Classification and characterization of hemocytes from two Asian horseshoe crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda

In present study, transmission electron microscopy and flow cytometry were utilized to investigate the classification, characterization and immune functions of hemocytes from horseshoe crab, Tachypleus tridentatus and Carcinoscorpius rotundicauda. Three types of hemocytes were distinguished respectively: the granular cell, the semi-granular cell and the hyaline cell by transmission electron microscopy, while three hemocyte subpopulations (Gate 1 cell, Gate 2 cell, Gate 3 cell) were classified by flow cytometry. Hyaline cell was the major cell type with the highest nuclear-cytoplasmic ratio and granular cell and semi-granular cell showed lower ratios. Immune parameters of hemocytes in horseshoe crabs were investigated by flow cytometry. Different hemocyte subpopulations respond for diverse functions. Lysosomal contents and hemocyte mortality in Gate 3 cell subpopulation were higher than that in other subpopulations, while reactive oxygen species, phagocytosis and non-specific esterase, in Gate 1 cell subpopulation, were higher than those in other subpopulations. The hemocyte types between the two species had no significant differences in staining or morphology.

nucleus occupied most of the room in cells, indicating the largest nuclear-cytoplasmic ratio (N/C). Among the two species of both male and female, their hemocyte sub-population in common shared uniform characters.
Total hemocyte count (THC). Hemocyte size frequency distributions were evaluated by coulter counter, and the size of hemocyte in T. tridentatus was more centralized than C. rotundicauda (Fig. 2). For the female T. tridentatus, the major peak of the hemocyte diameter was observed from 7.5 µm to 17 µm; the major peak of the hemocyte area was at 150 µm 2 -1000 µm 2 ; connected with the cell surface area, the hemocyte volume mainly peaked from 150 µm 3 to 2800 µm 3 . For the male T. tridentatus, the major peak of the hemocyte diameter was observed from 6 µm to 18 µm; the major peak of the hemocyte area was at 140 µm 2 -1000 µm 2 ; connected with the cell surface area, the hemocyte volume mainly peaked from 140 µm 3 to 2800 µm 3 . For the female C. rotundicauda, the major peak of the hemocyte diameter was observed from 7 µm to 20 µm; the major peak of the hemocyte area was at 180 µm 2 -1400 µm 2 ; connected with the cell surface area, the hemocyte volume mainly peaked from 200 µm 3 to 4800 µm 3 . For the male C. rotundicauda, the major peak of the hemocyte diameter was observed from 7.5 µm to 20 µm; the major peak of the hemocyte area was at 150 µm 2 -1400 µm 2 ; connected with the cell surface area, the hemocyte volume mainly peaked from 180 µm 3 to 4600 µm 3 . Among the two species of both male and female, the region and the peak were similar apart from the range of the surface area and volume. The volume of the hemocyte in C. rotundicauda was larger than those in T. tridentatus. Total hemocyte counts (THC) of MTT, FCR and MCR (50000-55000 mL −1 ) were significantly (p < 0.05) lower than FTT (ca.72000 mL −1 , Fig. 3A).
Hemocyte classification by flow cytometry. At least three subpopulations of hemocytes, in female T. tridentatus (FTT), male T. tridentatus (MTT) and female C. rotundicauda (FCR), male C. rotundicauda (MCR), were classified, according to the criteria of cell size (FSC) and the cell complexity (SSC, Fig. 4). The gate 1(G1) composes of cells with low complexity and small size, which may mainly include HCs. More complex cells with middle size were classified into gate 2 (G2), which may mainly contain SGCs. Cells in gate 3 (G3) were the biggest and the most complex, probably the GCs, whose proportion of number was the least.
