Fish & Shellfish Immunology 36 (2014) 315e319

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Short communication

Application of monoclonal antibody against granulocytes of scallop Chlamys farreri on granulocytes occurrence at different developmental stages and antigenic cross-reactivity of granulocytes in five other bivalve species Jing Xing, Xiaoqian Tang, Yongqing Ni, Wenbin Zhan* Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, Qingdao 266003, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 August 2013 Received in revised form 1 November 2013 Accepted 4 November 2013 Available online 9 November 2013

A monoclonal antibody (MAb) 6H7 raised specifically against granulocytes of scallop (Chlamys farreri) was employed to observe granulocyte occurrence successively in blastulae, gastrulae, trochophore larvae, D-shape larvae, umbo-veliger larvae and creeping larvae of C. farreri by immunohistochemistry assay contrasted with H&E stain using semi-thin sections. Moreover, the reactivity of the MAb with granulocytes of C. farreri, Bay scallop Argopecten irradians, Japanese scallop Patinopecten yessoensis, Blue mussel Mytilus edulis, Pacific oyster Crassostrea gigas and Manila clam Ruditapes philippinarum, was detected by immunofluorescence assay (IFA) with differential interference contrast and fluorescent microscopy and flow cytometric immunofluorescence assay (FCIFA). The results showed that positive signals were first observed at D-shape larval stage, about 28 h post fertilization, after that, umbo-veliger larvae exhibited the positive cells with a diameter of 3e5 mm distributed in velum, digestive gland and esophagus. Then in creeping larvae, the number of positive cells increased with average diameter of 5 e7 mm, and widely distributed in foot, digestive gland, gills and adductor muscles. No positive signal was found in blastulae, gastrulae and trochophore larvae. The results of IFA and FCIFA showed MAb 6H7 reacted to granulocytes of C. farreri, A. irradians, P. yessoensis and C. gigas, and the positive percentage reactivity were 53  2.5%, 15  2.5%, 12  2.1% and 19  2.1%, respectively, however, no cross-reaction was detected in hemocytes of R. philippinarum and M. edulis. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Chlamys farreri Monoclonal antibody Granulocyte Occurrence Cross-reactivity

1. Introduction Granulocytes, as one type of the bivalve hemocytes, generally have abundant cytoplasmic granules containing a mixture of hydrolytic enzymes and contribute to intracellular killing and are more phagocytic than hyalinocytes, which are smaller and have fewer or no granules in their cytoplasm with lower nucleus to cell ratio [2,8,16,19]. Some reports have shown granulocyte numbers significantly vary response to pathogen infection [4,7,15,18], pollution [3,6,10] and environmental factor fluctuation [13,14,16], illustrating that granulocytes are closely related to stress. However, the analysis of ontogenesis and antigenic homology of bivalve granulocytes is still limited. Using monoclonal antibody (MAb) against hemocytes of Chlamys farreri, hematopoietic tissue of C. farreri juvenile scallops was localized to the vesicle tissues

* Corresponding author. Tel./fax: þ86 532 82032284. E-mail address: [email protected] (W. Zhan). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.11.001

around adductor [23,25]. Moreover, granulocytes of the soft shell clam Mya arenaria were detected to originate from connective tissue [22]. The basophilic granulocyte of mussel Mytilus edulis occurred in trochophore and veliger larvae [21], whereas granulocytes of the oyster Ostrea edulis only appeared at the juvenile stage [26]. Besides the consensus of hemocytes types in morphological features in bivalve, hemocytes were found to be antigenically similar among scallop (C. farreri), bay scallop (Argopecten irradians), oyster (Crassostrea talienwhanensis), asiatic hard clam (Meretrix meretrix), monila clam (Ruditapes philippinarum), purplish washington clam (Saxidomus purpuratus) and horny ark (Scapharca subcrenta) [24]. Chinese scallop C. farreri, is one of the most important scallops widely cultured in the northern coastal provinces of China [27]. Our previous work has produced MAb specifically against granulocytes of C. farreri [19]. This paper describes the occurrence of the granulocytes during C. farreri developmental stages and the antigenicity similarity among other five bivalve species using the MAb, with the purposes of better understanding the ontogenesis of C. farreri

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granulocytes and identification of granulocyte in five other bivalve species.

culture supernatant was used as the primary antibody for IHC, IFA, and FCIFA.

