Morphological characterization of hemocytes of the brown mussel Perna perna: An update.

Considering the importance of hemocyte characterization for immunological studies, this work aimed to characterize the hemocyte types of Perna perna mussels combining transmission electron microscopy and flow cytometry with the classical optical microscopy. The results indicated four type of hemocytes: hyalinocytes, semigranulocytes, granulocytes and blast-like cells.

Copper exposure alters the metabolism of the blue crab Callinectes sapidus submitted to osmotic shock.

Copper (Cu) is an essential metal capable to alter many metabolic and physiological processes in animal species, depending on the environmental concentration and salinity. The present study evaluated the effects of Cu exposure on the metabolism of the blue crab Callinectes sapidus under different osmotic situations. Crabs were acclimated at two different salinities conditions (30 and 2). Subsequently, they were exposed to Cu during 96 h at each salinity and under hypo-osmotic shock. Results demonstrated that Cu exposure increased whole-body oxygen consumption. In addition, the activity of LDH decreased while citrate synthase increased in anterior gills from animals submitted to hypo-osmotic shock. This scenario indicates extra stress caused by sudden environmental osmotic changes, as commonly observed in estuarine environments, when combined with copper exposure. Therefore, the activity of LDH and citrate synthase enzymes might be sensitive indicators for aquatic toxicology studies approaching Cu contamination in estuarine environments.

Melatonin as a signaling molecule for metabolism regulation in response to hypoxia in the crab Neohelice granulata.

Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO2) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab-1) or 2 mgO2·L-1 (200 pmol·crab-1). Since luzindole (200 nmol·crab-1) did not significantly (p > 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab-1) increased the levels of glucose and lactate in crabs exposed to 6 mgO2·L-1, and luzindole (200 nmol·crab-1) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO2. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO2·L-1 significantly (p < 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab-1), 2 (200 and 20,000 pmol·crab-1) and 0.7 (200 or 20,000 pmol·crab-1) mgO2·L-1. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.

The effects of UV radiation on the visual system of the crab Neohelice granulata: a protective role of melatonin.

The first and main target-structure of ultraviolet (UV) radiation in animals is the body surface, including the skin and eyes. Here, we investigated cell damage in the visual system of the crab Neohelice granulata acclimated to constant light and exposed to UVA or UVB at 12:00 h for 30 min. The reactive oxygen species (ROS) production, antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO) damage, catalase (CAT) activity, and the melatonin immunohistochemical reactivity in the eyestalks were evaluated. The animals that received melatonin and were exposed to UVA and UVB radiation showed a decreased ROS concentration (p<0.05).The ACAP test showed a decrease (p<0.05) in their values when the animals received 2 pmol/crab of melatonin (physiological dose) before the exposure to UVA radiation. The animals exposed to UVB radiation after receiving the same dose of melatonin showed an increase (p<0.05) in the ACAP test compared with the animals exposed to UVB radiation after receiving only crab physiological saline. The CAT activity increased (p<0.05) in the animals that received melatonin and were exposed to UVA and UVB radiation. Animals exposed to UVA and UVB displayed an increase (p<0.05) in the LPO levels, whereas animals treated with melatonin showed lower (p<0.05) LPO levels when irradiated. The results indicate that the specific oxidative parameters altered by UV radiation can be modulated by a physiological dose of melatonin. Moreover, the melatonin regularly produced by virtually all eyestalk cells suggests that it may function to modulate the noxious effects of radiation, at least in the crab N. granulata.

Influence of the dark/light rhythm on the effects of UV radiation in the eyestalk of the crab Neohelice granulata.

Crustaceans are interesting models to study the effects of ultraviolet (UV) radiation, and many species may be used as biomarkers for aquatic contamination of UV radiation reaching the surface of the Earth. Here, we investigated cell damage in the visual system of crabs Neohelice granulata that were acclimated to either 12L:12D, constant light, or constant dark, and were exposed to UVA or UVB at 12:00h (noon). The production of reactive oxygen species (ROS), antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO) damage, catalase activity, and pigment dispersion in the eye were evaluated. No significant differences from the three groups of controls (animals acclimated to 12L:12D, or in constant light, or not exposed to UV radiation) were observed in animals acclimated to 12L:12D, however, crabs acclimated to constant light and exposed to UV radiation for 30min showed a significant increase in ROS concentration, catalase activity, and LPO damage, but a decrease in ACAP compared with the controls. Crabs acclimated to constant darkness and exposed to UV for 30min showed a significantly increased ROS concentration and LPO damage, but the ACAP and catalase activity did not differ from the controls (animals kept in the dark while the experimental group was being exposed to UV radiation). Pigment dispersion in the pigment cells of eyes of animals acclimated to constant light was also observed. The results indicate that UVA and UVB alter specific oxidative parameters; however, the cell damage is more evident in animals deviated from the normal dark/light rhythm.

Effect of melatonin in the antioxidant defense system in the locomotor muscles of the estuarine crab Neohelice granulata (Decapoda, Brachyura).

