The impact of Roundup® Original DI on the hemolymph metabolism and gill and hepatopancreas oxidative balance of Parastacus promatensis (Crustacea, Decapoda, Parastacidae).

In Brazil, glyphosate is present in more than 130 commercial formulations, and its toxic effects have already been tested in different species to understand its impact on biota Decapod crustaceans are widely used as experimental models due to their biology, sensitivity to pollutants, ease of collection, and maintenance under laboratory conditions. We evaluated the changes in metabolism (hemolymph) and oxidative balance markers (gill and hepatopancreas) of a crayfish (Parastacus promatensis) after exposure to Roundup® (active ingredient: glyphosate). The crayfish were captured in the Garapiá stream within the Center for Research and Conservation of Nature Pró-Mata, Brazil. We collected adult animals outside (fall) and during (spring) the breeding season. The animals were transported in buckets with cooled and aerated water from the collection site to the aquatic animal maintenance room at the university. After acclimatization, the animals were exposed to different concentrations of glyphosate (0, 65, 260, 520, and 780 µg/L). The results showed a significant variation in the hemolymph glucose, lactate, and protein levels. We observed variations in the tissue antioxidant enzymatic activity after exposure to glyphosate. Finally, the increase in oxidative damage required a high energy demand from the animals to maintain their fitness, which makes them more vulnerable to stress factors added to the habitat.

Emotional behavior in aquatic organisms? Lessons from crayfish and zebrafish.

Experimental animal models are a valuable tool to study the neurobiology of emotional behavior and mechanisms underlying human affective disorders. Mounting evidence suggests that various aquatic organisms, including both vertebrate (e.g., zebrafish) and invertebrate (e.g., crayfish) species, may be relevant to study animal emotional response and its deficits. Ideally, model organisms of disease should possess considerable genetic and physiological homology to mammals, display robust behavioral and physiological responses to stress, and should be sensitive to a wide range of drugs known to modulate stress and affective behaviors. Here, we summarize recent findings in the field of zebrafish- and crayfish-based tests of stress, anxiety, aggressiveness and social preference, and discuss further perspectives of using these novel model organisms in translational biological psychiatry. Outlining the remaining questions in this field, we also emphasize the need in further development and a wider use of crayfish and zebrafish models to study the pathogenesis of affective disorders.

El sueño en el crustáceo acocil.

Sleep is defined as a state of unconsciousness, reduced locomotive activity and rapid awakening, and is well established in mammals, birds, reptiles and teleosts. Commonly, it is also defined with electrical records (electroencephalogram), which are only well established in mammals and to some extent in birds. However, sleep states similar to those of mammals, except for electrical criteria, appear to occur in some invertebrates. Currently, the most compelling evidence of sleep in invertebrates has been obtained in the crayfish. In mammals, sleep is characterized by a brain state that is different from that of wakefulness, which includes a change to slow waves that has not been observed in Drosophila or bees. Herein, we show that the crayfish enters a brain state with a high threshold to vibratory stimuli, accompanied by a form of slow wave activity in the brain, quite different from that of wakefulness. Therefore, the crayfish can enter a state of sleep that is comparable to that of mammals.

El sueño es definido como un estado de inconciencia, reducción en la actividad locomotora y despertar rápido, el cual está bien identificado en mamíferos, aves, reptiles y teleosteos. Comúnmente también es definido con registros eléctricos (electroencefalograma), los cuales solo están bien establecidos en mamíferos y, en cierta manera, en aves. Sin embargo, estados de sueño similares a los de mamíferos, excepto por los criterios eléctricos, parecen ocurrir en algunos invertebrados. Actualmente la mejor evidencia de sueño en invertebrados ha sido obtenida en el acocil. En los mamíferos, el sueño se caracteriza por un estado cerebral diferente al de la vigilia, que incluye un cambio a ondas lentas, lo que no se ha visto en Drosophila o abejas. Aquí mostramos que el acocil tiene un estado cerebral con umbral elevado a estímulos vibratorios, acompañado por una forma de actividad de ondas lentas en el cerebro, muy diferente al de la vigilia. Por lo tanto, el acocil puede experimentar un estado de sueño comparable al de los mamíferos.

