Flow cytometric characterization of hemocytes of the flat oyster, Ostrea chilensis

Fish and Shellfish Immunology 97 (2020) 411–420

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Flow cytometric characterization of hemocytes of the flat oyster, Ostrea chilensis

T

Anne Roltona,∗, Lizenn Delislea, Jolene Berrya, Leonie Venterb, Stephen Charles Webba, Serean Adamsa, Zoë Hiltona a

Cawthron Institute, Private Bag 2, Nelson, 7042, New Zealand Aquaculture Biotechnology Research Group, School of Science, Faculty of Health and Environmental Sciences, Auckland University of Technology, Private Bag 92006, Auckland, 1142, New Zealand

b

A R T I C LE I N FO

A B S T R A C T

Keywords: Flow cytometry Ostrea chilensis Flat oyster Hemocytes Hyalinocytes Granulocytes Bivalve

The flat oyster, Ostrea chilensis, native to New Zealand (NZ) and Chile is considered an important ecological, cultural and fisheries resource. Currently, commercial landings of this species in NZ are restricted due to low population numbers caused by ongoing mortalities resulting from the presence of the haplosporidian parasite, Bonamia exitiosa. More recently, the arrival of B. ostreae in NZ led to major mortalities in farmed stocks. To understand how diseases caused by Bonamia spp. affect this oyster species, a more complete understanding of its biology, physiology and immune system is needed. The present study characterized, for the first time, hemocytes of adult O. chilensis, from the Foveaux Strait, NZ, using flow cytometry (FCM) and histology. Based on the internal complexity of the hemocytes, two main circulating hemocyte populations were identified: granulocytes and hyalinocytes (accounting for ~30% and ~70% of the total circulating hemocyte population, respectively). These were further divided into two sub-populations of each cell type using FCM. A third sub-population of granulocytes was identified using histology. Using FCM, functional and metabolic characteristics were investigated for the two main hemocyte types. Granulocytes showed higher phagocytic capabilities, lysosomal content, neutral lipid content and reactive oxygen species production compared to hyalinocytes, indicating their important role in cellular immune defence in this species. Methods of hemocyte sampling and storage were also investigated and flow cytometric protocols were detailed and verified to allow effective future investigations into the health status of this important species.

1. Introduction The flat oyster (Ostrea chilensis) is native to New Zealand (NZ) and Chile (O'Foighil et al., 1999) and also exists where it was deliberately introduced into the Menai Strait, UK, in the 1960s, to investigate its suitability to replace the dwindling O. edulis population [1,2]. In both Chile and NZ, the native O. chilensis fishery is a small but high valued industry, important to regional economies [3,4]. Currently, NZ production is exclusively from a highly-regulated seasonal dredge fishery in the Foveaux Strait, near the port of Bluff. From approximately 1985 to 1993, large-scale mortalities resulted in an estimated 90% reduction in the oyster populations of the Foveaux Strait [5,6] and caused the temporary closure of the fishery. The mortalities were attributed to the haplosporidian parasite, Bonamia exitiosa, believed to be endemic to NZ [7]. Similar mass mortalities observed as far back as 1964 were attributed to the same parasite [5]. Since the reopening of the fishery,

∗

there have been further epizootics attributed to B. exitiosa including from 2000 to 2016 [8]. Commercial landings of O. chilensis have further declined from ~13,000 tonnes in 2013 to 7000 tonnes in 2019 [4]. During this period, in addition to the presence of the endemic B. exitiosa, in 2014, major mortalities (60–95%) were reported in O. chilensis from the Marlborough Sounds, and in 2015 those oysters were confirmed to be infected by the related haplosporidian parasite B. ostreae, which had not previously been reported from NZ or anywhere else in the southern hemisphere [9]. Up to 2015, aquaculture production of O. chilensis in NZ had been increasing in value and importance; however, in 2017, when B. ostreae was detected in farmed O. chilensis in Stewart Island, near the Foveaux wild fishing grounds, the NZ government ordered the removal of all O. chilensis farms in an effort to control the spread of B. ostreae to uninfected wild populations [10]. Understanding the mechanisms underlying the immune response of O. chilensis to parasitic infection with Bonamia spp. will allow improved management

Corresponding author. E-mail address: [email protected] (A. Rolton).

https://doi.org/10.1016/j.fsi.2019.12.071 Received 16 October 2019; Received in revised form 18 December 2019; Accepted 21 December 2019 Available online 23 December 2019 1050-4648/ © 2019 Published by Elsevier Ltd.

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94.7 ± 3.2 g, mean ± SE, n = 48) were commercially fished by dredging from the Foveaux Strait, NZ (Lat. −46.7, Long 168.2) over a 5-week period from May until June 2019, during which time, average water temperatures were 10.6–13.7 °C. Freshly dredged oysters were stored in a chiller overnight (9–10 °C), before being packed on ice in a polybin with cardboard to stop any direct contact with ice, and transported to the Cawthron Institute, Nelson, NZ (~24 h on ice). Oysters were then held in 1 μm filtered UV-treated seawater at a temperature of 11–12.6 °C and salinity of 35–37 for 24–48 h before sampling.

