Chinese Crab Eriocheir Sinensis Research Articles

Risks and critical issues related to the discovery on the market of unauthorized live alien species on the Italian territory: Chinese crab (Eriocheir sinensis).

Eriocheir sinensis, Chinese Crab or Chinese Mitten Crab is a catadromous species belonging to the Varunidae family, native to river and estuarine areas of North and South East China and Korea. At European level, E. sinensis is widespread in the main water basins of Central and Northern Europe and, since 2016, it has been included in the list of invasive species important for the European Union and subjected to confinement and eradication measures which include the prohibition of collection, transit and placing on the market of live specimens (Regulation (EC) N° 1143/2014). The Chinese Crab can represent a significant danger for the local ecosystem and for the native biota as well as contributing to the appearance of hydrogeological instability phenomena resulting from the intense excavation and erosion of the riverbanks.The first finding of 5 kg of live specimens of Eriocheir sinensis was recorded in the official control by the UFS (Functional Simple Unit) veterinary public health and food safety of the ASL Toscana Centro at an ethnic catering establishment. The specimens were subjected to seizure, photographed, identified morphologically, and subjected to euthanasia and destruction in accordance with the European requirements for welfare and management of animal by-products.From the sanitary point of view, the dangers associated with the consumption of this crab are mainly biological and chemical therefore, risk communication is fundamental, not only at the level of the competent authorities in the sector, but also for the food business operators.

Classification of Hemocytes from Four Crustaceans and Cross-Reactivity of Their Antisera

Common antigens and analyzed the hemocytes of the lady crab (Charybdis japonica) were studied. Chinese mitten crab (Eriocheir sinensis), Dungeness crab (Cancer magister), and red king crab (Paralithodes camtschaticus) using a laser scanning confocal microscope (LSCM), flow cytometry (FCM), Western blotting (WB), and transmission electron microscopy. In this paper analyzed hemocyte classification and immunological characteristics of the whole proteins in these species and their antisera. Four antisera positively cross-reacted, to varying degrees, with hemocytes of the four species. Two subpopulations were classified in C. japonica, P. camtschaticus, and E. sinensis and three in C. magister. The LSCM results showed that four antisera were positive with the hemocytes of the four crustacean species, respectively. Dot fluorescent signals on the hemocyte membranes show a cricoid intensive distribution. FCM analysis showed a positive rate of cross-reaction, in which the reaction of the antisera with its own hemocytes was higher than that with other species' hemocytes. The positive rate of granulocytes was always higher than that of hyalinocytes. Western blotting results showed that the four antisera mainly recognized antigens with molecular weights of about 75 or 80 kDa in the hemocytes of C. japonica, 80 or 90 kDa in the hemocytes of P. camtschaticus, 30 and 82 kDa in the hemocytes of E. sinensis, and 43 or 70 kDa in the hemocytes of C. magister. More lanes reacted between hemocytes of E. sinensis and C. magister and their antisera. The results indicate antigenic similarities among the hemocytes of the four crustacean species.

Effect of salinity on the metabolism of female Chinese crabs, Eriocheir sinensis

We evaluated the effect of salinity on the metabolism of female Chinese crabs(Eriocheir sinensis).The crabs,body weight(55.59 ± 6.24) g,were held at salinities of 0,6,12,18 and 24,(16 ± 1)℃,and we measured oxygen consumption,CO2 production,and ammonia-N excretion.Using this data,we calculated the respiratory quotient,oxygen-nitrogen ratio,energy consumption rate,and metabolic substrate ratio.Salinity had a significant effect on the oxygen consumption rate and ammonia–N excretion rate(P0.05).Between salinity 0–24,the rates of oxygen consumption and ammonia–N excretion both decreased as salinity increased.We observed a similar pattern for CO2 production and energy consumption,but the differences were not significant relative to the control group at salinity 6(P0.05).Between salinity 12–24,CO2 production and energy consumption were significantly lower than in the control group.Salinity had no effect on the respiratory quotient and oxygen–nitrogen ratio(P0.05).Our results suggest that lipids are used preferentially as an energy source.Furthermore,proteins were utilized to a much greater extent than carbohydrates.The proportion of lipids in the metabolic substrate increased as salinity increased.Conversely,the carbohydrate metabolic substrate ratio decreased as salinity increased,whereas the protein metabolic substrate ratio remained constant.At salinity 24,the ratio of protein,carbohydrate,and lipid used as metabolic substrate was 6.4:14.6:79.0.During the reproductive migration,the female Chinese crab migrates from fresh water into the Yangtze River estuary.Thus,individuals must adjust to the higher salinity and modify their energy metabolism.Our results suggest that energy consumption decreases after entering salt water,allowing female crabs to divert energy into reproduction.

