Effects of histamine on survival and immune parameters of the Chinese mitten crab, Eriocheir sinensis.
Article Type: Report
Subject: Crabs (Health aspects)
Histamine (Health aspects)
Immune response (Research)
Authors: Zhao, Liulan
Yang, Xiaozhen
Cheng, Yongxu
Liang, Pan
Zhang, Jinbiao
Hong, Yuhang
Wang, Chun
Yang, Zhigang
Pub Date: 08/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2012 Source Volume: 31 Source Issue: 3
Topic: Event Code: 310 Science & research
Product: Product Code: 0913040 Crabs NAICS Code: 114112 Shellfish Fishing SIC Code: 0913 Shellfish
Geographic: Geographic Scope: China Geographic Code: 9CHIN China
Accession Number: 303011408
Full Text: ABSTRACT In the current study, we examined the effects of histamine on survival and immune parameters of the Chinese mitten crab, Eriocheir sinensis. The crabs infected with the common bacteria pathogen Aeromonas hydrophila were injected with 0 [micro]g histamine/g/crab, 1 [micro]g histamine/g/crab, 50 [micro]g histamine/g/crab, or 100 [micro]g histamine/g/crab, and their survival after 96 h was recorded. The results showed that histamine alone had no influence on crab survival but can decrease survival of the crabs challenged with A. hydrophila after 36 h. In addition, the survival of the crabs injected with the high level (100 [micro]g/g) of histamine was significantly greater than that of the lower level (50 [micro]g/g) after 60-96 h (P < 0.05), but not different from that of the control. Immune parameters of the hemolymph in crabs, such as the total hemocyte count (THC), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), and superoxide dismutase activity (SOD), were measured at 6 h, 12 h, and 24 h after histamine injection. Some immune parameters, such as PO and SOD, showed an increase, whereas THC, ACP, and AKP levels declined at 6 b. Surprisingly, after the injection of histamine, the crabs quickly released their pereiopods and began to twitch, and the number of the crabs with autotomy and spasm increased with the increasing histamine concentration (P < 0.05). This is the first report that indicates that histamine could lead to crab autotomy. The overall results of this study indicate that the effects of histamine on survival and the immune parameters of E. sinensis infected with A. hydrophila depend on its concentration.

KEY WORDS: histamine, survival, Eriocheir sinensis, Aeromonas hydrophila, hemocyte counts, phenoloxidase, acid phosphatase, alkaline phosphatase, superoxide dismutase activity, autotomy


The Chinese mitten crab (Eriocheir sinensis) is an important cultured species in China (Chen et al. 2007). Production increased dramatically from 8,000 t in 1991 to approximately 570,000 t in 2009 (China Fisheries Yearbook 2010). The crabs are usually fed with trash fish, which decay rapidly, leading to deteriorating water quality and increasing risk of disease outbreak (Edwards et al. 2004, Wu et al. 2007). Stale trash fish contain high level of biogenic amines such as histamine. Histamine and other biogenic amines, derivative of amino acids, act as neurotransmitters, hormones, and modulators (Cheng et al. 2005, Li et al. 2005, Cheng et al. 2006). Histamine is one common biogenic amine, formed by the decarboxylation of L-histidine, and has a high detection rate in fishmeal and seafood sold in many countries, including India, Thailand, the Philippines, and the Netherlands (Kennedy & Karunasagar 2004, Tao et al. 2011). In these products, histamine levels exceeding 200-500 [micro]g/g were often detected (Kennedy & Karunasagar 2004, Tao et al. 2011). Moreover, fed with high histamine content diet could cause poisoning in humans, and gastric diseases in chickens and aquatic animals. Based on the assessment of poisoning case, the U.S. Food and Drug Administration suggested a safe consumption level of histamine content in seafood. Therefore, histamine is often used as an important food quality criterion for seafood (Veciana 1990, Ricque-Marie et al. 1998).

