Haemolymph protein concentration of Scylla serrata : assessment of quantitative methods and intra-individual variability

The suitability of micro-Kjeldahl, biuret and Folin-Ciocalteu methods of protein determination in analysing the haemolymph protein concentration of Scylla serrata was assessed. It was found that biuret method is simple, reliable and the results are consistent. Intra-individual factor as a function of time of day is also responsible for individual variability in the haemolymph protein concentration of the crab.     

Mobilization of copper among tissues in the estuarine crab Scylla serrata (Forskal) under imposed starvation

The quantitative changes in copper free and bound to proteins in haemolymph and different forms of copper in muscle and hepatopancreas under imposed starvation were studied in the estuarine mud crab Scylla serrata. During the course of starvation, both haemolymph copper free and bound to proteins significantly declined and the regression analyses of these data further revealed that the haemolymph copper-free proteins were more affected than copper-bound proteins. The multiple stress condition namely injury and exsanguination along with starvation resulted in an earlier release and/or degradation of both these proteins. Hepatopancreas periodically accumulates and releases copper during starvation. The copper levels in haemolymph and hepatopancreas during different days of starvation showed a close inverse relationship between these two tissues. These changes in hepatopancreas were predominantly reflected in the copper that exists in association with low molecular weight substances. It is found that the copper thus accumulated was partly released back into haemolymph and a fraction may be excreted. This study also indicates the major role played by the low molecular weight substances in accommodation, detoxification and mobilization of copper in the decapod hepatopancreas during imposed starvation.     

Fractionation of DNA from marine invertebrate (Maja crispata, Mytilus galloprovincialis) haemolymph by alkaline elution.

1. An alkaline elution procedure for the detection of DNA damage in marine invertebrate haemolymph has been developed. 2. Provided that three criteria are optimized, such as buffer composition, small filter pores (0.22 microns GVWP 025 00, Millipore), and optimal amounts of haemolymph applied, flow rates may be changed within the range of 0.2 ml/min to 0.05 ml/min without adverse back-pressure on the filter and without blocking filter pores. 3. Under optimal conditions, 70% of mussel haemolymph DNA, and 80% of crab haemolymph DNA will be retained on the filter after 6 hr of elution, indicating shorter DNA in mussel haemolymph. 4. The technique is applicable for testing the in vivo effects of different compounds on DNA in marine invertebrates, and to measurements of DNA damage in naturally exposed mussels. 5. This argues an important case for the use of alkaline elution technique for assessment of environmental genotoxicity, and especially for investigation of DNA damage in different marine organisms which cover a broad range in their DNA molecular weights.     

Hyperosmotic readjustment of the crab,Hemigrapsus edwardsi

Summary Daily changes in the composition of the haemolymph and muscle tissue ofHemigrapsus edwardsi, following transfer to hyperosmotic saline, were investigated. The haemolymph approached a new equilibrium, with respect to ionic constituents, within 48 h. The muscle fibre water content, expressed in kg/kg dry weight, fell initially and then rose. This was associated with a rise in the amount (per kg tissue dry weight) of intracellular ninhydrin-positive substances (mainly amino acids) which was substantially complete within four days. It was concluded that this crab is capable of relatively rapid intracellular readjustment to hyperosmotic stress.     

In vitro and in vivo effects of copper on haemocyanin-o2 binding in the shore crab,Carcinus maenas

1. When added in vitro to crab haemolymph at concentrations of 50 or 100 mg.1⁻¹, copper decreased both haemocyanin-O₂ affinity and the cooperativity of O₂ binding.2. In crabs contaminated by a lethal dose of waterborne copper (2mg.l⁻¹), haemolymph total concentration of the metal never reached levels that could affect O₂ binding properties directly.3. Exogenous copper added in vitro or entering the animal in contaminated water was found for the most part non filterable and thus probably bound to haemolymph proteins.     

Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus).

The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2'-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.     

