species (ROS). This study evaluated biochemical impairments related to the oxidative stress induced by UVA, UVB and UVA+UVB (solar simulator-SIM) in environmental doses, during five consecutive days of exposure, in the brain and eyestalk of the crab Ucides cordatus. We evaluated these regions by sampling on the 1st, 3rd and 5th days of UV exposure for lipid peroxidation (LPO), antioxidant capacity against the peroxyl radical (ACAP), and the activities of catalase (CAT), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). Immunohistochemical and immunoblotting assays were performed for anti-activated-caspase 3 in the brains. After the first day of exposure, LPO increased in the eyestalks and brains of the UV-exposed animals; ACAP, and CAT, GPX and GST activities also increased in the brains. On the third day, the LPO values in the eyestalk remained high in the UV-exposed groups, while ACAP decreased in the brain and eyestalk and CAT activity remained high in all irradiated groups in both regions. On the fifth day, LPO decreased in the eyestalk and brain of the UV-exposed groups. These results may have been a consequence of the antioxidant defense system (ADS) activity, since CAT activity was high in both regions, ACAP was high in the eyestalks of the SIM group, and GPX activity remained high in the eyestalks of the UVA and UVB groups. Immunohistochemical assays and immunoblotting showed that there was apoptosis in the brains of the UV-exposed crabs. In conclusion, environmental doses of UV can cause oxidative damage to the CNS cells, including apoptosis.

Effects of seasonality and moult cycle on the proliferation of nerve cells and on the labelling of ecdysone receptors in an estuarine crab.

Decapod crustaceans show proliferation of the nerve cells in the olfactory lobe throughout their lives. However, the regulation of this process is still poorly understood, since it may vary with endogenous and exogenous factors. The objective of the present investigation was to quantify the proliferation of nerve cells and number of nerve cells with ecdysone receptors in the clusters of the central olfactory system in Neohelice granulata, according to moult stages and in different seasons (summer and winter). Three injections of bromodeoxyuridine (BrdU) were administered to the crabs. Brains were sectioned by microtome and fixed on slides for immunohistochemistry with anti-BrdU and anti-EcR antibodies. The proliferation of nerve cells was higher in winter than in summer, probably because in winter the crabs do not breed and the premoult and postmoult periods are longer. Crabs in postmoult exhibited more BrdU-labelled cells than crabs in premoult or intermoult in winter, because of a greater number of mitoses related to an increase in body size and addition of olfactory receptor neurons. The number of EcR-labelled cells was higher in premoult than in postmoult or intermoult in winter. The proliferation of nerve cells is regulated seasonally and according to moult stages.