Immune parameter measurements. For the two horseshoe crabs of both genders, the number of G1 cell was the maximum among three hemocyte types. Then the G2 cell number was more than G3 cell. Among two horseshoe crab species, the proportions of three types of hemocytes displayed some differences. Between FTT and MTT, every group of hemocyte was discrepant, while it showed similarity between FCR and MCR among three types of hemocyte. For all of the species, G1 cells have the highest proportion. All the hemocytes showed phagocytosis capacities, while the proportion of G1 cells was much higher than that of the G3 cells, and G2 cells' was higher than G3 cells' but lower than G1 cells' (Fig. 3B). The esterase activity of males and females of hemocytes did not show prominent difference, while in each species males' were higher than females' . G3 cells' were quite higher than G2 cells' , and G2 cells were a bit higher than G1 cells (Fig. 3C). The G3 cells showed higher reactive oxygen species (ROS) and ROS of the G3 cells in MCR and FCR were higher than MTT and FTT (Fig. 3D). The three hemocyte subpopulations had some differences in lysosomal contents, the highest value in the G3 cells, a lower in G2 cells and the lowest in G1 cells (Fig. 3E). Between the two species and between males and females, every three hemocyte subpopulations are almost different. Based on flow cytometry, the total hemocyte mortality was not high, with a mean of less than 4.0%, the mortality of G1 cells measured was significantly higher than G2 and G3 cells (Fig. 3F).  www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Until now, hemocyte classification in aquatic invertebrate has not reached a uniform standard, but most agree on the classification of granular cells and hyaline cells 35 . Our result showed that the inner structures of hemocytes can be displayed by transmission electron microscope (TEM) and divided into granular cells, semi-granular cells and hyaline cells. According to the number and size of granule, the invertebrate hemocytes could be easily to classify 35 . Three main types of hemocytes were characterized: granular cells, semi-granular cells and hyaline cells in two horseshoe crabs, whose hemocyte types are similar to some other invertebrates, including some crustaceans [35][36][37] . Besides, the hyaline cells possessed a big nucleus and a few large granules among the Chinese horseshoe crab and mangrove horseshoe crab as some other shellfishes 28,38 . According to the method of optical microscopy, granular cells, semi-granular cells and hyaline cells with different shapes as exhibited were typed for the hemocytes of Chinese horseshoe crab and mangrove horseshoe crab 31 , which are similar with our results. For the two horseshoe crabs, the types and features of hemocytes were similar, and the similar phenomena were also showed in other studies 39 . Besides, hyaline cells were unique contrasting to other cells, which is tally with the study in cultivate horseshoe crab before 30 . Previously, scanning electron microscope) also has been used to view the morphology of the hemocytes in horseshoe crab 31 . Based on the different size and varied numbers of wrinkle on the surface, the hemocytes were classified into three types: granular cells semi-granular cells and hyaline cells 31 .
As an indicator representing the immune functions of hemocyte 40 , total hemocyte counts (THC) were determined in the male and female of the two horseshoe crab species. Among the two species with female and male, the similar results were obtained from the male Chinese horseshoe crab and both male and female of the mangrove horseshoe crab, while the female Chinese horseshoe crab showed some differences. Perhaps different species within the genus as well as different genders can show some differences in such parameter. For example, THCs between the white shrimps Litopenaeus vannamei and Litopenaeus stylirostris were quite different 41,42 . THCs were www.nature.com/scientificreports www.nature.com/scientificreports/ also analyzed in two edible crabs, the Jonah crab Cancer borealis and the brown crab Cancer pagurus, and there were some differences between the two crab species 27 .
In present study, hemocytes also were analyzed by flow cytometry and several indexes were obtained. Usually, the number of hemocyte types varies from species to species. Base on this method, hemocytes in some shellfishes were classified into two 43,44 , three 22,23 and four 45 types. FCS and SSC are sometimes not reflecting actual FCS/SCC in crustaceans by FCM 46 , thus the matches between microscopic images and FCM may not be reached exactly. In the present study, hemocytes of the Chinese horseshoe crab and mangrove horseshoe crab, for males and females, were classified into three groups (G1 cells, G2 cells and G3 cells, probably are hyaline cells, semi-granular cells and granular cells). However, the proportion of each type of cell shows differences between two horseshoe crabs.
