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Endoenzymes and mitochondrial membrane potential associated with hemocyte types in Macrobrachium rosenbergii.


Freshwater prawns Macrobrachium rosenbergii, having high commercial value and suitability for use in a variety of culture systems, are an important culture fishery species in China and in other Southeast Asian countries (Roustaian et al. 2000, Tidwell et al. 2000). The yearly production of cultured freshwater prawns has increased quickly since 2000 in China. With rapid development of intensive culture, some serious diseases have been reported in M. rosenbergii (Saurabh & Sahoo 2008, Liang et al. 2011). Hemocytes of crustacean play an important and central role in the internal defense. The defense reactions include the hemocytic process of encapsulation, prophenoloxidase-activating system, phagocytosis, and the microbicidal mechanism based on the production of cytotoxic reactive oxygen intermediates and antimicrobial peptides (Bachere et al. 1995).

Endoenzymes may play an important role to kill or digest the pathogen in the process of phagocytosis. Peroxidase (POD) can be discharged into the phagosomes in the process of phagocytosis and is a part of microbicidal system. So it is an important enzyme for killing invading microorganisms in invertebrates (Afonso et al. 1999). Phenoloxidase (PO) can catalyze oxidation of phenol to form the melanin. The enzymatic reactions in turn produce a set of intermediate products such as quinones, diphenols, superoxide, hydrogen peroxide, and reactive nitrogen intermediates. These products are important during defense against bacterial, fungal, and viral agents. The enzyme has been demonstrated in various animal tissues and cell types, including melanocytes, neurons, and blood cells (Coles & Pipe 1994). Alkaline phosphatase (ALP) is an important constituent of the lysosomes of phagocytes (Olsen 1991). Information regarding the endoenzymes in hemocytes of freshwater prawn species is, however, scarce.

Mitochondrial membrane potential (MMP) is the central parameter that controls mitochondrial respiration, ATP synthesis, and [Ca.sup.2+] accumulation (Nicholls & Ward 2000) as well as the generation of reactive oxygen species (Boveris et al. 1972, Van Belzen et al. 1997). And the reactive oxygen species have long been known to be a component of the killing response of immune cells to microbial invasion. Therefore, the ability to determine MMP can provide important clues about the physiological status and function of the cells; however, little is known about MMP of the hemocyte in Macrobrachium rosenbergii.

The purpose of this study was to characterize the POD, PO, and ALP activities and the MMP in hemocyte subpopulations of Macrobrachium rosenbergii.



Macrobrachium prawns, Macrobrachium rosenbergii, with lengths of 11-14 cm, were obtained from Huangsha Live Seafood Wholesale Market (Guangzhou, Guangdong Province, People's Republic of China) and were maintained in the laboratory in 150-1 aquaria supplied with a constant flow of fresh water with controlled light (12 h light/12 h dark) and temperature (23-25[degrees]C) regime. The prawns in intermoult stage were used for all experiments. Hemolymph samples were drawn from the pericardial sinus using a 25-gauge needle and a 1-ml syringe containing an equal volume of modified Alsever solution (27 mM sodium citrate, 336 mM NaCl, 115 mM glucose, 9 mM ethylenediaminetetraacetic acid, and pH 7.0) used as anticoagulant. This buffer also prevented melanization and kept hemocytes in a quiescent state (Rodriguez et al. 1995).

Hemocyte Types

Hemocytes were directly analyzed after collection using a flow cytometer (BD FACSCalibur, Becton Dickinson). Two-parameter cytograms based on forward scatter height (FSC) and size scatter height (SSC) of unlabeled cells were designed. For each hemocyte sample, at least 10,000 events were counted. Results were expressed as dot plot or density cytograms indicating the size (FSC value) and the complexity (SSC value). Morphologically different subpopulations including hyalinocytes, semigranulocytes, and granulocytes were defined by their relative size and subcellular complexity. These subpopulations were electronically gated and sorted, and sedimented for the light microscope examination after Wright staining.

