Social context shapes cognitive abilities: associative memories are modulated by fight outcome and social isolation in the crab Neohelice granulata.

Cognitive abilities of an animal can be influenced by distinct social experiences. However, the extent of this modulation has not been addressed in different learning scenarios: are all tasks similarly affected by social experiences? In the present study, we analyzed the effect of social dominance in aversive and appetitive memory processes in the crab Neohelice granulata. In addition, we studied the influence of social isolation on memory ability. Social dominance experiments consisted of an agonistic phase immediately followed by a memory phase. During the agonistic phase, matched pairs of male crabs were staged in 10-min encounters and the dominant or subordinate condition of each member of the dyad was determined. During the memory phase, crabs were trained to acquire aversive or appetitive memory and tested 24 h later. Results showed that the agonistic encounter can modulate long-term memory according to the dominance condition in such a way that memory retention of subordinates results higher than their respective dominant. Remarkably, this result was found for both aversive and appetitive memory tasks. In addition, we found that isolated animals showed no memory retention when compared with animals that remained grouped. Altogether this work emphasizes the importance of social context as a modulator of cognitive abilities.

Integrative assessment of the ecological risk of heavy metals in a South American estuary under human pressures.

Biomonitoring of heavy metal pollution through the use of biomarkers could be a difficult task since the organisms' physiological changes could shift regarding natural factors (i.e., the season of the year) and due to the anthropogenic pressures of the environment. In the Southwest Atlantic Ocean, where most industrial and developing countries are settled, it is essential to address these concerns to generate information for the stakeholders and monitoring programs that aim to use biochemical biomarkers as early warning signals to detect heavy metal pollution. The present study intended to determinate the heavy metal concentrations in sediments and the hepatopancreas of the crab species Neohelice granulata as well as the ecological risk through the use of biomarkers and geochemical indices in sites with different anthropogenic pressures of the Bahía Blanca estuary (SW Atlantic Ocean) during the warm and cold season. The results showed low to moderate heavy metal pollution in the sediments by Cu with possible effects on the biota in a site with sewage waters' discharges. Except for GST that was explained by Cd, the biomarkers employed were not useful to assess spatial heavy metal pollution, and they might be ruled out by physiological seasonal variations rather than anthropogenic constraints, or another type of pollutants in the area.

Identification of a Novel Retrieval-dependent Memory Process in the Crab Neohelice granulata.

Fully consolidated associative memories may be altered by alternative retrieval dependent memory processes. While a brief exposure to the conditioned stimulus (CS) can trigger reconsolidation of the original memory, a prolonged CS exposure will trigger memory extinction. The conditioned response is maintained after reconsolidation, but is inhibited after extinction, presumably by the formation of a new inhibitory memory trace. In rats and humans, it has been shown that CS exposure of intermediate duration leave the memory in an insensitive or limbo state. Limbo is characterised by the absence of reconsolidation or extinction. Here we investigated the evolutionary conserved nature of limbo using a contextual Pavlovian conditioning (CPC) memory paradigm in the crab Neohelice granulata. In animals with fully consolidated CPC memory, systemic administration of the protein synthesis inhibitor cycloheximide after 1 CS presentation disrupted the memory, presumably by interfering with memory reconsolidation. The same intervention given after 320 CSs prevented CPC memory extinction. Cycloheximide had no behavioural effect when administered after 80 CS presentations, a protocol that failed to extinguish CPC memory. Also, we observed that a stronger CPC memory engaged reconsolidation after 80 CS instead of limbo, indicating that memory strength affects the parametrical conditions to engage either reconsolidation or limbo. Altogether, these results indicate that limbo is an evolutionary conserved memory process segregating reconsolidation from extinction in the number of CSs space. Limbo appears as an intrinsic component of retrieval dependent memory processing, with a key function in the transition from memory maintenance to inhibition.

Diet composition and long-term starvation do not affect crustacean hyperglycemic hormone (CHH) transcription in the burrowing crab Neohelice granulata (Dana, 1851).

