The First Morphologic and Functional Characterization of Hemocytes In

Fish and Shellfish Immunology 81 (2018) 423–429

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Fish and Shellﬁsh Immunology

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Short communication The ﬁrst morphologic and functional characterization of hemocytes in Hong Kong oyster, Crassostrea hongkongensis T

Jun Lia,b,1, Yuehuan Zhanga,b,1, Fan Maoa,b, Yue Lina,b, Shu Xiaoa,b, Zhiming Xianga,b, ∗∗ ∗ Haitao Maa,b, Yang Zhanga,b, , Ziniu Yua,b, a CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, PR China b South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, PR China

ARTICLE INFO ABSTRACT

Keywords: Hemocytes are the first line of defence of the innate immune system of molluscs. For the first time hemocytes of Crassostrea hongkongensis Crassostrea hongkongensis were morphologically and functionally characterized, identifying circulating cell types Hemocytes and studying their involvement in immune responses. In the present study, two main populations, hyalinocytes ROS and granulocytes, were characterized based on the presence or absence of cytoplasmic granules, using light and Phagocytosis electron microscopy (TEM), and flow cytometry analyses. Granulocytes are 7–13 μm in diameter and present Antibacterial activity evident cytoplasmic granules, and hyalinocytes, 6–15 μm in diameter, with a few or no granules. The mean number of circulating hemocytes in the hemolymph was 2.52 × 106 cells/mL. Flow cytometry indicated that both granulocytes and hyalinocytes showed cell phagocytosis and reactive oxygen species (ROS) production. However, phagocytosis and spontaneous production of reactive oxygen species (ROS) in granulocytes are much more active compared with hyalinocytes, which demonstrated that the granulocytes are the main hemocytes involved in the immune response of Hong Kong oyster. Moreover, the cell-free hemolymph showed antibacterial activity against Vibrio alginolyticus. Our results provide the basic information of hemocytes population of Hong Kong oyster for further investigations associated with innate immunity.

1. Introduction Although there is controversy concerning morphological descrip- tions of hemocytes types, two main hemocytes types, granulocytes and It is generally accepted that invertebrates lack an acquired immune hyalinocytes (agranulocytes), are generally recognized in bivalves system, but they do possess innate immunity, which consists of both based on the presence or absence of cytoplasmic granules [13,14]. cellular and humoral components of the circulatory system [1]. The Hyalinocytes contain few or no granules, and a round well-centred invertebrate innate immune system is the first line of defense against nucleus, whereas granulocytes, which contain granules and an ec- infection by a variety of pathogens [2]. The circulating hemocytes play centric, round to ovoid nucleus [15]. From a functional point of view, a central role in innate immunity. They are involved in recognition of both hyalinocytes and granulocytes can form pseudopodia, aggregate, invading foreign substances, encapsulation responses, phagocytosis, phagocyte particles and produce reactive oxygen species. However, production of reactive oxygen intermediates and secretion of in- granulocytes show higher phagocytic and higher levels of ROS flammatory cytokines [3–7]. In addition to their role in host defense, [7,16–18]. On the contrary, some authors have suggested the presence invertebrate hemocytes also perform various important physiological of only one cell type in mollusk hemolymph. Ottaviani et al. found only functions, including nutrient digestion, transportation, and distribution, one cell type in two different stages is present in Mytilus gallopro- digestion, wound healing, detoxification processes, shell mineralization vincialis, consistently with the Mix's one-cell-type model [19]. In Mac- and excretion [8–12]. Therefore, it is fundamentally important to rocallista nimbosa, flow cytometrically characterized hemocytes ap- characterize the circulating hemocytes in order to further understand peared as a unique population, both in terms of morphology and its role in oysters. intracellular parameters, but displayed high inter-individual variability

∗ Corresponding author. South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, PR China. ∗∗ Corresponding author. South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, PR China. E-mail addresses: [email protected] (Y. Zhang), [email protected] (Z. Yu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.fsi.2018.05.062 Received 16 February 2018; Received in revised form 26 May 2018; Accepted 31 May 2018 Available online 01 June 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved. J. Li et al. Fish and Shellfish Immunology 81 (2018) 423–429