In vertebrates, many studies verified different effects of melatonin in the antioxidant defense system (ADS). In crustaceans, few studies have been conducted to verify this possibility. We verified the melatonin effects in the crab Neohelice granulata using low (0.002 and 0.02 pmol/crab) and high (2.0 and 20.0 pmol/crab) melatonin dosages in short-term (0.5h) and long-term (9.5h) experiments. We analyzed the antioxidant capacity against peroxyl radicals (ACAP), reactive oxygen species (ROS) concentration, levels of by products of lipid peroxidation (LPO), oxygen consumption (VO(2)), the activity of glutamate cysteine ligase (gamma-GCL) and catalase (CAT) and glutathione content (GSH). Finally, the effects of exogenous melatonin were verified in terms of melatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) content in the muscles of N. granulata. In short-term experiment and low dosages, melatonin increased the VO(2), gamma-GCL activity and GSH content (p<0.05) and decreased melatonin content (p<0.05) without effects in ROS, ACAP and LPO (p>0.05). Possibly, melatonin is acting in the ADS increasing its efficiency and/or acting in mitochondrial activity and/or through signaling muscles to increase its consumption. AFMK was only detected in the eyestalk and cerebroid ganglia. In high dosages melatonin effects decreased, possibly by the desensitization of their receptors. In long-term experiment, melatonin decreased ACAP (p<0.05), and CAT activity (p<0.05) in low dosages. In high dosages melatonin reduced VO(2) (p<0.05) and increased ACAP (p<0.05), possibly stimulating others components of the ADS. In conclusion, melatonin in the locomotor muscles of N. granulata affects the antioxidant/pro-oxidant balance in a time and dosage dependent manner.

Effects of melatonin in connection with the antioxidant defense system in the gills of the estuarine crab Neohelice granulata.

Numerous studies have shown that melatonin exerts some influence on the antioxidant defense system (ADS) in vertebrates, but for crustaceans no such effect has been demonstrated till now. However, earlier reports did show a similar profile of daily variations in the ADS of the gills and the melatonin content of the eyestalk in the crab Neohelice granulata and, thus, the aim of this study was to take a closer look at the effects of melatonin in the gill ADS of N. granulata. Gill ADS is to a minor extent modulated by reactive oxygen species (ROS), because only the nonproteic sulfhydryl (NP-SH) content increases (p<0.05) in the presence of hydrogen peroxide (H(2)O(2)). No significant differences (p>0.05) were observed in the melatonin content of the hemolymph between intact and eyestalkless crabs. Gills from intact and eyestalkless crabs injected with physiological saline showed a daily variation in the total peroxyl radical scavenging capacity (TPRSC) (p<0.05) with two peaks, one at the photophase and another at the scotophase. However, in the gills of eyestalkless crabs injected with melatonin (2 x 10(-12)mol crab(-1)), the daily variation in TPRSC values was abolished (p>0.05). This molecule did not change the NP-SH content (p>0.05) in vitro, but decreased (p<0.05) the oxygen consumption in gills when incubated for 120 min. In the in vivo experiments melatonin also decreased (p<0.05) the oxygen consumption in eyestalkless crabs after 390 min. The results suggest that melatonin does not act directly on the ADS of the gills of N. granulata, but decreases the aerobic metabolism possibly involved in variations of tissue ADS.

Participation of nitric oxide in the color change induced by UV radiation in the crab Chasmagnathus granulatus.

The ability of UV radiation to stimulate color change in vertebrates is well known; however, the signaling pathway involved is not fully explained. Since nitric oxide (NO) is among the candidates for this role, in this study the participation of NO signaling in the pigment migration induced by UV radiation in melanophores of the crab Chasmagnathus granulatus was investigated. When the NO donor, SIN-1, was incubated with pieces of epidermis, there was an induction of a dose-dependent pigment dispersion (in vitro assays). When male adults were exposed to different doses of UVA and UVB, N(G)-nitro-l-arginine-methyl-ester, an NO synthase (NOS) blocker produced a decrease of the pigment dispersion induced by UV (in vivo assays). However, in similar assays, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger, decreased only the pigment dispersion induced by UVA. Interestingly, buthionine sulfoximine did not produce any change in pigment dispersion induced by UVA (in vivo assays) and SIN-1 (in vitro assays). Our results using NADPH-diaphorase histochemistry and immunocytochemistry against nNOS indicated the production of NO by epidermal cells. In conclusion, we suggest that NO is a key molecule for the induction of pigment dispersion in the melanophores of Chasmagnthus granulatus, and also that NOS activation is a fundamental step for this process.

Daily variation of melatonin content in the optic lobes of the crab Neohelice granulata.

Melatonin is a biogenic amine, known from almost all phyla of living organisms. In vertebrates melatonin is produced rhythmically in the pinealocytes of the pineal gland, relaying information of the environmental light/dark cycle to the organism. With regard to crustaceans only a handful of studies exist that has attempted to identify the presence and possible daily variation of this substance. We set out to investigate whether in the crab Neohelice granulata melatonin was produced in the optic lobes of these animals and underwent rhythmic fluctuations related to the daily light/dark cycle. Our experimental animals were divided into three groups exposed to different photoperiods: normal photoperiod (12L:12D), constant dark (DD), and constant light (LL). The optic lobes were collected every 4 hours over a 24-h period for melatonin quantification by radioimmunoassay (RIA). N. granulata kept under 12 L:12D and DD conditions, showed daily melatonin variations with two peaks of abundance (p<0.05), one during the day and another, more extensive one, at night. Under LL-conditions no significant daily variations were noticeable (p>0.05). These results demonstrate the presence of a daily biphasic fall and rise of melatonin in the eyestalk of N. granulata and suggest that continuous exposure to light inhibits the production of melatonin synthesis.