Sleep in the crustacean crayfish

Sleep is defined as a state of unconsciousness, reduced locomotive activity and rapid awakening, and is well established in mammals, birds, reptiles and teleosts. Commonly, it is also defined with electrical records (electroencephalogram), which are only well established in mammals and to some extent in birds. However, sleep states similar to those of mammals, except for electrical criteria, appear to occur in some invertebrates. Currently, the most compelling evidence of sleep in invertebrates has been obtained in the crayfish. In mammals, sleep is characterized by a brain state that is different from that of wakefulness, which includes a change to slow waves that has not been observed in insects. Herein, we show that the crayfish enters a brain state with a high threshold to vibratory stimuli, accompanied by a form of slow wave activity in the brain, quite different from that of wakefulness. Therefore, the crayfish can enter a state of sleep that is comparable to that of mammals.

Sleep in the crustacean crayfish.

Sleep is defined as a state of unconsciousness, reduced locomotive activity and rapid awakening, and is well established in mammals, birds, reptiles and teleosts. Commonly, it is also defined with electrical records (electroencephalogram), which are only well established in mammals and to some extent in birds. However, sleep states similar to those of mammals, except for electrical criteria, appear to occur in some invertebrates. Currently, the most compelling evidence of sleep in invertebrates has been obtained in the crayfish. In mammals, sleep is characterized by a brain state that is different from that of wakefulness, which includes a change to slow waves that has not been observed in insects. Herein, we show that the crayfish enters a brain state with a high threshold to vibratory stimuli, accompanied by a form of slow wave activity in the brain, quite different from that of wakefulness. Therefore, the crayfish can enter a state of sleep that is comparable to that of mammals.

Involvement of Melatonin in the Regulation of the Circadian System in Crayfish.

Melatonin (MEL) is an ancient molecule, broadly distributed in nature from unicellular to multicellular species. MEL is an indoleamine that acts on a wide variety of cellular targets regulating different physiological functions. This review is focused on the role played by this molecule in the regulation of the circadian rhythms in crayfish. In these species, information about internal and external time progression might be transmitted by the periodical release of MEL and other endocrine signals acting through the pacemaker. We describe documented and original evidence in support of this hypothesis that also suggests that the rhythmic release of MEL contributes to the reinforcement of the temporal organization of nocturnal or diurnal circadian oscillators. Finally, we discuss how MEL might coordinate functions that converge in the performance of complex behaviors, such as the agonistic responses to establish social dominance status in Procambarus clarkii and the burrowing behavior in the secondary digging crayfish P. acanthophorus.

Left-right asymmetry in firing rate of extra-retinal photosensitive neurons in the crayfish.

The purpose of this paper is to explore the firing rate of the caudal photoreceptors (CPRs) from the sixth abdominal ganglion of the crayfish Cherax quadricarinatus. We use simultaneous extracellular recordings on left and right CPR in the isolated ganglion (n = 10). The CPRs showed an asymmetry in the spontaneous activity and light-induced response. In darkness, we observed one subgroup (70%) in which the left CPR (CPR-L) and right CPR (CPR-R) had spontaneous firing rates with a median of 18 impulses/s and 6 impulses/s, respectively. In another subgroup (20%), the CPR-R had a median of 15 impulses/s and the CPR-L had 8 impulses/s. In both groups, the differences were significant. Furthermore, the CPRs showed an asymmetrical photoresponse induced by a pulse of white light (700 Lux, 4 s). In one subgroup (30%), the CPR-L showed light-induced activity with a median of 73%, (interquartile range, IQR = 51), while the CPR-R had a median of 41%, (IQR = 47). In another subgroup (70%), the CPR-R showed a median of 56%, (IQR = 51) and the CPR-L had a median of 42%, (IQR = 46). In both groups, the differences were significant. Moreover, we observed a differential effect of temperature on CPR activity. These results suggest a functional asymmetry in both activities from left and right CPRs. These CPR activity fluctuations may modulate the processing of information by the nervous system.