Enzyme Cytochemical Staining

POD Stain

Hemocyte smears were incubated for 35 min at 20[degrees]C in a medium (pH 7.6), containing 0.5 mg/ml diaminobenzidine (Sigma), 0.2 M Tris-HCl buffer, and 0.02% hydrogen peroxide, and then washed in water, counterstained with Wrights stain (Ogawa 1989). Negative control samples were incubated in Tris-HCl buffer. The positive rate (PR) of POD for the whole hemocytes was evaluated in 20 randomly selected microscope fields in the slides. The PR of POD for the hyalinocyte (HPR) was evaluated by examining 100 hyalinocytes. The PR of POD for the granulocyte (GPR) and for the semigranulocyte (SGPR) were evaluated as for HPR.

PO Stain

Hemocyte smears were fixed in 10% formalin for 1 h at room temperature, and then washed in running water for 3-4 min. The samples were transferred to and incubated in fresh PBS with 1 mg/ml L-3,4-dihydroxyphenylalanine (l-DOPA; Sigma) for 90 min at 30[degrees]C, and then washed in distilled water (Zhang & Li 2000). The negative control sample was incubated in PBS without l-DOPA. The PR of PO was calculated as for POD.

ALP Stain

Alkaline phosphatase was demonstrated by using NBT/BCIP as substrate (Sambrook et al. 1999). Hemocyte smears were incubated in a mixture medium of NBT/BCIP (Sigma), containing 33 mg NBT and 16.5 mg BCIP in 10-ml ALP buffer (100 mM sodium chloride, 5 mM magnesium chloride, 100 mM Tris-HCl, and pH 9.5), at 30[degrees]C for 20 min. The slides were washed with PBS, counterstained with Wrights stain. Negative control samples were incubated in ALP buffer without substrate. The PR of ALP was calculated as for POD.

Biochemical Analysis

Hemolymph of 20 prawns was pooled. Hyalinocytes, semi-granulocytes, and granulocytes were sorted with the flow cytometry (FCM), and collected. The cell suspensions of each of the hemocyte types were adjusted to a concentration of about [10.sup.5] cells/ml for biochemical analysis of endoenzymes.

Activity of POD

POD enzyme assay was carried out as described by Hammerschmidt et al. (1982). For the assay, 100 [micro]l of cell suspension was mixed with 200 [micro]l of 0.01% hydrogen peroxide and 1 ml of 0.05% diaminobenzidine 4HC1 and incubated at 37[degrees]C for 1 h, then kept at room temperature for 30 min. The reaction was measured spectrophotometrically at 470 nm. The POD activity was expressed as the absorbance increase per min ([DELTA]OD470/min). As a standard for the POD assay, known concentrations of horseradish POD (Gibco) were assayed using the same conditions as those for the cell suspensions.

Activity of PO

PO activity was assayed according to procedures described by Sung et al. (1994) using l-DOPA as a substrate. For the assay, 100 [micro]l of cell suspension were mixed with 500 [micro]l l-DOPA (3 mg/ml) and 500 [micro]l Tris-HCl (50 mM, pH 7.5). Then, the absorbance or optical density was measured at a wavelength of 490 nm. For these experiments, 1 unit of enzyme activity was defined as an increase in absorbance of 0.001/min/mg protein (Soderhall & Unestam 1979). Protein contents of the hemocyte suspensions were determined by the Lowry assay, with bovine serum albumin as a standard.

Activity of ALP

ALP activity was determined as described by Feng (1996). Briefly, 20 [micro]l of cell suspension and 1 ml of N-methyl-D-glucamine buffer (560 mM N-methyl-D-glucamine, 78.6 mM NaCl, 0.56 mM magnesium acetate tetrahydrate, and pH 10.1) were mixed at 37[degrees]C. Immediately after the addition of 100 [micro]l p-nitrophenyl phosphate (Sigma) as substrate, the absorbance at 405 nm was measured by spectrometer for 300s. The absorbance increase per min (AOD405/min) was used to calculate the activity of ALP.

MMP Assessment

In this experiment, 24 samples of Macrobrachium rosenbergii were used. Three hemocyte subpopulations were electronically gated, allowing the evaluation of MMP assessment for each cell type. The cationic lipophilic dye, 3',3'-dihexyloxacarbocyanine iodide (DIOC6; Sigma), had been used to probe MMP in living cells (Chen 1988). This cyanine dye could accumulate in the mitochondrial matrix under the influence of MMP. The DIOC6 was added to hemocyte suspension at a final concentration of 2 mM. After incubation for 5 min at room temperature, hemocytes were analyzed by FCM. The results were reported as fluorescence histograms.