of this oyster species in the face of continued major impacts from these parasites. Bivalve molluscs have an innate immune system and the response of bivalves to stressors such as disease are at least partially mediated by hemocytes, circulating within the hemolymph of the oyster [11–13]. Hemocytes are also involved in other homeostatic processes including tissue repair [14], detoxification [15] and nutrient digestion and distribution [12,16]. Two main sub-populations of hemocytes have been identified in bivalve molluscs; granulocytes, containing many granules within the cytoplasm, and hyalinocytes, which contain very few or no granules; however, several other hemocyte types may exist within these broad categories [11,13,17]. Using flow cytometry (FCM), the hemolymph of the European flat oyster, O. edulis, was found to contain three hemocyte types: small and large hyalinocytes and granulocytes [18]. [19] identified four types of hemocytes in the heart imprints of O. chilensis using transmission electron microscopy (TEM), which could be further sub-divided on the basis of size. Several studies have attempted to elucidate how Bonamia spp. parasites infect the hemocytes of Ostrea species [19–30]. Most studies have focused on the infection of the European flat oyster, O. edulis by B. ostreae, which is believed to have been introduced into Europe from North America with shipments of live oysters in the late 1970s [31]. For this host-parasite interaction, B. ostreae is thought to enter O. edulis hemocytes by host-specified phagocytosis [20,23] and, although found in all hemocyte types [21,30], is mainly found in large hyalinocytes and granulocytes [26,30]. In susceptible oysters, B. ostreae may actively increase its internalization within the hemocytes by suppression of esterase activity and ROS production [26] and may inhibit apoptotic pathways, which are thought to play an important role in the destruction of B. ostreae, within the hemocytes [24,29]. Work examining the response of O. chilensis haemocytes to B. exitiosa has been carried out by Refs. [19,25]. Using TEM and ultracytochemistry to study the interaction of B. exitiosa with hemocytes from heart imprints of O. chilensis, the authors found that mainly agranular hemocytes, which did not have a lysosomal system capable of injuring the parasite, were involved in phagocytosis of B. exitiosa (see Ref. [19]. There is, however, an incomplete understanding of how the hemocytes of O. chilensis respond to exposure of B. exitiosa, and to date, no information on how they respond to B. ostreae. To understand how the different hemocyte types are involved in functions such as the immune response of the O. chilensis, the morphological classification of hemocyte types requires additional determination of their functional and metabolic activities. Indeed, the characterization of hemocytes under natural, ambient conditions is essential to further understand cell-mediated responses of bivalves to stressors such as exposure to Bonamia spp. Flow cytometry has proved to be an effective tool for analysis of hemocytes of many bivalve mollusc species, enabling determination of not just different hemocyte types but also their functional and metabolic characteristics (e.g. Refs. [18,32–38]. To obtain accurate results, it is important that hemolymph samples are handled and stored appropriately prior to FCM analysis. Hemocyte aggregation, which is triggered by various stimuli including hemolymph removal from the bivalve [39] can lead to inaccuracies in FCM analysis. Hemocyte aggregation can be reduced using an appropriate antiaggregant solution or maintaining samples at a low temperature [39,40]; however, it is important to assess if using antiaggregant solutions has an effect on the hemocyte samples. The present study aimed to 1) evaluate the optimal methods of hemolymph sampling and storage from adult O. chilensis and 2) characterize the hemocytes within the hemolymph using flow cytometry and histology.

2.1. Preparing oysters for hemolymph extraction Oysters were prepared for hemolymph sampling by either making a small notch in the shell using wire cutters, or, myorelaxed using magnesium chloride (MgCl2) to allow access to the internal structures (n = 4 per method). To relax oysters, batches of 12–24 oysters were placed for 1–2 h in a water bath containing 2 L of reverse osmosis purified fresh water, 3 L of 1 μm filtered UV-treated seawater (resulting in a salinity of 20) and 150.0 g of analytical grade MgCl2 (Merck) for a final concentration of 30.0 g L−1 at 12 °C according to Refs. [41,42]. Prior to hemolymph collection, the live weight of the oyster being processed was recorded to the nearest 0.1 g, followed by the shell length measurement of the widest part of the shell to the nearest 0.1 mm. Hemolymph was extracted from oysters prepared using both methods with a 24-gauge needle and 1 mL syringe which had been held on ice prior to use. Between 400 and 1000 μL of hemolymph was removed from the adductor muscle sinus of each individual. Extracted hemolymph was added to microcentrifuge tubes containing autoclaved 0.2 μm filtered seawater (FSW) which had been kept on ice, resulting in a 1.25X dilution of the sample. Samples were held on ice until analysis. The total hemocyte count, hemocyte viability and reactive oxygen species (ROS) production were determined on the total hemocyte population of the eight individuals sampled (n = 4 for notch grinding and n = 4 for MgCl2 preparation) using the assays described in section 2.3.1.1. 2.2. Comparison of hemolymph diluent To prevent cell aggregation, hemolymph extracted from oysters (n = 5) was diluted 1.25X in either FSW which had been kept on ice as described in section 2.1 or, in Modified Alserver's Solution (MAS; 7.9 g L−1 sodium citrate; 19.6 g L−1 sodium chloride; 20.7 g L−1 glucose; 3.4 g L−1 ethylenediaminetetraacetic acid (EDTA) in deionised water (DI) and pH adjusted to 7 [43]; which had been kept on ice. Fresh, diluted hemolymph was observed under a microscope to determine purity prior to further analyses. To assess if the diluent had any effect on the hemocytes, the following parameters were determined on the total hemocyte population withdrawn from the same individual: total cell count, viability, esterase activity, phagocytic capability and ROS production. The assays used to determine these parameters are described in section 2.3.1. 2.3. Hemocyte characterization 2.3.1. Flow cytometry Hemolymph was diluted 1.25X in ice-cold FSW and held on ice until analysis as detailed in section 2.1. Flow cytometry analyses were performed on a Guava® easyCyte 5HT Benchtop flow cytometer (EMD Millipore, USA). Incubation of hemolymph samples with dyes were carried out in the dark at room temperature (16–18 °C). All assays were run at a final volume of 200 μL and data were acquired for 30 s at a flow rate of 0.12 μL s−1. The cellular assays performed on hemolymph and the total number of individuals analysed for specific parameters are described below. Data were analysed using Incyte™ software (EMD Millipore) and unless stated, relative fluorescence values are expressed in arbitrary units. All analyses were carried out within 4.5 h of