Phagocytes play a vital role in the innate immune responses in invertebrates 47 . The phagocytosis of hemocytesis poorly defined, it is possible that different types of hemocytes in invertebrates possess different capacities in phagocytosis 48 or some hemocytes were not phagocytes 22,49 . In the present study, it is obvious that the G1 cells (possible hyaline cells) with less granular were the main phagocytes, whereas G2 and G3 cells had lower phagocytosis, and in the study of Mats 50 , the dominance of hyaline cells in phagocytosis was also observed. While the Chinese mitten crab Eriocheir sinensis 43 showed a different result that G3 cells (possible granulacytes) were the main phagocytes. In our study, G1 cells of the four kinds of horseshoe crab were the main immunocompetent hemocytes, if phagocytosis activity was considered as the major indicator of immunity 50 . Lacking of intracellular prophenoloxidase, hyaline cells could be stimulated by the extracellular prophenoloxidase system, and degranulation has deeply connection with recognition function to foreign objects 51 . The similar result of the phagocytosis parameter was also showed in the mussel Mytilus edulis 33 .
The esterase activity of marine invertebrates, like shrimps 52 , mussels 53,54 , and clams 55 has been extensively reported using flow cytometry. As a kind of hydrolase enzyme, it hydrolyzes some choline esters. This enzyme thus plays an irreplaceable role in the immune defense of marine invertebrates 56,57 , and the mortality of hemocytes increases when esterase activity decreases according to some studies 55,58 . In some studies, the granular cells showed higher esterase activity than hyaline cells and were regarded to the major cell in defense 40,52 , indicating the importance of its granular cells with esterase activity 59 . This was the first time for studying esterase of hemocytes in the Chinese horseshoe crab and mangrove horseshoe crab. For both species, the esterase in G3 cells was higher than that in G1 and G2 cells. That is to suggest that cells with more granules should be more active in esterase activity. Semi-granular cells possess stronger ability for exocytosis, playing an important part in immunity 50,60 .
ROS production, induced by phagocytosis in the process of elimination 61 , is regarded as an important mechanism for microbicidal capacities 62 . ROS is usually used to determine immunity in marine invertebrates, such as bivalves 48,63 and crustaceans 43,64 . When hemocytes were stimulated (like exposure in vitro), respiratory burst occurred and toxic reactive oxygen species were released 65,66 . The most significant substances include superoxide anion (O 2− ), hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ) and hydroxyl (OH) 67 . In the present study, the ROS production of G3 cells was higher than other hemocytes. The ROS of granulocytes was also higher than that in hyaline cells in the crab Eriocheir sinensis 43 . In the hemocytes of Crassostrea gigas 23,68 , higher ROS production was detected in granular cells.
Lysosome is an organelle in cells, and lysozymes released from lysosome can non-specifically kill microorganisms by hydrolyzing cell walls 69 . Therefore, lysosome plays key roles in host defenses of microorganisms 70 . In the inactivation of invading microbes, lysozyme participates among the hemolymph constituents 71 . Many studies on hemocytes of marine invertebrates measured lysozyme in hemocyte, such as crabs 43 , oysters 23,72 and mussels 22,49 . It was found in the green-lipped mussel P. viridis 40 that the granular cells contained higher lysosomal content than other groups of hemocytes. Similarly in the present study, lysosomal content in G3 cells was higher than that in G1 cells in the two horseshoe crab species. Cytosolic lysosomes release various enzymes in vesicles, which combines with the production of ROS to destroy the foreign particles 73,74 . Meanwhile, there were some differences in G1 cells among the four kinds of horseshoe crabs, although all of them had lower lysosomal content.
