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Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(S1): 171-184, March 2021 (Published Mar. 30, 2021)

Cytocentrifugation as an additional method to study echinoderm

coelomocytes: a comparative approach combining living cells,

stained preparations, and energy-dispersive x-ray spectroscopy

Vinicius Queiroz

Vincenzo Arizza

Mirella Vazzana

Enrique E. Rozas

Marcio R. Custódio

1,4

1. Departamento de Fisiologia Geral, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brasil;

vinicius_ufba@yahoo.com.br (*Correspondence).

2. Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Universittá degli Studi di Palermo,

Palermo, Italy; mirella.vazzana@unipa.it; vincenzo.arizza@unipa.it

3. Departamento de Engenharia Química, Escola Politécnica, Universidade de São Paulo, São Paulo, Brasil;

isoquir@gmail.com

4. Núcleo de Pesquisa em Biodiversidade Marinha da Universidade de São Paulo, São Paulo, Brasil; mcust@usp.br

Received 28-VI-2020. Corrected 28-X-2020. Accepted 10-XI-2020.

ABSTRACT

Introduction: Echinoderm coelomocytes have traditionally been investigated through a morphological approach

using light microscopy, which relies on the idea of constant cell shape as a stable character. However, this can be

affected by biotic or abiotic conditions. Objective: To analyze if the consistency in cell morphology offered by

the cytocentrifugation method, might be used as a convenient tool to study echinoderm coelomocytes. Methods:

Cells of Echinaster (Othilia) brasiliensis (Asteroidea), Holothuria (Holothuria) tubulosa (Holothuroidea),

Eucidaris tribuloides, Arbacia lixula, Lytechinus variegatus, and Echinometra lucunter (Echinoidea) were

spread on microscope slides by cytocentrifugation, stained, and analyzed through light microscopy. Additionally,

fluorescence microscopy, scanning electron microscopy, and energy-dispersive x-ray spectroscopy were applied

to cytospin preparations, to complement the analysis of granular and colorless spherulocytes of Eucidaris

tribuloides. Results: Altogether, 11 cell types, including phagocytes, spherulocytes, vibratile cells, and progeni-

tor cells were identified in the samples analyzed. The granular spherulocyte, a newly-described cell type, was

observed in all Echinoidea and was very similar to the acidophilic spherulocytes of Holothuria (Holothuria)

tubulosa. Conclusions: Cytocentrifugation proved to be versatile, either as the main method of investigation

in stained preparations, or as a framework on which other procedures may be performed. Its ability to maintain

a constant morphology allowed accurate correspondence between live and fixed/stained cells, differentiation

among similar spherulocytes as well as comparisons between similar cells of Holothuroidea and Echinoidea.

Key words: comparative cell morphology; echinoderm physiology; energy-dispersive x-ray spectroscopy;

invertebrate immunology; spherulocytes; vibratile cells.

Queiroz, V., Arizza, V., Vazzana, M., Rozas, E.E., &

Custódio, M.R. (2021). Cytocentrifugation as an

additional method to study echinoderm coelomocytes:

a comparative approach combining living cells,

stained preparations, and energy-dispersive x-ray

spectroscopy. Revista de Biología Tropical, 69(S1),

171-184. DOI 10.15517/rbt.v69iSuppl.1.46348

DOI 10.15517/rbt.v69iSuppl.1.46348

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Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(S1): 171-184, March 2021 (Published Mar. 30, 2021)

Regardless of the scientific scope (e.g.

physiology, environmental monitoring), the

identification and classification of echinoderm

coelomocytes –the circulating cells present

in the coelomic fluid– has been traditionally

based on morphological data (Matranga et

al., 2005; Arizza, Giaramita, Parrinello, Cam-

marata, & Parrinello, 2007). Morphological

characterization of coelomocytes primarily

relies on two techniques: transmission electron

microscopy (TEM) and/or light microscopy

(LM) (Smith, 1981).

TEM analyses have provided fine struc-

tural details and information of cytoplasmic

components, providing insights on cell physiol-

ogy and function (Queiroz & Custódio, 2015;

Magesky, Oliveira-Ribeiro, Beaulieu, & Pel-

letier, 2017). However, such analyses require

sophisticated infrastructure, and considerable

time must be spent in the preparation of sam-

ples. By contrast, based on faster and cheaper

procedures, LM allows the observation of

cell behavior and detection of morphological

traits in living cells (Matranga et al., 2005).

With this technique, gross morphology and

chemical properties of fixed cells can be inves-

tigated (Queiroz & Custódio, 2015; Vazzana,

Siragusa, Arizza, Buscaino, & Celi, 2015).