Statistical Analyses

The SPSS software and the Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA) were used in the statistical analysis. The abundance of the hemocyte subpopulations was analyzed using Cell Quest software. Differences in MMP among the three different hemocyte subpopulations were assessed using a one-way analysis of variance with the Student-Newman-Keuls test. Data were presented as mean [+ or -] SD, and P < 0.05 was used for significance.


Hemocyte Types

Three different morphologic subpopulations could be defined based on the relative size and subcellular organelle characteristics (complexity). The FSC alone (linear scale) could distinguish a group of small cells (R1), having FSC values less than half of those of the larger cells. The two groups of larger cells could be resolved further by SSC (logarithmic scale) with the upper group (R3) having a relative higher SSC value than the lower group (R2) (Fig. 1). Cells electronically gated were also sorted for histological examination. Wright staining results showed that granulocytes (R3) with the small nucleus and plenty of cytoplasm were the most complex and usually oval or spherical in shape and larger than hyalinocytes. Flyalinocytes (R1) were spherical and the smallest with centrally placed and prominent nucleus and thin cytoplasm. A distinct population of cells had morphological characteristics, which were intermediate between granulocytes and hyalinocytes, and herein designated as semigranulocytes (R2) that were usually spindleshaped with an expanded cytoplasm and larger than hyalinocytes (Fig. 2). The abundance of the hemocyte subpopulations was analyzed using Cell Quest software of FCM. The mean percentage (n = 12) of hyalinocytes, semigranulocytes, and granulocytes were 53.586 [+ or -] 4.7698, 15.509 [+ or -] 1.7846, and 16.946 [+ or -] 2.3621, respectively.

Enzyme Cytochemistry

POD Stain

Sites of POD activity occurring throughout the cytoplasm of all three types of hemocytes appeared dark brown in color (Fig. 3). No brown staining was found in the samples incubated in negative control, and the numbers of POD-positive cells were variable in three types of hemocytes. The PR of POD was 51%, comprising 10% hyalinocytes, 19% semigranulocytes, and 22% granulocytes (Fig. 4). The FIPR of POD was 25%, SGPR was 52%, and GPR was 55% (Fig. 5).

PO Stain

Hemocytes stained positive for PO appeared a fine gray to black granular deposit within the cytoplasm by using L-DOPA as substrate (Fig. 6). Unstained cells appeared a faint brown because of the presence of the chromogenic substance. The PR of PO was 36%, comprising 6% hyalinocytes, 18% semigranulocytes, and 12% granulocytes (Fig. 4). The HPR of PO was 10%, SGPR was 53%, and GPR was 47% (Fig. 5).

ALP Stain

The blue color existing within the cytoplasm of hemocytes reflected the activity of ALP (Fig. 7). No staining was found in the control samples. The PR of ALP was 55%, comprising 11 % hyalinocytes, 20% semigranulocytes, and 24% granulocytes (Fig. 4). The HPR of ALP was 25%, SGPR was 42%, and GPR was 56% (Fig. 5).

Biochemical Analyses

Activities of POD, PO, and ALP occurred in all three types of hemocyte. Activity of POD was 0.2 ng/ml in hyalinocytes, 0.8 ng/ml in semigranulocytes, and 1.2 ng/ml in granuloctyes. The PO activity was 0.5 units in hyalinocytes, 7 units in semigranulocytes, and 8 units in granulocytes. Activity of ALP was 3 U/l in hyalinocytes, 9 U/l in semigranulocytes, and 15 U/l in granulocytes.

Mitochondrial Membrane Potential

Circulating hemocytes from 24 prawns were analyzed for MMP. Hemocytes displayed either one peak or two peaks in the green fluorescence histograms (Fig. 8). The fluorescence intensity was directly correlated to the cell complexity. Among the hemocytes, more granular cells displayed higher fluorescence intensity than less granular hemocytes (Fig. 9). The DIOC6 fluorescence level in granulocytes was greater than that in semigranulocytes, and the latter was greater than that in hyalinocytes (Fig. 10). The fluorescence levels of the three groups were significantly different from each other based on one-way analysis of variance with the Student-Newman-Keuls test.


