Flow cytometric characterization of hemocytes of the solitary ascidian, Halocynthia roretzi

Accepted Manuscript Flow cytometric characterization of hemocytes of the solitary ascidian, Halocynthia roretzi Ludovic Donaghy, Hyun-Ki Hong, Kyung-Il Park, Kajino Nobuhisa, Seok-Hyun Youn, Chang-Keun Kang, Kwang-Sik Choi PII:

S1050-4648(17)30243-7

DOI:

10.1016/j.fsi.2017.05.009

Reference:

YFSIM 4562

To appear in:

Fish and Shellfish Immunology

Received Date: 19 November 2016 Revised Date:

27 April 2017

Accepted Date: 1 May 2017

Please cite this article as: Donaghy L, Hong H-K, Park K-I, Nobuhisa K, Youn S-H, Kang C-K, Choi KS, Flow cytometric characterization of hemocytes of the solitary ascidian, Halocynthia roretzi, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.05.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Flow Cytometric Characterization of Hemocytes of the Solitary Ascidian, Halocynthia

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roretzi.

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Ludovic Donaghy1, Hyun-Ki Hong1,5, Kyung-Il Park4, Kajino Nobuhisa1, Seok-Hyun Youn2,

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Chang-Keun Kang3, and Kwang-Sik Choi1* 1

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School of Marine Biomedical Science (BK21 PLUS), Jeju National University 102,

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Jejudaehakno, Jeju 63243, Republic of Korea

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Technology, Gwangju 61005, Republic of Korea

Department of Aquatic Life Medicine, College of Ocean Science and Engineering, Kunsan

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School of Environmental Science & Engineering, Gwangju Institute of Science and

National University, Gunsan 54150, Republic of Korea 5

Research Institute for Basic Sciences, Department of Oceanography, Chonnam National University, Gwangju 61186, Republic of Korea

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Fishery and Ocean Information Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea

Submitted to Fish & Shellfish Immunology

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As a Research Article

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Running Title: Hemocyte characterization of ascidian Halocynthia roretizi

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* Corresponding author: K.-S. Choi

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Email: [email protected]; Tel: +82-64-756-3422; Fax: +82-64-756-3493

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ABSTRACT Internal defense of ascidians relies, at least partially, on cells circulating in body

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fluids and infiltrating in tissues, referred to as hemocytes, although structure and composition

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of ascidian hemocytes still remain unclear. In the present study, we investigated hemocyte

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types and their functions of the solitary ascidian Halocynthia roretzi using flow cytometry.

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Based on morphology, cellular activities and intracellular parameters from the flow cytometry,

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we identified eight hemocyte types including, three granulocytes (Gr-1, Gr-2, and Gr-3), 4

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hyalinocytes (Hy-1, Hy-1’, Hy-2, and Hy-3) and lymphocyte-like (Ly-like) cells. The

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granulocyte Gr-1 accounted for 30% of the total circulating hemocytes and exhibited highest

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density of lysosomes and oxidative activity. Gr-1 was deeply involved in phagocytosis and

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degradation of foreign material. Hyalinocytes consist of two main populations, Hy-1 and Hy-

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2, and each accounted for 30% of the circulating hemocyte. Hy-1 displayed lysosomal

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content, an inducible oxidative activity, and no proteases, while Hy-2 expressed highest

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density of intracellular proteases, no lysosomes and a low oxidative activity. It was believed

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that Hy-2 may represent an important link between cellular and humoral immune reactions.

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Hy-1 did not show phagocytosis activity. Hy-3 and the Ly-like cells presented a similar

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profile except for their size and complexity, and Hy-3 may represent an intermediate

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differentiation/maturation step between Ly-like cells and other hemocyte populations. This

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first characterization of the hemocyte populations of H. roretzi provides a solid basis to

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investigate further their respective roles and functions in physiological and pathological

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contexts.

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Key words

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Ascidians, Halocynthia roretzi, hemocytes, phagocytosis, lysosomes, proteases, oxidative

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activity, flow cytometry

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1. INTRODUCTION Ascidians are considered a key group in chordate phylogeny [1] and have been

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suggested the sister group of vertebrates [2,3]. Immune system of ascidians has been

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investigated at the molecular level, as a model for the evolution of innate immune system of

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vertebrates [4,5]. However, the structure and composition of ascidian immune cells, referred

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to as hemocytes, still remain unclear. Hemocytes are involved in immune response [6-8] as

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well as in physiological functions such as tunic and tissue repair, scavenging of dead cells,

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nutrient acquisition and distribution, and accumulation and excretion of waste metabolites [9-

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14]. Hemocytes are, therefore primordial for maintenance of homeostasis and survival of

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ascidians. Several studies have aimed to classify hemocytes of ascidians by the means of light

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and electron microscopy [15-17]. However, even within a single species, different studies

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have reported a different number of hemocyte types, ranging from 4 to 12 types of cell

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populations [18-22]. Identification and quantification of ascidian hemocyte populations by

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the means of microscopy can therefore be extremely challenging, and has led to numerous

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inconsistencies and misinterpretations, partially due to subjectivity of the visual analysis.

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To understand hemocyte types and their involvement in maintenance of homeostasis

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in ascidians, it is necessary to determine their functional and metabolic activities. For this

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purpose, flow cytometry has previously proved successful in detailed characterization of

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cellular and intracellular parameters of hemocytes from marine invertebrates. However, while

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flow cytometry has extensively been applied to the study of hemocytes of marine bivalves

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and gastropods [23-26], it has scarcely been applied to ascidians. For instance, in the solitary

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ascidian Halocynthia aurantium, Sukhachev et al. [27] discriminated between five hemocyte

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populations based on cytomorphology and applied a panel of ten monoclonal antibodies

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(mAbs) directed against human leukocyte adhesion molecules. From that panel, only two

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mAbs showed cross-reactivity and no functional activity was further investigated. A first

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ACCEPTED MANUSCRIPT attempt to characterize hemocytes of H. roretzi was also performed using flow cytometry [28].

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However, the study was poor with the determination of only three basic hemocyte

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populations and no functional characterization. Further investigations are therefore, necessary

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to understand the hemocyte populations of ascidians and their respective roles and functions.