Effects of atrazine on osmoregulation in the Chinese mitten crab,Eriocheir sinensis

This study integrates results from acute contamination with atrazine of isolated perfused gills, and from in vivo chronic contamination of euryhaline Chinese mitten crabs, Eriocheir sinensis, acclimated to freshwater. Atrazine 1 mg/l in contact with the basolateral membrane (IN) increases the transepithelial potential difference (TEP) from -20.8 +/- 4.9 to -29.7 +/- 3.8 mV in isolated perfused posterior gills (P < 0.01). This effect is only partially explained by a modification of Na(+) and Cl(-) active influxes. No TEP modification is detected when atrazine is added (OUT) indicating that molecular mechanisms located on the basolateral membrane are likely to be the only ones affected. Another explanation would be that cuticular barrier prevents atrazine penetration into the gill. Haemolymph osmolarity, Na(+) and Cl(-) concentrations of crabs living in freshwater contaminated with atrazine 1 mg/l during 14 days are not significantly modified. We conclude that although atrazine can disturb osmoregulatory mechanisms of isolated gills, this pollutant would be of minor importance in affecting osmoregulatory capacities of the Chinese mitten crab in natural conditions.     

Acute tolerance and metabolic responses of Chinese mitten crab (Eriocheir sinensis) juveniles to ambient nitrite

The lethal concentration of nitrite to the Chinese mitten crab Eriocheir sinensis was tested by exposing the animals to 17.78, 23.71, 31.62, 42.17, and 56.23 mg NaNO2 L(-1) at 20 degrees C for 24, 48, 72, and 96 h. The corresponding LC50 value for each time exposure was 43.87 (38.70-51.70), 40.24 (34.88-46.01), 38.87 (33.72-46.01) and 38.87 (33.72-46.01) mg NaNO2 L(-1) or 29.25 (25.80-34.47), 26.83 (23.25-30.67), 25.91(22.48-30.67), 25.91(22.48-30.67) mg NO2-N L(-1), respectively. The physiological response of the crab to nitrite toxicity was further investigated by exposing the crab to 0, 10, 20, 30 and 40 mg NaNO2 L(-1) for 2 d. The changes of nitrogenous compounds in haemolymph, oxyhemocyanin and metabolism were measured at 3, 6, 24 and 48 h upon exposure. Haemolymph nitrite was significantly enhanced by the increase of nitrite from 10 to 40 mg NaNO2 L(-1) during the 2-day exposure. The concentrations of nitrate, urea and glutamate in haemolymph increased concomitantly with the exposing time and ambient nitrite levels, suggesting that the formation of nitrate, urea and glutamine may be the possible end products of nitrite detoxification in crabs. The diffusion of nitrite caused a reduction of oxyhemocyanin, resulting to hypoxia in tissues. Under a hypoxia condition, crabs increased energy demand for metabolism as indicated by the elevated levels of glucose and lactate in haemolymph. Our data showed that ambient nitrite could affect oxygen carrying capacity through oxyhemocyanin reduction and the increase of energy catabolism in crabs. This study suggests that nitrite could be detoxified through the pathway of nitrate, urea and glutamine formation in crabs.     

Antibacterial activity in four marine crustacean decapods

A search for antibacterial activity in different body-parts of Pandalus borealis (northern shrimp), Pagurus bernhardus (hermit crab), Hyas araneus (spider crab) and Paralithodes camtschatica (king crab) was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid, and further extracted and concentrated on C18 cartridges. Eluates from the solid phase extraction were tested for antibacterial, lysozyme and haemolytic activity. Antibacterial activity against Escherichia coli, Vibrio anguillarum, Corynebacterium glutamicum and Staphylococcus aureus was detected in extracts from several tissues in all species tested, but mainly in the haemolymph and haemocyte extracts. V. anguillarum and C. glutamicum were generally the most sensitive micro-organisms. In P. borealis and P. bernhardus most of the active fractions were not affected by proteinase K treatment, while in H. araneus and P. camtschatica most fractions were sensitive to proteinase K treatment, indicating antibacterial factors of proteinaceous nature. In P. bernhardus the active fractions were generally heat labile, whereas in H. araneus the activities were resistant to heat. Differences between active extracts regarding hydrophobicity and sensitivity for heat and proteinase K treatment indicate that several compounds are responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in some fractions and haemolytic activity against human red blood cells could be detected in haemolymph/haemocyte and exoskeleton extracts from all species tested.     