Salinity regulates N-methylation of phosphatidylethanolamine in euryhaline crustaceans hepatopancreas and exchange of newly-formed phosphatidylcholine with hemolymph

Phosphatidylcholine (PC), the main phospholipid in eukaryotes, is synthesized via two different routes, the phosphatidylethanolamine N-methyl transferase (PEMT) and the CDP-choline pathways. We previously showed in euryhaline fish that salinity impacts the relative contribution of the two pathways for PC biosynthesis, with PEMT pathway being activated in the liver of sea water (SW)-adapted animals. To address the occurrence of such phenomenon in other animals we performed in vivo metabolic studies in two crustacean species: the Chinese crab (Eriocheir sinensis) and the green crab (Carcinus maenas). In both species, the levels of PC and phosphatidylethanolamine in hepatopancreas and hemolymph were not modified by SW-adaptation. In E. sinensis, SW-adaptation activated PC labeling from L-(U-(14)C)-serine in the hepatopancreas and resulted in an increased ratio of PC specific activities between hemolymph and hepatopancreas. In C. maenas, incorporation of L-(3-(3)H)-serine and L-(2-(14)C)-ethanolamine into PC of hepatopancreas was strongly inhibited after acclimation to fresh water (FW). The results show that PC synthesis via the PEMT pathway and its subsequent release into hemolymph are both activated in SW- compared to FW-adapted animals. SW-adaptation also resulted in increased tissue concentrations of betaine and labeling from L-(U-(14)C)-serine, suggesting that the PEMT-derived PC is used for the synthesis of organic osmolytes. The physiological relevance of these observations is discussed.

Effect of salinity on hemolymph osmotic pressure, sodium concentration and Na+-K+-ATPase activity of gill of Chinese crab, Eriocheir sinensis

The effects of salinity on hemolymph osmotic pressure, Na+ concentration and Na+-K+-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na+ concentration increased significantly (P 0.05); However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1 d, and then tended to be stable from day 1 to day 15.

Tissue-specific cadmium accumulation and metallothionein-like protein levels during acclimation process in the Chinese crab Eriocheir sinensis

Aquatic organisms chronically exposed to cadmium can increase their resistance to a subsequent elevated exposure. In order to investigate mechanisms involved in acclimation process in the Chinese crab Eriocheir sinensis, we compared Cd level as well as metallothionein-like protein (MTLP) content in different tissues after direct acute exposure (i.e. 500 μg Cd L −1 for 3 days), and after acute following chronic (i.e. 10 or 50 μg Cd L −1 for 30 days) exposure. Cadmium accumulation occurred in the following order: anterior gill>hepatopancreas>posterior gill>carapace>hemolymph>muscle. As high concentrations as 188 μg Cd g −1 w.w. were reported in anterior gills and seem to reach a saturation level. In these gills, the highest MTLP induction was observed after a direct acute exposure, for which a correlation with Cd content occurred. However, the Cd-binding potential by MTLPs was exceeded for any exposure condition. In hepatopancreas, the highest Cd level was reported for crabs acclimated during 30 days to 50 μg Cd L −1 before challenging with an acute exposure. Moreover, we showed that MTLPs were induced during the acclimation process. In this organ, MTLPs are theoretically sufficient to bind all Cd. These results suggest that during a chronic exposure to 50 μg Cd L −1, Chinese crabs acquire the capacity to hold more cadmium in hepatopancreas where it can be sequestrated by MTLPs. On the contrary, MTLP induction seems to be a rapid response to acute exposure in anterior gill, but is not sufficient to sequester all Cd. Other sequestration and/or detoxification mechanisms must take place in anterior gill to cope with high Cd levels.