A few studies have reported that dietary histamine can affect crustacean growth but does not have any effect on survival. These studies included the in blue shrimp Litopenaeus stylirostris (Tapia-Salazar et al. 2001) and Litopenaeus vannamei (Ricque-Marie et al. 1998). Other studies have reported that histamine decreases significantly the survival of Mysis sp., Neomysis awatschensis, and Neomysis japonica (Yang et al. 2010). Moreover, in vertebrates, histamine can improve the survival of mice and regulate their immune cytokines (Hornyak et al. 2005). Apparently, there are diverse effects of histarnine on immune regulation because of differential expression of 4 types of histamine receptors in vertebrates (Jutel et al. 2002). The invertebrates lack adaptive immunity and rely on innate immunity based on cellular and humoral components (Ratcliffe et al. 1985). Although studies have proved the existence of histamine receptors in invertebrates, such as in snails and flatworms (El-Shehabi et al. 2009), studies of the effects of histamine on the immunology of invertebrates are lacking. Previous studies of histamine on the immune response in crustaceans focused only on survival, not on underlying immunology, such as hemocytes and immune enzymes. It is known that injected histamine can be carried in the bloodstream in rainbow trout (Kennedy & Karunasagar 2004). Cebada et al. (2006) reported that histamine was detected in the hemolymph of crayfish, and the levels fluctuated from 25 250 [micro]M during the 24-h cycle. Therefore, hemocytes played an essential role in the effect of histamine. Among the hemocytes and enzymes participating in immune defenses, the to hemocyte counts (THC), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), and superoxide dismutase (SOD) have been used in Eriocheir sinensis (Li et al. 2005, Chang et al. 2007).

The primary goals of this study were to determine the effect of histamine on the survival of E. sinensis infected with Aeromonas hydrophila, a common bacterial pathogen of the crab, and to determine the immune response of E. sinensis injected with histamine, including the variations in THC and other immune parameters. During the current study, we found, unexpectedly, autotomy and spasm of the crabs after histamine injection. Autotomy is a reflex severance of an appendage from a fixed breakage point (Wood & Wood 1932). The autotomy rate has been suggested to be an indication of physiological stress from factors such as low temperature, starvation, and the presence of foreign matter (Wood & Wood 1932, Rome et al. 2005), and biogenic amine has been related closely to a protostress response (Ottaviani & Franceschi 1996). Spasm is a sudden, involuntary contraction of a group of leg muscles (Ohtsuka et al. 2001).


Culture ofAeromonas hydrophila

The pathogenic strain of A. hydrophila was obtained from the Bank of Aquatic Animals and Plants Pathogens of the Ministry of Agriculture in Shanghai Ocean University. The pathogen A. hydrophila was inoculated asceptically into tryptic soy broth medium from tryptic soy agar and was cultured overnight at 27[degrees]C. Then, the broth cultures were centrifuged at 6,000g for 30 min at 4[degrees]C. The supernatant fluid was removed, and the bacterial pellet was resuspended in saline solution (0.85% NaCl).

Healthy crabs averaging 1.01 [+ or -] 0.10 g in weight were collected from a commercial crab farm on Chongming Island in Shanghai and were maintained for 2 wk in aerated aquaria supplied with continuously flowing seawater. During all acclimation and experimentation periods, the culturing conditions were as follows: temperature, 24.6 [+ or -] 0.75[degrees]C; pH, 8.39 [+ or -] 0.06; [NH.sup.+.sub.4] , 0.1-0.2 mg/L; nitrite, <0.005 mg/L; and photoperiod, 12 h light/12 h dark. Only intermolt juvenile crabs were used in the experiments and were fed an artificial diet.

Histamine (Sigma product no. H7125) was dissolved in crab saline (NaCl, 496 mM; KCl, 9.52 mM; MgS[O.sub.4], 12.8 mM; Ca[Cl.sub.2], 16.2 mM; Mg[Cl.sub.2], 0.84 mM; NaHC[O.sub.3], 5.95 mM; HEPES, 20 mM; pH, 7.4) to concentrations of 0.1 mg/mL, 5 mg/mL, and 10 mg/mL before injection.

Experiment 1

Survival of Histamine-Injected E. sinensis Infected with A. hydrophila

The Chinese mitten crab, E. sinensis, was injected individually, at the base of the third pereiopod, with 0.1 mg/mL, 5 mg/mL, and 10 mg/mL histamine solution (approximately 20 [micro]L) to reach doses of 1 [micro]g/g/crab, 50 [micro]g/g/crab, and 100 [micro]g/g/crab, respectively. Challenge tests were conducted 1 h after the injection of 20 [micro]L bacterial suspension (3.6 x [10.sup.10] cfu/mL/crab) at the base of the third pereiopod, resulting in 7.2 x [10.sup.8] cfu/crab. The crabs that received no histamine but were infected with A. hydrophila at a level of 7.2 x [10.sup.8] cfu/crab served as the challenge control. The crabs that received histamine at 100 [micro]g/g/crab and then saline (20 [micro]L) served as the unchallenged control (Table 1). Two experimental or initial crabs were kept in each of the 27 x 20 x 18-cm plastic casings containing 4 L freshwater. There were a total of 5 treatments, each with 15 replicate casings (30 crabs). Crab survival was observed every 12 h throughout the experimental period of 96 h. The autotomy rate and spasm rate of the crabs during the experiment were calculated as follows:

Autotomy crab rate = Number of autotomy crabs/Number of intact crabs x 100

Autotomy pereiopod rate

= Number of autotomy pereiopods/Number of total pereiopods x 100

Spasm rate = Number of spasm crabs/Number of normal crabs x 100

Experiment II

Immune Parameters of E. sinensis Injected with Histamine

The procedure for E. sinensis injection was similar to that for experiment I. There were 4 treatments--saline (group 1), 1 [micro]g/g histamine/crab (group 2), 50 [micro]g/g histamine/crab (group 3), and 100 [micro]g/g histamine/crab (group 4)--with 3 replicate groups per treatment. Six crabs were sampled at each of the 3 times (6 h, 12 h, and 24 h) for each treatment, with a total of 18 crabs. In addition, 6 crabs without any treatment were used as the initial group (group 0). Two crabs were placed in a separate casing (average weight, 1.01 [+ or -] 0.10 g). The experimental setup of the initial group was similar to that for experiment I.

At 6 h, 12 h, and 24 h after the injection, 5 crabs from 3 casings in each group were sampled randomly. The hemolymph from the 5 crabs in each group was pooled, and there were 3 samples for each treatment. The immune parameters THC, PO, ACP, AKP, and SOD of hemolymph of E. sinensis were measured. The immune parameters at 0 h were measured from the initial group (group 0).

Hemolymph (200 [micro]L) was withdrawn from the base of the third pereiopod of each crab using a 1-mL sterile syringe; added quickly to a 1-mL Eppendorf centrifuge tube containing 200 [micro]L anticoagulant solution (27 mmol/L sodium citrate, 336 mmol/L NaCl, 115 mmol/L glucose, and 9 mmol/L EDTA, at a pH of 7.0) (Rodriguez et al. 1995); and then homogenized using a Whirlpool vortex mixer. The diluted hemolymph was placed into a hemocytometer and THC was recorded using a microscope (Leica DM2500).

An aliquot of the hemolymph was collected without anticoagulant and kept at 4[degrees]C for 5 min. The resulting clot was broken up using a micropestle and was centrifuged repeatedly at 6,000g rpm at 4[degrees]C for 20 min. The supernatant was stored at -80[degrees]C for later analysis of the immune parameters.

The PO activity of the hemolymph was recorded by measuring the L-3, 4-dihydroxyphenylalanine (L-DOPA) conversion to dopachrome at 492 nm. Briefly, 20 [micro]L supernatant was preincubated with 20 [micro]L trypsin (1 mg/mL) for 30 min at 37[degrees]C in 96-well microtiter plates. To each sample, 20 [micro]L L-DOPA (3 mg/mL) was added, and then each sample was incubated for 5 min at 37[degrees]C. Distilled water (260 [micro]L) was added to slow the reaction. The activity of PO was detected spectrophotometrically using a microplate reader, measuring the formation of the red pigment DOPA-chrome after 5 min, 10 min, 20 min, and 60 min of absorbance at 492 nm. For the control, L-DOPA alone was incubated with 0.45 M NaCl, and spontaneous oxidation was monitored. The PO activity was expressed as units per milligram protein.

The activities of ACP, AKP, and SOD were assayed using the corresponding detection kits (Nangjing Jiancheng Bioengineering Institute, China) according to the manufacturer's guidelines.

The total protein content was determined using the Bradford method, with bovine serum albumin as the standard (Bradford 1976). All the immune parameters were expressed as values relative to the total protein content.

Statistical Analysis

The data (mean [+ or -] SEM) from the control and treatment groups were subjected to a 1-way analysis of variance (ANOVA) and compared using Tukey's test (P < 0.05). All statistics were performed using the SPSS 11.0 software package.