A morphological study on gills of a crab acclimated to fresh water

The gills of the fully euryhaline Chinese crab Eriocheir sinensis were studied by light and electron microscopy. In these Phyllobranchiates, the gills consist of a double row of lamellae extending laterally from a central shaft. Haemolymph flow pattern inside the gill is described and the existence of a complex secondary vascularization inside the platelets is reported. It is shown that important differences exist between the ultrastructure of the three anterior and the three posterior pairs of large gills. The epithelium of the posterior gills is much thicker and possesses an extensive elaboration of the plasma membranes in the form of infoldings, crypts and interdigitations, along which are packed numerous mitochondria. The presence of such a complex membrane system opening to the extracellular space and closely associated with mitochondria is common to all salt-transporting tissues. This study corroborates the idea that the posterior pairs of gills of Eriocheir sinensis are the only ones implicated in active Na+ uptake when the crab lives in dilute aquatic environment. The epithelium of anterior gills is much thinner and the cells poor in intracellular organelles. It seems to be involved essentially in respiration. Thus this work clearly corroborates the existence already suggested by physiological approach of a functional difference between the different pairs of E. sinensis branchiae with respect to their participation in the respiration and in the regulation of the blood ions content. Common to both types of gills is the presence of a lamellar septum separating the haemolymph space into two compartments. The part played by that structure in determining the pattern of haemolymph flow, together with periodic bridges forming pillars across the haemolymph space, is emphasized.     

Bacterial diseases of crabs: a review

Bacterial diseases of crabs are manifested as bacteremias caused by organisms such as Vibrio, Aeromonas, and a Rhodobacteriales-like organism or tissue and organ tropic organisms such as chitinoclastic bacteria, Rickettsia intracellular organisms, Chlamydia-like organism, and Spiroplasma. This paper provides general information about bacterial diseases of both marine and freshwater crabs. Some bacteria pathogens such as Vibrio cholerae and Vibrio vulnificus occur commonly in blue crab haemolymph and should be paid much attention to because they may represent potential health hazards to human beings because they can cause serious diseases when the crab is consumed as raw sea food. With the development of aquaculture, new diseases associated with novel pathogens such as spiroplasmas and Rhodobacteriales-like organisms have appeared in commercially exploited crab species in recent years. Many potential approaches to control bacterial diseases of crab will be helpful and practicable in aquaculture.     

Oxygen and carbon dioxide transport by the haemocyanin of an amphibious crab,Holthuisana transversa

Summary The oxygen and carbon dioxide transporting properties of the haemolymph from an amphibious Australian crab,Holthuisana transversa were investigated. Within the temperature range 15 to 35°C increasing temperature markedly decreased oxygen affinity (H=–54 kJ·mol^–1). The Bohr effect was small at all temperatures with a mean value of –0.13. Over the temperature range 15–35°C there was a significant increase in the cooperativity of oxygen binding. Changing the concentration of Ca,l-lactate or haemocyanin in the haemolymph could elicit no significant change in either O₂ affinity or cooperativity of O₂ binding. There was no evidence in support of a specific effect of CO₂ on oxygen affinity of either non-dialysed or dialysed haemolymph.The amount of CO₂ that could be carried byH. transversa haemolymph was significantly reduced by increased temperature (approx. 14 to 12.5 mmol·l^–1 CO₂). Comparisons of oxygenated and deoxygenated haemolymph at a fixed pH were unable to demonstrate the presence of a significant Haldane effect. Combining data from oxygenated and deoxygenated haemolymph the buffer value was calculated to be in the range –6.2 to –8.5 mmol·l^–1 HCO ₃ ^– ·pH unit^–1.The insensitivity ofH. transversa haemocyanin function to all modulating influences except temperature is discussed with respect to the ecology of this crab.     