[20]. In C. rhizophorae, the different subpopulations of hemocytes may 2.3. Light microscopy in reality be different stages of one type of cell, which accumulates granules and loses complexity as it degranulates in response to en- Immediately after withdrawal from C. hongkongensis,30μl whole vironmental stress [21]. On the other hand, even the existence of three hemolymph were loaded onto acid alcohol cleaned micro-scope slides. types of hemocytes has been hypothesized. Hemocytes of the sub- After adhesion to slides, hemocytes were fixed for 20 min in 4% par- tropical oysters Saccostrea kegaki, Ostrea circumpicta, C. ariakensis and aformaldehyde dissolved in PBS and then air-dried, mounted and ob- Hyotissa hyotis were classified into three main types: granulocytes, served under Olympus BH-2 microscope equipped with a video camera hyalinocytes and blast-like cells [22,23]. In addition, three populations (JVC 3-CCD) and ImageNT and Microimage software. of hemocytes (granulocytes, hyalinocytes and intermediate cells) and (hyalinocytes, granulocytes and serous cells) were observed in C. vir- 2.4. Subpopulations analysis of hemocytes ginica and Pinctada imbricate, respectively [24,25]. C.hongkongensis, is an endemic oyster species along the coastal of Flow cytometry analysis was performed as previously described the South China, with a cultivation history of a couple of centuries [29]. After withdrawal, hemolymph was diluted 1:1 in anti-clotting [26,27]. It is found in low-salinity estuaries from the Fujian to Guangxi solution and analyzed by a “FACSCalibur” flow cytometer (Becton provinces, with populations centered in the Guangdong province. Dickinson Biosciences, USA), equipped with a laser providing excitation However, another oyster, Crassostrea gigas is a eurythermal and eur- at 488 nm. Hemocytes populations were defined based on their forward yhaline species which widely distributes around the world and adapted scatter (FSC, correlated to size) and the side scatter (SSC, correlated to to the low-temperate and high-salinity waters of northern China. Due to internal complexity) properties. A total of 10,000 events were acquired its high economic and ecological value, C. hongkongensis aquaculture for each hemolymph sample. The analysis was identically repeated on 5 has been one of the largest coastal industries in this area, and the an- adult oysters. Data were processed using Cell Quest Software” (Becton nual production has recently reached almost one million tons. To pre- Dickinson Immunocytometry Systems, USA) and were displayed as dot vent disease associated mortality and subsequent management of the plot. Hong Kong oyster farming, understanding the immune system of C. hongkongensis is crucial. Hemocytes are thought to play a central role in 2.5. Phagocytic capability of hemocytes the recognition and elimination of foreign pathogens. The characterization of hemocytes is essential to understand its immune system of C. Flow cytometry was used to measure the phagocytic activity of hongkongensis as well as its resistance to pathogenic infection. hemocytes of the Hongkong oyster as described previously [29]. To our knowledge, despite the availability of studies on the mor- Briefly, the collected hemolymph was incubated with fluorescent (FITC) phology and function of hemocytes in other molluscan species, no latex beads (1 μm, sigma, USA) for 1 h at 20 °C. After incubation, excess systematic information concerning circulating hemocytes morphology, of particles were removed by gently washing twice with PBS. Cell as- function and their involvement in immune response in the C. hon- sociated fluorescence was measured using flow cytometry (BD FACS- gkongensis has previously been reported. With the aim of providing the Calibur) at 530 nm (FL1). Hemocytes cell populations were defined first description of hemocytes from this oyster species, we characterized based on their size (FSC) and granularity (SSC) properties, and a total of C. hongkongensis hemocytes on the basis of their morphology and 10,000 events were acquired for each sample. The mean fluorescent functions, particularly potential immunological activities including the intensity in different populations was determined using the Cell Quest production of ROS, phagocytosis ability and antibacterial activity. Software.