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In the present study, using flow cytometry, we aimed at investigating the hemocytes

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of the solitary ascidian H. roretzi, which is one of the valuable marine shellfish resources in

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Korea. In 2016, the Korean ascidian aquaculture industry produced 31,353 metric tons of H.

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roretzi with a value of approximately 50 million US dollars [29]. On the south coast, wild

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populations of H. reretzi mainly inhabit subtidal rocky bottoms, where they are also culture

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using suspended long-line method in small bays. Characterization of populations and sub-

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populations of H. roretzi hemocytes in this study was based not only on their cytomorphology,

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but also on cellular functions and intracellular parameters including phagocytosis capacities,

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lysosomal density, basal and stimulated oxidative activity, and intracellular proteases activity.

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2. MATERIALS AND METHODS

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2.1. Animals

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From June to August 2016, adults H. roretzi were supplied from a H. roretzi

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aquaculture farm located in Jinhae bay on the southeast coast of Korea. After being

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transported to the laboratory, ascidians were maintained in aerated seawater (salinity 32 ± 1

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psu; temperature 21 ± 1 °C) for 24 hours before analysis of hemocyte parameters. A total of

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80 individuals have been used in the present study.

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2.2. Hemolymph collection Using a 1 mL syringe fitted with a 22 G x 1 1/4˝ needle and filled with 500 µL of ４

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[19]), approximately 500 µL of hemolymph were collected from each individual.

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Hemolymph was withdrawn from the visceral cavity by puncture through the tunic matrix.

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Each hemolymph sample was visually assessed for purity (i.e., absence of gametes and food)

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under a light microscope. From each individual, 150 µL hemolymph harvested were

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transferred in microtubes containing an equal volume of 3% formalin and maintained on ice.

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The remaining hemolymph was centrifuged at 315 g for 10 min at 4 °C, and the supernatant

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was discarded, while the pellet was re-suspended in 800 µL of EDTA solution. To determine

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phagocytosis activity, the hemocytes were re-suspended in 800 µL of FSW. The tubes

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containing the hemolymphs were kept on ice during incubations with flow cytometry probes

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and chemicals. The reaction was carried out for 10 min.

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2.3. Flow cytometry analyses

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Hemocyte parameters were analyzed using a FACS Calibur flow cytometer (Becton-

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Dickinson, USA) equipped with a 488 nm Argon laser. Analyses were conducted on

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individual samples. Each investigated parameter has been determined on a minimum of 15

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animals. From 5,000 to 10,000 cells were analyzed in each parameters. Data were processed

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and analyzed using Flowing Software 2.5.1.

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2.3.1. Cytomorphology of hemocyte populations Cytomorphology of H. roretzi hemocyte populations was investigated using SYBR

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Green I (Invitrogen, USA) staining procedure. SYBR Green I is a cell permeant dye which

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binds the double stranded DNA and allows discrimination of single cells, aggregates and

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debris. Morphology of single cells was determined based upon relative flow cytometric

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parameters, Forward Scatter (FSC) and Side Scatter (SSC). FSC and SSC respectively ５

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measure the particle size and the internal complexity. For the analysis, a 100 µL either fresh

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or formalin-fixed hemocytes was mixed with 400 µL of FSW containing SYBR Green I (final

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concentration 10x) and incubated for 20 minutes in the dark at room temperature.

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2.3.2. Light and scanning electron microscopy

Morphology of hemocytes populations were characterized using light microscopy

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and scanning electron microscopy (SEM). BD FACSAria III flow cytometer (Becton

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Dickinson, USA) equipped with a 488 mm laser discriminated the formalin-fixed H. roretzi

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hemocytes according to the cell size (FSC) and the granularity (SSC). The sorted hemocyte

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sub-populations were centrifuged, and the hemocyte monolayers were stained with

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Hamacolor reagents (Merck, Germanry) for light microscopy.

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For SEM, the discriminated hemocyte sub-populations were fixed in 2%

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glutaraldehyde at 4 °C for overnight. The fixed cells were then filtered through a 0.2 µm

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membrane filter (Isopore, Ireland), and the filtered hemocytes were dehydrated in graded

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series of ethanol (50, 70, 85, 95, and 100 %). The filters containing the dehydrated hemocytes

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were dried using the critical point method using liquid CO2, then coated with gold in Ion

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sputter (E-1045, Hitachi, Japan). Finally, morphological features of the hemocytes were

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examined using a scanning electron microscope (Horiba EX-250, Hitachi, Japan).

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2.3.3. Mortality of hemocytes Hemocyte mortality was determined on fresh harvested hemocytes using a double

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staining procedure including SYBR Green I and propidium iodide (PI; Sigma-Aldrich, USA).

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As previously described, SYBR Green I allows for discrimination between single cells,

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aggregates and debris. Membrane of viable cells does not allow PI to penetrate, whereas

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altered membranes are permeable to PI. Dead hemocyte cells are characterized by loss of ６

ACCEPTED MANUSCRIPT membrane integrity and are, therefore, double stained by SYBR Green I and PI. A 100 µL

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sample of fresh hemocytes was mixed with 400 µL of FSW containing SYBR Green I (final

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concentration 10x) and incubated for 10 min in the dark at room temperature. PI (final

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concentration 20 µg.ml-1) was then added to each tube and incubated for 10 more minutes in

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the same conditions. Hemocyte mortality was determined as the percentage of SYBR Green I

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(FL1 detector)/ PI (FL3 detector)-positive cells.

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2.3.4. Lysosomal content

The presence and relative amount of lysosomes in hemocytes was determined on

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formalin-fixed hemocytes using LysoTracker Red (Invitrogen, USA), a membrane permeable,

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fluorescent probe that accumulates within the lysosomal compartments. Formalin-fixed

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hemocytes was diluted in FSW (1:5) containing LysoTracker Red (final concentration 1 µM)

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and incubated 90 min in the dark, at room temperature. Relative intracellular lysosomal

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quantity is expressed as the level of red fluorescence (FL3 detector) in arbitrary units (A.U.).

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2.3.5. Intracellular oxidative activity

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Intracellular oxidative metabolism was determined on fresh hemocytes using 2’7’-

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dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Invitrogen, USA), a membrane

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permeable, non-fluorescent probe. Inside hemocytes, the –DA radical is first hydrolyzed by

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esterase enzymes. Intracellular reactive oxygen species (ROS), nitrite radicals and various

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oxidase and peroxidase enzymes then oxidize DCFH to the fluorescent DCF molecule. DCF

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green fluorescence is thus proportional to the intracellular oxidative activity of hemocytes.