To perform this procedure, two methods have

been employed. The first one consists of fixing

cells in suspension just after their collection

(Vazzana et al., 2015), while the second one

uses the natural ability of living cells to spread

and attach themselves to flat surfaces (Branco,

Borges, Santos, Junior, & Silva, 2013).

Light microscopy is an easy and quick

method, but it has some disadvantages as well.

This procedure is based on cell morphology as

a stable character, but coelomocyte morphol-

ogy can be affected by biotic or abiotic condi-

tions. Phagocytes can switch between petaloid

and filiform conformations (Edds, 1977, 1993;

Canicattì, D’Ancona, & Farina-Lipari, 1989),

while spherulocytes may transit between round-

er and more elongated shapes (Matranga et al.,

2005). Similarly, temperature can influence

the spreading ability of phagocytes (Branco

et al., 2013). Due to this morphological vari-

ability, the accurate identification of different

cell types by LM may be compromised. For

this reason, a method capable of maintaining a

consistent cell morphology would be preferred.

Cytospin may be defined as a centrifuga-

tion technique that uses centrifugal force to

attach cells directly onto microscope slides

(Rathert, Roth, & Soloway, 1993; Gill, 2013).

Cytocentrifuges are commonly used in clini-

cal medicine to analyze cells suspended in

low-concentrated fluids (e.g. peritoneal and

bronchoalveolar; Bibby, 1986; Fleury-Feith,

Escudier, Pocholle, Carre, & Bernaudin, 1987).

They are used to spread cells in a single layer,

thus concentrating them on a small area and

preserving morphological details (Qing-fan,

1986). Although cytocentrifugation dates from

the mid-1980s and is a well-established method

for cell analyses in clinical medicine, its use is

not so widespread among invertebrate scholars,

and even less so among echinoderm research-

ers (Raftos, Gross, & Smith, 2004; Majeske,

Oleksyk, & Smith, 2013b). To the best of our

knowledge, only three (recent) studies have

used cytospin preparations to analyze coelo-

mocyte morphology in Echinodermata (Grand,

Pratchett, & Rivera-Posada, 2014; Queiroz &

Custódio, 2015; Taguchi, Tsutsui, & Naka-

mura, 2016).

Considering the inherent advantages of

cytocentrifugation, as stated by Qing-fan

(1986), and the scarcity of studies using this

method to analyze coelomocyte morphology,

this work aims to address three main questions:

1) Is cytocentrifugation a satisfactory meth-

od to investigate coelomocyte morphology in

Echinodermata? 2) Could cytospin slides be

used in conjunction with other techniques? 3)

Would cytocentrifugation be useful in compar-

ing cell morphology of different echinoderm

groups? Considering all analyzed groups, i.e.

Asteroidea, Holothuroidea, and Echinoidea, we

found eleven cell types, which were observed

in both live and stained preparations. Cyto-

centrifugation was used in combination with

other methods (e.g. fluorescence microscopy),

providing additional data on the spherulo-

cytes of Eucidaris tribuloides. Moreover, this

method allowed comparisons between similar

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coelomocytes belonging to different classes

of Echinodermata.

MATERIALS AND METHODS

Specimen maintenance and bleeding

procedures: Five specimens of Echinaster

(Othilia) brasiliensis (Asteroidea), Eucidaris

tribuloides, Lytechinus variegatus, and Echi-

nometra lucunter (Echinoidea) were collected

at Praia Grande (23°49’24” S & 45°25’01”

W), São Sebastião (SP), SE Brazil, while five

individuals of Holothuria (Holothuria) tubu-

losa (Holothuroidea) and Arbacia lixula (Echi-

noidea) were collected in the Gulf of Palermo

(38°06’ N & 13°30’ E), Sicily, SW Italy. Brazil-

ian echinoderms were acclimated for one week

at 23-25 °C in a running seawater aquarium and

Italian species were acclimated at 12-15 °C.

Echinaster (Othilia) brasiliensis was fed with

dead crabs, sea urchins, or small fish collected

at the same locality, while sea urchins were

fed with frozen algae, as described by Queiroz

(2018). Echinoderms from Italy were fed with

commercial invertebrate food (Azoo, Taikong

Corp., Taiwan).

Coelomocytes were collected following

specific procedures according to the species.