2. Materials and methods 2.6. ROS production of hemocytes

2.1. Animals and hemolymph collection ROS were measured with the non-fluorescent probe 2′,7 ′- dichlorofluorescein diacetate (DCFH-DA) as previously described [30]. Healthy C. hongkongensis (two-year-old, shell height 10.00 cm ± Hemocytes were then incubated with 10 μmol/L DCFH-DA in the dark 0.05 cm) were obtained from an oyster culture farm in Zhuhai, at ambient temperature for 20 min according to the manufacturer's Guangdong province, China. The oysters were maintained in aerated instructions. DCFH-DA can passively diffuse into cells and be deacety- sand-filtered seawater at 23–25 °C for one week prior to the experiment. lated by esterases to form nonfluorescent 2′,7′-dichlorofluorescein During the acclimation period, the oysters were fed twice daily with (DCFH). In the presence of ROS, DCFH reacts with ROS to form green Tetraselmis suecica and Isochrysis galbana. fluorescent product DCF, which is trapped inside the cells. DCF fluor- The hemolymph was collected from the posterior adductor muscle escence was detected by flow cytometry (BD FACSCalibur); and a total of C. hongkongensis with a plastic syringe. The samples were stored in of 10,000 events were acquired for each sample. ice and added to an equal volume of marine anticoagulant (MAC1; 0.1 M glucose, 15 mM trisodium citrate, 13 mM citric acid, 10 mM 2.7. Transmission electron microscopy (TEM) EDTA, 0.45 M NaCl, pH 7.0) to prevent clotting. All subsequent analyses were performed on individual samples. Total hemolymph from at least 10 oysters was pooled for this assay. The collected hemolymph was immediately mixed and fixed using 4% 2.2. Total hemocytes count (THC) and viability assay paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1.5 h at 4 °C. The suspension was centrifuged (750 g, 10 min) and ViaCount assay was used with Guava easyCyte 5HT Flow cytometer washed three times with 0.1 M phosphate buffer. Following this, pellets (Guava Technologies, Inc., Hayward, CA, USA) to evaluate total he- were postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for mocytes count and viability of hemocytes as previously described [28]. 1 h at 4 °C. After post-fixation, the pellets were washed in the same Briefly, 20 μL of hemocytes suspension were mixed with 180 μLof buffer and the hemocytes were embedded in melted 2% agar and then Guava ViaCount reagent, and incubated for 5 min at room temperature. centrifuged (750 g, 10 min). After dehydration in a graded series of Sample acquisition and data analysis were performed using the Via- ethanol and absolute acetone, the samples were embedded in Epon. Count software module. Also, Total hemocyte count (THC) have be Ultrathin sections were mounted on the copper grid and counterstained done by hemocytometer counting and complement the cytometer data. with uranyl acetate and lead citrate. Finally, the sections were observed The 50 μl of hemolymph-anticoagulant mixture was filled into hemo- using transmission electron microscopy (Hiachi H-7650B, Japan) cytometer (Malassez chamber) and examined on light microscope. equipped with a digital camera (Morada, Soft Imaging System) and

424 J. Li et al. Fish and Shellfish Immunology 81 (2018) 423–429 image analysis software (iTEM).

2.8. Antibacterial activity

Antibacterial activity of the hemolymph was investigated using the growth curve of Vibrio alginolyticus as previous described with some modiﬁcations [31]. Brieﬂy, all bacteria were grown in LB medium at 30 °C to mid-logarithmic phase and then diluted with fresh LB medium − to 104 colony forming units (CFU) mL 1. Then, hemolymph was ex- tracted into equal volume of marine anticoagulant and then centrifuged at 300 g for 10 min to remove cells from suspension. Aliquots (100 μL) of cell-free hemolymph were added to the diluted bacteria. An equal volume of sea water was used as a control. The samples were incubated at optimum temperature and the OD600 was measured every hour for 8h.