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Fresh hemocytes was diluted in FSW (1:5) containing DCFH-DA (final concentration 10 µM)

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+/- Phorbol 12-myristate 13-acetate (PMA; final concentration 10 µg.mL-1; Sigma-Aldrich,

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USA), an inducer of ROS production. After 60 min in the dark at room temperature, tubes

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were transferred on ice to stop the oxidative metabolism and proceeded to flow cytometry

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analysis. Relative intracellular oxidative activity was expressed as the level of green

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fluorescence (FL1 detector) in arbitrary units (A.U.).

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2.3.6. Intracellular proteases

Presence and relative activity of proteases in the hemocytes was determined using

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EnzCheck Protease Assay (Invitrogen, USA), which consists of casein derivatives heavily

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labelled with the pH-insensitive green-fluorescent dye. In its native form, fluorescence of the

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casein conjugate is quenched, and protease-catalyzed hydrolysis releases green fluorescence.

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The accompanying increase in fluorescence is therefore proportional to the protease activity.

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Briefly, fresh hemocytes was diluted in FSW (1:5) containing Bodipy FL casein (Invitrogen,

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USA, final concentration 5 µg.mL-1) and incubated in the dark at room temperature. After 60

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minutes, the assay tubes were transferred on ice to stop the enzymatic activities and run flow

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cytometry analysis. Relative intracellular protease activity was expressed as the level of green

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fluorescence (FL1 detector) in arbitrary units (A.U.).

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2.3.7. Phagocytosis capacity

Evaluation of phagocytosis capacities was based on the ingestion of fluorescent latex

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microbeads (2.0 µm, Polysciences Inc., USA) by the hemocytes. PMA was used to stimulate

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phagocytosis of hemocytes. Incubation of fresh hemocytes with FSW (1:5) containing

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microbeads +/- PMA (Final concentration 10 µg.mL-1) was performed at room temperature

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for 120 min. Phagocytosis reaction was minimized by transferring the tubes on ice.

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Phagocytic capacities were defined as (i) the percentage of hemocytes that ingested one or

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more microbeads, and (ii) the average number of microbeads per phagocytic hemocytes.

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2.4. Statistics Prior to the analyses, the flow cytometry data were transformed as arcsin of the

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square root. Shapiro-Wilk test and Cochran’s test were performed to test normality and

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homogeneity of variance of the data, respectively. When variances were homogeneous, data

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were analyzed by one-way ANOVA; if not, data were compared using the non-parametric

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Kruskal-Wallis test. Following comparison of variances, Dunn’s test was performed to

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determine pairwise differences between data groups. One-tailed t-test was used to compare

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oxidative activity in hemocytes with and without PMA stimulation. For all statistical analyses,

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differences were considered significant at p < 0.05. Statistical analyses were performed using

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MaxStat (MyCommerce, USA).

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3. RESULTS

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3.1. Hemocyte populations based on cytomorphology

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Using flow cytometry, SYBR Green I staining first allowed discrimination of single

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cells from the aggregates and debris. Hemocyte populations circulating in H. roretzi

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hemolymph were then determined based on size (FSC) and internal complexity (SSC) (Fig. 1,

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Table 1). Circulating hemocyte populations were investigated in formalin-fixed (Fig. 1A) and

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fresh (Fig. 1B) hemocytes. In formalin-fixed hemocytes, a total of 7 different hemocyte

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populations were identified based on cytomorphological discrimination: 3 sub-populations of

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granulocytes (Gr 1 to 3), 3 sub-populations of hyalinocytes (Hy 1 to 3), and 1 population of

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lymphocyte-like (Ly-like) cells (Fig. 1A, Table 1). Granulocytes (Gr 1 to 3) were

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characterized by high internal complexity (SSC), while hyalinocytes (Hy 1 to 3) displayed

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lower internal complexity with a larger range of sizes, compared to the granulocytes (FSC).

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Lymphocyte-like cells were characterized by smaller size and internal complexity than all

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other hemocyte populations. In the fresh hemocytes (Fig. 1B), only 4 different hemocyte

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populations could be discriminated based on cytomorphology: 1 population of granulocytes,

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2 populations of hyalinocytes (Hy 1 and Hy 2 + 3), and lymphocyte-like cells. In the formalin-fixed smaples, 3 distinct hemocyte populations could be recognized,

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including Gr 1, Hy 1 and Hy 2, which accounted for 26.8 ± 3.8, 26.7 ± 2.8 and 28.3 ± 3.0 %

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of the total circulating hemocytes, respectively (Table 2). Granulocyte populations Gr 2 and

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Gr 3 accounted respectively for 5.3 ± 1.6 and 5.1 ± 1.5 % of the total hemocytes. The

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hyalinocyte population Hy 3 and lymphocyte-like cells accounted respectively for 4.2 ± 0.5

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and 3.6 ± 0.8 % of the total hemocytes. In the fresh hemocytes (Table 2), granulocytes

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accounted for 40.1 ± 3.8 %, while the hyalinocyte Hy 1 and Hy 2 + 3 accounted for 16.9 ±

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1.7 and 38.2 ± 3.3 % of the total hemocytes, respectively. Lymphocyte-like cells accounted

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for 4.9 ± 1.0 % of the total hemocytes. Both in the formalin-fixed and fresh hemocytes,

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granulocytes represented between 35 and 40 % of the total cells, hyalinocytes accounted for

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55 to 60 %, and lymphocyte-like cells represented less than 5 % of the total circulating

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hemocytes (Table 1).

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3.2. Microscopic observation of hemocyte populations The flow cytometry successfully discriminated different sub-populations of H. roretzi

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hemocytes, and allowed to visualize the seven hemocyte sub-populations. Under a light

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microscope, Gr 1 and Gr 2 appear as round and oval, containing numerous granules in the

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cytoplasm (Fig. 2A). Several vacuoles can be seen in the cytoplasm of Gr 3 (Fig. 2B, C).