Effects of Histamine on the Survival of E. sinensis Infected with A. hydrophila

There was no mortality in the unchallenged control groups; however, mortality occurred after challenged with A. hydrophila. After 12-36 h of histamine injection, the survival of bacteria-infected crabs was not significantly different from those in the control group, whereas a difference was observed among the 3 histamine treatments. At 12 h, the survival of the l-[micro]g histamine/g/crab group decreased by 24% (P < 0.05) relative to that of the group with 100 [micro]g histamine/g/crab. Among the histamine groups, survival increased as the histamine concentration increased. Later, at 24 h and 36 h, survival was not significantly different among the histamine-treated groups. Beginning at 48 h, the survival in the groups of 1 [micro]g histamine/g/crab and 50 [micro]g [micro]g histamine/g/crab was significantly different from that of the control group. At 48 h, the survival in the group with 50 lag histamine/g/ crab decreased dramatically by 43.75% (P < 0.05), compared with the control. From 60-96 h, survival in the groups of 1 [micro]g histamine/g/crab and 50 [micro]g histamine/g/crab was significantly lower than that of the control group (P < 0.05), and the survival in the group of 100 [micro]g histamine/g/crab was significantly higher than that of the 50 [micro]g histamine/g/crab (P < 0.05). From 12-96 h, survival of the 100-[micro]g histamine/g/ crab group was not different from that of the control group (Table 1).


After the injection of histamine, some of the crabs quickly released their pereiopods and began to twitch; this phenomenon was not observed in the control group. The number of the crabs with autotomy and spasm increased with increasing histamine concentration (Table 2). After 3 h, the crabs received an injection of 1 [micro]g/histamine/g stopped spasm and returned to normal, but the spasm rates of the challenged groups of 50 [micro]g histamine/g/crab and 100 [micro]g histamine/g/crab were still higher (66.67% and 80.00%, respectively) than that of the control. All crabs returned to normal at 5 h (Table 2).

Effects of Histamine on the Immune Parameters of E. sinensis

After 6 h, THC decreased in groups 3 (50 [micro]g/g histamine) and 4 (100 [micro]g/g histamine). In particular, THC of group 3 was significantly different from that of the saline control (group l; P < 0.05). After 12 h, THC in groups 2 and 3 decreased significantly by 25.94% and 22.62%, respectively, whereas that of group 4 increased dramatically--3-fold--compared with group 1. No significant difference in THC was observed among the 4 treatments at 24 h. Mean THC was 20.7 [+ or -] 4.01 x [10.sup.6] cells/ mL in group 0 (Fig. 1A).

Although THC in the groups of crabs injected with 1 [micro]g/g histamine (group 2), 50 [micro]g/g histamine (group 3), and 100 [micro]g/g histamine (group 4) increased compared with that of the control (group 1) at 6 h, 24 h, and 12 h, respectively, there was no significant difference, except for the 100-[micro]g/g group, which increased dramatically at 12 h (Fig. 1A).

At each measurement time, the histamine-injected groups had significantly greater PO activity compared with that of the saline control (group 1; P < 0.05). In addition, at 6 h and 12 h, groups 2 and 4 showed dramatically elevated levels compared with those of group 3 (Fig. 1B). At 24 h, PO activity in the histamine-treated groups correlated negatively with the histamine concentration.

At 6 h after histamine injection, ACP activity in crabs in all histamine-treated groups decreased by 55.02% (group 2), 73.71% (group 3), and 8.11% (group 4) compared with the control (group 1), and there were marked differences among the histamine-treated groups. At 12 h, ACP activity showed a dose-dependent pattern in the histamine-treated groups (Fig. 2A). There was no difference between the histamine-treated groups and the saline control at 24 h.

A similar trend was observed for AKP activity of the crabs at 6 h, whereas AKP activity showed no difference from the saline control at 12 h. AKP activity exhibited no significant trends at 24 h. The ACP and AKP activity of all other groups were significantly less than the initial values of 7.77 [+ or -] 0.56 U/mg protein and 4.61 [+ or -] 0.38 U/mg protein (Fig. 2A, B).


At each measurement time, all the histamine-treated groups had greater SOD activity than that of the control group. At 6 h after the injection of histamine, the SOD activity of groups 3 and 4 increased significantly (P < 0.05) compared with group 1, and SOD activity increased as the histamine concentration increased. At 12 h and 24 h, SOD activity increased (P < 0.05) significantly in groups 2 and 4 compared with the control group. SOD activity in all histamine-treated groups at 24 h was significantly lower than that of the initial value of 7.22 [+ or -] 0.94 U/mg protein (Fig. 2C).