Energy reserves mobilization: Strategies of three decapod species.

In food deprivation assays, several different responses have been observed in crustaceans. However, studying energy reserves utilization among more than one species during the same starvation period has not yet been performed, particularly to discern whether the responses are due to intrinsic and/or environmental factors. We hypothesize that decapod species with similar feeding habits have the same strategies in the use of energetic reserves during starvation, even though they inhabit different environments. The aim of this study was to compare the energy reserves mobilization of three decapods species (Cherax quadricarinatus, Palaemon argentinus and Munida gregaria) with similar feeding habits, exposed to similar food deprivation conditions. The crayfish, shrimp and squat-lobster were experimentally kept at continuous feeding or continuous starvation throughout 15 days. Every 3rd day, the midgut gland index (MGI), and the glycogen, lipid and protein contents were measured in the midgut gland (MG) and pleon muscle. Palaemon argentinus mobilized more reserves during starvation, followed by C. quadricarinatus, and the last M. gregaria. The starved shrimps presented low MGI, whereas MG showed a reduction in glycogen (from day 6 to 15), lipid (from day 3 to 15), and protein levels (at day 9 and 15) while in their muscle, lipid reserves decreased at days 3 and 6. In C. quadricarinatus, the most affected parameters in the MG were MGI, glycogen (from day 6 to 15), and lipids (at day 12 and 15). In the MG of M. gregaria only the glycogen was reduced during fasting from 3 to 15 days. Even though the three studied species have similar feeding habitats, we found that their energetic profile utilization is different and it could be explained by the habitat, life span, temperature, organ/tissue, and metabolism of the species. Our results may be useful to understand the several different responses of crustaceans during starvation.

Circadian rhythm in melatonin release as a mechanism to reinforce the temporal organization of the circadian system in crayfish.

Melatonin (MEL) is a conserved molecule with respect to its synthesis pathway and functions. In crayfish, MEL content in eyestalks (Ey) increases at night under the photoperiod, and this indoleamine synchronizes the circadian rhythm of electroretinogram amplitude, which is expressed by retinas and controlled by the cerebroid ganglion (CG). The aim of this study was to determine whether MEL content in eyestalks and CG or circulating MEL in hemolymph (He) follows a circadian rhythm under a free-running condition; in addition, it was tested whether MEL might directly influence the spontaneous electrical activity of the CG. Crayfish were maintained under constant darkness and temperature, a condition suitable for studying the intrinsic properties of circadian systems. MEL was quantified in samples obtained from He, Ey, and CG by means of an enzyme-linked immunosorbent assay, and the effect of exogenous MEL on CG spontaneous activity was evaluated by electrophysiological recording. Variation of MEL content in He, Ey, and CG followed a circadian rhythm that peaked at the same circadian time (CT). In addition, a single dose of MEL injected into the crayfish at different CTs reduced the level of spontaneous electrical activity in the CG. Results suggest that the circadian increase in MEL content directly affects the CG, reducing its spontaneous electrical activity, and that MEL might act as a periodical signal to reinforce the organization of the circadian system in crayfish.

Desensitization and recovery of crayfish photoreceptors. Dependency on circadian time, and pigment-dispersing hormone.

In this work, we studied the characteristics of recovery from desensitization of the light-elicited current of crayfish. Applying a two-flash protocol, we found that the first flash triggers a current that activates with a noticeable latency, reaches a peak value, and thereafter decays along a single exponential time course. In comparison with the first-elicited current, the current elicited by the second flash not only presents an expected smaller peak current, depending on the time between flashes, but it also displays a different latency and decay time constant. Recovery of the first flash values of these current parameters depends on the circadian time at which the experiments are conducted, and on the presence of pigment-dispersing hormone. Our data also suggest the existence of distinctive desensitized states, whose induction depends on circadian time and the presence of pigment-dispersing hormone.