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Contrary to the granulocytes (i.g., Gr 1, 2, and 3), Hy 1 (Fig. 2D), Hy 2 (Fig. 2E, F), and Hy 3

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(Fig. 2G) exhibit no granules in the cytoplasm. Although Hy 1, 2, and 3 are similar in shape,

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sizes of these 3 hyalinocytes sub-populations are different. Ly-like cell is small and round,

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with very thin cytoplasm (Fig. 2H).

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lymphocyte-like cell. In SEM, the granulocyte is characterized with presence of numerous

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microvilli on the cell surface (Fig. 3A), while the microvilli are absent or scarce on the

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hyalinocyte cell surface (Fig. 3B). Compared to the granulocyte or hyalinocyte, the

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lymphocyte-like cell is comparatively smaller in size, smooth cell surface and spherical (Fig.

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3C).

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3.3. Mortality of hemocyte populations

Mortality of H. roretzi hemocyte determined using flow cytometer ranged from 0.45

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to 6.37 %, with a mean value of 2.0 ± 0.8 % (N=15, Fig. 4B). Mortalities of the granulocyte

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(Gr 1+2+3) and hyalinocyte populations (Hy 2+3) were similar to the total hemocyte

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mortality (Fig. 4B). Hyalinocyte Hy 1 population and lymphocyte-like cells exhibited lower

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mortality than other sub-populations, respectively 0.6 ± 0.2 % and 0.3 ± 0.2 % (Fig. 4B,

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Table 1).

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3.4. Lysosomal content

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The relative content in lysosomes was investigated on formalin-fixed hemocytes.

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Based on intracellular lysosomal content and complexity (SSC), 8 different hemocyte

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populations

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cytomorphologically (Gr 1 to 3, Hy 1 to 3 and Ly-like), plus 1 hyalinocyte sub-population

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(Hy 1’). The sub-population Hy 1’ could not be discriminated from Hy 1 based on size and

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complexity but contained less quantity of lysosomes. Based on the quantity of lysosomes, the

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sub-populations could be grouped into 3 categories (Fig. 5A): low content, including

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granulocyte populations Gr 2 and Gr 3, and Ly-like cells, medium content, including

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hyalinocyte populations Hy 1’ and Hy 2, and high content, including the populations Gr 1, Hy

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1 and Hy 3. Comparison of lysosomal content among different hemocyte sub-populations may

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however be affected by size of the cells, since bigger hemocyte may contain larger quantity

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of lysosome, without necessarily an actual higher density of lysosomes and higher

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intracellular digestion capacities. To overcome this bias, we determined density of the

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lysosomes as the ratio between fluorescence intensity (FL3) and cell size (FSC) (Fig. 5B,

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Table 1). Based on that ratio, the population Gr 1 actually expressed a higher concentration of

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lysosomes than all other hemocyte populations, suggesting a higher involvement in the

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intracellular digestion. Also, density of the lysosomes in lymphocyte-like cells is actually

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similar to Hy 1 and Hy 3 populations. Regarding the density of lysosomes, hemocyte

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populations could be divided into three groups (Fig. 5B, Table 1): low density, including

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granulocytes Gr 2 and Gr 3, and hyalinocytes Hy 1’ and Hy 2, medium density, including

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hyalinocyte populations Hy 1 and Hy 3, and Ly-like cells, and high density represented by the

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granulocyte population Gr 1.

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3.5. Intracellular oxidative activity

Intracellular oxidative activity was determined on fresh hemocytes. On these samples,

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the populations of Gr 3 and Hy 3 could not be discriminated by cytomorphological

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parameters. Although Gr 1 and Gr 2 populations showed similar FSC and SSC parameters

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(Fig. 1B), they could be discriminated based on intracellular oxidative activity. As for

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lysosomal content, to avoid bias due to different size of cells and to accurately compare

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intracellular oxidative activity, we calculated the ratio of fluorescence intensity (FL1) to the

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cell size (FSC) (Fig. 6A, Table 1). Basal intracellular oxidative activity (i.e., without PMA

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stimulation) was the highest in the granulocyte population Gr 1, followed by populations Gr 2

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ACCEPTED MANUSCRIPT and Hy 1, and was the lowest in Hy 2 and Ly-like cells (One-way ANOVA and Dunn, p<

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0.05). After PMA stimulation, the oxidative activity was also the highest in Gr 1, followed by

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Gr 2 and Hy 1, and was the lowest in Hy 2 and Ly-like cells. To illustrate extent of increase in

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the oxidative activity before and after the PMA stimulation, ratios of the basal oxidative

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activities (i.e. NoPMA) to PMA stimulated cell oxidative activities are determined and

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plotted on Fig. 6B. As Fig. 6B showed, the ratios in Gr 1 and 2 and Hy 1 ranged 2.5 to 3,

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indicating that the oxidative activities of these cell populations increased 2.5 to 3 fold after

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MPA stimulation, although the ratios among these 3 cell populations were not significantly

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different. Contrary to Gr 1 and 2 and Hy 1, the ratio of the basal oxidative activity to the

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PMA stimulated oxidative activities in Hy 2 and Ly-like hemocytes ranged 1.5 to 1.7, and

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these ratios were significantly lower that the ratios observed from Gr 1 and 2 and Hy 1 (one-

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way ANOVA and Dunn, p<0.05).

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3.6. Activity of intracellular proteases

Activity of intracellular proteases was determined on fresh hemocytes, and the ratio

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between fluorescence intensity (FL 1) and cell size (FSC) was calculated to compare the

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proteases activity among different hemocyte populations (Fig. 7, Table 1). While intracellular

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protease activity of hemocyte Gr 3 could not be observed, populations Hy 1’ and Hy 3 could

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be discriminated based on fluorescence intensity (FL 1) and internal complexity (SSC)

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parameters. The hyalinocyte population Hy 2 displayed significantly higher activity of

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intracellular proteases compared with all other hemocyte populations. The protease activity in

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Gr 2 was significantly higher than Gr 1, Hy 1, Hy 2, Hy 3, and Ly-like cells (One-way

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ANOVA; Dunn; p < 0.05).

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ACCEPTED MANUSCRIPT Phagocytosis capacities of H. roretizi hemocytes were expressed as the percentage of

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cells that ingested at least one bead, and the mean number of beads per cell that performed

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phagocytosis. Hyalinocytes and Ly-like cells did not show any phagocytosis activity, while

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the granulocytes appeared to be involved in phagocytosis (Fig. 8A, Table 1). However,

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cytomorphological modifications due to the engulfment of microbeads did not allow for an

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accurate determination of the sub-populations of granulocytes involved in the phagocytosis.