All the unchallenged control crabs survived for 96 h, suggesting that histamine alone had no influence on the survival of the crabs. In other studies, all the white shrimp (Litopenaeus vannamei) injected with dopamine or noradrenaline also survived (Li et al. 2005, Cheng et al. 2006). However, some of the crabs infected with A. hydrophila died after they were injected with histamine. From 60-96 h, the survival of crabs in the histamine-treated groups (1 [micro]g histamine/g and 50 [micro]g histamine/g) showed a significant reduction compared with that of the control, which indicates that histamine decreased survival of the A. hydrophila-infected crabs. Similar results were observed using other biogenic amines, such as dopamine and noradrenaline, in the white shrimp L. vannamei (Li et al. 2005, Cheng et al. 2006).

Furthermore, the survival rate was not different between the histamine-treated group and the control from 12-36 h. After 36 h, a difference was seen between the treatment and control groups. Our results suggest that the effect of histamine on the survival of crabs required more than 36 h. This time may be related to metabolism and the action of histamine. Histamine metabolizes slowly and accumulates in the tissue (Stuart et al. 2007). However, Kennedy and Karunasagar (2004) showed that mortalities at 48 h were similar in the tiger shrimp Penaeus monodon infected with Vibrio alginolyticus fed with a histamine-containing diet for 25 days (long term) and 3 days (short term). Some of the histamine in the feed may be lost through leaching. Tapia-Salazar et al. (2001) found that histamine levels in the diet were reduced by 72-75% after 1 h. In the current study, histamine was injected directly into the crabs. No difference in the survival of the crabs with 100 [micro]g histamine/g was observed compared with that of the control from 12-96 h, although the survival rate was higher than that of crabs with 50 gg histamine/g from 48-96 h. No similar phenomenon has been reported in other crustaceans. Therefore, histamine may act in different pathways to regulate physiological events from the other biogenic amines in crustaceans (Sainath & Reddy 2010).


In the current study, interesting findings in the group of the 100-[micro]g/g dose suggest that histamine concentration is very important. Hornyak et al. (2005) reported that high levels of histamine can improve survival in mice by regulating cytokines and by blocking immune suppression. Similarly, Tripathi et al. (2011) observed that high-dose (200 [micro]g/kg) histamine can increase the serum levels of AKP and aspartate aminotransferase levels in immunized rabbits. In humans, high concentrations of histamine have been shown to trigger the respiratory burst of neutrophils (Benbarek et al. 1999). In contrast, low concentrations of histamine alone did not stimulate superoxide anion production in neutrophils (Seligmann et al. 1983), and these immune responses had a close relationship with health. So, the histamine concentration was proved to be an important factor in affecting the survival of vertebrates.

In invertebrates, the effect of biogenic amine on physiological function exhibits concentration dependency. Octopamine (OA) has different effects on hemocyte function in different concentrations in insects (Dunphy & Downer 1994). Low-dose OA decreased hemocyte number, and high-dose OA increased hemocyte number in the insect Galleria mellonella (Dunphy & Downer 1994). Tapia-Salazar et al. (2001) also found that different concentrations of histamine have different effects on the growth of L. stylirostris. Weight gain of the shrimp was maximum at 1,200 mg/kg and 2,400 mg/kg dietary histamine, whereas less gain was found at higher doses (Tapia-Salazar et al. 2001). Available evidence indicates that high levels of histamine could increase the release of nitric oxide to protect organisms from bacteria (Peh et al. 2001), and nitric oxide has various effects on immunological functions, such as lymphocyte adhesion, cytotoxicity, and cytokine production, in both vertebrates and invertebrates (Bolton et al. 2000). Moreover, resistance of insects to infection increased when the pathogen was co-incubated with OA (Baines et al. 1992). In the current study, whether the histamine was co-incubated with bacteria is unknown.


Interestingly, in the current study, autotomy was observed in the histamine-treated crabs. No corresponding phenomena have been reported for other biogenic amines. The results suggest that histamine can lead the crabs to release pereiopods. Previous studies have reported that autotomy was a known response mechanism to avoid predators and limit wounds (Wood & Wood 1932), and the autotomy rate was suggested to be an indication of physiological stress (Wood & Wood 1932, Rome et al. 2005). In the current study, the autotomy rate increased with increasing histamine concentration, indicating that high levels of histamine were linked to severe physiological stress. Through autotomy, energy may be saved, and vital activities may be maintained for longer survival (Rome et al. 2005). Autotomy could explain the short-term survival observed. The suitability of limb regeneration as a biomarker has been proposed previously, and the rate of pereiopod regeneration can serve readily as an assay for the presence of environmental contaminants (Clare et al. 1992). However, further study is needed to determine whether the autotomy of crabs can be used as a biomarker for monitoring histamine levels.

