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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
1
*
Vincenzo Arizza
2
Mirella Vazzana
2
Enrique E. Rozas
3
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
6
cells/mL by dilute coelomic fluid
using the anticoagulant solution. Subsequently,
60 µL of the sample (6 x 10
4
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 = 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).