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In absence of PMA stimulation, the percentage of cells performing phagocytosis accounted

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for 6.8 ± 1.5 % of total hemocytes and 40.3 ± 7.2 % of granulocytes (Fig. 8C). Granulocytes

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ingested a mean number of 4.0 ± 0.5 beads per cell (Fig. 8C). PMA stimulation did not

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significantly modify the phagocytosis index and the mean number of ingested beads per cell.

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4. Discussion

The flow cytometry used in this study enabled us to discriminate 7 different types of

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hemocytes circulating in body fluids of the solitary ascidian H. roretzi; 3 populations of

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granulocytes (Gr 1 to 3), 3 populations of hyalinocytes (Hy 1 to 3) and 1 population of

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lymphocyte-like cells (Ly-like). While 7 hemocyte cell populations could be observed from

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samples fixed right after withdrawal, only 4 populations were distinguishable from the fresh

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body fluids; 1 population of granulocyte, 2 populations of hyalinocyte and the lymphocyte-

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like cell. In marine invertebrates, fewer types of hemocytes are commonly observed from

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fresh samples compared to the fixed samples [24, 30]. Previous studies also have reported

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that fixed hemocytes appear larger and more complex than fresh hemocytes in flow

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cytometry [28, 30]. Degranulation, exocytosis, reorganization of intracellular organelles and

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cytoskeleton, as well as potential osmolarity-driven cell volume adjustment may occur during

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centrifugation and maintenance in suspension before analysis.

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Flow cytometry revealed that in the hemolymph of H. roretzi, granulocytes accounted for 35 to 40 % of the circulating cells, while the hyalinocytes represented 55 to 60 %

358

of the total hemocytes, and Ly-like cells remained less than 5 % of the total cells. Hemocyte

359

populations of H. roretzi have been previously investigated by the means of light and electron

360

microscopy. Although an attempt for a standardized nomenclature of hemocytes in H. roretzi

361

was made in 1993 [16], almost all investigators have reported a different classification

362

scheme with different cell names and distribution frequencies, ranging from 5 [19] to 12

363

different cell populations [20]. While identification and quantification of hemocyte types by

364

microscopy can lead to inconsistencies and misinterpretations, direct comparison with flow

365

cytometrically defined cell populations may be difficult, both in terms of cell types and

366

frequencies [31, 32]. Indeed, the flow cytometric parameter “granularity” or “internal

367

complexity” (SSC) varies with numerous parameters such as the types and quantity of

368

organelles, roughness of plasma and intracellular membranes, or inclusions of extracellular

369

material. Classification based on the flow cytometric SSC parameter may therefore not fully

370

match with the observation of vacuoles or granules, one of the main visual classification

371

criteria. Furthermore, cells displaying similar SSC level may actually belong to different sub-

372

populations. To accurately characterize and discriminate between cell populations, it is

373

necessary to determine cellular activities and intracellular metabolisms.

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Phagocytosis is the most widely investigated cellular activity of marine bivalve

375

hemocytes, which is deeply involved in immune defense against pathogens [33-35].

376

Phagocytosis, however is primordial for defense against all kind of invading foreign

377

substances, complementary acquisition of nutrients, removal of natural and pathologic dead

378

cells as well as remodeling of tissues such as post-spawning gonadic tissues (reviewed in

379

[36,37]). Environmental- or anthropogenic-driven alteration of phagocytosis capacities of

380

hemocytes would therefore, impair homeostasis of ascidians, threatening sustainability of １５

ACCEPTED MANUSCRIPT wild or cultured animals. In H. roretzi, only granulocytes demonstrated phagocytosis

382

capacities against latex microbeads. Unfortunately, ingestion of microbeads modified

383

cytomorphological parameters, not allowing for accurate identification of the granulocyte

384

sub-populations that perform phagocytosis.

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Phorbol 12-myristate 13-acetate (PMA) has been reported to modulate the

386

phagocytosis capacity, either by inhibiting uptake of specific targets [38], or by inducing

387

maturation of the immune cells and stimulating their phagocytosis activity [39]. In the present

388

study, stimulation of hemocytes of H. roretzi with PMA did not modify their phagocytosis

389

capacity. A few studies have investigated phagocytosis activities of H. roretzi hemocytes

390

using light microscopy, reported that 1-2 types of H. roretzi hemocytes are involved in the

391

activities, although those studies did not define or classified the hemocyte types. Based upon

392

microscopic observation of phagocytosis activity of H. roretzi hemocyte, Sawada et al. [40]

393

reported 2 main phagocytic populations, namely as phago-amoebocyte (9.4% of the total

394

hemocytes) and fusogenic phagocytes (34% of the total hemocytes), and one sub-population

395

of the vacuolated cells that appeared to have very low level of phagocytosis capacities.

396

Contrastingly, Othake et al. [20] observed only one main population of H. roretzi hemocyte

397

performing phagocytosis, named as small granular amoebocytes, while a population of

398

fibroblastic hemocyte able to phagocytose only small particles (<1 µm). Engulfing of small

399

particles may be pinocytosis rather than phagocytosis [41,42]. More recently, Choi et al. [28]

400

reported phagocytosis activity in hemocytes of H. roretzi analyzed using flow cytometry. In

401

their study, no information about the cell populations performing the phagocytosis was

402

provided, while they reported that up to 80% of the H. roretzi hemocytes are involved in the

403

phagocytosis. Although these authors used zymosan A as the phagocytosis substrate, such

404

values is considerably higher than the previous study [40] and our own results. Nevertheless,

405

phagocytosis activity may depend on the substrate and it cannot be excluded that other

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hemocyte sub-populations of H. roretzi may be involved in phagocytosis of smaller particles

407

or specific targets such as, for instance, bacteria, apoptotic cells or post-spawning gonadic

408

tissue. Lysosomes are organelles involved in the intracellular degradation of engulfed

410

materials through acidification and release of hydrolytic enzymes into the phagocytic

411

vacuoles [43]. Based on lysosomal content, we identified 8 different populations of H. roretzi

412

hemocytes in this study. It was noticeable that the sub-population Hy 1’ could not be

413

discriminated from Hy 1 based on size and complexity, although they appeared functionally

414

different from Hy 1. Such similar hemocyte sub-populations could be discriminated by

415

differential lysosomal contents, as Donaghy et al. [25] reported in case of the gastropod

416

Turbo cornutus. In this study we determined density of the lysosomes in different sub-

417

populations of the hemocytes, by calculating a ratio of the fluorescence intensity associated

418

with quantity of the lysosomes to size of the hemocytes, in order to compare lysosomal

419

content among the different cell populations without bias of cell sizes. As the ratio indicated,

420

the granulocyte population Gr 1 showed a much higher density of lysosomes than all other

421

populations, suggesting that Gr 1 is the major hemocyte population involved in phagocytosis.