2. Materials and methods

2.4. Immunohistochemistry assay

2.1. Embryos and larvae of scallop (Chlamys farreri) collection and semi-thin section preparation

Sections of embryos and larvae at different developmental stages were antigen retrieved by heating in a microwave oven at 500 W for 2 min in 10 mM sodium citrate buffer, pH 6.0, and cooled at room temperature, then transferred to PBS containing 0.5% Tween-20 (PBS-T, pH 7.4). After that, they were immersed in 0.5 M EDTA for 20 min to eliminate endoenzyme activity [29]. After three washes with PBS-T, the sections were blocked in 5% bovine serum albumin for 30 min at 37  C, and then washed again and incubated with MAb 6H7 as the primary antibody for 1 h at 37  C. Afterwards, sections were washed in PBS-T again for three times and incubated with biotin conjugated goat anti-mouse IgG (Sigma, Germany) diluted at 1:200 as the secondary antibody for 45 min at 37  C, followed by another wash, the sections were then incubated with alkaline phosphatise (AP) conjugated streptavidin (Sigma) diluted at 1:200 for 45 min at 37  C. Following three washes, color reactions were developed using an AP-Red kit (Merck, Germany), together with hematoxylin counterstain. Finally, sections were washed, mounted in glycerin and observed under a light microscope (Olympus, Japan). Negative controls were incubated with supernatant of myeloma cells as the primary antibody.

Embryos and larvae of C. farreri at different stages were produced at a scallop hatchery in Weihai, Shandong province. One hour of post fertilization (PF), the embryos and larvae including blastulae(9h PF), gastrulae(16h PF), trochophore larvae(21h PF), Dshaped larvae(26h-2D PF), umbo-veliger larvae(4-5D PF), and creeping larvae(13-14D PF), were identified microscopically and collected successively. Then samples were fixed in 4% formaldehyde and titrated from paraformaldehyde in phosphate buffered saline (PBS; 0.14 M NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) at 4  C for 5 h. After rinsing in PBS, all samples were dehydrated and embedded in epoxy resin, then sectioned with thickness of 2 mm. After that, the sections were finally deresinated, and used in hematoxylin and eosin (H&E) stain or immunohistochemistry assay (IHC). 2.2. Six species bivalve collection and hemocyte suspension preparation Adult bivalves including scallop (C. farreri and A. irradians), oyster (C. gigas), clam (M. meretrix, R. philippinarum, and S. purpuratus) and horny ark (S. subcrenta) were purchased from Qingdao Harbor, and kept in a circulating seawater system in the laboratory until the next day. Hemolymph from each bivalve was withdrawn from the adductor muscle sinus with sterilized syringes, simultaneously mixed (1:1, v/v) with PBS containing EDTA (PBS-E; PBS containing 19.1 mM EDTA, pH 7.4), then pooled and centrifuged at 700  g for 10 min at 4  C. The pellet was washed twice, resuspended in PBS and the hemocyte suspension was adjusted to a final concentration of 107 cells/ml and used for immunofluorescence assay (IFA) and flow cytometric immunofluorescence assay (FCIFA). 2.3. Monoclonal antibody (MAb) MAb 6H7 specific against granulocytes of C. farreri was previously produced in our lab [19]. In present study, the hybridoma