Active Absorption of Cl– and Na+ in Posterior Gills of Chinese Crab Eriocheir Sinensis: Modulation by Dopamine and cAMP

Abstract Isolated posterior gills from the Chinese crab Eriocheir sinensis acclimated to freshwater (FW) were used for perfusion. Dopamine and dibutyryl cAMP (db-cAMP) induced a significant increase of 36Cl– influx. When amiloride, a specific sodium selective channels blocker, was added to the incubation saline, it induced a dramatic decrease of 22Na+ influx, and such an effect could be reversed by dopamine or db-cAMP. In the same experimental condition, we observed that ouabain added to the serosal side induced a marked decrease of the stimulating effect of db-cAMP on 22Na+ uptake across the epithelium to the hemolymph, suggesting that dopamine stimulates Na+ and Cl– influxes via cAMP and Na+/K+-ATPase pathways.

High pressure and glycolytic flux in the freshwater Chinese crab, Eriocheir sinensis

The hexose part of glycolysis has been studied in the freshwater Chinese crab Eriocheir sinensis exposed to high pressure (101 ATA, i.e. 1000 m depth) at 14°C and in normoxic conditions. Glycolytic fluxes (from glucose, J A and from Glucose 6 Phosphate, J B) have been determined using NADH depletion during the conversion of dihydroxy acetone phosphate into α-glycerol phosphate. Measurements have been performed at 14 and 19°C. Pressure exposure induces an increase of glycolytic flux and a decrease of the time needed for the transition from aerobic to anaerobic glycolysis. As a consequence pressure-exposed crabs have a higher potential to increase glycolytic flux than control animals at atmospheric pressure. It is concluded that high pressure known to alter numerous enzymes individually, can also modify an overall metabolic pathway.

Active NaCl absorption across split lamellae of posterior gills of the chinese crab Eriocheir sinensis: stimulation by eyestalk extract.

Split lamellae of the posterior gills of freshwater-adapted Chinese crabs (Eriocheir sinensis) were mounted in a modified Ussing-type chamber, and active and electrogenic absorption of Na(+) and Cl(-) were measured as positive (I(Na)) or negative (I(Cl)) short-circuit currents. Haemolymph-side addition of eyestalk extract stimulated I(Cl) by increasing both the transcellular Cl(-) conductance and the electromotive force for Cl(-) absorption. The effect was dose-dependent. Boiling the eyestalk extract did not change its effectiveness. The stimulating factor passed through dialysis tubing, indicating that it has a molecular mass of less than 2 kDa. R(p)cAMPS, a blocker of protein kinase A, reduced the stimulated I(Cl). Eyestalk extract stimulated I(Na) by increasing the transcellular Na(+) conductance at constant electromotive force. Amiloride-induced current-noise analysis revealed that stimulation of I(Na) was accompanied by an increase in the apparent number of open apical Na(+) channels at a slightly reduced single-channel current. In addition to the electrophysiological experiments, whole gills were perfused in the presence and in the absence of putative transport stimulators, and the specific activities of the V-ATPase and the Na(+)/K(+)-ATPase were measured. Eyestalk extract, theophylline or dibutyryl-cyclic AMP stimulated the activity of the V-ATPase, whereas the activity of the Na(+)/K(+)-ATPase was unaffected. The simultaneous presence of R(p)cAMPS prevented the stimulation of V-ATPase by eyestalk extract or theophylline.

Active NaCl absorption across split lamellae of posterior gills of Chinese crabs ( Eriocheir sinensis) adapted to different salinities

Split lamellae of posterior gills of Eriocheir sinensis adapted to fresh water, brackish waters (9 or 18‰) or seawater (36‰) were mounted in Ussing chambers, and transepithelial short-circuit currents and conductances were measured with salines, containing approximately in vivo-like NaCl concentrations. Active sodium and chloride absorption ( I Na and I Cl), the transcellular conductances and the leak conductance were identified with external amiloride and/or DIDS. Split gill lamellae of crabs adapted to fresh water displayed similar magnitudes of I Na and I Cl with 10 mmol l −1 NaCl in the external medium (internally haemolymph-like NaCl saline). Augmenting external NaCl (50 mmol l −1) resulted in an increase of I Cl, whereas I Na decreased. Split gill lamellae of crabs adapted to brackish waters (external NaCl of 125 and 225 mmol l −1, respectively) showed lower currents than preparations of freshwater crabs (50 mmol l −1 external NaCl). With split gill lamellae of seawater crabs no currents were detected (450 mmol l −1 NaCl on both sides). The transcellular conductances showed similar changes as the currents. The leak conductance of split gill lamellae of crabs adapted to fresh or brackish waters was low (0.3–0.8 mS cm −2), whereas it was much higher (7 mS cm −2) with preparations of seawater crabs.