3. Results and discussion

3.1. Cell count and viability assay Fig. 1. Light micrographs of fixed hemocytes. Granulocytes are indicated with Total hemocytes count of C. hongkongensis varied considerably black arrows and hyalinocytes with white arrows. among individuals. The mean number of circulating hemocytes in the hemolymph of C. hongkongensis (n = 10) was 2.52 ± 1.1 × 106 cells/ analysis was determined by flow cytometry [20,23,36,50]. Flow cyto- mL. As previously reported for other bivalve species, great inter-in- metry is a well-established technique in the hemocyte subpopulation dividual variability in circulating hemocytes numbers has been ob- analysis in bivalves [24]. In the present study, flow cytometry has also served. The THC of C. hongkongensis was higher than that of some bi- been employed to detect the subpopulations of hemocytes in Hong valves, such as C. gigas (up to 1.6 × 106 cells mL-1) [32], Cerastoderma Kong oyster (Fig. 2). Two distinct populations were recognized based glaucum (5.5 × 105 cells mL-1) [17], Callista chione (1.2 × 106 cells mL- on the two-parameter plots, size (FSC) and granularity (SSC). The he- 1) [33] and Tridacna crocea (0.3–2.6 × 106)[34], but it was lower than mocytes with high granularity (Region 1) were designated as granulo- that of Scylla olivacea (16.2 × 106 cells mL-1) [35], P. fucata (4.9 × cytes, and the Region 2 with low SSC value was haylinocytes. 106 cells mL-1) [36], Argopecten irradians (37.5 × 106 cells mL-1) [18], and M. edulis (5.68 × 106 cells mL-1) [37]. Various factors, both exogenous (water temperature and salinity, pollutants) and endogenous 3.3. Phagocytosis assay (age, sex and reproductive period of animals), have been suggested to influence THC in bivalves [38–40]. The differences of THC between Phagocytosis is commonly considered to be one of the most im- these species may also be caused by the different experimental proce- portant cell-mediated immune reactions in bivalves [51]. Hemocytes of dures used to determine circulating hemocytes mean number. bivalves are known to phagocytize exogenous materials such as bac- The hemocytes viability has been quantified using ViaCount assay, teria, broken cells or own necrotic cells [52]. In the present study, to which can distinguishes between viable and nonviable cells, based on estimate phagocytosis activity of hemocytes of Hong Kong oyster, the their differential permeability to two fluorescent nucleic acid stains phagocytic ability of hemocytes was measured using retention of (Annexin V and 7-AAD) in the reagent. Hemocytes in freshly drawn fluorescent FITC-latex beads in the cells. The results indicated that both hemolymph showed 92% viability and the cell viability is almost un- granulocytes and hyalinocytes showed phagocytic capacities. In line changed after 1 h from the withdrawal. with the light microscopy observation, hemocytes of C. hongkonggenesis were able to engulf fluorescent latex beads (Fig. 3A). Moreover, 3.2. Morphological and subpopulations analysis of hemocytes

With regard to circulating hemocytes types, two major hemocytes types granulocytes and hyalinocytes, were distinguished by light microscopy in the Hong Kong oyster based on the presence or absence of intra-cytoplasmic granules, respectively (Fig. 1). Granulocytes re- present 38 ± 1% of the total hemocyte populations. They vary in size from 7 to 13 μm and contained numerous granules in the cytoplasm. The hyalinocytes varied in diameter from 6 to 15 μm and were characterized by very few or no granules in the cytoplasm. Hyalinocytes have the ability for amoeboid movement. Some Hyalinocytes appeared round when attached to a glass slide, but others extended filopodia and spread over the slide. The hyalinocytes show little colored granules and a high nucleus/cytoplasm ratios. Both hemocytes populations have been identified in the oysters C. rhizophorae [41], C. gigas [42], O. edulis [42], C. virginica [43], C. ariakensis [7], S. glomerata [44], the clams R. philippinarum [45], T. crocea [34], Mercenaria mercenaria [46], and the scallop Argopecten irradians [18]. More hemocytes types have been observed bivalves, such as Perna viridis [47], Cristaria plicata [48] and Fig. 2. Flow cytometric determination of the hemocytes of C. hongkongensis. Chlamys farreri [49] in previous studies using various methods, in- Two main hemocytes populations were distinguishable: hyalinocytes and cluding light microscopy, electron microscopy, and flow cytometry. granulocytes. Two main hemocytes populations are identified on the basis of In previous works, lots of bivalves hemocytes subpopulation their FSC and SSC values: hyalinocytes and granulocytes.

425 J. Li et al. Fish and Shellfish Immunology 81 (2018) 423–429

Fig. 4. ROS production of C. hongkongensis hemocytes. Representative histogram of fluorescence showing the ROS production of two main hemocytes populations (B) Histogram of fluorescence in FL1-H channel representing the ROS production recorded in the hyalinocytes and granulocytes. Vertical bars represented the means ± S.D. (N = 5). The asterisk (**p < 0.01) indicates significant differences in hyalinocytes and granulocytes group.