2.5. Immunofluorescence assay and flow cytometric immunofluorescence assay Hemolymph from ten individuals of each species was pooled, 1 ml of hemocyte suspension was applied to a 24-well culture plate and incubated with 1 ml of MAb 6H7 as the primary antibody for 1 h at 37  C. After centrifuging at 700  g for 5 min in PBS, the hemocyte pellet was resuspended and incubated with 500 ml of fluorescein isothiocyanate conjugated goat anti-mouse Ig (GAM-FITC, Sigma) diluted at 1:200 as the secondary antibody for 45 min at 37  C in dark, then centrifuged again and resuspended in 500 ml of PBS. One drop of each resulting suspension was applied to a clean slide and observed under a fluorescence microscope contrasted with differential interference contrast (DIC) imaging (Olympus, Japan). The remaining suspension was analyzed by a flow cytometer (Becton, America). Three parameters FS, SS and FL, corresponding to cell size, cytoplasmic granularity and fluorescence intensity respectively, were employed to evaluate the percentage reactivity (PR) of MAb

Fig. 1. Hematoxylin and eosin (H&E) stain and immunohistochemistry (IHC) of C. farreri larvae. a, d: D-shape larva, bar ¼ 15 mm; b, e: Ambo-veliger larva, bar ¼ 15 mm; c,f: Creeping larva, bar ¼ 25 mm; a, b and c: Hematoxylin and eosin (H&E) stain; d, e and f: Test group of IHC incubated with MAb 6H7.

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6H7 with the hemocytes of six species bivalve. Data were analyzed using WinMDI2.9 software. Negative controls were the same as in IHC. This experiment was performed in quadruplicate. 3. Results 3.1. Immunohistochemistry Under microscopy after H&E stain, C. farreri blastula were spherical in shape with a diameter of 30  5 mm, and had many short cilia around the surface and a blastocoel in the embryonic center in vivo; gastrula had archenteron and blastopore formed by embryonic epiboly and invagination; trochophore larva was 60  8 mm in diameter containing a tuft of cilia and a thick flagellum (Data not shown). D-shaped larva was brown with shell height of 120  10 mm, had numbers of peripheral cilia and a distinguishable visceral mass in the center covered with transparent shells like a capital letter “D” (Fig. 1(a)); as for umbo-veliger larva, their visceral

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mass swelled, and the umbo formed in the center of the shells (Fig. 1(b)); following that, about 13 days after fertilization, creeping larva with a shell length of 180 mm was developed, the byssal gland began to secrete the byssus, and the shells became thick and dark in color (Fig. 1(c)). The IHC results showed positive signals (in red), which were first observed in D-shape larvae, and the weak red deposits distributed around the velum, digestive gland and esophagus, however, granulocytes were hard to distinguish, (Fig. 1(d)); in the umbo-veliger larvae, more intense positive signals appeared. Distinguishable positive cells 3e5 mm in diameter could be observed, and they were localized at mantle, digestive gland, esophagus, particularly in base of velum (Fig. 1(e)); after that, in creeping larvae, the number of positive cells increased with average diameter of 5e7 mm, and widely distributed in foot, digestive gland, gills and adductor muscles (Fig. 1(f)). No positive signal was found in blastulae, gastrulae and trochophore larvae and the controls (Data not shown).

Fig. 2. Detection of MAb 6H7 reactivity to the hemocytes of C. farreri, A. irradians, P. yessoensis, and C. gigas by immunofluorescence assay and flow cytometric immunofluorescence assay(FCIFA). (a2, b2, c2 and d2): Differential interference contrast imagings showed identical views with fluorescence microscopy, bar ¼ 10 mm; (a3, b3, c3 and d3): Test group of FCIFA which were incubated with MAb 6H7 as primary antibody; (a4, b4, c4 and d4): Control group of FCIFA which were incubated with supernatant of myeloma cells as primary antibody. G: granulocytes; H: hyalinocytes; N: negative; PG: positive granulocytes; PH: positive hyalinocytes. a: C. farreri, b: A. irradians, c: P. yessoensis, d: C. gigas.