422

The flow cytometry revealed that all H. roretzi hemocyte populations, including the cells

423

which are not performing phagocytosis contain lysosomes. Indeed, besides degradation of

424

ingested material, lysosomes are also involved in other intracellular processes, including

425

exocytosis, plasma membrane repair, cell signaling and energy metabolism [44], and may

426

therefore be found in virtually all the hemocyte populations.

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Unstimulated intracellular oxidative activity, including the production of ROS and

428

RNS, is sometimes incorrectly assumed as arising from NADPH oxidases and reflecting the

429

immune capacities of the cells (reviewed in [45]). Basal oxidative activity, however, does not

430

reflect any immune activity of hemocytes. Indeed, in an absence of any stimulation, oxidative １７

ACCEPTED MANUSCRIPT activity of hemocytes spontaneously arises from several intracellular sources, of which

432

mitochondria may be the major [46], followed by endoplasmic reticulum and peroxisomes

433

(reviewed in [45]). Unstimulated intracellular oxidative activity therefore, reflects cellular

434

metabolism, including catabolism, respiration and anabolism.

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The granulocyte population Gr 1 showed a very active intracellular metabolism,

436

higher than all other cell types. Hemocyte populations Gr 2 and Hy 1 displayed an

437

intermediate unstimulated intracellular oxidative activity, while the hyalinocyte population

438

Hy 2 and the Ly-like cells showed a very low level of metabolism. Differences in basal

439

intracellular oxidative activity are commonly reported among hemocyte populations of

440

marine invertebrates. Cell metabolism is usually the highest in granulocytes, while Ly-like

441

cells, also referred to as Blast-like cells, are defined by a very low metabolism [46-51]. Basal

442

oxidative metabolism cannot, however, denote the capacity of hemocytes to increase their

443

intracellular production of ROS upon specific stimulation.

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To understand nature of ROS, PMA has commonly been used for its capacity to elicit

445

ROS production from various intracellular components, depending on cell types. In Human

446

neutrophils, PMA stimulation induces an oxidative burst by activation of the type 2 NADPH

447

oxidase (Nox2; [52]), resulting in a very high increase in ROS production, up to a 20-fold

448

increase, within a short period [53, 54]. In other cell types, PMA stimulation may result in a

449

moderate (up to 5-fold) increase in ROS production through several distinct mechanisms. For

450

instance, PMA may activate type 5 NADPH oxidase (Nox5) [55], induce translocation of

451

different types of protein kinase C to mitochondria [52], or stimulate ROS production in

452

several intracellular organelles [56]. In the solitary ascidian H. roretzi, PMA stimulation of

453

hemocytes resulted in an increase of oxidative activity in all hemocyte types, with two main

454

patterns: a moderate increase (2.5- to 3-fold) in the granulocytes and the hyalinocyte

455

population Hy 1, and a low increase (1.5-fold) in the hyalinocyte population Hy 2 and the

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ACCEPTED MANUSCRIPT lymphocyte-like cells. Different mechanisms of PMA-induced activation of ROS production

457

may therefore, occur in these different hemocyte populations, suggesting different roles for

458

the produced ROS. In granulocytes, which are capable of phagocytosis, increased ROS

459

production may be related to pathway of destruction of ingested material [52]. The

460

hyalinocyte population Hy 1 however, does not seem able of phagocytosis. Activated ROS

461

production in Hy 1 may therefore, be involved in other processes such as cell signaling, gene

462

expression or regulation of cell growth [57-59]. In the hyalinocyte population Hy 2 and Ly-

463

like cells, the low increase of oxidative metabolism may only reflect mitochondrial

464

dysfunction because of the translocation of PKC to the mitochondria after activation by PMA

465

[55].

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Proteases are a broad class of enzymes present in all types of cells, capable of cleave

467

proteins and peptides, and involved in numerous and various functions in organisms. In the

468

present study, a high density of intracellular proteases was observed in the hyalinocyte Hy 2

469

and, at a much lesser extent, in the granulocyte Gr 2. In the granulocyte populations,

470

proteases may exert intracellular functions, potentially in link with phagocytosis processes.

471

The hyalinocyte Hy 2 however, does not demonstrate phagocytosis capacity and proteases are

472

therefore, more likely to be excreted upon specific stimuli. Indeed, Azumi et al. [19] reported

473

the release of proteolytic activities from hemocytes of H. roretzi upon chemical stimulation.

474

One of the substrate specificity of the released proteolytic activities matched with proteasome

475

activity. While proteasome is mainly known as the major pathway for intracellular protein

476

degradation, numerous studies have reported existence of the extracellular proteasome and its

477

potential activity in antimicrobial and inflammatory responses [60, 61]. In the solitary

478

ascidian H. roretzi, extracellular proteasome has also been characterized as implicated in the

479

fertilization processes [62,63]. While characterizing further extracellular proteolytic activities

480

in H. roretzi, Azumi et al. [64] suggested that some excreted proteases may be involved in

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ACCEPTED MANUSCRIPT producing immunoactive peptides. Indeed, both intra- and extracellular proteolytic cascades

482

control several immune reactions in marine invertebrates, including clotting, melanisation,

483

activation of Toll-like receptors, and complement-like reactions [65]. The hyalinocyte Hy 2

484

may therefore, represent an important link between cellular and humoral immune reactions.