The survival of the crabs with 1 [micro]g histamine/g, 50 [micro]g histamine/g, and 100 [micro]g histamine/g correlated positively with the number of crabs with autotomy and the rate of pereiopod loss at 12 h. These results suggest that the crabs could maintain homeostasis by releasing pereiopods to cope with high levels of histamine, and increased their survival at 12 h compared with the control. Schmiege et al. (1992) observed the ultrastructure of autotomy-induced atrophy of muscles in the crab Carcinus maenas, such as extensive myofilament erosion and loss of organelles. Furthermore, the effect of histamine could be cumulative, and eventually affects the survival of the animals (Yang et al. 2010). These results suggest that survival has an important relationship with autotomy and histamine concentration.

Immune Parameters

The functions of hemocytes include recognition, phagocytosis, melanization, cytotoxicity, and cell-cell communication. Therefore, the total cell counts (THC) demonstrate a change with immunological parameters (Ellis et al. 2011, Matozzo et al. 2011). PO is the key enzyme in the melanization process, as the terminal enzyme acting in the recognition and effect or component of the arthropod defense system (Matozzo et al. 2011). ACP is a typical hydrolase that plays a key role in digesting extracellular invaders. AKP is a polyfunctional enzyme that plays a role in immune defense (Ellis et al. 2011, Matozzo et al. 2011, Tripathi et al. 2011). SOD is the first and most important line of defense against scavenging reactive oxygen species (ROS), and it protects tissues from oxidative damage (Lee & Soderhall 2002, Medina et al. 2009). Therefore, THC, PO, ACP, AKP, and SOD activity are essential components of the immune response in crabs.

There is a dynamic flux between circulating hemocytes and noncirculating hemocytes (Coles et al. 1995). In the current study, THC decreased at 6 h after the histamine (50 [micro]g and 100 [micro]g) injection. Previous studies demonstrated that quick hemocyte proliferation was difficult, and a great number of hemocytes was more likely to mobilize immediately from the hemolymph to heal the damaged location promptly (Matozzo et al. 2011). Interestingly, in the current study, we also found that histamine can lead the crabs to autotomy in a short time. Hemocytes were responsible in part for repairing the wound caused by autotomy and, therefore, THC of the circulation was less than that of the saline control at 6 h after histamine injection.

In mammals, histamine has been reported to modulate hematopoiesis, possibly during the immune response, and has a relationship with proliferation and apoptosis (Dy & Schneider 2004). Therefore, histamine could regulate the immune response through the availability of hemocytes in vertebrates. In invertebrates, there was a positive correlation between THC and PO activity in biogenic amine studies (Seligmann et al. 1983, Cheng et al. 2005). Matozzo and Marin (2010) also reported that the hemocytes of Carcinus aestuarii participated in PO production. Previous studies demonstrated a decrease of PO activity followed a decrease of THC in white shrimp (L. vannamei) injected with biogenic amines such as dopamine or noradrenaline (Cheng et al. 2005, Cheng et al. 2006). However, in the current study, PO activity increased with decreasing THC, indicating that the increase in PO activity did not stem from THC, but may be assumed to come from other tissues. It has been demonstrated that the prophenoloxidase transcript was expressed in the hepatopancreas, heart, mid gut, and other locations, and PO activity was also found in these locations (Wang et al. 2006). It is well known that declawed crabs showed significantly greater PO activity in their hemolymph after 1 day and 3 days; PO was produced to deposit melanin around the damaged tissues and participated in wound repair (Matozzo & Marin 2010). Contrary to our expectations, the particular response of autotomy, similar to declawing, was observed in the histamine-treated groups in the current study, differing from the results with other biogenic amines. Therefore, the damage resulting from autotomy may be a key explanation for elevated PO activity.

Studies have found that ACP and AKP have an important role in immune defense (Ellis et al. 2011, Matozzo et al. 2011). In the current study, ACP and AKP activity of the crabs in all the histamine-treated groups was significantly less at 6 h than that seen in the control group. However, in another study, during the incubation of Tetrahymena pyriformis in histamine-treated cultures, phosphatase activity increased significantly, and histamine was identified as a phagocytosis stimulation that affected the phosphatase synthesis and phagocytosis of hemocytes, which was a part of immune defense (Kovacs & Csaba 1990, Ellis et al. 2011, Matozzo et al. 2011). In immunized rabbits, all the histamine-treated groups showed a different increase in the AKP profiles (Tripathi et al. 2011). The results suggested that histamine could change ACP and AKP activity. These differences may be attributed to species specificity.

