Effects of high temperature and LPS injections on the hemocytes of the crab Neohelice granulata.

Temperature fluctuations, particularly elevated temperatures, can significantly affect immune responses. These fluctuations can influence the immune system and alter its response to infection signals, such as lipopolysaccharide (LPS). Therefore, this study was designed to investigate how high temperatures and LPS injections collectively influence the immune system of the crab Neohelice granulata. Two groups were exposed to 20 °C (control) or 33 °C for four days. Subsequently, half were injected with 10 µL of physiological crustacean (PS), while the rest received 10 µL of LPS [0.1 mg.kg-1]. After 30 min, the hemolymph samples were collected. Hemocytes were then isolated and assessed for various parameters using flow cytometry, including cell integrity, DNA fragmentation, total hemocyte count (THC), differential hemocyte count (DHC), reactive oxygen species (ROS) level, lipid peroxidation (LPO), and phagocytosis. Results showed lower cell viability at 20 °C, with more DNA damage in the same LPS-injected animals. There was no significant difference in THC, but DHC indicated a decrease in hyaline cells (HC) at 20 °C following LPS administration. In granular cells (GC), an increase was observed after both PS and LPS were injected at the same temperature. In semi-granular cells (SGC), there was a decrease at 20 °C with the injection of LPS, while at a temperature of 33 °C, the SGC there was a decrease only in SGC injected with LPS. Crabs injected with PS and LPS at 20 °C exhibited higher levels of ROS in GC and SGC, while at 33 °C, the increase was observed only in GC and SGC cells injected with LPS. A significant increase in LPO was observed only in SGC cells injected with PS and LPS at 20 °C and 33 °C. Phagocytosis decreased in animals at 20 °C with both injections and exposed to 33 °C only in those injected with LPS. These results suggest that elevated temperatures induce changes in immune system parameters and attenuate the immune responses triggered by LPS.

Mortality, metabolic rate, and oviposition of Gryllus (Gryllus) assimilis (Fabricius, 1775) (Orthoptera: Gryllidae) females under constant and fluctuating warm temperatures.

The global average temperature will increase by up to 5.7 °C, under high greenhouse gas emissions, consequently increasing the frequency of heatwaves, according to recent IPCC forecasts. These especially impacts ectotherms, such as insects, which are the most susceptible animals to changes in environmental temperature, affecting their physiology and reproduction. Thus, we investigated the effects of a 96-h exposure to constant temperatures (CT: 27, 30.5, 34, 39, 41, or 43 °C) and fluctuating temperatures (FT: 27/34 °C, 12/12 h) on the survival, metabolic rate, and oviposition of the female cricket Gryllus (Gryllus) assimilis (Orthoptera: Gryllidae). Mortality, body mass and water content of females and males were quantified and compared. It was found that CT27, CT34 and FT27/34 do not cause mortality in females of G. (G.) assimilis. CT30.5 (average temperature between 27 and 34), despite causing mortality of 5.0 ± 3.5%, do not differ from CT27, CT34 or FT27/34. CT39 causes a mortality of 8.3 ± 5.5%. Estimated lethal temperature for 50% of the population of females (LT50Temp) is 40 °C, and 43 °C promotes 100% mortality in 96 h. Comparing mortality between sexes, females present higher LT50Temp and thermotolerance than males. In addition, FT27/34 and CT34 do not differ in the metabolic rate, but both have higher values than CT27. CT34 strongly reduces oviposition in females, however FT27/34 does not. We suggest that CT34 reduces oviposition in females in two ways: by affecting the endocrine system related to egg production, or by causing behavioral egg retention, as a strategy to survive thermal stress. Moreover, females had a higher wet body mass and present a lower average weight loss than males. In conclusion, despite females present a higher mortality at temperatures above 39 °C, they are more thermotolerant than males. Furthermore, CT34 is detrimental to the oviposition of G. (G.) assimilis.

Impact of warmer constant and fluctuating temperatures in the male Jamaican field cricket, Gryllus assimilis (Fabricius, 1775) (Orthoptera: Gryllidae).