The cellular mortality is usually accompanied by necrotic and apoptotic courses 75 , thus hemocyte mortality is a useful indicator for immune system after being stressed in marine invertebrates 54,76 . There were some studies of hemocytes mortality in mussels 40,63 and clams 55 . From the present study, hemocyte mortality was lower in G3 cells and higher in G1 cells. Combined with the lysosomal content, higher lysozyme safeguarded lower mortality in G3 cells, and in G1 cells lower lysozyme caused higher mortality, likely that lysosome is relevant to hemocyte mortality tightly owing to the high phagocytosis of G1 cells (hyaline cells) hydrolyzing foreign objects after endocytosis 72 . For example, in the green-lipped mussel P. viridis 40 , the mortality of hyaline cells was higher than granular cells, indicating that hyaline cells were not positive as granular cells in immune defense. It is possible that different subpopulations are responsible for different functions 48 .
Same as other crustaceans, the immune system of horseshoe crab lacks of immunoglobulin and removes exogenous object and pathogen through phagocytosis activity, esterase activity, ROS production and lysosome, belonging to innate immune 77 . Crustacean hemocytes play important roles in the host immune response including recognition, phagocytosis, melanization, cytotoxicity and cell-cell communication 50,77,78 . Classification of the hemocyte types in decapods crustaceans is based mainly on the presence of cytoplasmic granules in hyaline cells, semi-granular cells, and granular cells 79 . Studies on crustacean have shown that different types of hemocytes play different roles in immunity 50,80 . Each cell type is active in defense reactions, for example, in crayfish, the hyaline cells are chiefly involved in phagocytosis, the semi-granular cells are the cells active in encapsulation, while the granular cells participate in storage and release of the prophenoloxidase and cytotoxicity 50,77,78 .
Apart from those, hemocytes in horseshoe crab need more deeply study. This study has demonstrated that the hemocytes of C. rotundicaud and T. tridentatus possess many morphological and functional characteristics as other marine invertebrates, some immune parameters in every types of hemocyte were different to other invertebrates. More studies on immune functions of different subpopulations of hemocytes in horseshoe crab should be further investigated.
After the adaptation period, six horseshoe crabs of each gender were sampled to obtain hemolymph for both species. Hemolymph was collected from the joint between bent breastplate and plastron using a 1.0 mL plastic syringe with 22 G needle and all samples obtained were stored in tubes with pre-chilled (187 USP unit ml −1 ) heparin 81 . Transmission electron microscopy (TEM). Five mL of hemolymph were prepared in the solution of 4% paraformaldehyde, 0.3 M sucrose in a 0.1 M cacodylate buffer (pH 7.2) and 2.5% glutaraldehyde for 10 minutes. Then, prepared cells were centrifuged at 400 g at ambient temperature for 10 minutes and the supernatant was discarded. The pellets were resuspended and encased in 3% molten agar. Then the embedded cells were whittled into 1 mm 3 small blocks and immobilized by fresh fixative at 4 °C overnight. After being washed using cacodylate buffer, cells were fixed with 1% osmium tetroxide (OsO 4 ) in 0.1 M cacodylate buffer (pH 7.2) at 4 °C for one hour. Samples were douched in the same cacodylate buffer and distilled water, dehydration in ethanol solutions of six different grades (30%, 50%, 70%, 80%, 95% and 100%) and transferred into acetone. Later before embedding, the samples were infiltrated gradually in resin. Ultrathin sections (60-90 nm) were made by using a Leica Ultracut UCT ultra microtome (Austria). Ultrathin sections were mounted onto coated collodion with 100-mesh copper grids. A section was dyed with 2% uranyl acetate aqueous solution for fifteen minutes before stain with Reynold's lead citrate for ten minutes. Sections were observed using a FEI/Philips Tecnai 12 BioTWIN transmission electron microscope (Netherlands) operated at 80 kV.

Total hemocyte counting (THC). An electronic particle counter/size analyser (Multisizer ™ 3 Coulter
Counter, Beckman Coulter) was used to evaluate the hemocyte concentration in hemolymph. The size frequency distribution and hemocyte concentration (number of cells per milliliter) were determined. Before sample running, 0.5 mL of hemolymph was added into 9.5 mL of Isoton ® II solution to crank out the mixture, and every time 1000 µL of the mixed solution were counted.

Parameter measurements by flow cytometry.