2. Methods Adult Ostrea chilensis (shell length 84.4 ± 5.6 mm, live weight 412

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and is expressed as a percentage of metabolically active hemocytes relative to the total cell population. To validate this method of staining, a sub-sample of hemolymph from 10 individuals which were to be analysed for esterase activity, were held at 70–90 °C for 20 min to render cells metabolically inactive, before the above staining procedure was carried out on both ‘active’ and ‘inactive’ cells.

hemolymph extraction after determination of longevity (described in section 2.3.1.1). 2.3.1.1. Cell concentration, morphology, viability, longevity and preservation. Hemolymph samples from 30 individuals were diluted 10X with room temperature FSW before staining with SYBR® Green (Invitrogen, 10X final concentration of stock) and propidium iodide (PI, Invitrogen, final concentration 10 μg mL−1) for 5 min. The dye, SYBR® Green I, stains double stranded DNA emitting green fluorescence (FL1 detector) whereas PI enters only cells with compromised membranes. This allows differentiation between cells which are alive (stained green) and those that are considered non-viable or dead (stained both green and red). Using forward scatter (FSC) and side scatter (SSC) light (which correspond to the relative size and internal complexity, or granularity of the hemocytes, respectively), the concentration and morphology of different hemocyte populations were investigated. Hemocyte viability (% of alive cells) was determined on the total hemocyte population and hemocyte sub-populations which were identified as above. Viability is expressed as the percentage of hemocytes stained only green (alive) relative to those stained and both green and red (dead). To validate the above method of double staining, a sub-sample of hemolymph from 10 individuals which were to be analysed for viability, were held at 70–90 °C for 20 min to kill cells. The above staining procedure was then carried out on both ‘live’ and ‘heat-killed’ hemocytes from the same individuals and the percentages of viable hemocytes were expressed as a proportion of the total hemocyte population. To assess the longevity of the hemolymph, the total hemocyte concentration and hemocyte viability (expressed as a proportion of the total hemocyte population) was determined at 2, 4.5 and 6 h post hemolymph extraction (n = 5). In addition, the hemolymph from 5 individuals was preserved in formalin (3.3% final concentration [36], to compare hemocyte concentration and morphological changes of fresh cells and those following 1- and 9-days preservation.

2.3.1.5. Phagocytic capabilities of cells. The uptake of fluorescent latex beads (Fluoresbrite yellow-green microspheres, 2 μm, Polysciences) was used to determine the phagocytic activity of the hemocytes from 20 individuals. This was determined on the total hemocyte population and sub-populations. Hemolymph was diluted 4X with room temperature FSW and 6 μl of 50X diluted bead solution was then added for 30 min prior to analysis. To validate this method, cytochalasin B (Sigma-Aldrich), which inhibits phagocytosis, was used as control for bead adherence without ingestion. Samples from all 20 individuals were run in duplicate, one treated as above and the other with the addition of cytochalasin B at a final concentration of 10 μg mL−1 [45]. The phagocytic capabilities of hemocytes were calculated in twoways: 1) the difference between the proportion of the cell population containing 1 bead or more between duplicate tubes, one treated with cytochalasin B; and 2) expressing the proportion of hemocytes containing 3 or more beads relative to the entire cell population [35]. 2.3.1.6. Reactive oxygen species (ROS) production. The non-fluorescent dye 2′,7′-Dichlorodihydro-fluorescein diacetate (DCFH-DA, Sigma) was used to measure the production of reactive oxygen species (ROS) within the hemocytes. DCFH-DA penetrates hemocytes where it is hydrolysed into DCFH which is retained within the cell. The presence of ROS within the cell oxidises DCFH into fluorescent 20,70-dichlorofluorescein (DCF) in a quantitative manner which is detected on the green, FL1 detector. Hemolymph (n = 25) was diluted 4X with room temperature FSW and DCFH-DA was added at a final concentration of 10 μM for 30 min before analysis [34]. Relative oxidative activity was determined for the total hemocyte population and sub-populations and expressed as the level of green fluorescence. The hemolymph from 10 individuals was used to validate this method. Three aliquots from the same hemolymph sample were treated as above and two were used as a negative and positive control respectively. The negative control was additionally exposed to 10 μM final concentration of CCCP (Carbonyl cyanide 3-chlorophenylhydrazone; Sigma-Aldrich) for the 30 min incubation period of staining (modified from Ref. [46]. The CCCP is an uncoupler of oxidative phosphorylation which can inhibit ROS production. The positive control was additionally exposed to 8 μL of 200X diluted hydrogen peroxide (H2O2, 30% (w/w); Sigma-Aldrich [47]; for the 30 min incubation period. Hydrogen peroxide is a major ROS within the cell and H2O2 added to cells crosses the cell membranes and oxidises DCFH into DCF. Results were calculated for the total hemocyte population only and are expressed as percent increase or decrease relative to the untreated sample (containing no CCCP or hydrogen peroxide).