Potential of active excretion of ammonia in three different haline species of crabs

Isolated perfused gills of stenohaline crabs Cancer pagurus adapted to seawater, brackish water-adapted euryhaline shore crabs Carcinus maenas and freshwater-adapted extremely euryhaline Chinese crabs Eriocheir sinensis were tested for their capacity to excrete ammonia. Gills were perfused with haemolymph-like salines and bathed with salines equal in adaptation osmolality. Applying 100 μmol · l−1 NH4Cl in the perfusion saline and concentrations of NH4Cl in the bath that were stepwise increased from 0 to 4000 μmol · l−1 allowed us to measure transbranchial fluxes of ammonia along an outwardly as well as various inwardly directed gradients. The gills of all three crab species were capable – to different extents – of active excretion of ammonia against an inwardly directed gradient. Of the three crab species, the gills of Cancer pagurus revealed the highest capacity for active excretion of ammonia, being able to excrete it from the haemolymph (100 μmol · l−1 NH+4) through the gill epithelium against ambient concentrations of up to 800 μmol · l−1, i.e. against an eightfold gradient. Carcinus maenas and E. sinensis were able to actively excrete ammonia against approximately fourfold gradients. Within the three crab species, the gills of E. sinensis exhibited the greatest capacity to resist influx at very high external concentrations of up to 4000 μmol · l−1. We consider the observed capacities for excretion of ammonia against the gradient as ecologically meaningful. These benthic crustaceans protect themselves by burying themselves in the sediment, where, in contrast to the water column, concentrations of ammonia have previously been reported that greatly increase haemolymph levels. Electrophysiological results indicate that the permeabilities of the gill epithelia are a clue to understanding the species-specific differences in active excretion of ammonia. During the invasion of brackish water and freshwater, the permeabilities of the body surfaces greatly decreased. The gills of marine Cancer pagurus exibited the greatest permeability (ca. 250 mS cm−2), thus representing practically no influx barrier for ions including NH+4. We therefore assume that C. pagurus had to develop the strongest mechanism of active excretion of ammonia to counteract influx. On the other hand, freshwater-adapted E. sinensis exhibited the lowest ion permeability (ca. 4 mS cm−2) which may reduce passive NH+4 influxes at high ambient levels.

Industrialized Culture of Chinese Crab (Eriocheir sinensis) originated from the Yangtze River

长江水系中华绒螯蟹,在满足其营养及较适水质环境的前提下,用工厂化恒温条件饲养,从大眼幼体起经141d可养成成蟹,个体大小达到或超过池养的2秋龄蟹,性成熟率达到89%,可作为亲本进行人工繁殖。;Large-eye-larva of Chinese crab(Eriocheir sinensis) originated in the Yangtze River can be raised to adult in 141 days under an industrialized stable water temperature while the conditions of nutrition and water quality should be met up with. Its weight can be higher than that of the two-year-old Chinese crab rasied usually in ponds. The rate of mature of the industrially-cultured crab is up to 89% which can be used for artificial breeding.

Dopamine as a Modulator of Ionic Transport and NA + /K + -Atpase Activity in the Gills of the Chinese Crab Eriocheir sinensis

Dopamine as a Modulator of Ionic Transport and NA + /K + -Atpase Activity in the Gills of the Chinese Crab Eriocheir sinensis

DOPAMINE AS A MODULATOR OF IONIC TRANSPORT AND NA+/K+-ATPASE ACTIVITY IN THE GILLS OF THE CHINESE CRAB ERIOCHEIR SINENSIS

ABSTRACT Dopamine and dibutyryl cAMP induced a significant increase of 22Na+ influx in salt-transporting posterior gills isolated from the Chinese crab Eriocheir sinensis acclimated to fresh water. When isolated gills were incubated and perfused in Cl--free saline (Cl replaced by gluconate), dopamine or dibutyryl cAMP still produced a significant increase of 22Na+ influx. After 1-h perfusion, dopamine increased intracellular cAMP content and Na+/K+-ATPase activity as compared with control perfusions without dopamine.