Cells from E. brasiliensis were collected as

described by Coteur, DeBecker, Warnau, Jan-

goux and Dubois (2002), by cutting the tip of

one arm and draining 1 mL of coelomic fluid

inside an Eppendorf previously filled with

1 mL of anticoagulant solution (20 mM ethyl-

enediamine tetraacetic acid (EDTA), sodium

chloride 460 mM, sodium sulfate 7 mM, potas-

sium chloride 10 mM, 4-(2-hydroxyethyl)-1-pi-

perazineethanesulfonic acid (HEPES) 10 mM,

pH 8.2 – based on Dunham & Weissman,

1986). This solution was originally developed

for marine sponges, but has shown good results

also for echinoderms (Queiroz & Custódio,

2015; Queiroz, 2020). Cells from H. tubulosa

were collected following the protocol of Vaz-

zana et al. (2015). Briefly, a 2 cm-long inci-

sion on the anterodorsal side of the specimens

was made using a sterile scalpel, then 3 mL of

coelomic fluid were drained into 15 mL falcon

tubes containing 3 mL of anticoagulant solu-

tion. Echinoid coelomocytes were collected

according to Queiroz and Custódio (2015), by

inserting a syringe needle preloaded with 0.5

mL of isosmotic anticoagulant solution into the

peristomial membrane and 0.5 ml of coelomic

fluid was withdrawn from each sea urchin.

Cytological preparations: Live cells

were observed just after collection by plac-

ing drops of coelomic fluid on a microscopic

slides, and covered with glass coverslips. For

cytological analyses, live cells were depos-

ited on microscope slides using a FANEN

248 simultaneous fluid removal cytocentri-

fuge. Firstly, the cell density was adjusted

to 1 x 10

cells/mL by dilute coelomic fluid

using the anticoagulant solution. Subsequently,

60 µL of the sample (6 x 10

cells) were

added in each spot and centrifuged by 5 min

at 80 x g. Afterwards, slides were fixed for

45 min in formaldehyde sublimate (Custódio,

Hajdu, & Muricy, 2004; Queiroz & Custódio,

2015), stained with toluidine blue (TB) or

Mallory’s trichrome (MT) (Behmer, Tolosa, &

Freitas-Neto, 1976) and mounted using Entel-

lan mounting medium (Merck).

Assays with fluorescence microscopy

(FM), scanning electron microscopy (SEM),

and energy-dispersive x-ray spectroscopy

(EDS) were performed to investigate additional

application to cytospin preparations. Granular

and colorless spherulocytes of E. tribuloides

were used as a model to analyze other char-

acteristics of echinoderm coelomocytes. Cells

stained with MT were used for FM assays, by

analyzing the natural fluorescence of acid fuch-

sin (excitation: 540 nm (green); emission: 630

nm (red); Sabnis, 2010). For SEM and EDS

analyses, live cells were deposited on round

coverslips and fixed in formaldehyde sublimate

as described above. Afterwards, the cover-

slips were washed once in Milli-Q water for

40 minutes, air-dried, attached to stubs using

small pieces of double-sided tape, and stored

at room temperature in a closed container with

silica gel. Just before SEM analyses, the cov-

erslips were sputter-coated with a 40-60 nm

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thick layer of gold and observed in a Sigma

VP (Zeiss) scanning electron microscope. For

EDS analyses, the coverslips on stubs were

analyzed with no further preparations, with the

aid of energy-dispersive X-ray spectroscopy

coupled to desktop SEM (Phenon world). The

results were shown as the mean percentage ±

standard deviation (M ± SD) to each element

of five cells, and Student’s t-tests (α = 0.05)

were used to analyze differences between dis-

tinct cell compartments inside the same cell

(e.g. Nucleus X Cytoplasm) or similar com-

partments in different cell types. To reduce

possible mistakes, common elements from

the glass coverslip found in the analyses, such

as oxygen, silicon, magnesium, and sodium

were removed from the results. Percentages

were then recalculated based on the remaining

elemental percentages.

Cell identification: Live cells of all spe-

cies were identified immediately after col-

lection based on morphological features.

Afterwards, we looked for cells with the same

aspect in fixed preparations (cytospin, MET,

and EDS) considering general morphology

and/or tinctorial characteristics. Phagocytes

were identified by their filopodial or petaloid

cytoplasmic expansions, while progenitor cells

and vibratile cells displayed a central nucleus

surrounded by a thin layer of cytoplasm, and

a remarkable flagellum respectively. In Holo-

thuroidea and Echinoidea, more than one type

of transparent spherulocyte was found. Thus,

based primarily on live preparations, general

cell morphology and vacuole (spherule) shape

were herein considered key characters to dis-

criminate subpopulations. Lastly, identification

was confirmed following specific literature

from each class/species: Asteroidea (Kanungo,

1984), Holothuroidea (Vazzana et al., 2015),

and Echinoidea (Johnson, 1969; Queiroz &

Custódio, 2015).

RESULTS

Cell observations: Analysis performed on

live and stained cells identified 11 cell types

in the three echinoderm classes, distributed

into four broad categories: phagocytes, pro-

genitor cells, vibratile cells, and spherulocytes

(Fig. 1, Fig. 2).