In crustaceans, a classification scheme has been commonly adopted with three types of circulating hemocytes (hyalinocytes, semigranulocytes, and granulocytes), usually identified with light microscopy. In this study, FCM with two simple parameters, FSC and SSC, could efficiently resolve the three subpopulations of Macrobrachium rosenbergii hemocytes. In the flow cytograms, SSC measured granularity, and FSC was a relative measure of cell size. In M. rosenbergii, granulocytes had higher relative values of SSC than did the semigranulocytes. Hyalinocytes had the smallest values of SSC. The results and flow cytograms were comparable to those of hemocytes from penaeid shrimps (Yip & Wong 2002). The present study on the abundance of the different hemocyte subpopulations showed that the hyalinocytes was the predominant cell type, followed by granulocytes and semigranulocyte. In some species of the penaeid shrimp, however, the semigranulocyte was the predominant cell type, followed by hyalinocytes and granulocytes (Yip & Wong 2002, Sun et al. 2010).

In this study, the endoenzymes were evaluated in three hemocyte subpopulations from Macrobrachium rosenbergii. Enzymes POD, PO, and ALP existed in three types of hemocytes observed by cytochemical methods. The PR of POD, PO, and ALP of semigranulocytes and granulocytes in total were relatively higher than those of hyalinocytes. Furthermore, higher SGPR and GPR of the three enzymes than HPR suggested that these enzymes were more commonly associated with semigranulocytes and granulocytes than hyalinocytes. Some cells of three types, however, appeared to lack activity, probably because hemocytes or these enzymes were in different maturation stages. In addition, biochemical analysis showed that these enzyme activities were greater in the semigranulocyte and granulocyte populations compared with the hyalinocyte population. This result might be due to the apparent presence of more granules in the cytoplasm of semigranulocytes and granulocytes. Because of higher activities of POD, PO, and ALP associated with semigranulocytes and granulocytes, these two cell types might have a stronger immunocompetence than hyalinocytes. The outcome might suggest functional differences among the hemocytes in the prawn.

Penetration of the fluorescent dye, DIOC6, within mitochondria had been used to measure the MMP in hemocytes from Eisenia foetida (Cossarizza et al. 1995) and Ostrea edulis (Xue et al. 2001) and Penaeus vannamei (Sun et al. 2010), at the single cell level by FCM techniques. This analysis provided informationon such as mitochondria number and mitochondria activity. Even though it was not clear that all mitochondria within one cell cytoplasm displayed identical membrane potential, the mitochondria-related fluorescence of DIOC6 was directly correlated to the mitochondrial activity. The present study showed that though mitochondria existed in the three types of the prawn hemocytes, there was more mitochondrial activity in the granulocytes and semigranulocytes than in the hyalinocytes.


This study was partially supported by Fund Planning Project from Tianjin Municipal Education Commission (20090621), Scientific Program of Tianjin City (15JCZDJC34000), Innovation Teams of Higher Learning Institutions of Tianjin (TD12-5018), Transformation Fund for Agricultural Science and Technology Achievements (2013GB2E000365), and Project of Department of Education of Guangdong Province (CXZD1114).


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(1) Key Laboratory of Ecology and Environment Science of Higher Education Institutes, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, 55 Zhongshan Avenue West, Tianhe District, Guangzhou 510631, China; (2) Tianjin Key Laboratory of Aqua-Ecology and Aquaculture, College of Fisheries, Tianjin Agricultural University, 22 Jinjing Road, Xiqing District, Tianjin 300384, China

* Corresponding authors. E-mails: or wanganl@

DOI: 10.2983/035.035.0230


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Author:Sun, Jingfeng; Wang, Anli; Xian, Jian-An; Lv, Aijun; Shi, Hongyue; Zhang, Shulin
Publication:Journal of Shellfish Research
Geographic Code:9CHIN
Date:Aug 1, 2016
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