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3.2. Immunofluorescence assay and flow cytometric immunofluorescence assay Under a fluorescence microscope, MAb 6H7 showed a positive reaction with granulocytes of C. farreri, A. irradians, P.yessoensis and C. gigas (Fig. 2(a1)e(d1)). Contrasted with DIC (Fig. 2(a2)e(d2)), the fluorescent foci were presented all over the granulocytes of four species bivalve, and no fluorescence was labeled on hyalinocytes. However, the fluorescent foci weren’t present at the hemocytes of M. edulis and R. philippinarum, no positive signal was observed. Analyzed by flow cytometry, hyalinocytes and granulocytes of each species formed two areas according to their cell size and cytoplasmic granularity in the dotplot, and their fluorescence intensity in the histogram. Compared with controls, the PR of MAb 6H7 with C. farreri granulocytes was 53  2.5% (Fig. 2(a3)), it was 15  2.5% to A. irradians granulocytes (Fig. 2(b3)), 12  2.1% to P. yessoensis granulocytes (Fig. 2(c3)) and 19  2.1% to C. gigas granulocytes (Fig. 2(d3)). MAb 6H7 was negative with hyalinocytes of these bivalve species. And also negative will all cells from M. edulis and R. philippinarum.

4. Discussion Prior to the experiments, the characterization of antigranulocyte MAb (6H7) was confirmed by FCIFA, immunoelectron microscopy and western blot assay [19]. Moreover, IHC results in adult scallop showed MAb 6H7 had no cross-reaction with other cells in gill, mantle, gonad, visceral mass, kidney and adductor muscle except for granulocytes [19]. In this study, positive signals occurred in the larvae confirming that the epitope which MAb 6H7 recognizes on granulocytes is presented from the D-shape larval stage and increases with the development of larval stage. In order to get a distinguishable observation on the inner structures of embryos and larvae, resin-embedded semi-thin (2 mm) sections were applied in this paper, it shows superior preservation of cellular details compared with those embedded in paraffin and permits high resolution to be obtained. Furthermore, after the antigen retrieval was used, the immunological staining gave both better intensity of positive signal and better ultrastructure preservations of the tissue [5,28]. It has been reported that using MAb, basophilic granulocytes first appear in trochophore larva of mussel M. edulis [12]. However, in oyster O. edulis [26], granulocytes only appeared in 30e32 days old juveniles. This discrepancy may be explained by differences in experimental species [17] or granulocyte epitopes that MAb recognized. In this study, we found that granulocytes were mainly located in velum, digestive gland and esophagus, which may suggest that larval granulocytes are already involved in food capture, digestion and nutrient transport like adult granulocytes [1]. Moreover, granulocytes were also distributed in gill and mantle where scallops contact environmental factors and pathogenic microorganisms, which might highlight the potential role of the larval granulocytes in the interactions between larvae and waterborne microorganisms at the gill and mantle with possible involvement in physiological functions such as microorganisms recognization and elimination [9,11,26]. As the development of larvae proceeds, the size, number and density of positive cells increased, and they are more widely extended. This trend was consistent with hemocytes of O. edulis larvae [26]. Interestingly, granulocytic hemocytes was reported that may be directly involved in shell crystal production for oyster, and they increase in abundance relative to other hemocytes following experimentally induced shell regeneration [20]. In our study, we found larval shells and positive granulocyte signals