2.3.1.2. Neutral lipid content. The green lipophilic fluorescent dye BODIPY® 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4aDiaza-s-Indacene, Invitrogen) was used to stain neutral lipids within the hemocytes of 15 individuals. One microlitre of 1 mM BODIPY® was added to 50 μL of room temperature FSW before 50 μL of hemolymph was added. Following 30 min of incubation, samples were diluted with 150 μL of FSW before analysis. Relative neutral lipid content was determined on the total hemocyte population and sub-populations and is expressed as the level of green fluorescence (FL 1 detector). 2.3.1.3. Lysosomal content. LysoTracker® Red DND-99 (Invitrogen) is a red-fluorescent dye used for labelling acidic organelles in live cells and accumulates within the lysosomal compartments. The hemolymph from 15 individuals was diluted 4X with room temperature FSW and LysoTracker® Red was added at 1 μM final concentration [44] and incubated for 30 min. The relative intracellular lysosomal quantity was determined on the total hemocyte population and sub-populations and is expressed as the level of red fluorescence (FL3 detector).

2.3.2. Histology Following hemolymph sampling for flow cytometry, oysters were carefully shucked, and a 3–4 mm thick cross section of the whole oyster was taken following the method of Howard et al. (2004). Sections were fixed in 4% formalin in filtered seawater for 48 h before being transferred to 70% ethanol for at least 48 h. Fixed sections were processed using standard histological techniques and stained with haematoxylin and eosin before being cover-slipped (e.g. Howard et al., 2004). Hemocytes present within the blood vessels in the connective tissue and gill tissue were observed using an Olympus BX51 light microscope at 1000X magnification (oil immersion) to determine different cell types.

2.3.1.4. Esterase activity. Fluorescein diacetate (FDA, Invitrogen) was used to measure the non-specific esterase activity of hemocytes. This dye permeates metabolically active hemocytes, wherein intracellular esterases hydrolyse the FDA into fluorescein which emits a green fluorescence (FL1 detector). Hemolymph (n = 25) was diluted 10X with room temperature FSW and stained with FDA (final concentration 1.25 mg L−1) for 10 min. Hemocytes were divided into those showing esterase activity (corresponding to high green fluorescence) and those in which no esterase activity was detected (low green fluorescence). Esterase activity was determined on the total hemocyte population only 413

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3.3.2. Histology Visual observations of histologically preserved hemocytes present in the blood spaces of the gill and connective tissue allowed distinction of five different hemocyte types based on their size, granularity, nucleus: cytoplasm (N:C) ratio and eosinophilic or basophilic staining. Large, highly eosinophilic granulocytes (Fig. 2A–C), could be distinguished from large, less eosinophilic semi-granulocytes (Fig. 2A, D and E), smaller granulocytes (Fig. 2A–C), hyalinocytes (Fig. 2A, G-I) and small basophilic hyalinocytes or blast-like cells (Fig. 2A and I).

2.4. Statistical analyses Statistical analyses were performed using the program SPSS statistics 23 software and results were considered significant when p ≤ 0.05. Where percent data were obtained (i.e. viability), data were arcsine square-root transformed to ensure normality. Levene's test was used to test the homogeneity of variances. A one-way ANOVA was used to compare differences in the total hemocytes concentration and viability of hemocytes over time. Independent t-tests were conducted for analysing the differences between the two methods of oyster preparation for hemolymph extraction, hemolymph diluent, the phagocytic capabilities of different hemocyte types and assay validation methods. All data are presented as mean ± standard error (SE).

3.3.3. Cell preservation Compared to fresh hemolymph samples, the total hemocyte concentration did not change significantly at days 1 and 9 following formalin fixation (p = 0.448, n = 5); however, different cell types were not distinguishable after 24 h of formalin fixation.

3. Results

3.4. Functional and metabolic characteristics

3.1. Preparation of oysters for hemolymph extraction

3.4.1. Cell viability and longevity Of the entire hemocyte population, the proportion of cells that were alive ranged from 75.0 to 96.6% (89.5 ± 0.9%, mean ± SE, n = 30). Dividing the total hemocyte population into the 2 sub-populations (hyalinocytes and granulocytes) as described above, the mean ± SE proportion of the granulocyte population that was viable was 84.4 ± 1.8% and was 89.4 ± 1.3% for hyalinocytes (Table 1). Subsamples of hemolymph from the same individual which had been exposed to 70–90 °C for 20 min, showed a reduction in viable hemocytes from the mean of 89.5 ± 1.6% to 2.6 ± 1.0% (Fig. 3A, n = 10). Hemolymph samples from the same individuals which had been held on ice for 2, 4.5 and 6 h before assessment of the total hemocyte concentration and hemocyte viability, showed a significant reduction in both parameters at 6 h compared to 2 h and 4.5 h post-bleeding (p = 0.04 and p ≤ 0.001 respectively, n = 5, Fig. 4).