The burrowing crab Neohelice granulata is a key omnivorous species in intertidal areas along the southwestern Atlantic from southern Brazil to northern Argentinean Patagonia. This crab is adapted to starvation and can endure natural periods of food deprivation. The metabolic adjustments during starvation depend on the type of diet the crabs were fed previously. Since eyestalk-crustacean hyperglycemic hormone (CHH) is the principal regulator of glucose homeostasis in decapods, we investigated whether CHH transcription was affected by diet composition and starvation. Crabs were maintained in the laboratory for two weeks and subsequently divided in two groups. One received a high carbohydrate (HC) diet, and the other was fed a high protein (HP) diet. After this period, they were starved for four weeks. The full-length cDNA sequence of N. granulata CHH was determined and aligned with CHH sequences of other crabs. Levels of circulating glucose and glycogen were higher in the hepatopancreas and muscle of the HC-fed group and decreased after starvation. Glucose and glycogen concentrations were not altered by starvation in the HP group. Triglyceride levels within the hemolymph were not altered by diet or starvation. However, triglycerides concentration was higher in the hepatopancreas of HC compared to HP-fed group. Long-term starvation and diet composition did not affect CHH transcription.

A baseline study of microplastics in the burrowing crab (Neohelice granulata) from a temperate southwestern Atlantic estuary.

Growing evidence has demonstrated that microplastics (MPs) are available for a wide range of marine organisms, with filter-feeding bivalves and crabs being especially vulnerable. The crab Neohelice granulata is considered a key and structuring species in the Bahía Blanca Estuary (BBE) (SW Atlantic) and its ecological role makes this species especially vulnerable to several pollutants. In this study, male specimens of N. granulata and water samples were collected at three sites in the BBE for the presence of MPs. Different types of MPs were found in all the crabs and the water column samples, although the most frequent were fibers <500-1500 µm, mainly blue. This is the first study to identify MPs in the gills and digestive tract of N. granulata. Moreover, gills presented higher total abundances of MPs than the digestive tract, which suggests that in this case the main uptake of MPs would be by adherence to the gills.

Underwater acoustic communication during the mating behaviour of the semi-terrestrial crab Neohelice granulata.

In semi-terrestrial crabs, the production of sounds has been recognized to be related to courtship communication dynamics. The present study aimed to assess if the crab Neohelice granulata (Varunidae) was able to emit acoustic signals and if they played a role in the crab's behaviour. We also assessed the locomotor behaviours to examine these parameters in different mating contexts of crabs. The study was divided into two different experimental conditions: 'solitary experiment' (consisting of three combination layouts with male, unreceptive and receptive females alone) and 'group experiment' (consisting of mixed combinations layouts of males, unreceptive, and receptive females). Synchronized acoustic and video monitoring systems were used to record the acoustic signals and locomotor behaviours of alone and grouped specimens. The greatest values of locomotor behavioural parameters were observed in layouts with receptive females alone and with 2 males plus 1 receptive female, probably related to courtship behaviour. N. granulata produced two distinct signals, a multi-pulse rasp signal (highest numbers were recorded in layouts with male alone and with 2 males plus 1 receptive female) and a single rasp signal. These results may suggest that males use the multi-rasp signal to advertise their presence to other males or to attract receptive females.

Metabolic effects of epinephrine on the crab Neohelice granulata.

Although widely known for their involvement in the control of carbohydrate and lipid metabolism of vertebrates, the participation of catecholamines (CAs) in the metabolism of invertebrates is less understood. This study was designed to identify the physiological role of Epinephrine (E) in the intermediary metabolism of the burrowing crab Neohelice granulata and how E regulates the metabolism in crabs fed with a high-carbohydrate (HC) or a high-protein (HP) diet. To answer these questions, we evaluated in vivo the effects of E injections on glucose and triglycerides in the hemolymph and tissue glycogen levels of crabs fed with HC or HP diet. An in vitro investigation was carried out to assess the direct effects of E on glycogenolysis, lipolysis and glycolysis pathways in the hepatopancreas, mandibular muscle and anterior and posterior gills of this crab. E injections increased glucose and did not affect triglycerides levels in the hemolymph of either group of crabs, and E decreased glycogen in the hepatopancreas and mandibular muscle only in HP crabs, suggesting that these effects may be mediated by the crustacean hyperglycemic hormone (CHH). When the tissues were incubated with different concentrations of E, the concentration of glucose released to the medium decreased in the hepatopancreas and posterior gills, while glucose oxidation increased in the posterior gills of HP crabs. Incubation with E did not alter any parameter in tissues of HC crabs. These effects suggest that E may be involved in the metabolic response to osmotic stress.