both appeared at D-shape stage. The coincidence indicates granulocyte might be involved in shell mineralization. Granulocytes present morphological or structure similarities among many bivalve species, and in this paper, we show antigen similarity among granulocytes of C. farreri, A. irradians, P. yessoensis and C. gigas. The cross-reactivity demonstrates epitope recognition by MAb 6H7 in granulocytes of three other bivalves. However, the PR is variable probably because similar epitopes on granulocytes antigen of C. farreri aren’t completely the same amount as that of other species bivalve granulocytes. The MAb could partly react with one or some of the epitopes on the proteins of other bivalve species [24]. No cross-reactivity with granulocytes of R. philippinarum and M. edulis indicated this epitope was not presented in these two bivalve species. The cross reaction results suggested that MAb 6H7 has the potential to help understand granulocyte diversity and differentiation in bivalve species. Acknowledgments This research was supported by Key Technology R&D Program of China (No. 2012BAD17B02), NCET (No.10-0763), and SRF for ROCS, SEM (No. [2011]1139). References [1] Aladaileh S, Nair SV, Birch D, Raftos DA. Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function. J Invertebr Pathol 2007;96: 48e63. [2] Auffret M. Bivalve hemocyte morphology. Am Fish Soc Spec Publ 1988;18: 169e77. [3] Bado-Nilles A, Gagnaire B, Thomas-Guyon H, Le Floch S, Renault T. Effects of 16 pure hydrocarbons and two oils on haemocyte and haemolymphatic parameters in the Pacific oyster, Crassostrea gigas (Thunberg). Toxicol In Vitro 2008;22:1610e7. [4] Barcante TA, Barcante JMP, Fujiwara RT, Lima WS. Analysis of circulating haemocytes from Biomphalaria glabrata following Angiostrongylus vasorum infection using flow cytometry. J Parasitol Res 2012. 314723. [5] Bmrsen SH, Nguyen GH. Increased level of immunogold labeling of epoxy sections by rising the temperature significantly beyond 100 degrees C in the antigen retrieval medium. Micron 2001;32(4):591e7. [6] Bouilly K, Bonnard M, Gagnaire B, Renault T, Lapègue S. Impact of diuron on aneuploidy and hemocyte parameters in Pacific oyster, Crassostrea gigas. Arch Environ Contam Toxicol 2007;52:58e63. [7] Canesi L, Gallo G, Gavioli M, Pruzzo C. Bacteria-hemocyte interactions and phagocytosis in marine bivalves. Micros Res Tech 2002;57:469e76. [8] Carballal MJ, López C, Azevedo C, Villalba A. In vitro study of phagocytic ability of Mytilus galloprovincialis Lmk haemocytes. Fish Shellfish Immunol 1997;7: 403e16. [9] Carballal MJ, Villalba A, López C. Seasonal variation and effects of age, food availability, size, gonadal development, and parasitism on the hemogram of Mytilus galloprovincialis. J Invertebr Pathol 1998;72:304e12. [10] Donaghy L, Hong HK, Lee HJ, Jun JC, Park YJ, Choi KS. Hemocyte parameters of the pacific oyster Crassostrea gigas a year after the Hebei spirit oil spill off the west coast of Korea. Helgol Mar Res 2010;64:349e55. [11] Donaghy L, Lambert C, Choi KS, Soudant P. Hemocytes of the carpet shell clam (Ruditapes decussatus) and the Manila clam (Ruditapes philippinarum): Current knowledge and future prospects. Aquaculture 2009;297:10e24. [12] Dyrynda EA, Pipe RK, Ratcliffe NA. Sub-populations of haemocytes in the adult and developing marine mussel, Mytilus edulis, identified by use of monoclonal antibodies. Cell Tissue Res 1997;289:527e36. [13] Gagnaire B, Frouin H, Moreau K, Thomas-Guyon H, Renault T. Effects of temperature and salinity on haemocyte activities of the Pacific oyster, Crassostrea gigas (Thunberg). Fish Shellfish Immunol 2006;20:536e47. [14] Goedken M, Morsey B, Sunila I, Dungan C, De Guise S. The effects of temperature and salinity on apoptosis of Crassostrea virginica hemocytes and Perkinsus marinus. J Shellfish Res 2005;24:177e83. [15] Hégaret H, da Silva PM, Wikfors GH, Lambert C, De Bettignies T, Shumway SE, et al. Hemocyte responses of Manila clams, Ruditapes philippinarum, with varying parasite, Perkinsus olseni, severity to toxic-algal exposures. Aquat Toxicol 2007;84:469e79. [16] Hégaret H, Wikfors GH, Soudant P. Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation II. haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst. J Exp Mar Biol Ecol 2003;293:249e65. [17] Hine PM. The inter-relationships of bivalve haemocytes. Fish Shellfish Immunol 1999;9:367e85.

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Application of monoclonal antibody against granulocytes of scallop Chlamys farreri on granulocytes occurrence at different developmental stages and antigenic cross-reactivity of granulocytes in five other bivalve species.

A monoclonal antibody (MAb) 6H7 raised specifically against granulocytes of scallop (Chlamys farreri) was employed to observe granulocyte occurrence s...
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