The total hemocyte concentration from oysters which had been relaxed using MgCl2 was significantly higher than for those from which hemolymph was extracted after cutting a notch in the shell (1.3 × 106 ± 2.0 × 105 cells mL−1 vs 4.1 × 105 ± 1.5 × 105 cells mL−1, p = 0.011, n = 4). No significant differences were recorded in the viability or ROS production of the hemocytes between oysters prepared for extraction using either notch-cutting or relaxation using magnesium chloride (p = 0.197 and p = 0.513 respectively, n = 4). Further hemolymph characterization was done on oysters relaxed using MgCl2. 3.2. Comparison of hemolymph diluent No significant differences were recorded in the total hemocyte concentration (p = 0.774), hemocyte viability (p = 0.103), esterase activity (p = 0.816), phagocytic capability (p = 0.976) or ROS production (p = 0.566, n = 5) between hemolymph which had been diluted in either FSW or MAS before analysis. Although the above analyses were determined considering the hemolymph as one total hemocyte population, different hemocyte sub-populations could be determined. More hemocyte sub-populations were observed when using FSW as a diluent, and therefore, further analyses were performed on hemocytes which had been diluted in FSW.

3.4.2. Neutral lipid content Two hemocyte populations could be distinguished when assessing neutral lipid content of the hemocytes, granulocytes and hyalinocytes as described above. Granulocytes contained approximately 50% more neutral lipid than hyalinocytes (Table 1, n = 15). 3.4.3. Lysosomal content Of the two hemocyte populations that could be distinguished when determining lysosomal content, similarly to neutral lipid, the granulocytes contained 52.3% more lysosomes than hyalinocytes (Table 1, n = 15).

3.3. Cell concentration and morphology

3.4.4. Esterase activity Two clear populations couldn't be distinguished for the esterase assay, so values are given as a % of the total hemocyte population. The mean proportion of the hemocyte population showing esterase activity was 81.4 ± 2.3%, mean ± SE, n = 25 (Table 1). A sub-sample of hemolymph from the same 10 individuals which had been exposed to 70–90 °C for 20 min showed a reduction in the mean proportion of hemocytes demonstrating esterase activity (from 80.7 ± 2.5% to 3.9 ± 1.1%, Fig. 3B).

3.3.1. Flow cytometry The concentration of the total hemocyte population ranged from 5.2 × 105–3.2 × 106 cells mL−1 (mean ± SE 1.2 × 106 ± 1.1 × 105 cells mL−1, n = 30). Flow cytometry allowed discrimination of up to four different hemocyte sub-populations circulating in the hemolymph, based on their internal complexity or granularity (Fig. 1). The two least complex or granular cell populations (‘A’ and ‘B’ in Fig. 1 and ‘Hyalinocytes 1’ and ‘2’ in Table 1) were often merged and were more abundant than the two more complex populations (‘C’ and ‘D’ in Fig. 1 and ‘Granulocytes 1’ and ‘2’ in Table 1). The middle population (‘C’ on histograms and also in red on cytograms, Fig. 1) was often small and hard to distinguish from the more complex population (‘D’ on histograms and also in green on cytograms, Fig. 1), therefore; for further analysis in which different cell types could be distinguished, the hemolymph was divided into granulocyte and hyalinocyte populations, which corresponded to the 2 most- and 2 leastcomplex populations respectively (shown on far-right cytograms in green and yellow, Fig. 1). Granulocytes and hyalinocytes comprised approximately 27.9% and 72.1% of the hemolymph, respectively (Table 1).

3.4.5. Phagocytic capabilities of cells Hemocytes showed phagocytic capabilities, ingesting phagocytic beads within the 30 min incubation period (Fig. 5A and B), which were clearly shown on a green fluorescence histogram (Fig. 5C). The mean ± SE percentage of the total hemocyte population that exhibited phagocytosis defined as (1) the difference between the proportion of the cell population containing 1 bead or more between duplicate tubes, one treated with cytochalasin B; and (2) expressing the proportion of hemocytes containing 3 or more beads relative to the entire cell population, was 6.6 ± 1.9% (method 1) and 4.7 ± 1.0% (method 2) 414

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Fig. 1. Histograms and cytograms showing the hemocyte populations within the hemolymph of two Ostrea chilensis adults (a & b) based on their size and complexity. a: Individual 1. from left; a histogram showing the total hemocyte population sub-divided into 4 populations based on their internal complexity (A-D = least to most complex); cytograms showing the total hemocyte population from the same individual; the A-D groupings of the hemocyte sub-populations as part of the hemocyte population as a whole (A = yellow, B = blue, C = red and D = green); and, hemocytes grouped only as granulocyte (green) and hyalinocyte (yellow) populations. b: Individual 2, as above. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion

(n = 20 Fig. 3C). Dividing the cell population into two groups as described in section 3.3.1, granulocytes showed higher phagocytic capabilities compared with hyalinocytes (12.7 ± 2.1% vs 6.9 ± 2.4% using method 1 and 11.3 ± 1.7% vs 1.1 ± 0.5% using method 2, mean ± SE, n = 20, Fig. 3C, Table 1). No significant differences were recorded between the two analysis methods when considering the total hemocyte population (p = 0.775) or just the granulocytes (p = 0.725); however, the hyalinocytes analysed using method 1 showed significantly higher levels of phagocytosis than those analysed using method 2 (6.9 ± 2.4% vs 1.1 ± 0.5% respectively, p = 0.03, Fig. 3C, n = 20).