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The present study is the first flow cytometric characterization of hemocyte

486

populations in the hemolymph of H. roretzi, based on the cellular activities and intracellular

487

metabolism. To summarize, 2 main hemocyte types can be observed; granulocytes and

488

hyalinocytes. The granulocytes consist of one major population, Gr 1, which accounts for

489

about 30% of the total circulating hemocytes. The population Gr 1 expresses the highest

490

density of lysosomes, as well as the highest unstimulated and stimulated intracellular

491

oxidative activity, and is the most active hemocyte involve in the phagocytosis and

492

degradation of foreign material. The population Gr 2, which accounts for about 5% of the

493

circulating cells, does not express any particular density of lysosomes but contains

494

intracellular proteases and displays an inducible oxidative activity. The potential roles and

495

functions of the granulocyte population Gr 2 are however, currently unknown. Hyalinocytes

496

consist of 2 main hemocyte types, Hy 1 and Hy 2, each accounting for about 30% of the total

497

circulating cells, with different cellular nature; Hy 1 displays lysosomal content as well as an

498

inducible oxidative activity without proteases, while Hy 2 expresses the highest density of

499

intracellular proteases with no lysosomes and a low oxidative activity. Although the Hy 1 did

500

not show phagocytosis activity in the present study, it may not be excluded a capacity to

501

internalize small size particles or specific targets such as, for instance, apoptotic cells. The

502

hyalinocyte Hy 3 and the Lymphocyte-like cells present a similar profile except for the size

503

and complexity. The Hy 3 may represent an intermediate maturation step between Ly-like

504

cells and other hemocyte populations. This first functional characterization of the hemocyte

505

populations of the solitary ascidian H. roretzi provides a solid basis to investigate further their

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respective roles and functions in physiological and pathological contexts.

507 508

510

Acknowledgments

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The authors thank to staffs of Shellfish Research and Aquaculture Laboratory at Jeju National University, for their technical support. This research was supported by a grant from

512

National Institute of Fisheries Science of Korea (R2017052). This study was also, in part,

513

supported by the funding from the Ministry of Oceans and Fisheries of Korea, through a

514

project, “Long-term changes of structure and function in marine ecosystems of Korea (2017)”.

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where? J. Leukoc. Biol. 94 (2013) 657-670. [53] G. Rothe, G. Valet, Flow cytometric analysis of respiratory burst activity in phagocytes with Hydroethidine and 2’,7’-Dichlorofluorescin, J. Leukocyte Biol. 47 (1990) 440-448. [54] J.P. Robinson, W.O. Carter, P.K. Narayanan, Oxidative Product Formation Analysis by Flow Cytometry, Methods Cell Biol. 41 (1994) 437-447.

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[55] D. Cosentino-Gomes, N. Rocco-Machado, J.R. Meyer-Fernandes, Cell Signaling

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through Protein Kinase C Oxidation and Activation. Int. J. Mol. Sci. 13 (2012) 10697-

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[56] B.C. Dickinson, C. Huynh, C.J. Chang, A palette of fluorescent probes with varying

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emission colors for imaging hydrogen peroxide signaling in living cells, J. Am. Chem.

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Soc. 132 (2010) 5906-5915.

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[57] D. Oyanedel, R. Gonzalez, K. Brokordt, P. Schmitt, L. Mercado, Insight into the

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messenger role of reactive oxygen intermediates in immunostimulated hemocytes from

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the scallop Argopecten purpuratus, Dev. Comp. Immunol. 65 (2016) 226-230.

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[58] W. Dröge, Free radicals in the physiological control of cell function, Physiol. Rev. 82

[59] K. Bedard, K.H. Krause, The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology, Physiol. Rev. 87 (2007) 245-313.

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[60] S.U. Sixt, B. Dahlmann, Extracellular, circulating proteasomes and ubiquitin - Incidence and relevance. Biochim. Biophys. Acta, Mol. Basis Dis. 1782 (2008) 817-823.

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[61] I. Bochmann, F. Ebstein, A. Lehmann, J. Wohlschlaeger, S.U. Sixt, P.M. Kloetzel, B.

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Dahlmann, T lymphocytes export proteasomes by way of microparticles: a possible

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mechanism for generation of extracellular proteasomes, J. Cell Mol. Med. 18 (2014) 59-

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[62] H. Sawada, N. Sakai, Y. Abe, E. Tanaka, Y. Takahashi, J. Fujino, E. Kodama, S. ２７

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Takizawa, H. Yokosawa, Extracellular ubiquitination and proteasome-mediated

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degradation of the ascidian sperm receptor, PNAS 99 (2002) 1223-1228.

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[63] N. Sakai, H. Sawada, H. Yokosawa, Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi: isolation and characterization, Dev. Biol.

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264 (2003) 299-307.

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and their involvement in invertebrate immunity, Trends Biochem. Sci. 35 (2010) 575-583.

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[65] L. Cerenius, S.H. Kawabata, B.L. Lee, M. Nonaka, K. Söderhäll, Proteolytic cascades

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ascidian hemocytes by treatment with calcium ionophore, Zool. Sci. 13 (1996) 365-370.

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[64] K. Azumi, H. Yokosawa, Characterization of novel metallo-proteases released from

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ACCEPTED MANUSCRIPT List of Figures

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Figure 1. Hemocyte populations of Halocynthia roretzi in fixed and fresh hemocytes. Flow

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cytometric dot plots of size (FSC) against internal complexity (SSC) of a representative

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sample of hemocyte populations observed in formalin-fixed (A), and fresh (B) hemocytes.

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Seven hemocyte populations were observed: Granulocytes Gr 1 to Gr 3, Hyalinocytes Hy 1 to

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Hy 3 and lymphocyte-like cells (Ly-like).

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Figure 2. Light microscopy of H. roretzi hemocyte sub-populations. A, granulocyte type 1

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(Gr 1), B, granulocyte type 2 (Gr 2), C, granulocyte type 3 (Gr 3), D, hyalinocyte type 1 (Hy

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1), E, hyalinocyte type 2 (Hy 2), F, hyalinocyte type 3 (Hy 3), G, lymphocyte-like cell (Ly-

700

like). All the scale bars represent 5 µm.

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Figure 3. SEM images of H. roretzi hemocytes. A, Gr 1, granulocyte type 1, B, Gr 2,

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granulocyte type 2, C, Hy 1, hyalinocyte type 1, D, Hy 2, hyalinocyte type 2,

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lymphocyte-like cells.

E, Ly-like,

All the scale bars in the SEM images represent 2 µm.