Are semi-terrestrial crabs threatened by human noise? Assessment of behavioural and biochemical responses of Neohelice granulata (Brachyura, Varunidae) in tank.

This study examined the effects of human lab-generated noise (sweep tone) on the behaviour and biochemistry of a semi-terrestrial crab (Neohelice granulata). The experiment was carried out in tanks equipped with video- and audio-recording systems on a total of seventy-eight specimens. In total, 42 experimental trials with sweep-tone exposure and control conditions were performed using crabs in single and group layouts. After a habituation period of 30 min, the locomotor and acoustic (sound signals emitted by the crabs) behaviours were monitored for 30 min. During this time, the animals in sweep-tone conditions were exposed to ascending sweeps in a bandwidth range of 2.5-25 kHz. Exposure to sweep-tone noise produced significant changes in the number of signals emitted, locomotor behaviours and plasma parameters, such as haemolymph total haemocyte count and glucose, lactate and total protein concentrations, revealing that human noise could represent a disturbance for this crustacean species.

Heavy metal bioconcentration factors in the burrowing crab Neohelice granulata of a temperate ecosystem in South America: Bahía Blanca estuary, Argentina.

An extensive population of the burrowing crab, Neohelice granulata, inhabits the intertidal areas of the Bahía Blanca estuary, a moderately polluted temperate ecosystem located to the southwest of the province of Buenos Aires, Argentina. In order to determine the metal-accumulating ability of this species, concentrations of Cd, Cu, Pb, Ni, Zn, Mn, Cr, and Fe in soft tissues of adult specimens were measured. Subsequently, the bioconcentration factors (BCFs) of all heavy metals were determined using levels of concentrations previously obtained in intertidal sediments. The results showed concentrations above the detection limit in soft tissues of male and female crabs for all metals except Pb and Cr. BCF > 1 were obtained for Cd, Cu, and Zn, indicating that these metals are accumulated and biomagnified. However, BCF values < 1 were found for the rest of the metals (Mn, Ni, and Fe). The findings of metal accumulation in soft tissues of N. granulata is of great importance taking into account that this is a key species within this temperate ecosystem, playing a major role in the transference of pollutants to higher trophic levels.

Metal biomonitoring in a Patagonian salt marsh.

Patagonian salt marshes are not affected by pollution, but historical mining wastes are a continuous source of metals to salt marsh in San Antonio Bay. The present study evaluated the concentration of metals in sediments and used the halophyte Spartina spp. and the crab N. granulata as biomonitors. The levels of metals in sediment and organisms in SAB remained at levels corresponding to a slight enrichment or contamination. The highest levels corresponded to innermost sites of the Encerrado channel and close to the mining wastes. Spartina is a phytostabilizer so its aboveground tissues do not reflect the concentrations in the sediment; although, it retains the metals in its belowground tissues and in the rhizosediment. N. granulata showed to be a useful biomonitor for Pb, but not for the other metals.

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata: Effects of osmotic stress.

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG.

A baseline study of the metallothioneins induction and its reversibility in Neohelice granulata from the Bahía Blanca Estuary (Argentina).

Contamination by heavy metals causes serious effects in marine systems. Nowadays, the combination of chemical and biological data is recommended in monitoring programs. Metallothioneins (MT) are early-warning signals of metal exposure and are widely used in biomonitoring. The present research evaluates the heavy metals levels in sediments and the MT synthesis in the crab Neohelice granulata from the Bahía Blanca Estuary (BBE). Then, the recovery capabilities of N. granulata followed by a depuration phase are assessed. Results demonstrate a slow decline in the level of metals in the study area. In relation to MT, female crabs showed elevated MT when compared to males. Furthermore, MT synthesis diminished after the depuration phase. These data constitute the first MT information in organisms from the BBE and may be considered as baseline for future studies in this matter. Although pollution level found was low, further biomonitoring considering both types of data is recommended.

Metals from mine waste as potential cause of oxidative stress in burrowing crab Neohelice granulata from San Antonio bay.