不同盐度条件下中华绒螯蟹亲蟹行为及血淋巴生理变化

设定淡水对照组、盐度18适应组、盐度30骤变组(18→30)和盐度0骤变组(18→0),采用视频记录分析法研究了不同盐度条件下中华绒螯蟹雌性亲蟹的8项行为学指标变化,并测定了血淋巴渗透压及离子、血蓝蛋白含量。结果表明:中华绒螯蟹亲蟹封闭反应行为仅发生于盐度组(盐度18组和盐度30骤变组),且盐度30骤变组封闭反应时间显著高于盐度18组(P<0.05);腹部开合行为仅见于盐度0骤变组;盐度组第一触角回缩时间显著高于对照组(P<0.05);其他5项行为学指标活动频率均于盐度0骤变组最高。各实验组亲蟹血淋巴渗透压及离子浓度均高于外界实验水体,且均随盐度升高而增大。血蓝蛋白含量随盐度的降低而升高,盐度0骤变组血蓝蛋白含量显著高于盐度30骤变组(P<0.05)。分析认为,中华绒螯蟹亲蟹在0～30盐度范围内进行高渗透压调节,腹部开合行为是其在低盐度环境下暴露尾肠吸收水中离子的一种行为策略,封闭反应有助于机体减少高盐度坏境下水的吸收及扩散失盐。     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

Drinking and osmoregulation in the mangrove crab Ucides cordatus following exposure to benzene

In benzene-exposed Ucides cordatus acclimated for 96 h to 9 and 34 per thousand SW, haemolymph, urine and gastric juice are isosmotic with each other, but differ significantly in osmolality from external media. In both salinities, under benzene action, urine K+ excretion and calcium absorption are increased significantly, whereas Na+ absorption and Mg2+ excretion show U/B ratios similar to control values. In 9 per thousand SW, some ionic exchanges via benzene-exposed gills are possibly hastened. Benzene exposure decreases significantly branchial chamber water osmolality, [Na+] and [K+], whereas [Ca2+] and [Mg2+] are unaffected. However, faster medium exchange presumably occurs in 34 per thousand SW, both crab groups show branchial chamber water osmotic and ionic concentrations similar to surrounding medium. Benzene exposure unaffected gastric juice composition. In both media, [Ca2+] and [Mg2] accumulate several times higher than surrounding media, and [Na+] and [K+] are significantly hypo-ionic to haemolymph. Na+ and K+ G/H ratios are lower in crabs acclimated to 34 per thousand SW than in crabs acclimated to 9 per thousand SW. Drinking rates are enhanced by benzene exposure and are higher at 34 per thousand SW than in seawater isosmotic with the haemolymph (26 per thousand SW). Benzene exposure affects significantly osmoregulatory capability, slowing haemolymph dilution after transfer to clean 9 per thousand SW. Lower haemolymph dilution rate accounts for higher osmolality, but 48 h after transfer there is no recovery like in control crabs. Haemolymph transfusion experiments suggest an interaction among effects of benzene and hormonal factors, possibly on water influx.     

Characterization of red sternum syndrome in mud crab farms from Thailand

Samples of abnormal mud crabs, Scylla serrata (Forsk?l, 1755) (Decapoda: Portunidae), were collected from crab farms in Samutsongkhram Province, Thailand. These crabs had hard carapaces, red chelipeds and joints, pale hepatopancreas, gills, and soft muscles. They were almost immobile and finally died. The haemolymph revealed three stages of the syndrome, namely orange, orange-white, and milky-white in colors. The haemolymph, integument, hepatopancreas, gills, abdominal and claw muscle, stomach, and heart were dissected and histologically examined using transmission and scanning electron microscopy. Closer examinations found infection with rod-, curve rod-, or coccus-shape bacteria with thin and thick cell walls in all investigated organs and haemolymph. Isolation of the microorganisms from the infected tissues of red sternum syndrome crabs resulted in five types of bacteria. No microorganism growth was observed in normal crabs. Interestingly, the types of isolated bacteria can be classified according to the severity of the disease. Additionally, the degree of bacterial infection found was consistent with the stages of the disease. It was postulated that the bacteria entered the crabs via the gills, and then migrated through circulating haemocytes, before reaching the internal organs.     

Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs?

Sites of synthesis and release patterns of crustacean hyperglycaemic hormone precursor-related peptide (CPRP) were investigated with those of crustacean hyperglycaemic hormone (cHH), in order to determine whether this precursor-related peptide satisfies certain criteria necessary for its definition as a secretable, circulating hormone. Using the edible crab, Cancer pagurus, sites of CPRP synthesis were determined by immunohistochemistry and release patterns of both peptides were determined in vivo and in vitro by radioimmunoassay of haemolymph and eyestalk superfusates. Both peptides were co-released from sinus glands (SGs) following potassium-evoked depolarization of isolated eyestalk preparations. However, stress-evoked in vivo release resulted in apparent non-stoichiometric circulating peptide profiles. This phenomenon is explained by notable differences in clearance rates of the peptides in haemolymph. In contrast to cHH, CPRP is very slowly degraded in vivo. Although CPRP is clearly a circulating peptide, whose release is concomitant with that of cHH, physiologically pertinent roles for this molecule remain to be discovered.     