Ectotherms, such as insects, are susceptible to climate changes because their body temperature is not physiologically regulated. Forecasts indicate a worldwide temperature rise of 1.4-6 °C by 2100, and an increase in heatwave frequency is expected. This study investigated the effects of constant temperatures (CT; 27, 34, 39, 40 and 41 °C) and fluctuating temperature (FT; 27/34 °C: 12/12 h) on the survival, metabolic rate, locomotor activity, gas exchange pattern, heat loss and water content of the male Jamaican field cricket, Gryllus assimilis (Fabricius, 1775) (Orthoptera: Gryllidae). It was found that 39 °C was the estimated lethal temperature for 50% of the population and that 41 °C was considered the thermal limit, causing 100% mortality in 96 h. Furthermore, FT caused slightly higher mortality (8.9 ± 3.8%) than CT27 (0%) and CT34 (1.43 ± 1.43%). FT caused a greater increase in the metabolic rate and locomotor activity than CT27. It was found that G. assimilis males had a continuous gas exchange as a standard at CT27; however, CT34 changed the gas exchange pattern from continuous to cyclic in 27% of crickets. FT decreased heat loss in crickets more than CT34; however, no significant differences were found in locomotor activity and metabolic rate. In addition, no significant differences between CT27, CT34 and FT were observed in terms of water content, thus suggesting no difference in water loss. Thus, it is suggested that FT, despite involving a modest warming, increased the climate sensitivity of G. assimilis males and led to a change in their optimum temperature, pushing it beyond its usual thermal limits. However, higher mortality in FT compared to CT27 (control) and CT34 must be interpreted with caution. In addition, the risk of higher mortality of G. assimilis males is predicted, especially in South America, where this cricket is widely distributed.

Effect of Illicium verum (Hook) essential oil on cholinesterase and locomotor activity of Alphitobius diaperinus (Panzer).

The aim of this work was to test the insecticidal effect of the essential oil of Illicium verum (Hook) by observing the survival, biochemical parameters (acetylcholinesterase (AChE) activity, glutathione s-transferase (GST) activity and the concentration of reactive oxygen species (ROS)) and locomotor capacity of the Coleoptera Alphitobius diaperinus (Panzer), a pest of beef poultry. The sublethal concentrations (100% survival of A. diaperinus during 96 h of exposure) of I. verum essential oil selected for analysis were 0.5% and 1%. The selected sublethal concentrations did not show significant increases in ROS levels after 24 h of exposure to the essential oil. However, increases in GST activity were seen following exposure to 0.5% I. verum essential oil, while decreases in AChE activity were observed following exposure to concentrations of 0.5% and 1%. These results correlate with the observed behavior of A. diaperinus; when placed into an arena, these insects typically demonstrate aversion to stimuli and refuge-seeking behavior. Following exposure to 0.5% I. verum essential oil, the insects showed loss of refuge-seeking capacity and, following exposure to a concentration of 1%, loss of locomotor capacity. Overall, these results indicate that I. verum essential oil can be used as an alternative to conventional insecticides.

Oxygen sensing in crustaceans: functions and mechanisms.

Animals that live in changing environments need to adjust their metabolism to maintain body functions, and sensing these changing conditions is essential for mediating the short- and long-term physiological and behavioral responses that make these adjustments. Previous research on nematodes and insects facing changing oxygen levels has shown that these animals rapidly respond using atypical soluble guanylyl cyclases (sGCs) as oxygen sensors connected to downstream cGMP pathways, and they respond more slowly using hypoxia-inducible transcription factors (HIFs) that are further modulated by oxygen-sensing prolyl hydroxylases (PHs). Crustaceans are known to respond in different ways to hypoxia, but the mechanisms responsible for sensing oxygen levels are more poorly understood than in nematodes and insects. Our paper reviews the functions of and mechanisms underlying oxygen sensing in crustaceans. Furthermore, using the oxygen sensing abilities of nematodes and insects as guides in analyzing available crustacean transcriptomes, we identified orthologues of atypical sGCs, HIFs, and PHs in crustaceans, including in their chemosensory organs and neurons. These molecules include atypical sGCs activated by hypoxia (Gyc-88E/GCY-31 and Gyc-89D/GCY-33) but not those activated by hyperoxia (GCY-35, GCY-36), as well as orthologues of HIF-α, HIF-ß, and PH. We offer possible directions for future research on oxygen sensing by crustaceans.

Emersion behavior of the semi-terrestrial crab Neohelice granulata during hypoxic conditions: Lactate as a trigger.

Climate changes affecting aquatic environments are increasing, and the resultant environmental challenges require animals to adopt alternative compensatory behavioral and physiological strategies. In particular, low levels of dissolved O2 are a regular problem for estuarine animals, leading to activation of a series of behavioral and physiological responses. This study on the semi-terrestrial crab Neohelice granulata examined patterns of emersion behavior under different levels of dissolved O2 availability and the role of lactate in this behavior. Emersion behavior was recorded for 4.5 h for crabs in water at four different levels of dissolved O2 (6, 3, 2, and 1 mg O2/L) and with free access to air. Oxygen consumption and hemolymphatic lactate levels were measured using the same experimental design. Emersion behavior was also recorded for 70 min in normoxic water after lactate or saline injections. Crabs increased their emersion behavior only in severe hypoxia (1 mg O2/L), and O2 consumption decreased under more severe hypoxic conditions. Despite the increase in emersion behavior, which leads to higher O2 availability, an increase in hemolymphatic lactate levels indicates that the animals still need to resort to anaerobic pathways to fulfill their metabolic demand. Furthermore, animals injected with lactate showed higher emersion behaviors than animals injected with a saline solution even in normoxia. These results suggest that the increase in hemolymphatic lactate can act directly or indirectly as a trigger for the increase in emersion behavior in the semi-terrestrial crab N. granulata.