The Natural Protected Area San Antonio bay is of particular importance for its congregation of migratory shorebirds and it has been declared one of the Western Hemisphere Shorebird Reserve Network International site (WHSRN). Present study represents the first assessment of variation on oxidative stress biomarkers in male crab Neohelice granulata from San Antonio bay (Río Negro, Argentina) under field conditions, associated mainly to metal contamination coming from passive mining wastes. Three sites were sampled once every three months from November 2012 to August 2013 within this sea inlet (Pile, Fishery and Port) and a control site at the southeast of the bay (Punta Perdices). Accumulation of Ni, Zn, Cr and Al varied only with seasons although without a constant trend, meanwhile Cd, Cu and Pb also varied among sites being highest in Pile and Port. Biochemical results indicated that variations in catalase activity was only site specific being maximum in Pile; meanwhile lipid radical, α-tocopherol and metallothioneins were only seasonal specific being higher in autumn and winter. Seasonal variation was also found for total thioles, being the content higher in summer and autumn than in winter. Correlation analysis revealed that malondialdehyde and α-tocopherol have a positive association with Al and negative with Ni, meanwhile GST has a positive association with Fe. Crabs from the closest area to the waste pile did not exhibit a differentiated oxidative pressure despite the higher accumulation of metals. It is possible that crabs from contaminated areas have developed a tolerance to metals, indicating a strong ecotoxicological selective pressure. More studies are needed to assess whether there is a transfer of metals through the food chain.

Serotonin effects in the crab Neohelice granulata: Possible involvement of two types of receptors in peripheral tissues.

In crustaceans, serotonin (5-HT) controls various physiological processes, such as hormonal secretion, color changes, reproduction, and metabolism. Since 5-HT injections cause hyperglycemia, this study was designed to further investigate this action of 5-HT in the crab Neohelice granulate, fed with a high-carbohydrate (HC) or a high-protein (HP) diet. The effects of pre-treatment with mammalian 5-HT receptor antagonists, cyproheptadine and methiothepin, were also investigated. A series of in vivo experiments with (3)H-5-HT was carried out in order to investigate the presence of putative receptors in peripheral tissues. Since gills were the tissue with the highest labeling in in vivo experiments, in vitro studies with isolated anterior and posterior gills were also conducted. Cyproheptadine blocked the hyperglycemic effect of 5-HT in HP-fed crabs. Methiothepin reduced glycogen levels in the anterior gills of HP crabs and partially blocked the 5-HT-like posture. The injection of (3)H-5-HT identified specific binding sites in all the tissues studied and revealed that the binding can be influenced by the type of diet administered to the crabs. Incubation of the anterior and posterior gills with (3)H-5-HT and 5-HT confirmed the specificity of the binding sites. Both antagonists inhibited (3)H-5-HT binding. In conclusion, this study highlights the importance of serotonin in the control of glucose homeostasis in crustaceans and provides evidences of at least two types of 5-HT binding sites in peripheral tissues. Further studies are necessary to identify the structure of these receptors and their signaling pathways.

Grazing Scar Characteristics Impact Degree of Fungal Facilitation in Spartina alterniflora Leaves in a South American Salt Marsh

Grazing scars of burrowing crabs and Hemiptera insects were simulated on leaves of the salt marsh grass Spartina alterniflora. Simulations of crab feeding generated two-fold higher fungal (ergosterol) content in leaves in comparison to that generated by insect scar simulations (1.26 ±0.55 and 0.57 ±0.25 µg per cm², respectively). This study provided evidence that herbivory could facilitate microbial infection by fungi in dominant South American salt marsh plants and indicated that specific feeding mechanisms used by different herbivores might differentially impact the strength of this interaction. .

Natural occurrence of White spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus in Neohelice granulata crab.

White spot syndrome virus (WSSV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are two infectious agents associated to economic losses in shrimp aquaculture. As virus spread occurs through vectors and hosts, this study sought to verify the presence of WSSV and IHHNV in Neohelice granulata crab from Lagoa dos Patos estuary in Brazil and nearby shrimp farms. DNA extractions were performed with phenol/chloroform protocol. Molecular diagnosis was carried out by nested PCR for WSSV and one-step PCR for IHHNV. Results showed the presence of WSSV on crabs of both Lagoa dos Patos and farms, while IHHNV was found only on crabs collected in estuary. This is the first study to report IHHNV presence in N. granulata. Moreover, as analyzed crabs had no clinical symptoms or showed in situ mortality, we suggest its use as a bioindicator for virus occurrence in aquatic environments.