Asteroidea: Only phagocytes were

observed in live preparations of E. (Othilia)

brasiliensis, showing a central nucleus and a

largely vacuolated cytoplasm (Fig. 1A). Our

analysis of microscope slides revealed that live

cells spread immediately after collection, and

that live and stained phagocytes display a very

similar morphology (Fig. 1D). Progenitor cells

and colorless spherulocytes were observed in

stained preparations (data not shown), howev-

er, they were not found in living preparations.

Holothuroidea: Five cell types were

found in H. (Holothuria) tubulosa: phago-

cytes (filopodial and petaloid morphotypes),

progenitor cells, spherulocytes, morula cells,

and acidophilic spherulocytes. Live filopodial

phagocytes show a central body with several

filopodia (Fig. 1B), while petaloid phagocytes

display several lamellipodia (Fig. 1C), and

the same general patterns were observed in all

cytospin preparations (Fig. 1E, 1F). Progenitor

cells show a prominent nucleus surrounded by a

thin cytoplasmic layer not always visible in live

cells (Fig. 1G), which became more evident

in TB stained preparations (Fig. 1K). Spheru-

locytes had a prominent nucleus and round

finely granular vacuoles (Fig. 1H) that showed

affinity to methyl blue in MT preparations (Fig.

1L), indicating the presence of mucopolysac-

charides in the spherules. Also, we verified

that the cells usually called “morula” (Vazzana

et al., 2015) are actually coelomocytes con-

taining spherules with irregular profile (from

round to more elongated). They also stain with

methyl blue in MT preparations, evidencing

its mucopolysaccharide content (Fig. 1I, Fig.

1M). Acidophilic spherulocytes bear round

spherules, with affinity to acid fuchsin in MT

preparations, which indicates a protein-moiety

(Fig. 1J, Fig. 1N).

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Echinoidea: Five coelomic subpopula-

tions were observed in all analyzed species:

phagocytes, vibratile cells, red and colorless

spherulocytes, and one new cell type: the

granular spherulocyte. Phagocytes showed a

prominent cell body with petal-like cytoplas-

mic expansions (Fig. 2A’). In cytospin prepa-

rations, these cells were irregular in shape,

ranging from elongated to roundish, with a

subcentral nucleus and a vacuolated cytoplasm

Fig. 1. Live (A-C and G-J) and stained (D-F and K-N) coelomocytes of Echinaster (Othilia) brasiliensis (A and D) and

Holothuria (Holothuria) tubulosa (B, C, and E-N). A-F. Phagocytes. G, K. Progenitor cell. H, L. Spherulocytes. I, M.

Morula cell. J, N. Acidophilic spherulocyte. D-K. Toluidine Blue. L-N. Mallory’s trichrome. Arrow = nucleus. Scales: A.

For cells of Echinaster (Othilia) brasiliensis (10µm), B. For cells of Holothuria (Holothuria) tubulosa (10µm).

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as key features (Fig. 2A, 2F, 2K, 2P). Vibratile

cells are round flagellated spherule-filled coe-

lomocytes (Fig. 2B’), which stains light blue in

MT (Fig. 2B, 2L, 2Q) or purplish in TB prepa-

rations (Fig. 2G), the latter indicating the pres-

ence of glycosaminoglycans in the spherules.

Live red spherulocytes were readily noticed

by the round red spherules present in live cells

(Fig. 2C’, 2U, 2V, 2W). These cells do not stain

in MT slides but present a characteristic brown-

ish color (Fig. 2C, 2H, 2M, 2R), probably due

to echinochrome. Even in fixed but not stained

preparations, the brownish color is character-

istic. Lastly, two types of transparent spheru-

locytes were identified in Echinoidea, named

colorless and granular spherulocytes. Live cells

of the first type displayed transparent spher-

ules of variable shape (ranging from roundish

to elongated - Fig. 2D’), while live granular

spherulocytes showed round uniform spher-

ules (Fig. 2E’). In MT preparations, colorless

spherulocytes stained bluish (Fig. 2D, 2I, 2N,

2S), and granular spherulocytes stained pink-

ish, indicating that their vacuoles are filled with

mucopolysaccharides and protein, respectively.

Analysis of granular and colorless

spherulocytes of Eucidaris tribuloides using

FM, SEM, and EDS: Fluorescence analysis

of granular and colorless spherulocytes, previ-

ously observed in brightfield microscopy (Fig.

3A), showed an intense red fluorescence in the

MT preparation due to the affinity of acid fuch-

sin for granules, which was not observed in the

nucleus (Fig. 3B). On the other hand, colorless

spherulocytes stained with MT did not show

fluorescence (Fig. 3B). SEM analyses allowed

a detailed observation of spherulocyte mor-

phology (Fig. 3C, 3D). While granular spheru-

locytes showed the usual spherical cell shape

and regular-sized spherules (Fig. 3C), colorless

spherulocytes showed a variable shape, ranging

from spherical to more elongated, which was

also observed in the spherules (Fig. 3D).