Active and electrogenic absorption of Na + and Cl- across posterior gills of Eriocheir sinensis: influence of short-term osmotic variations

Split lamellae of posterior gills of Chinese crabs (Eriocheir sinensis) acclimated to fresh water were mounted in a modified Ussing-type chamber, and the transepithelial short-circuit current and conductance were measured. The epithelium shows independent active and electrogenic absorption mechanisms for Na+ and Cl- that can be measured as positive and negative short-circuit currents, respectively, in the absence of the counter ion. Increasing the osmolarity of the haemolymph-side saline by addition of sucrose resulted in a marked decrease in active uptake of both Na+ and Cl-. In contrast, increasing the internal osmolarity by addition of urea or moderately decreasing the haemolymph-side osmolarity resulted in a marked increase in Na+ as well as Cl- transport. Circuit analysis revealed that Na+ current changes are mostly due to alterations in the apical amiloride-sensitive Na+ conductance, while Cl- current changes are caused not only by alterations in the transcellular conductance but also by changes in the electromotive force for Cl- absorption. Osmotic perturbations in the external bath induced current changes in the same directions, but the magnitudes of the effects were smaller than those after internal osmotic variations, indicating that the external barrier has a lower water permeability than the internal barrier. Short-term osmotic perturbations did not significantly affect the leak conductance, which is not associated with active transport and which may mostly reflect the paracellular conductance.

Uptake of mercury by the gills of the fresh water chinese crab Eriocheir sinensis (Milne-Edwards)

An in vitro perfused preparation of gills isolated from the euryhaline Chinese crab Eriocheir sinensis has been used to investigate the uptake of mercury. The results show that mercury crosses the epithelio-cuticular complex of the anterior respiratory gills and of the posterior salt-transporting gills. Mercury passes down its concentration gradient and it may reasonably be concluded that the heavy metal enters the crab via diffusion. However, the mercury influx through the epithelio-cuticular complex is significantly increased when the concentration gradient of mercury is nullified by the addition of mercury to the internal perfusion medium. A mechanism such as exchange-diffusion could then be involved in the transepithelial movements of mercury in crab gills, but this remains a matter of speculation. It is also demonstrated that the metabolic inhibitor 2,4-dinitrophenol, applied in the perfusion medium, drastically and immediately inhibits the influx of mercury through the anterior gills, substantiating the idea that a mechanism requiring metabolic energy is involved in the transepithelial movements of mercury. The nature of the non-diffusional component of mercury transport remains a matter of controversy.

Effects of hydrostatic pressure and temperature on the energy metabolism of the Chinese crab (Eriocheir sinensis) and the yellow eel (Anguilla anguilla)

Abstract Freshwater eel ( Anguilla anguilla ) and crab ( Eriocheir sinensis ) have been exposed to increases in hydrostatic pressure (101 ATA) or water temperature (+5°C) while being kept in normoxic conditions. Their overall oxygen consumption, ṀO 2 , and the adenylic nucleotides content of their muscles have been measured and compared. A temperature increase results in an increase in oxygen consumption by both species studied. However, when taking into consideration the strong increase in critical oxygen partial pressure, crabs appear to be much more sensitive to hypoxia. In contrast, with the temperature influence, exposure to 101 ATA hydrostatic pressure induces quite similar general effects in both crab and fish: following a transient increase, the oxygen consumption ṀO 2 progressively decreases down to −40% of its control values after 4 days' pressure application. The results establish that both species studied, Eriocheir as well as Anguilla , are quite able to acclimate to high pressure. However, if one considers that, due to their life cycle, eels might likely be preadapted to hydrostatic pressure, crabs then appear as a better model in which to investigate the biological effects of the physical parameters.