Single and repeated low-dose UVB radiation exposures affect the visual system.

The visual system is an important biological indicator of effects induced by ultraviolet (UV) radiation. However, research has extensively investigated the effects of high-dose UV radiation in a single exposure, thus, the differential of this work was to investigate the effects of UVB radiation in low doses in single and repeated exposure. Therefore, we investigated the effects of repeated exposure to environmental UVB doses (0.09 J/cm2) on the retina and optic lobes of the crab Neohelice granulata. We evaluated the reactive oxygen species (ROS) concentration, antioxidant capacity against peroxyl radicals (ACAP) levels, catalase (CAT) and glutathione S-transferase (GST) activities and lipoperoxidation (LPO) levels and performed histological analysis. The crabs were exposed to UVB radiation for 1 or 60 days, while the control group was exposed to visible light. In the retina region, increases in ROS concentration and CAT and GST activities after the single exposure were observed. After 60 days of exposure, we observed an increase in ACAP levels. In the optic lobes, we observed an increase in GST activity and a decrease in LPO levels after the single exposure. However, we observed an increase in ROS concentration after 60 days of exposure. Moreover, after 60 days of exposure, infiltrating hemocytes in the retina and disorganization in neuron cell bodies of the external medulla were observed. In this sense, single and repeated exposure to low doses of UVB radiation induced changes in oxidative status and inflammatory process in the visual system of the crab Neohelice granulata.

High temperature acclimation alters the emersion behavior in the crab Neohelice granulata.

An increase in environmental temperature can deleteriously affect organisms. This study investigated whether the semiterrestrial estuarine crab Neohelice granulata uses emersion behavior as a resource to avoid thermal stress and survive higher aquatic temperatures. We also examined whether this behavior is modulated by exposure to high temperature; whether, during the period of emersion, the animal loses heat from the carapace to the medium; and whether this behavior is altered by the temperature at which the animal has been acclimated. The lethal temperature for 50% of the population (LT50) was determined through 96-h mortality curves in animals acclimated at 20 °C and 30 °C. The behavioral profile of N. granulata during thermal stress was based on monitoring crab movement in aerial, intermediary, and aquatic zones. Acclimation at a higher temperature and the possibility of emersion increased the thermotolerance of the crabs and the synergistic effect of acclimation temperature. The possibility of leaving the hot water further increased the resistance of these animals to thermal stress. We observed that when the crab was subjected to thermal stress conditions, it spent more time in the aerial environment, unlike under control conditions. Under the experimental conditions, it made small incursions into the aquatic environment and stayed in the aerial environment for a longer time in order to cool its body temperature. The animals acclimated at 20 °C and placed into water at 35 °C remained in the aerial zone. The animals acclimated and maintained at 30 °C (control) that were placed in water at 35 °C with the possibility of emerging into hot air transited more frequently between the aquatic and aerial zones than did the animals that were put in water at 35 °C with the possibility of emerging into a cooler air environment. We conclude that emergence behavior allows N. granulata to survive high temperatures and that this behavior is influenced by acclimation temperature.

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.

Effects of hypoxia and reoxygenation on the antioxidant defense system of the locomotor muscle of the crab Neohelice granulata (Decapoda, Varunidae).

Crustaceans often occur in areas with variations in oxygen and experience situations known as hypoxia and reoxygenation. Consequences of such situations are increased levels of reactive oxygen species. To avoid oxidative damage intertidal crabs appear to possess an efficient antioxidant defense system (ADS). However, to date, studies have not addressed the strategies that are adopted by the crabs when exposed to hypoxia/reoxygenation cycles. Towards this end we evaluated the ADS and the role of melatonin as an antioxidant in the locomotor muscle of the crab Neohelice granulata under conditions of severe hypoxia and reoxygenation. Total antioxidant capacity against peroxyl radicals and the enzymes superoxide dismutase, catalase, glutathione peroxidase (GPx), and glutathione-S-transferase as well as the key enzyme of glutathione synthesis, glutamate cysteine ligase (GCL), were evaluated. Furthermore, GSH, GSH/GSSG index as well as hemolymph and cellular melatonin levels were evaluated. During hypoxia, increased GPx and GCL activity and decreased GSH and mitochondrial melatonin levels were observed, but during reoxygenation catalase activity increased and cytosolic melatonin levels decreased. It appears that the ADS in the locomotor muscle of N. granulata exert a modulating effect when being confronted with hypoxia and reoxygenation to avoid oxidative stress. During hypoxia, the ADS appear to target GPX activity as well as GSH and mitochondrial melatonin. During reoxygenation, however, evidence suggests that catalase and cytosolic melatonin are involved in the recovery of the locomotor muscle from oxidative damage and the suppression of further damage.