Coelomocyte characterization by SEM

was essential to study the elemental composi-

tion of the nucleus (Nu) and cytoplasm (Cy) in

both cells using EDS (Fig. 3E). For granular

spherulocytes, in both nucleus and cytoplasm

compartments, the most representative ele-

ments were carbon, nitrogen, and sulfur, while

phosphorus and calcium were the less com-

mon (Table 1). The colorless spherulocyte fol-

lowed a similar pattern, with some differences.

Carbon, nitrogen, and calcium were the most

representative elements, and phosphorus and

sulfur were the less common (Table 1). Statis-

tical analyses corroborated the differences in

elemental composition between distinct com-

partments in the same cell, or between similar

compartments in different cells (Fig. 3E). Thus,

the elemental composition of nitrogen, sulfur,

and calcium was different between the nucleus

and cytoplasm of granular spherulocytes, while

sulfur differed in the colorless spherulocyte

(Fig. 3E). Comparisons between both spheru-

locytes showed differences in the composition

of nuclear carbon and sulfur, and cytoplasmic

carbon, nitrogen, sulfur, and calcium (Fig. 3E).

TABLE 1

Percentage (%) of the main chemical elements in the colorless and granular spherulocytes of Eucidaris tribuloides.

Values are presented as mean ± standard deviation (M ± SD). Legend: C = Carbon, N = Nitrogen,

P = Phosphorus, S = Sulfur, Ca = Calcium

Chemical element

Colorless Spherulocytes Granular Spherulocytes

Nucleus Cytoplasm Nucleus Cytoplasm

C 51.90 ± 6.61 57.30 ± 9.18 43.80 ± 3.57 43.70 ± 4.91

N 27.16 ± 5.02 24.80 ± 5.23 27.20 ± 5.97 42.42 ± 5.78

P 3.16 ± 1.66 1.64 ± 1.23 5.76 ± 5.67 0.46 ± 0.30

S 8.12 ± 1.51 3.36 ± 1.56 16.90 ± 4.99 10.34 ± 2.17

Ca 9.66 ± 1.64 12.70 ± 6.88 6.26 ± 2.26 3.08 ± 1.40

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Fig. 2. Live and stained coelomocytes of Echinoidea. A’-E’, A-E. Eucidaris tribuloides (ET). F-J. Arbacia lixula (AL).

K-O. Lytechinus variegatus (LV). P-T. Echinometra lucunter (EL). U-W. Live red spherulocytes of Arbacia lixula,

Lytechinus variegatus, and Echinometra lucunter. A’, A, F, K, P. Phagocytes. B’, B, G, L, Q. Vibratile cells. C’, C, H, M,

R. Red spherulocytes. D’, D, I, N, S. Colorless spherulocytes. E’, E, J, O, T. Granular spherulocyte. A-F, H-J, and L-T.

Mallory’s trichrome. G, K. Toluidine blue Arrow = nucleus. Arrowhead = flagellum. Scales = 10µm; A’, A. For live and

stained cells respectively of Eucidaris tribuloides. F. For stained cells of Arbacia lixula. K. For stained cells of Lytechinus

variegatus. P. For stained cells of Echinometra lucunter.

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Fig. 3. Granular and colorless spherulocytes of Eucidaris tribuloides studied under different methods. A, B. Light and

fluorescence microscopy of Mallory’s trichrome-stained granular and colorless spherulocytes. C, D. Detailed morphology

of the granular and colorless spherulocytes respectively, under scanning electron microscopy. E. Elemental composition of

cytoplasm and nucleus of granular and colorless spherulocytes in energy-dispersive x-ray spectroscopy, and morphology of

granular and colorless spherulocytes in EDS analysis (Inset). C = Carbon. N = Nitrogen. S = Sulfur. P = Phosphorous. Ca

= Calcium. CS = Colorless spherulocyte. GS = Granular spherulocyte. Red asterisk = P < 0.05. Black asterisk = P < 0.01.

White arrow = Granular spherulocyte nucleus. Scale: A, B, E = 10µm; C, D = 5 µm.

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DISCUSSION

In the present study, we confirmed that: 1)

cytocentrifugation was satisfactory to analyze

coelomocyte morphology in Echinodermata,

revealing 11 cell types in the six species ana-

lyzed; 2) cytospin slides can be used along with

other techniques, providing additional relevant

data, and 3) cytocentrifugation allowed us to

compare coelomocytes of different echino-

derms classes. Considering these results, it

can be suggested that cytocentrifugation is an

improved method for additional analysis in

coelomocyte studies in Echinodermata.