SOD is considered to eliminate ROS during an immune response (Lee & Soderhall 2002, Medina et al. 2009). In the current study, SOD activity of the histamine-treated crabs increased significantly compared with that of the controls. This increase in SOD activity is thought to be the result of the increased production of superoxide anions (Medina et al. 2009). It has been reported that histamine could suppress ROS generated on phagocytic cells (Lee & Soderhall 2002). In the current study, with an increasing concentration of injected histamine, the SOD activity increased at 6 h. These results show that histamine could improve SOD activity to diminish ROS and protect the body from damage. Among other biogenic amine studies, Aladaileh et al. (2008) also found that noradrenaline can increase the production of the superoxide anion and peroxide in the hemocytes of the Sydney rock oyster. However, our study showed that the value of SOD activity after injection decreased significantly compared with that of the initial group. Published information shows that the endogenous (tissue) histamine level can increase with the exogenous histamine (dietary) level, as seen in N. japonica (Yang et al. 2010). These variations may explain the finding that the accumulated histamine damage the ability to respond to reactive oxygen intermediates with time, which subsequently cause oxidative damage and led to the observed decline. However, further research is needed to support this hypothesis.

Crabs with histamine concentrations of 1 [micro]g/g and 100 [micro]g/g showed markedly elevated immune parameters compared with those with the 50-[micro]g/g treatment, especially those with high levels of histamine (100 [micro]g/g). At 12 h, high-level histamine (100 [micro]g/g) increased THC by almost 3-fold, and PO and SOD activity reached their peak levels. ACP activity of the crabs with 100 [micro]g/g histamine showed an abrupt increase at 12 h. These results suggest that histamine demonstrates transient immuno-stimulation, which may explain why the survival rate of the crabs with high levels of histamine was similar to that of the control crabs. However, limited information is available concerning the histamine immune response in this crustacean species. Other biogenic amines (such as OA) also had different effects on hemocyte function in insects (Dunphy & Downer 1994). In previous studies, histamine elevated the growth of shrimp (L. vannamei and L. stylirostris) to a moderate level (Tapia-Salazar et al. 2001, Ricque-Marie et al. 1998). Therefore, we speculate that histamine concentration must reach a critical point to influence the immune functions of the crabs, which helps to choose the appropriate quantity of trash fish to feed E. sinensis and control the disease.

In conclusion, histamine alone had no influence on the survival of normal crabs, but histamine increased the survival of the crabs infected with A. hydrophila. Moreover, histamine did modulate some of the tested immune parameters, with increased PO and SOD activity and decreased THC, ACP, and AKP activity at 6 h. Autotomy of the crabs after histamine injection was seen, which might be a key explanation for the difference between histamine and other biogenic amines in their regulation of patterns of immune function. However, further study is needed to determine whether the autotomy of crabs can be used as a biomarker for monitoring histamine levels and whether there is critical histamine concentration to influence the immune functions of crabs.


This research was supported by a grant from the Shanghai Post-graduate Education Innovation Program (B-9400-100006-2) and the Innovation Research Group Developing Project at the University of Shanghai and NSFC (no. 30871927). We thank Peng Fan and Lili Yang for their support during the sampling. We Mr. Shen, Manager, Muyu Crab Farm, for help with the sample supply.


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Key Laboratory of Exploration and Utilization of Aquatic Resources and Aquaculture Division, E-Institute of Shanghai Universities, Shanghai Ocean University, 999 Huchenghuan Road, Lingang New District, Shanghai 201306, China; College of Fisheries and Life Science, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, 999 Huchenghuan Road, Shanghai, 201306, China

([dagger]) These authors contributed equally to this work.

* Corresponding author. E-mail: chengyongxucrablab@hotmail.com

DOI: 10.2983/035.031.0329
Effect of histamine administration on the survival of E. sinensis
challenged with A. hydrophila.