Recently collected hemocytes within unwrought crude hemolymph were analyzed for immune parameters by a BD FACS Calibur flow cytometer equipped with an air-cooled argon laser and offers a laser excitation at a spot of 488 nm. A FSC limitation (>150) was defined to eliminate bacteria and cell debris. Datum was described as cell cytograms pointing the granularity (SSC value), the proportional size (FSC value) and the fluorescence channels identifying with the markers used. For each hemolymph sample, 20000 events were obtained in total, while the flow rate was corrected to maintain the whole events below 300 every second. The fluorescent frequency distribution column diagram of the hemocyte population was subsequently acquired. The type of fluorescence channel relied on the parameter monitored: hemocyte mortality was evaluated by FL2 (greenish orange emission for 585 nm), while reactive oxygen species (ROS), enzymes and phagocytosis were measured using FL1 (green emission for 530 nm). Data were analyzed by FlowJo ® 10.0 software. Hemocyte mortality was detected by propidium iodide (PI), which is a type of fluorescent dye that only enters and stains dead hemocytes. 10 μL of solution of PI (Siama Aldrich) at a concentration of l mg·mL −1 was added into 400 μL hemolymph, and the mixture was incubated for 30 minutes in the dark at 4 °C before analyzed by flow cytometry. Hemocyte mortality was analyzed as the percentage of hemocytes showing PI fluorescence relative to the whole hemocyte counts.
Phagocytosis was detected using fluorescent microspheres as a phagocytic goal by an in italic assay, and estimated as the percentage of cells having internalized at least three fluorescent beads 40,82 . 400 μL of hemolymph was incubated for 1 hour at ambient temperature in the dark with 10 μL of the Fluorospheres ® carboxylate-modified microspheres with 1/10 dilution (yellow-green fluorescent, 1 μm diameter, Invitrogen). Finally, the fluorescent beads concentration was 10 8 mL −1 , and the beads/hemocytes ratio was 100/1.
Activity of non-specific esterase was detected using non-specific lip soluble ground substance fluorescein diacetate (FDA, Sigma). FDA stock solutions (0.04 mM) were prepared in dimethyl sulphoxide (DMSO) and reserved at −20 °C. Working solutions of FDA (400 μM) were drew up by 1/10 diluting the stock with strained and sterile seawater. 400 microliters of hemolymph with 2 μL FDA solution was incubated in the dark for 15 minutes at ambient temperature. The percentage of cells expressing enzymatic activity was defined based on the percentage of fluorescent cells among all cells.
The cellular reactive oxygen species (ROS) were measured by 2′7′-dichlorofluorescein diacetate (DCFH-DA; Sigma). DCFH-DA, a nonfluorescent fluorescein analogue, can disseminate into hemocytes, and then is hydrolysed into 2′,7′-dichlorofluorescein (DCFH). Enzymatic activity was defined based on fluorescent cells among the whole cells. A 10 mM DCFH-DA stock was added to DMSO and is generally diluted to 10% in strained sterile seawater as a working fluid. Each analysis required mixture with four hundred microliters of hemolymph and 4 μL of DCFH-DA, and the mixture was then incubated in the dark for 15 minutes at ambient temperature.
Lysosomal content was measured by a merchant LysoTracker ® Yellow HCK-123 (1 mM in DMSO, Invitrogen). 400 μL hemolymph mixed with 1 μL of LysoTracker were incubated took place at ambient temperature for two www.nature.com/scientificreports www.nature.com/scientificreports/ hours in the dark and the reaction was ceased on ice. LysoTracker fluorescence in the hemocytes was described in arbitrary units (A.U.).

Statistics analysis.
Prior to the analysis, data were checked for homogeneity of variance with Levene's test and normality with the Shapiroe-Wilk's test using statistical software SPSS 18.0. Percentage data were arcsine transformed. One-way analysis of variance (ANOVA) and Tukey's test were used to compare the differences of immune parameters among granular cell, semi-granular cell and hyaline cell identified by flow cytometry. For all analysis, the results are expressed as the means ± SD of the data and significant differences were known as p < 0.05.