Through cytocentrifugation, coelomocytes

are forced to spread onto slides in short runs,

binding more efficiently and faster than when

allowed to adhere under passive conditions.

Incubating cells from suspensions and waiting

for them to settle, attach, and spread, has been

the most usual procedure to attach coelomo-

cytes on flat surfaces (Bertheussen & Seljelid,

1978; Romero, Novoa, & Figueras, 2016). This

poses three main problems: 1) The ability to

attach and spread on surfaces is not observed

in some coelomic populations (e.g., vibratile

cells), which are usually lost during slide pro-

cessing (Romero et al., 2016); 2) The timespan

to achieve cell adhesion may be very long

(Romero et al., 2016), which can lead to the

degradation and loss of cell characters; 3) The

ability to attach and spread on the glass surface,

and consequently the cell morphology, can be

affected by environmental conditions, such as

temperature (Branco et al., 2013). We under-

stand that in some specific situations where the

preservation of the natural shape of coelomo-

cytes is necessary (e.g. Edds, 1993; Majeske,

Bayne, & Smith, 2013a), cytocentrifugation

may not be the best protocol to access cell mor-

phology. However, cytospin preparations are

more efficient compared to the usual method,

since all cell types can be spread onto common

microscope slides without further preparation

(e.g. Poly-L lysine; Majeske et al., 2013b),

within a short period, and their morphology did

not vary considerably.

To the best of our knowledge, only three

studies have used cytocentrifugation to analyze

echinoderm coelomocyte morphology (Grand

et al., 2014; Queiroz & Custódio, 2015; Tagu-

chi et al., 2016), and all of them showed a high

potential to improve cell analyses in Echino-

dermata. Only phagocytes and small spherical

cells were previously known to the sea star

Acanthaster planci (Boolotian, 1962). Howev-

er, in a study using cytocentrifugation, Grand et

al. (2014) described four distinct coelomocytes

to this species, showing that its coelomocyte

diversity may have been underestimated. Simi-

larly, a new spherulocyte was also detected in

E. tribuloides (Queiroz & Custodio, 2015), and

for the first time, spherulocytes were report-

ed to be able to phagocyte foreign particles

(Tagushi et al., 2016). In the same way, we

also obtained an unequivocal correspondence

between living and fixed cells of all analyzed

species, and for the first time, we report a fifth

cell type –the granular spherulocyte– in the

well-studied echinoids A. lixula, L. variegatus,

and E. lucunter (Branco et al., 2013). Thus,

our data show that this method can improve

morphological descriptions by relating living

and fixed cells. It was also able to demonstrate

that the newly-described granular spherulocyte

(Queiroz & Custodio, 2015) is not restricted to

E. tribuloides, and may be widespread among

regular echinoids.

Another important result obtained herein

was the possibility to differentiate spheru-

locyte subpopulations in fixed preparations.

These subpopulations are identified due to

their spherule-filled cytoplasm and named after

the characteristics of living cells, but are yet a

poorly understood cell type in Echinodermata

(Ramírez-Gómez & García-Arrarás, 2010).

Still, even when alive, spherulocyte identifi-

cation may be imprecise because more than

one type of “transparent” spherulocyte may be

present, as we report herein. Consequently, the

correct identification of live or fixed cells is

still difficult. However, we demonstrate herein

that cytocentrifugation is a useful alternative

method to discriminate spherulocytes since

it allows a good correlation between live and

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fixed/stained cells. For example, three different

subpopulations were unequivocally identified

in live and fixed preparation of H. (Holothuria)

tubulosa, E. tribuloides, A. lixula, L. variega-

tus, and E. lucunter, and differentiated by cell

profile and vacuoles shape (when alive), and its

stain affinity (in fixed preparations). By con-

trast, the correspondence between live and fixed

spherulocytes of Holothuria (Selenkothuria)

glaberrima (see Fig. 2A-C in Ramírez-Gómez,

Aponte-Rivera, Méndez-Castaner, & García-

Arrarás, 2010) and Echinometra mathaei (see

Fig. 5 in Piryaei, Ghavam-Mostafavi, Gha-

vam-Mostafavi, & Pooshang-Bagheri, 2018)

leaves some doubts. We strongly believe that

correspondence between live and fixed prepa-

rations could be increased if cytocentrifuga-

tion had been used to prepare the samples in

these studies.

Although cellular characteristics obtained

by light and even electron microscopy may fail

to identify homologous cell types between dis-

tinct phyla, these methods might be a useful tool

between closer species (Arendt, 2008). Thus,

the morphological approach presented here can

be used to compare cells from different echino-

derm classes, and the acidophilic and granular

spherulocytes provide one interesting example.