                                  Cumulative Survival (%),
                                  Time after Challenge (h)
Histamine          Bacteria
([micro]g/g)      (cfu/crab)                 12

100                 Saline        100.00 [+ or -] 0.00 (a)
Saline         7.2 x [10.sup.8]    76.67 [+ or -] 0.06 (bc)
1              7.2 x [10.sup.8]    63.33 [+ or -] 0.06 (a)
50             7.2 x [10.sup.8]    73.33 [+ or -] 0.05 (bc)
100            7.2 x [10.sup.8]    83.33 [+ or -] 0.10a (b)

               Cumulative Survival (%), Time after Challenge (h)
([micro]g/g)              24                         36

100            100.00 [+ or -] 0.00 (a)   100.00 [+ or -] 0.00 (a)
Saline          70.00 [+ or -] 0.00 (b)    60.00 [+ or -] 0.00 (b)
1               60.00 [+ or -] 0.00 (b)    46.67 [+ or -] 0.06 (b)
50              60.00 [+ or -] 0.00 (b)    50.00 [+ or -] 0.08 (b)
100             70.00 [+ or -] 0.10 (b)    60.00 [+ or -] 0.00 (b)

               Cumulative Survival (%), Time after Challenge (h)
([micro]g/g)              48                          60

100            100.00 [+ or -] 0.00 (a)    100.00 [+ or -] 0.00 (a)
Saline          53.33 [+ or -] 0.12 (bc)    50.00 [+ or -] 0.17 (b)
1               33.33 [+ or -] 0.06 (cd)    23.33 [+ or -] 0.10 (cd)
50              30.00 [+ or -] 0.08 (d)     16.67 [+ or -] 0.05 (d)
100             56.67 [+ or -] 0.06 (b)     46.67 [+ or -] 0.06n (bc)

               Cumulative Survival (%), Time after Challenge (h)
([micro]g/g)              72                           84

100            100.00 [+ or -] 0.00 (a)    100.00 [+ or -] 0.00 (a)
Saline          46.67 [+ or -] 0.15 (b)     43.33 [+ or -] 0.11 (b)
1               10.00 [+ or -] 0.10 (c)     10.00 [+ or -] 0.10 (c)
50              10.00 [+ or -] 0.00 (c)     10.00 [+ or -] 0.00 (c)
100             40.00 [+ or -] 0.10 (b)     30.00 [+ or -] 0.10 (b)

               Cumulative Survival (%), Time after Challenge (h)
([micro]g/g)              96

100            100.00 [+ or -] 0.00 (a)
Saline          43.33 [+ or -] 0.12 (b)
1               10.00 [+ or -] 0.10 (cd)
50               6.67 [+ or -] 0.055 (d)
100             30.00 [+ or -] O.10 (bc)

The data in the challenged groups in the same column with
different superscripts are significantly different (P < 0.05)
among treatments.  The values are the mean [+ or -] SE (n = 30
crabs in each case).

Effects of autotomy rates and spasms in E. sinensis after the
histamine injections.

                                          After Injection:
                    Before Injection        Autotomy Crab
                      (no. intact)            Rate (%)

100 + saline               30          50.00 [+ or -] 0.10 (c)
Saline + bacteria          30           0.00 [+ or -] 0.00 (a)
1 + bacteria               30          16.67 [+ or -] 0.06 (b)
50 + bacteria              30          50.00 [+ or -] 0.00 (c)
100 + bacteria             30          53.33 [+ or -] 0.06 (c)

                    Before Injection      After Injection:
                     (total no. of       Autotomy Pereiopod
                      pereiopods)             Rate (%)

100 + saline              300          12.00 [+ or -] 0.01 (c)
Saline + bacteria         300           0.00 [+ or -] 0.00 (a)
1 + bacteria              300           1.67 [+ or -] 0.01 (b)
50 + bacteria             300           9.67 [+ or -] 0.02 (c)
100 + bacteria            300          12.67 [+ or -] 0.04 (c)

                                      Spasm Rate
                                   after Injection

                             3 h                     5 h

100 + saline        60.00 [+ or -] 0.00 (b)   0.00 [+ or -] 0.00
Saline + bacteria    0.00 [+ or -] 0.00 (a)   0.00 [+ or -] 0.00
1 + bacteria         0.00 [+ or -] 0.00 (a)   0.00 [+ or -] 0.00
50 + bacteria       66.67 [+ or -] 0.12 (b)   0.00 [+ or -] 0.00
100 + bacteria      80.00 [+ or -] 0.10 (c)   0.00 [+ or -] 0.00

The data in the challenged groups in the same column with
different superscripts are significantly different (P < 0.05)
among treatments.

The values are the mean t SE (n = 30 crabs in each case).

Autotomy Crab Rate = Number of autotomy crabs/Number of intact
crabs x 100%.

Autotomy Pereiopod Rate = Number of autotomy pereiopods/Number of
total pereiopods x 100%.

Spasm Rate = Number of spasm crabs/Number of normal crabs x 100%.
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