Long-Term Starvation and Posterior Feeding Effects on Biochemical and Physiological Responses of Midgut Gland of Cherax quadricarinatus Juveniles (Parastacidae).

We investigated the effect of long-term starvation and posterior feeding on energetic reserves, oxidative stress, digestive enzymes, and histology of C. quadricarinatus midgut gland. The crayfish (6.27 g) were randomly assigned to one of three feeding protocols: continuous feeding throughout 80 day, continuous starvation until 80 day, and continuous starvation throughout 50 day and then feeding for the following 30 days. Juveniles from each protocol were weighed, and sacrificed at day 15, 30, 50 or 80. The lipids, glycogen, reduced glutathione (GSH), soluble protein, lipid peroxidation (TBARS), protein oxidation (PO), catalase (CAT), lipase and proteinase activities, and histology were measured on midgut gland. Starved crayfish had a lower hepatosomatic index, number of molts, specific growth rate, lipids, glycogen, and GSH levels than fed animals at all assay times. The starvation did not affect the soluble protein, TBARS, PO levels and CAT. In starved juveniles the lipase activity decreased as starvation time increased, whereas proteinase activity decreased only at day 80. The histological analysis of the starved animals showed several signs of structural alterations. After 30 days of feeding, the starved-feeding animals exhibited a striking recovery of hepatosomatic index, number of molts, lipids and glycogen, GSH, lipase activity and midgut gland structure.

Compensatory Growth in Juveniles of Freshwater Redclaw Crayfish Cherax quadricarinatus Reared at Three Different Temperatures: Hyperphagia and Food Efficiency as Primary Mechanisms.

Feeding restriction, as a trigger for compensatory growth, might be considered an alternative viable strategy for minimizing waste as well as production costs. The study assessed whether juvenile redclaw crayfish Cherax quadricarinatus (initial weight 0.99 ± 0.03 g) was able to compensate for feeding restriction at different temperatures (23 ± 1, 27 ± 1 and 31 ± 1 ° C). Hyperphagia, food utilization efficiency, energetic reserves, and hepatopancreas structure were analyzed. Three temperatures and two feeding regimes (DF-daily fed throughout the experiment and CF- 4 days food deprivation followed by 4 days of feeding, intermittently) were tested. The restriction period was from day 1 to 45, and the recovery period was from day 45 to 90. The previously restricted crayfish held at 23, 27, and 31 ± 1 ° C displayed complete body weight catch-up through compensatory growth following the restriction period with depressed growth. The mechanisms that might explain this response were higher feed intake (hyperphagia), and increased food utilization efficiency. Hepatopancreatic lipids were used as a metabolic fuel and hepatosomatic index was reduced in the previously restricted crayfish, but recovery at the same level of unrestricted crayfish occurred after the shift to daily feeding. The highest temperature affected adversely growth, feed intake, food efficiency, and metabolism of crayfish, whereas the lowest temperature and feeding restriction induced a more efficient growth of the crayfish.

Glial fibrillary acidic protein (GFAP) shows circadian oscillations in crayfish Procambarus clarkii putative pacemakers.

Although several studies of glia have examined glial fibrillary acid protein (GFAP) and its relationship to the circadian rhythms of different organisms, they have not explored the daily GFAP oscillations in the putative pacemakers of the crayfish Procambarus clarkii or in other crustaceans. In this study we investigated the daily variations in GFAP concentrations in the eyestalk and brain, which are considered to be putative pacemakers in adult P. clarkii. In both structures, the glial GFAP was quantified using the indirect enzyme-linked immunosorbent assay (ELISA), and double labeling immunofluorescence was used to detect it and its co-localization with protein Period (PER), an important component of the circadian clock, in various regions of both structures. The ELISA results were analyzed using Cosinor and one-way ANOVA with Bonferroni and Scheffé's post hoc tests. The results of this analysis showed that the GFAP levels present circadian oscillations in both structures. Moreover, GFAP was localized in different structures of the eyestalk and brain; however, co-localization with PER occurred only in the lamina ganglionaris, specifically in the cartridges of the eyestalk and in some of the cluster 9 brain cells. These results suggest that as in other invertebrates and vertebrates, glial cells could be involved in the circadian system of P. clarkii; however, thus far we cannot know whether the glial cells are only effectors, participate in afferent pathways, or are part of the circadian clock.