4.1. Hemocyte populations In the present study, two main sub-populations of hemocytes were identified by FCM and histology based on their morphology: granulocytes and hyalinocytes. The classification of bivalve hemocytes into 2 main groups (1) granulocytes, with granules in the cytoplasm and typically a low nucleus: cytoplasm (N:C) ratio and, (2) hyalinocytes, containing few or no granules within the cytoplasm and a higher N:C ratio, is well established (e.g. Refs. [17,48]. Indeed, these hemocyte types have been identified in many bivalve species (e.g. Refs. [32–36,49,50]; including O. chilensis [19]; however, this is the first time different hemocyte types and their functional and metabolic characteristics have been quantified in O. chilensis. FCM allowed discrimination of up to four different types of hemocytes whilst observations of histologically preserved hemocytes identified five different hemocyte types split between these hyalinocyte and granulocyte populations. A previous study by Ref. [19]; used transmission electron microscopy (TEM) to morphologically distinguish four types of hemocytes in the heart imprints of O. chilensis. The large and

3.4.6. Reactive oxygen species (ROS) production Granulocytes showed higher ROS production than hyalinocytes (Table 1, n = 25). When analysing the hemocyte population as a whole, hemocytes from the same individuals which were exposed to either hydrogen peroxide or CCCP showed an average 222.6 ± 32.0% increase or 29.5 ± 3.4% reduction in ROS production respectively (mean ± SE, n = 10) (Fig. 3D).

Table 1 A summary of the structural, functional and metabolic characteristics of the hemocyte populations of adult Ostrea chilensis. Mean values ( ± SE) or relative quantity indicated by +, are presented per hemocyte sub-population where possible. FSC = size, SSC = internal complexity, ROS = reactive oxygen species, n values given in table.

Hyalinocytes 1 Hyalinocytes 2 Granulocytes 1 Granulocytes 2

FSC

SSC

% of total population n = 30

Viability %

Neutral lipid content n = 15

Lysosomal content n = 15

Esterase activity % n = 25

Phagocytic capacity n = 20

ROS production n = 25

+ +++ +++ +++

+ ++ +++ ++++

28.9 ± 1.7 43.3 ± 1.3 9.4 ± 0.6 18.3 ± 1.2

72.1 ± 1.4

89.4 ± 1.3

+

+

81.4 ± 2.3

+

+

27.9 ± 1.4

84.4 ± 1.8

++

++

++

++

415

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did not change significantly following 9 days preservation in 3.3% formalin. However, different hemocyte types became harder to distinguish after 24 h fixation. Several authors have reported changes in the morphology of hemocyte populations [35,52] and even an increase in the number of distinguishable hemocyte populations in formalin fixedcompared with fresh samples [44]. Nonetheless, the preservation of the total hemocyte population, without distinction between sub-populations is still of practical use; allowing future analysis of structures such as lysosomes [44], the presence of neutral lipids, and alleviating the need for immediate analysis of the hemolymph after sampling. Indeed, the total hemocyte concentration and viability of hemocytes was significantly reduced after 4.5 h ‘holding’ on ice, indicating a decline in the ‘quality’ of the hemolymph and a need to complete analysis of ‘fresh’ samples as quickly as possible after hemolymph extraction. Using FCM on fresh samples, hyalinocytes accounted for ~70% of the circulating hemocytes; ~40% of which were the larger ‘hyalinocyte 2’ population and ~30% the smaller, ‘hyalinocyte 1’ population. Granulocytes accounted for the other ~30% of circulating hemocytes; ~20% and 10% of which were the more complex ‘granulocytes 2’ and less complex ‘granulocytes 1’ population respectively. Studying hemocytes of O. edulis [18,49], similarly found that hyalinocytes were more abundant than granulocytes. In the study of [18] using FCM, the small and large hyalinocytes accounted for ~65% and 24% of the hemocytes population and ~10% were granulocytes. Various biotic (e.g. species, age, relative susceptibility) and abiotic factors (e.g. location, season) may cause substantial variation in the percentages of different hemocyte types found [13,33,53]. Despite the difficulties in the classification of sub-populations of hyalinocytes and granulocytes, such classification, if possible, is important, as sub-populations of both cell types have been found to be functionally heterogeneous [13]. In the current study, the four types of hemocytes identified using FCM were not present or clearly distinct in every oyster [18,54]. similarly observed variations in the number of hemocyte populations in O. edulis and Mytilus edulis respectively. In the present study however, as there was always a clear distinction between cells that were more complex (granulocytes) and those that were less complex (hyalinocytes), further characterization was done on these two cell types only. The analysis of the following functional and metabolic parameters confirmed this as a good divisor.