important role in the cellular defense through their microbicidal capacities [53–55]. Under this circumstance, spontaneous production of ROS was measured in circulating hemocytes of the hongkong oysters using the oxidation of nonfluorescent DFCH to the highly fluorescent DCF. Numerous studies have reported that granulocytes are the main Fig. 3. Phagocytic ability of C. hongkongensis hemocytes. (A) Light micrographs hemocyte population involved in ROS production in marine bivalves of hemocytes that have engulfed latex beads with green fluorescent. (B) [45,56–58]. When revealed by the DCFH-DA staining method, the Histogram of fluorescence in FL1-H channel representing the phagocytic ac- oxidative activity determined from each hemocytes population in- tivity recorded in the hyalinocytes and granulocytes. Vertical bars represented dicated that granulocytes showed significantly higher ROS production the means ± S.D. (N = 5). The asterisk (**p < 0.01) indicates significant as compared with the hyalinocytes (Fig. 4). Such difference in ROS ff di erences in hyalinocytes and granulocytes group. production between these two types of cells could be associated with the differential involvement of ROS production pathways of the two through flow cytometry analysis, the phagocytosis of latex beads was hemocytes [7,16]. Further efforts would be required to get better un- detected in the hemocytes as an increase of the fluorescent level re- derstanding on the role of hemocytes in immunological process. gistered in the FL1-H channel (Fig. 3B). In agreement with previous studies with other bivalves [17,18], granulocytes are far more phagocytic than hyalinocytes (221.16 vs. 16.13). 3.5. TEM analysis of hemocytes

Based on the observation of cellular contents using TEM, the same 3.4. ROS production two major hemocytes types (granulocytes and hyalinocytes) were evident under TEM (Fig. 5). From the TEM, the granulocytes are small to Mollusc hemocytes have been reported to produce reactive oxygen medium in size and elongated, rounded, or spindle-shaped. The nucleus species (ROS) [7]. The production of ROS in hemocytes plays an generally presented in lobate. The granulocytes showed denser

426 J. Li et al. Fish and Shellfish Immunology 81 (2018) 423–429

0.25 Haemolymph Sea water 0.2

0.15

0.1 OD 600 nm

0.05

0 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h

Vibrio alginolyticus

Fig. 6. Antibacterial activities of hemolymph from C. hongkongensis. The growth curve of V. alginolyticus exposed to hemolymph and sea water as a ne- gative control. Bacterial growth was recorded as absorbance at 600 nm. Each point in the graph represents the mean ± S.D. (N = 5).

3.6. Antibacterial activity of hemolymph

To further investigate the immune function of hemocytes of Hong Kong oyster, antibacterial activity was measured using the cell-free hemolymph. The growth of V. alginolyticus was signiﬁcantly greater and more rapid in the control group (Fig. 6), indicating some inhibition of bacterial growth in the presence of cell-free hemolymph, which is in good agreement with previous studies on O. edulis, M. galloprovincialis [63] and Scrobicularia plana [64]. The antibacterial activity in this species is probably due to the presence of small antibacterial peptides, similar to the ones isolated from hemolymph of mussels like M. edulis and M. galloprovincialis [65].

4. Conclusion

In conclusion, two main populations of hemocytes (granulocytes and hyalinocytes) were characterized from Hong Kong oyster by flow cytometry, light microscopy and transmission electron microscopy in the present study. Different levels of phagocytosis and ROS production in granulocytes and hyalinocytes suggested a differential involvement of each hemocytes population in physiological and immunological responses. In addition, the plasma showed antibacterial activity with V. alginolyticus. Further studies will provide basic information of the he- Fig. 5. Transmission electron micrographs of hemocytes in C. hongkongensis. matopoiesis and hemocytes maturational processes. (A) Granulocytes; (B) Hyalinocytes. N: Nucleus M: Mitochondria GR: granule ER: Endoplasmic reticulum. Acknowledgments cytoplasm, which contained different number of granules with different This work was supported by National Science Foundation of China size and electron-density. In these cells, large numbers of electron (No. 31502193), Guangdong Natural Science Foundation (No. granules of different sizes and some organelles, such as endoplasmic 2014A020208083) and Science and Technology Planning Project of reticulum and mitochondria were also visualized. Hyalinocytes, the Guangdong Province, China (No. 2017B030314052), Guangdong second hemocytes, were identified by their large nuclei with many Natural Science Funds for Distinguished Young Scholar's (No. chromatin clumps, and small amounts of endoplasmic reticulum and 2015A030306003), and the Guangdong Special Support Program of mitochondria. Chromatin is accumulated in electron-dense clumps in Youth Scientific and Technological Innovation (No. 510143757060). the periphery of nucleus. A few granules were also present in the cy- We also thank Dr. JianLin Zhang for his kind assistance with the flow toplasm of hyalinocytes. These hemocytes types were consist with the cytometry analysis. previous studies in Perna viridis [59], Octopus vulgaris [60], Ruditapes decussatus [61], Pomacea canaliculata [62], Callista Chione [33]. References

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