Effects of glyphosate on growth rate, metabolic rate and energy reserves of early juvenile crayfish, Cherax quadricarinatus M.

Early juveniles of the crayfish Cherax quadricarinatus were exposed for 60 days to 10 and 40 mg/L of pure glyphosate (acid form) in freshwater. Mortality was 33 % at the highest concentration, while no differences in molting were noted among treatments. After the first month of exposure, weight gain was significantly (p < 0.05) reduced in the 40 mg/L group. At the end of the assay, lipid levels in muscle, as well as protein level in both hepatopancreas and muscle were significantly (p < 0.05) reduced. These results suggest long-term utilization of both lipid and protein as main energetic reserves, likely in response to the chronic stress associated with herbicide exposure. Besides, the lower pyruvate kinase activity in muscle suggests a possible metabolic depression in this tissue. The hemolymphatic ASAT:ALAT ratio showed higher levels than the control at the highest glyphosate concentration, indicating possible damage to several tissues.

Population and reproductive features of the freshwater shrimp Macrobrachium jelskii (Miers, 1877) from São Paulo State, Brazil / Aspectos populacionais e reprodutivos do camarão de água doce Macrobrachium jelskii (Miers, 1877) do Estado de São Paulo, Brasil

The aim of this study was to describe the population structure and reproduction of Macrobrachium jelskii from a stream in the central region of São Paulo State. A total of 1,215 specimens was collected monthly during one year (February 2008 to January 2009), being 535 males, 578 females and 102 juveniles. The overall sex-ratio was not significantly different from the expected 1:1, but has significantly differed in some months. A total of 136 ovigerous females were collected and the reproductive period was continuous with peak of occurrence on the hottest months. An increase in juveniles following the highest frequency of ovigerous females was observed and characterized the recruitment period. The fecundity was low and varied from 1 to 56 eggs per female. The biological profile observed here matched, in general aspects, with the pattern developed by tropical and subtropical inland populations, with some particularities related with environmental characteristics.(AU)

O objetivo deste estudo foi descrever a estrutura populacional e a reprodução de Macrobrachium jelskii de um ribeirão na região central do Estado de São Paulo. Um total de 1215 espécimes foi coletado mensalmente durante o período de um ano (fevereiro de 2008 a janeiro de 2009), sendo 535 machos, 578 fêmeas e 102 juvenis. A razão sexual total não foi significantemente diferente da esperada 1:1, mas diferiu significativamente em alguns dos meses coletados. Um total de 136 fêmeas ovígeras foi coletado e o período reprodutivo foi considerado contínuo com pico de ocorrência nos meses mais quentes. Um aumento no número de juvenis foi identificado após a mais alta frequência de fêmeas ovígeras e caracterizou o período de recrutamento. A fecundidade foi baixa e variou de 1 a 56 ovos por fêmea. O perfil biológico observado aqui correspondeu, em aspectos gerais, com o padrão desenvolvido por populações continentais tropicais e subtropicais, com algumas particularidades relacionadas com as características do ambiente.(AU)

Adult neurogenesis: ultrastructure of a neurogenic niche and neurovascular relationships.