Fig. 2. A: Light micrographs of hemocytes present in blood vessel of the connective tissue of Ostrea chilensis showing several different hemocyte types. The dotted arrow and B and C show large eosinophilic granulocytes; black arrow and D and E show large semi-granulocytes; grey triangle and F show smaller granulocytes; white triangles and G and H show hyalinocytes and; black triangles and I show small basophilic hyalinocytes or blast-like cells. Scale bar = 10 μm.

small fine- and, coarse-granulocytes identified in that study may correspond to the large and smaller eosinophilic- and, large semi-granulocytes identified using histology and the two granulocyte populations identified using FCM in the present study. Similarly, the hyalinocyte population identified in the study of [19] may correspond to the two hyalinocyte populations identified using histological and FCM techniques in the present study. The identification of different cell types beyond granulocytes and hyalinocytes is challenging due, in part, to the unknown ontogeny of hemocytes. For example, the ‘hyalinocytes 1’ and small basophilic hyalinocyte population identified in the current study using FCM and histology may be blast-like cells; characterised as having a narrow rim of basophilic cytoplasm and a high N:C ratio and reported in other bivalve molluscs [13,51]. These cells are assumed to be hemocyte progenitors and may differentiate into several different hemocyte types. This may make distinction between maturing cells and truly separate cell populations difficult. Indeed, the smaller granulocytes identified by Ref. [19] and in the present study may develop into larger granulocytes and, the presence of ‘semi-granulocytes’ or granulocytes with fewer/finer granules identified in both studies, may be large/more complex granulocytes which have degranulated e.g. following phagocytosis [13,48]. The variation in the number of different hemocyte types identified above also highlights the difficulty in comparing results between hemolymph that was sampled, prepared and analysed using different experimental techniques [18]. identified three hemocyte populations from the hemolymph of the European flat oyster, O. edulis, using flow cytometry; small hyalinocytes, large hyalinocytes and granulocytes which, similarly to the present study, differed more in their internal complexity than their size. Light and electron microscopic analysis of O. edulis hemocytes however, revealed four different sub-populations; smaller and larger hyalinocytes and two-types of granulocytes, which differed in the morphology and size of the granules within the cytoplasm [49]. In the current study, the total hemocyte concentration in samples

4.2. Functional and metabolic characteristics The hyalinocytes and granulocytes of O. chilensis showed different functional and metabolic capacities. The phagocytic capability, lysosomal content, ROS production and neutral lipid content of the granulocytes were higher than for hyalinocytes. The higher phagocytic capabilities of granulocytes have been reported in several oyster species such as the Sydney rock oyster (Saccostrea glomerata) [32], Eastern oyster (Crassostrea virginica) [55], Pacific oyster (Crassostrea gigas) [56] and the closely-related O. edulis (Xue et al., 1998). Lysosomes are involved in the intracellular degradation of engulfed foreign material by secretion of enzymes. In keeping with the above results of granulocytes showing approximately 50% more phagocytic capability than hyalinocytes (using method 1), granulocytes also contained ~50% more lysosomes than hyalinocytes. This supports the commonly held belief that granulocytes are the main cell type involved in the cellular immune defence of bivalves [13,57,58]. Studying hemocytes of O. chilensis which specifically phagocytosed Bonamia sp. however [19], reported that hyalinocytes were the cell type actively phagocytosing Bonamia sp. and less than 1% of large granulocytes were found to do the same. In contrast, in response to the presence of another haplosporidian parasite, Mikrocytos roughleyi, only the granulocytes of the rock oyster, C. commercialis, were found to be involved in phagocytosis [59]. Phagocytosis may therefore depend on the type of particle encountered and lysosomal enzymes present in the hemocytes [19]. Indeed, in the present study, although granulocytes 416

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Fig. 3. Validation of flow cytometric assays for A: Cell viability, B: esterase activity, C: phagocytosis, D: ROS production. A: The proportion of viable (white) and dead hemocytes (grey) from the same individual which had either been analysed immediately (untreated) or, analysed following 20 min exposure to 70–90 °C (heatkilled). Viability was determined on the total hemocyte population, n = 10. B: Proportion of esterase + ve (white) and -ve hemocytes (grey) from the entire population taken from the same individual which had been either analysed immediately (untreated), or heat-killed as described above (heat-killed), n = 10. C: The % of hemocytes showing phagocytosis as determined by the uptake of fluorescent latex beads. White columns were calculated as the difference between the proportion of hemocytes containing 1 bead or more between duplicate tubes relative to negative controls (treated with cytochalasin B) and; grey columns were calculated by expressing the % of hemocytes containing 3 or more beads relative to the entire hemocyte population, n = 20. D: The reactive oxygen species production of hemocytes stained with DCFH-DA and the addition of either hydrogen peroxide or CCCP for 30 min. Results are displayed as a % increase or decrease in ROS production relative to untreated samples, n = 10. Data were compared using independent t-tests and treatments with the same letter in the same typeface were not significantly different (p > 0.05). All data are presented as mean ± SE.

showed higher phagocytic capabilities of 2 μm fluorescent beads, both cell types were capable of phagocytosis. The phagocytic capability of both granulocytes and hyalinocytes has been similarly reported in other bivalve species (e.g. Refs. [32,34,36]. The lower phagocytic capabilities of hyalinocytes seen in this study

when defining phagocytosis as three or more beads associated with each hemocyte, compared with the method which defined phagocytosis as the difference between two tubes, one of which was treated with the inhibitor cytochalasin B, was similarly reported by Ref. [36] when analysing the whole hemocyte population of C. virginica using the same

Fig. 4. A: The total hemocyte concentration and B: viability of hemocytes held on ice for up to 6 h after extraction from adult Ostrea chilensis. Differences between time points were compared using a 1-way ANOVA. * (p ≤ 0.05), *** (p < 0.001). Data are presented as mean ± SE. 417