中华绒螯蟹水瘪子病不同发病阶段的病理变化特点

为了解中华绒螯蟹(Eriocheir sinensis)“水瘪子”病发生发展的过程, 揭示疾病的本质, 对病蟹进行了较全面的病理特点分析研究。根据病蟹肝胰腺颜色, 将病蟹划分为浅黄、浅粉、乳白和灰白4个发病阶段。研究结果表明, 随着病情的发展, 病蟹肝胰腺与蟹重比逐渐减少, 腔液与蟹重比逐渐增加, 肝胰腺管直径逐渐缩小, 各发病阶段病蟹的这些指标都与健康蟹有极显著差异(P<0.01), 病蟹肝胰腺与蟹重比除乳白阶段与灰白阶段之间和腔液与蟹重比除浅粉阶段与乳白阶段和乳白阶段与灰白阶段之间以及肝胰腺管直径除乳白阶段与灰白阶段之间的差异均不显著(P>0.05)和浅粉阶段与乳白阶段之间的差异显著(P<0.05)外, 其他各发病阶段之间这些指标均有极显著差异(P<0.01); 4个发病阶段病蟹6种组织器官中肝胰腺、鳃、肌肉组织和心脏组织病理变化具有相似性, 浅黄阶段主要是以组织器官水肿和萎缩为主, 是一种可逆的组织病理过程, 但在浅粉阶段肝胰腺和肌肉组织出现细胞坏死, 以后发病阶段更为严重, 鳃和心脏组织在乳白阶段出现细胞坏死, 坏死是一种不可逆的组织病理过程, 神经团和肠道组织病理变化不明显。根据不同发病阶段的病理特点, 在未来确定病因后通过消除病因等方法, 只有在病理变化处于可逆状态的浅黄阶段, 病蟹才有恢复的可能, 才有防控和挽救价值, 而浅粉阶段以后的病蟹由于主要器官组织细胞崩解坏死处于不可逆的病理过程, 即使病因消除病蟹已不可能恢复, 这样的病蟹应及时清除, 减少继续养殖造成进一步的经济损失。     

Ammonia exposure of Chasmagnathus granulata (Crustacea, Decapoda) Dana, 1851: accumulation in haemolymph and effects on osmoregulation

In order to study lethal and sublethal effects of ammonia to the estuarine crab Chasmagnathus granulata in the presence of an additional stress factor such as salinity, we determined the LC₅₀ (96 h) of ammonia at 20‰ and in response to osmotic stress (5–40‰) and evaluated ammonia accumulation in the haemolymph of C. granulata and ammonia effects on osmo- and ion-regulation of this species through determinations of the haemolymph Na⁺, Ca²⁺, Cl⁻ and osmotic concentration. The LC₅₀ values (96 h) of total ammonia (NH₃+NH₄⁺) were 10.10, 17.85 and 14.0 mM for crabs maintained at 5, 20 or 40‰ salinity, respectively, suggesting that this crab is fairly resistant to ammonia. The haemolymph ammonia concentration augmented with ambient ammonia during a 6-h exposure to sublethal ammonia concentrations which were not enough to reach equilibrium between external and haemolymph ammonia. At 20‰ salinity, following a 96-h exposure to sublethal concentrations, a significant decrease (P<0.05) of haemolymphatic chloride concentration was registered at 3.3 and 5.5 mM of total ammonia. At 40‰ salinity, a significant increase (P<0.05) of the haemolymph osmotic pressure was apparent at 5.5 mM total ammonia. We postulate that C. granulata gives priority to NH₃ formation as a mechanism to eliminate it by simple diffusion. The differential Na⁺ and Cl⁻ regulation of crabs maintained at 20‰ salinity could modify the strong ion difference, augmenting pH, which in turn should lead the NH₄⁺/NH₃ equilibrium towards NH₃.