These two coelomocytes are identified by their

transparent color and spherule shapes (Vazzana

et al., 2015; Vazzana et al.,2018; Queiroz &

Custodio, 2015). Both spherulocytes showed

uniform spherules filled with a peptidic moiety,

as showed by the pinkish color produced by

the acid fuchsine present in MT solution. We

recognize that even though morphology and

chemical contents are similar, further studies

are still necessary. Only the discovery of spe-

cific cell markers - such as those expressed in

helper and cytotoxic lymphocytes (Taniuchi,

2018) - can elucidate this issue. However, the

approach used herein can improve the under-

standing of spherulocyte diversity and can

certainly be employed as an additional strategy

to understand the real nature of vibratile cells

in Echinodermata (Hetzel, 1963; Xing, Yang,

& Chen, 2008; Smith et al., 2018).

Cytocentrifugation is also quite useful as

a preparation method, alongside which other

procedures may be performed, mainly when

the aim is to analyze coelomocyte diversity.

Some studies used cytospin preparation as a

framework for FM (Clow, Raftos, Gross, &

Smith, 2004; Majeske et al., 2013b; Golcon-

da, Buckley, Reynolds, Romanello, & Smith,

2019) or SEM (Grand et al., 2014). How-

ever, only two of these works (Clow, Raftos,

Gross, & Smith, 2004; Grand et al., 2014)

analyzed cell morphology using cytocentrifu-

gation. Using cytospins, further aspects of the

granular spherulocyte were analyzed herein by

different techniques. FM revealed that, in addi-

tion to the morphological characteristics, the

red fluorescence of the protein-filled spherules

may be an identification marker, while SEM

corroborated the differences in the shape pat-

tern of cytoplasmic spherules observed in light

and fluorescence microscopy.

EDS has been commonly used to analyze

the presence of uncommon elements (e.g.

heavy metals) in invertebrate coelomocytes/

hemocytes (e.g. Tullius, Gillum, Carlson, &

Hodgson, 1980), but few studies have focused

on conventional cell elements (Scippa, De Vin-

centiis, & Zierold, 1990). This method revealed

specific differences in the elemental composi-

tion in different compartments of the same cell

as well as between similar compartments of

different cells, which could reflect organelle

functioning. For example, sulfur was higher

in the nucleus if compared to the cytoplasm of

the granular and colorless spherulocytes of E.

tribuloides, a pattern similar to that observed

in the morula cell of the urochordates Halo-

cynthia papillosa and Phallusia mammillata

(Scippa, Botte, Zierold, & De Vincentiis, 1985;

Scippa, De Vincentiis, & Zierold, 1993). By

constrast, although nitrogen has not been men-

tioned to other invertebrates (e.g. Scippa et al.,

1990; Scippa et al., 1993; Giamberini, Auffret,

& Pihan, 1996), this element was higher in

granular but not in colorless spherulocytes.

Higher nuclear sulfur can indicate higher

levels of Methionine, an amino acid univer-

sally associated with the initiation of protein

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synthesis (Kozak, 1983). Such an explanation

fits properly with the general function of the

morula cell of H. papillosa and spherulocytes

of E. tribuloides, which are cells adapted to

produce and store chemical compounds. In

contrast, higher cytoplasmic nitrogen corrobo-

rates the assumptions of a rich-protein moiety

observed through acid fuchsin affinity. Lastly,

if considered in conjunction, the two-fold lev-

els of nuclear sulfur and cytoplasmic nitrogen

observed in granular spherulocytes confirm the

engagement of this cell in protein production.

Therefore, our results suggest that sulfur and

nitrogen concentrations are different in color-

less and granular spherulocytes of E. tribuloi-

des, and could be used as a specific chemical

marker to differentiate these cells in other echi-

noids. This approach could provide important

results in the cells of other invertebrates.

Most studies addressing echinoderm

coelomocytes by FM, either using cytospin

preparations (Clow, Raftos, Gross, & Smith,

2004; Golconda et al., 2019) or other methods

(Brockton et al., 2008; Majeske et al., 2013a),

have focused on sea urchin phagocytes, while

spherulocytes, in general, have been poorly

studied (García-Arrarás et al., 2006; Falugi

et al., 2012). That may be due to the ease in

identifying phagocyte subpopulations. Even

with the use of specific markers to discrimi-

nate phagocyte subtypes, the identification is

still mostly based on morphology (Golconda

et al., 2019). To SEM, although Grand et al.