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obtain accurate results. Although no differences were recorded in the viability or ROS production of the hemocytes between the 2 methods of preparing oysters for hemolymph extraction, fewer circulating hemocytes were recorded from the oysters which had a notch cut in the shell to allow access to the adductor muscle. These hemocytes may have aggregated in response to the disturbance of the oyster during notchcutting and so did not pass through the flow cell during FCM analysis. The rapid aggregation of hemocytes in response to the mechanical disturbance of hemolymph sampling was also reported by Ref. [39]. In their study, mussel (Mytilus californianus) hemolymph was collected by inserting a plastic rod between the shells to prevent closing, before a cooled sterile syringe and needle were used to extract the hemolymph from the posterior adductor muscle [39]. In the present study, the extraction of the hemolymph was also easier in oysters relaxed using MgCl2, as gaping ensured easier access to the adductor muscle, further indicating this method of preparing flat oysters for hemolymph extraction as more suitable for future use. As hemocyte clumping is often a problem during the analysis of bivalve hemolymph, the use of two diluents to prevent cells from clumping were also tested (FSW and MAS) and again, no differences were recorded in the total concentration, viability, esterase activity, ROS production or phagocytic capabilities of the hemocytes. More hemocyte populations could be identified in hemolymph which had been diluted in ice-cold FSW compared to ice-cold MAS. Although Modified Alserver's solution contains sodium chloride and glucose to help maintain the morphology of hemocytes [70], for O. chilensis, FSW appears to be a more suitable diluent to maintain hemocyte morphology. In addition, a previous study by Ref. [70]; reported a reduction in the chemiluminescence of the hemocytes of M. galloprovincialis which had been diluted in an anti-aggregant solution containing EDTA. This chemical was found to scavenge calcium ions from the hemolymph which, although slowed hemocyte aggregation, also affected the calcium-dependent functions, such as ROS production [70], again indicating FSW as a more suitable diluent for FCM studies. By using the specific sampling and storage method described in the present study, the total hemocyte concentration and viability of O. chilensis hemocytes remained the same for approximately 4.5 h following extraction from the oyster, suggesting that analyses of hemocyte parameters should be completed within this time to obtain accurate results. It should be considered; however, that other functional and metabolic characteristics of hemocytes, such as ROS production and phagocytic capabilities, which were not measured when assessing hemocyte longevity in the present study, may significantly change prior to this time.

Fig. 5. Micrographs showing A: hemocyte and 2 μm green fluorescent bead separately (no ingestion) and B: hemocyte associated with 1 bead. Scale bar = 50 μm. C: histogram showing hemocytes (grey) which are associated with more than 1 green fluorescent bead and more than 3 beads. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

methods. The release of ROS by hemocytes, although considered a part of normal cellular respiration [60], is also part of an important mechanism of internal cellular defence in which pathogens are eliminated following phagocytosis [36,61–63]. The increased ROS production in granulocytes compared to hyalinocytes recorded in the present study, agrees with this, and has been similarly found in other bivalve species [33,36,64,65]. Neutral lipid within hemocytes is considered an important means of energy storage within the cell [66,67] and this energy may be mobilised by the cell to support an immune response. In the present study, the amount of neutral lipid was estimated to be again ~50% higher in granulocytes compared with hyalinocytes, and further supports the hypothesis that granulocytes may play a more important role in phagocytosis and the overall immune response. Esterase is responsible for the hydrolysis of several choline esters and plays a role in immune defence in bivalves [68]. In the current study, esterase activity was determined on the hemocyte population as a whole and ~80% of the population demonstrated esterase activity. Characterizing hemocyte sub-populations of the green-lipped mussel, Perna viridis [65], reported that granulocytes, in keeping with having higher phagocytic capabilities, lysosomal content and ROS production, also demonstrated higher proportions of cells with esterase activity, ~60% compared with ~3% in hyalinocytes. Given the same trends reported for phagocytosis, lysosomal content, ROS production and neutral lipid content in the present study, we could expect the same trend for esterase activity. The viability of the entire hemocyte population in the present study ranged from 73.5% to 96.6%, similar to the viability of hemocytes of O. edulis reported by Xue et al. (2001; 78.2%–99.3%). The recycling of dead hemocytes by other hemocytes in circulation is widely accepted as the reason for the small proportions of dead circulating hemocytes [12,36,69] and has been noted in histological preparations of several bivalve species (Webb, personal observation). Hemocyte viability is a reliable indicator of a stressed immune system in oysters [36] and in the present study, was used (along with other parameters) as an indicator of effective hemolymph sampling in O. chilensis.

4.4. Conclusion Using FCM, the present study identified four different hemocyte types circulating in the hemolymph of the flat oyster, O.chilensis, collected from the Foveaux Strait, NZ. These four cell types were divided into two main hemocyte populations (hyalinocytes and granulocytes) and were morphologically, functionally and metabolically characterised. Protocols for sampling and characterizing the hemocytes are detailed and provide a useful resource for those conducting research on the impact of stressors (e.g. exposure to parasites such as Bonamia spp.) on the functions of the hemocytes and subsequent immune response of flat oysters. Acknowledgements We thank Dr. Philippe Soudant and Nelly LeGoïc from the Institut Universitaire Européen de la Mer, France, for their flow cytometry advice; Joanna Copedo for technical assistance and Dr. Kate Hutson for review of the manuscript. This work was supported by funding from the NZ Government's Ministry for Business, Innovation and Employment contract CAWX1707.

4.3. Hemocyte sampling Effective hemocyte sampling, storage and handling are essential to 418

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