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Figure 4. Mortality of the hemocytes of H. roretzi. Mortality was determined as the

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percentage of SYBR Green I / Propidium iodide double-positive cells (A). Data is presented

708

as mean ± 95% confidence interval. N=33. Different letters (a, b, c) on histogram (B) indicate

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statistical difference between hemocyte populations (One-way ANOVA, p < 0.001; Post-

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comparison Dunn test, p < 0.05).

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Figure 5. Relative quantity of lysosomes in the hemocytes of H. roretzi. LysoTracker

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fluorescent dye accumulates in lysosomal compartments of hemocytes. The red fluorescence

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intensity (FL3) is proportional to the amount of lysosomes inside hemocytes. Fluorescence ２９

ACCEPTED MANUSCRIPT intensity (FL3) against internal complexity (SSC) dot plot of hemocyte populations stained

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with LysoTracker (A), allowing for discrimination between eight sub-populations. Histogram

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displaying the ratio between fluorescence intensity (FL3) and the size of hemocytes (FSC)

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(B). Data is presented as mean ± 95% confidence interval. N = 33. Different letters (a, b, c)

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on histogram (B) indicate statistical difference between hemocyte populations (One-way

720

ANOVA, p < 0.001; Post-comparison Dunn test, p < 0.05).

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Figure 6. Relative intracellular oxidative activity in the hemocytes of H. roretzi. None

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fluorescent DCFH-DA dye accumulates in cytoplasm of hemocytes and is oxidized to the

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fluorescent DCF molecule by intracellular reactive oxygen and nitrogen species. The green

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fluorescence intensity (FL1) is proportional to the oxidative activity in hemocytes. (A)

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Histogram displaying the ratio between fluorescence intensity (FL1) and the size of

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hemocytes (FSC), with or without PMA stimulation. (B) Histogram displaying the ratio

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between fluorescence intensity with and without PMA. Data is presented as mean ± 95%

729

confidence interval (N=32). Different letters (a, b, c) on histograms indicate statistical

730

difference between hemocyte populations (One-way ANOVA, p < 0.001; Post-comparison

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Post-comparison Dunn test, p < 0.05). Asterisks indicate statistical difference between No

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PMA and PMA stimulation (One-tailed t-test; *** = p < 0.001; ** = p < 0.01).

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Figure 7. Relative activity of intracellular proteases in the hemocytes of H. roretzi. None

735

fluorescent casein derivatives accumulate in cytoplasm of hemocytes. Protease-catalyzed

736

hydrolysis releases fluorescent dyes. The green fluorescence intensity (FL1) is proportional to

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the activity of proteases in hemocytes. Histogram displaying the ratio between fluorescence

738

intensity (FL1) and the size of hemocytes (FSC). Data is presented as mean ± 95%

739

confidence interval. N = 32. Different letters (a, b, c) on histogram indicate statistical ３０

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difference between hemocyte populations (One-way ANOVA, p < 0.001; Post-comparison

741

Dunn test, p < 0.05).

742

Figure 8. Phagocytosis capacities of hemocytes of H. roretzi. (A) Size (FSC) against internal

744

complexity (SSC) dot plot of hemocytes associated (black dots) or not (grey smear) with

745

fluorescent microbeads. (B) Histogram of the fluorescence intensity (FL1) associated with

746

ingested beads, which allowed determination of the mean number of beads per hemocyte.

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Peaks of fluorescence represent the quantity of beads associated with cells. The micrographs

748

show the granulocytes with the engulfed microbeads. (C) Phagocytosis index, defined as the

749

percentage of hemocytes that engulfed at least one bead among the total of hemocytes (% of

750

total) and among the granulocyte population (% of Gr), and mean number of beads per cell

751

that performed phagocytosis. Phagocytosis index and the mean number of beads per cell were

752

determined with and without PMA stimulation (N = 21).

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List of Tables

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Table 1. Summary of cytomorphological, functional and metabolic parameters of

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hemocyte populations of Halocynthia roretzi. FSC = size; SSC = internal complexity; % =

758

percentage of each population; Mort. = mortality; Lyso. = lysosomal density (FL3/FSC);

759

Oxid. act. - basal = intracellular oxidative activity (FL1/FSC) in absence of stimulation; PMA

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stim. = fold increase of intracellular oxidative activity after PMA stimulation; Phago. =

761

phagocytosis capacity; N/A = not applicable.

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Table 2. Percentages of hemocyte populations on formalin-fixed and fresh samples. Data

764

is presented as mean ± 95% confidence interval (CI 95%). Number of animal used in the

765

analysis was 27 and 33 in fixed and fresh hemocytes, respectively.

766

３１

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Figure 1.

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Figure 2.

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３３

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776 777

Figure 3.

781 782 783 784 785

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786 787

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Figure 4.

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Figure 5.

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Figure 6.

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Figure 7.

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Figure 8.

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Table 1.

804 805

Gr 1

++

++++

Gr 2

++

+++++

Gr 3

++

++++++

+++

+++

Hy 2

++

++

Hy 3

++

++

Ly-like

+

+

Hy 1

%

35-40

Lyso.

+

+

807 808 809

811

-

Fixed Mean

813

CI 95%

26.8

Gr 2

5.3

Gr 3

5.1

1.5

Hy 1

26.7

2.8

Hy 2

28.3

3.0

4.2

0.5

3.6

0.8

++

++

+

+

+

++

++

+

N/A

N/A

N/A

++

+

+

+++++

+

1.6

Proteases

+

+/-

++

N/A

N/A

+

++

+/-

+

+

Mean

CI 95%

3.8

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812

+++

Fresh

Gr 1

Ly-like

stim.

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Table 2.

Hy 3

- basal

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PMA

+

55-60

<5

Oxid. act.

++

+/-

Hy 1’

810

Mort.

40.1

3.8

16.9

1.7

38.2

3.3

4.9

1.0

４０

Phago.

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SSC

SC

FSC

+

-

-

ACCEPTED MANUSCRIPT Highlights

- A few studies have investigated structure and types of ascidian immune cells - We characterized hemocytes of the ascidian Halocynthia roretzi using flow cytometry

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- Eight types of hemocytes identified: 3 granulocytes, 4 hyalinocytes and lymphocyte-like cells

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- This study provides a basis to investigate further respective roles and functions of the ascidian hemocytes