Air exposure behavior of the semiterrestrial crab Neohelice granulata allows tolerance to severe hypoxia but not prevent oxidative damage due to hypoxia-reoxygenation cycle.

The air exposure behavior of the semi-terrestrial crab Neohelice granulata during severe hypoxia was studied. This study also verified whether this behavior mitigates possible oxidative damage, namely lipoperoxidation, caused by hypoxia and reoxygenation cycles. The lethal time for 50% of the crabs subjected to severe hypoxia (0.5 mgO2 · L(-1)) with free access to air was compared to that of crabs subjected to severe hypoxia without access to air. Crabs were placed in aquaria divided into three zones: water (when the animal was fully submersed), land (when the animal was completely emerged) and intermediate (when the animal was in contact with both environments) zones. Then the crabs were held in this condition for 270 min, and the time spent in each zone was recorded. Lipid peroxidation (LPO) damage to the walking leg muscles was determined for the following four experimental conditions: a--normoxic water with free access to air; b--hypoxic water without access to air; c--hypoxic water followed by normoxic water without air access; and d--hypoxic water with free access to air. When exposed to hypoxic water, N. granulata spent significantly more time on land, 135.3 ± 17.7 min, whereas control animals (exposed to normoxic water) spent more time submerged, 187.4 ± 20.2 min. By this behavior, N. granulata was able to maintain a 100% survival rate when exposed to severe hypoxia. However, N. granulata must still return to water after periods of air exposure (~ 14 min), causing a sequence of hypoxia/reoxygenation events. Despite increasing the survival rate, hypoxia with air access does not decrease the lipid peroxidation damage caused by the hypoxia and reoxygenation cycle experienced by these crabs.

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.

Antioxidant defense system rhythms in crustaceans and possible roles for melatonin.

Animals in their habitats are subject to many cyclical patterns for different environmental parameters, resulting in selective pressure to develop biological rhythms for metabolism. To avoid oxidative stress, a rhythmic variation in the antioxidant defense system (ADS) should be associated with aerobic metabolic rhythms. In this review, we summarize and discuss the latest findings on rhythmic variations of the ADS in different tissues of crustaceans, as well as possible mechanisms for their regulation. In vertebrates, melatonin has been shown to be an important molecule in the regulation of the ADS and to be a high-capacity scavenger of reactive oxygen species. Given that this indoleamine has been identified in crustaceans, we also discuss the possible implications of this molecule in crustacean ADS regulation.

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.

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.

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.

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.

Circadian rhythm of pigment migration induced by chromatrophorotropins in melanophores of the crab Chasmagnathus granulata.

The circadian rhythm of black pigment migration of melanophores of the crab Chasmagnathus granulata and the variation in responsiveness of these cells to pigment-dispersing hormone (beta-PDH), crustacean cardioactive peptide (CCAP), and red pigment-concentrating hormone (RPCH) were investigated. Melanophores of C. granulata possess an endogenous circadian rhythm of pigment migration, with black pigments staying more dispersed during the day period and more aggregated during the night period. This rhythm seems to be largely dependent on an endogenous release of neurohormones from eyestalks, and to a lesser extent on a primary response to illumination. beta-PDH was the most potent PDH isoform to induce pigment dispersion in both in vivo (EC50 = 0.4 pmol/animal) and in vitro (EC50 = 0.18 microM) assays. CCAP also induced pigment dispersion in vivo and in vitro assays (EC50 = 12 microM), but it was less potent than beta-PDH. In vivo, RPCH induced a low and nondose-dependent pigment aggregation, while in vitro, it had no effect on pigment migration. The responsiveness of melanophores of C. granulata to beta-PDH was significantly higher during the day period when compared to the night period in both assays, in vitro and in vivo. These results suggest that the endogenous circadian rhythm of black pigment migration is dependent on both endogenous circadian rhythm of beta-PDH synthesis and/or release from eyestalks and on an endogenous rhythm of responsiveness of melanophores to beta-PDH.