(2014) have used cytocentrifugation to analyze

A. planci coelomocytes, the authors did not

provide a more detailed correspondence among

the living, stained, and SEM preparations,

which impairs further comparisons. Regard-

ing EDS, as far as we know, there is no work

using cytocentrifugation to perform this tech-

nique. However, Falugi et al. (2012) used an

Environmental Scanning Electron Microscope

coupled with EDS to analyze the presence of

nanoparticles in smeared cells. In this latter

study, which certainly provided important data

on the effect of nanoparticles on echinoderm

coelomocytes, some additional aspects could

have been observed if cytocentrifugation was

used. For example, in addition to confirm-

ing the presence of Sn, Ce, and Fe inside

the coelomocytes, and observing impacts on

subcellular compartments, Falugi et al. (2012)

could have answered other relevant questions,

such as: 1) Do different cell types accumulate

nanoparticles equally? 2) Can nanoparticles be

found in the nucleus? 3) Which cell type was

most impacted at the subcellular level? Conse-

quently, our results show that cytocentrifuga-

tion could be a useful tool to improve studies

on coelomocyte in Echinodermata and other

invertebrates as well.

Cytocentrifugation proved to be versatile,

being useful as the main method to investi-

gate echinoderm cell morphology in stained

preparations or as a framework for other proce-

dures. Although stained phagocytes displayed

little variations if compared to live cells, cell

identification was not impaired. Therefore,

cytocentrifugation maintains constant cell mor-

phology, which allowed accurate correspon-

dence between live and fixed/stained cells,

correct identification of apparently similar

types of spherulocytes, as well as comparisons

between related species. Still, it is a fast, practi-

cal, and inexpensive method, useful to different

samples. This includes low concentrated cell

suspensions, which would allow the study of

species with little body fluid volumes such as

Crinoidea and Ophiuroidea. Finally, this meth-

od may be used either as a convenient strategy

able to improve cell morphology or coelo-

mocyte physiology studies. Thus, the present

work provides strong evidence that cytospin

preparations have a high potential to improve

coelomocyte analyses in Echinodermata and

other invertebrates.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

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ACKNOWLEDGMENTS

The authors thank Sheila Schuindt and

Márcio Cruz, Instituto de Biociências (IB-

USP), for their help in the SEM analyses. The

author is indebted to Daniel C. Cavallari for

the English improvements. This work was

supported by FAPESP (Proc. 2013/50218-2,

2015/21460-5, and 2018/14497-8) and Coorde-

nação de Aperfeiçoamento de Pessoal de Nível

Superior-Brazil (CAPES Finance Code 001),

and is a contribution of NP-BioMar (Research

Center for Marine Biodiversity, USP).

RESUMEN

Citocentrifugación como un método adicional para

estudiar celomitos de equinodermos: un enfoque

comparativo que combina células vivas,

preparaciones teñidas y espectroscopía de rayos-x

de dispersión de energía

Introducción: Los celomocitos de equinodermos

se han investigado tradicionalmente a través de un enfo-

que morfológico utilizando microscopía óptica, que se

basa en la idea de la forma celular constante como un

carácter estable. Sin embargo, esto puede verse afectado

por condiciones bióticas o abióticas. Objetivo: Analizar

si la consistencia en la morfología celular que ofrece el

método de citocentrifugación podría utilizarse como una

herramienta conveniente para estudiar los celomocitos de

equinodermos. Métodos: Células de Echinaster (Othi-

lia) brasiliensis (Asteroidea), Holothuria (Holothuria)

tubulosa (Holothuroidea), Eucidaris tribuloides, Arbacia

lixula, Lytechinus variegatus y Echinometra lucunter

(Echinoidea) se esparcieron en portaobjetos de microsco-

pio por citocentrifugación, se tiñeron y analizaron mediante

microscopía óptica. Adicionalmente, se aplicó microscopía

de fluorescencia, microscopía electrónica de barrido y

espectroscopía de rayos X con dispersión de energía a las

preparaciones de citoespina, para complementar el análisis

de los esferulocitos granulares e incoloros de Eucidaris

tribuloides. Resultados: En total, se identificaron en las

muestras analizadas 11 tipos de células, incluidos fagoci-

tos, esferulocitos, células vibrátiles y células progenitoras.

El esferulocito granular, un tipo de célula recién descrito,

se observó en todos los Echinoidea y fue muy similar a los

esferulocitos acidófilos de Holothuria (Holothuria) tubu-

losa. Conclusiones: La citocentrifugación demostró ser un

método bastante versátil, ya sea como el método principal

de investigación en preparaciones teñidas o como un marco

en el que se pueden realizar otros procedimientos. Su capa-

cidad para mantener una morfología constante permitió

una correspondencia precisa entre las células vivas y las

células fijas/teñidas, la diferenciación entre esferulocitos

similares, así como comparaciones entre células similares

de Holothuroidea y Echinoidea.

Palabras clave: morfología celular comparativa; fisiología

de equinodermos; espectroscopía de rayos-x de energía

dispersiva; inmunología de invertebrados; esferulocitos;

células vibrátiles.

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