The first-generation precursors producing adult-born neurons in the crayfish (Procambarus clarkii) brain reside in a specialized niche located on the ventral surface of the brain. In the present work, we have explored the organization and ultrastructure of this neurogenic niche, using light-level, confocal and electron microscopic approaches. Our goals were to define characteristics of the niche microenvironment, examine the morphological relationships between the niche and the vasculature and observe specializations at the boundary between the vascular cavity located centrally in the niche. Our results show that the niche is almost fully encapsulated by blood vessels, and that cells in the vasculature come into contact with the niche. This analysis also characterizes the ultrastructure of the cell types in the niche. The Type I niche cells are by far the most numerous, and are the only cell type present superficially in the most ventral cell layers of the niche. More dorsally, Type I cells are intermingled with Types II, III and IV cells, which are observed far less frequently. Type I cells have microvilli on their apical cell surfaces facing the vascular cavity, as well as junctional complexes between adjacent cells, suggesting a role in regulating transport from the blood into the niche cells. These studies demonstrate a close relationship between the neurogenic niche and vascular system in P. clarkii. Furthermore, the specializations of niche cells contacting the vascular cavity are also typical of the interface between the blood/cerebrospinal fluid (CSF)-brain barriers of vertebrates, including cells of the subventricular zone (SVZ) producing new olfactory interneurons in mammals. These data indicate that tissues involved in producing adult-born neurons in the crayfish brain use strategies that may reflect fundamental mechanisms preserved in an evolutionarily broad range of species, as proposed previously. The studies described here extend our understanding of neurovascular relationships in the brain of P. clarkii by characterizing the organization and ultrastructure of the neurogenic niche and associated vascular tissues.

Sleep in invertebrates: crayfish.

Although sleep is a very conspicuous behavior in all animals that we are frequently in contact with and possibly in many others, its scientific study was for many years restricted to very few of them. However, since the end of the XX century there have been studies about sleep in several animals and currently many of them attempt to found, first, if all animals sleep and second, if their sleep is similar to that of other animals. An important objective of this search is to identify the animal species in which sleep originated, which might gives us clues about the need that was fulfilled by such behavior. The search started with insects, among the most developed arthropods, but has now been expanded to include other invertebrates, among them crustaceans. In this work we review some aspects of sleep in invertebrates, focusing on the crustacean crayfish, animals in which both, behavioral and electrophysiological studies have been conducted and whose results show surprising similarities with sleep in mammals.

Crayfish brain-protocerebrum and retina show serotonergic functional relationship.

The results from various studies have indicated possible functional relationships between crayfish electroretinogram (ERG) rhythmic amplitude changes and the serotonergic pathways projecting from the central brain through the optic neuropils to the eye, but to date, this functional interaction has not been proven. Here, in a set of experiments using an isolated eyestalk-brain preparation, we investigated whether there is a circadian input from the brain to retina that regulates this rhythm. We sought to determine whether the protocerebral bridge (PB) stimulation affects the ERG amplitude in accordance with the zeitgeber time (ZT) and whether 5-HT modulates the associate input. Our results showed that photic stimulation of retina produced changes in both the amplitude and the frequency of spontaneous electrical activity in the protocerebral neuropils. In addition, electrical stimulation of the medial protocerebrum, particularly the PB, produced statistically significant changes in the ERG that depended on both the time of day and the level of serotonin. This suggests that pathways between retina and PB seem to be serotonergic.

GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons.

In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones.

In vitro effects of both dopaminergic and enkephalinergic antagonists on the ovarian growth of Cherax quadricarinatus (Decapoda, Parastacidae), at different periods of the reproductive cycle.

The in vitro effect of both spiperone (dopaminergic antagonist) and naloxone (enkephalinergic antagonist), was assayed on small pieces of ovary dissected from C. quadricarinatus females, with the eventual addition of some neuroendocrine organs, such as thoracic ganglion or eyestalk tissue. The incorporation of tritiated leucine by the ovary was measured in order to estimate the ovarian growth. During the post-reproductive period, both mentioned antagonists were able to significantly stimulate the ovary in the presence of thoracic ganglion, but did not produce any significant effect in the preparation containing ovary and eyestalk tissue, or only ovary. No significant effects of the assayed antagonists were noted during the pre-reproductive period. These results were in accordance with previous models describing the neuroendocrine control of crustacean reproduction, and represent new findings about the hormonal context in different periods of the reproductive cycle of crayfish. Besides, by means of the experimental combination of the tested antagonists with dopamine or met-enkephalin, a new model dealing with the interaction of these two neurotransmitters on the hormonal secretion of thoracic ganglion has been proposed.