Cl− influx across posterior gills of the Chinese crab (Eriocheir sinensis): potential energization by a V-type H+-ATPase

Abstract Cl− absorption across isolated, perfused gills of freshwater adapted Chinese crabs (Eriocheir sinensis) was analysed by measuring transepithelial potential differences (PDte) and radioactive tracer fluxes across isolated, perfused posterior gills. Applying hemolymph-like NaCl salines on both sides of the epithelium PDte amounted to −30±1 mV (n=14). Undirectional Cl− influxes of 470±38 and effluxes of 245±27 μmol·hr−1·g−1 wet weight (ww) (n=14) resulted in a Cl− net influx of 226±31 μmol·hr−1·g−1 ww. Symmetrical substitution of Na+ by choline resulted in a substantial hyperpolarisation of the gill. Cl− influx was unchanged under these conditions. However, net influx of Cl− decreased by 40%, due to an increase of the Cl− efflux. Nevertheless, a significant Cl− net influx remained which was independent of the presence of Na+. When 2 mmol/l ouabain were added to the internal perfusion medium, PDte increased, although the fluxes remained unchanged. Following external application of 1μmol/l of the V-type H+-ATPase inhibitor bafilomycin, Al PDte and Cl− effluxes were not significantly affected. However, Cl− influxes decreased. These findings suggest that Cl− can be taken up independently of Na+ and that active Na+ independent Cl− uptake across the posterior gill of Eriocheir sinensis is probably driven by a V-type H+-ATPase localized in the apical membrane.

A V-ATPase drives active, electrogenic and Na+-independent Cl- absorption across the gills of Eriocheir sinensis

Using biochemical and electrophysiological techniques, we have examined the proposal that an H+-ATPase is involved in Cl- uptake across the gills of the Chinese crab Eriocheir sinensis. Bafilomycin A1 (1 µmol l-1), a specific inhibitor of V-ATPases, was used to investigate the importance of this H+-translocating enzyme in Cl- transport across the gill. In homogenates of ion-transporting posterior gills, we found the activity of a bafilomycin-sensitive V-ATPase to be markedly higher than in the anterior gills, which are not involved in ion transport. A similar distribution was found for the Na+/K+- and the mitochondrial F1Fo-ATPase. After differential and density centrifugation, the specific activity of the V-ATPase was enriched by a factor of 5. Neither Na+/K+- and F1Fo-ATPase activities nor acid phosphatase activity copurified with the bafilomycin-sensitive ATPase activity, indicating that at least the major portion of V-ATPase activity is not of basolateral, mitochondrial or lysosomal origin. In fluorescence studies, using Acridine Orange or Oxonol V as dyes, membrane vesicles displayed ATP-dependent proton transport and membrane potential generation, which were markedly reduced in the presence of bafilomycin. In addition to these biochemical studies, we mounted split lamellae of posterior gills in an Ussing-type chamber and measured the negative short-circuit current (Isc), which was shown to reflect active, electrogenic, Na+-independent and ouabain-insensitive Cl- absorption. After the addition of 1 µmol l-1 bafilomycin to the external bath, this Isc was reduced to about 50­60 % of its original value. Concomitantly, the conductance of the preparation decreased by about 13 %. From these results, we conclude that an apical V-ATPase drives electrogenic Cl- uptake across the posterior gills of the Chinese crab.

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

In the present investigation we studied the carbonic anhydrase (CA) in various tissues of Chinese crabEriocheir sinensis which were acclimated to different salinities (0, 10, 20, 30‰). We found only negligible CA activity in haemolymph, heart, hypodermis, antennal gland, leg muscle and digestive gland, irrespective of the acclimation medium. However, high amounts of CA activity were found in the gills. In the case of the posterior gills, a strong dependence on the acclimatization of the animals was demonstrated; the highest activities were found in those adapted to tap water. To investigate the cellular distribution of the CA in the posterior gills, the additional enzyme activities were measured in all fractions of a differential centrifugation of the gill homogenate: Na+/K+-ATP'ase (a marker for the plasmamembrane); lactate dehydrogenase (LDH; as marker for the cytosol); and succinate dehydrogenase (SDH; as marker for mitochondria). Independent of the acclimation salinity (0 or 36‰ salinity), we found about 70% of CA associated with the highest level of the Na+/K+-ATP'ase in the second 100 000 g pellet (membrane fraction), while only 15% were found in the cytosolic fractions (associated with highest levels of LDH). We conclude that the carbonic anhydrase of posterior gills of the Chinese crab is mainly membrane-bound. Furthermore, the activity of CA shows a strong dependence on the salinity of the water in which the crabs were kept.