Paracentrotus coelomocytes

Queiroz, Vinicius, Arizza, Vincenzo, Vazzana, Mirella & Custódio, Márcio R., 2022, Comparative evaluation of coelomocytes in Paracentrotus sea urchins: Description of new cell types and insights on spherulocyte maturation and sea urchin physiology, Zoologischer Anzeiger 300, pp. 27-40 : 35-36

4.1. Paracentrotus coelomocytes and the identification of coelomic cells in other echinoids

From an immunological perspective, P. lividus is a relatively well-known species. Although studies on its humoral components are not uncommon (e.g. Stabili et al., 1996; Chiaramonte et al., 2020), investigations on its cellular effectors are more frequent (e.g. Arizza et al., 2007; Chiaramonte et al., 2019). By contrast, the immune aspects of P. gaimardi are completely unknown. In the present study, through a comparative evaluation of the coelomocytes in both species, we observed that their cells are similar. However, the number of subpopulations was underestimated since six cell types were revealed in both species. For the first time, we provided information on the coelomocytes of P. gaimardi , as well as a detailed characterization of red, colorless, and granular spherulocytes in both species, including the identification of a set of different morphotypes.

Total and differential cell counts in P. lividus were similar to those found in P. gaimardi and fit the pattern previously described for echinoids in the literature ( Bertheussen and Seljelid, 1978; Smith et al., 2010). Even for the red spherulocytes, which showed a higher percentage in P. gaimardi , the proportions are in line with the pattern already observed in P. lividus and other sea urchins ( Smith et al., 2010). Different factors have been pointed out as capable of influencing physiological or immune parameters, such as gender, age, or symbiotic associations ( McCaughey and Bodnar, 2012; Arizza et al., 2013; Queiroz, 2020a); the last two being able to affect cell counts ( McCaughey and Bodnar, 2012; Queiroz, 2020a). Thus, the difference observed in the red spherulocyte percentage can be related to factors other than interspecific differences.

Both species presented six cell types, unlike the four usual commonly described types in the literature (i.e. phagocytes, vibratile cells, and red and colorless spherulocytes – Branco et al., 2013; Chiaramonte et al., 2019; Work et al., 2020). Phagocytes and vibratile cells are easily identified in fresh or stained preparations, regardless of the approach, due to the presence of prominent cytoplasmic expansions (filiform or bladder-like), or a flagellum, respectively ( Work et al., 2020; Queiroz et al., 2021a; 2021b). Petaloid and filopodial phagocytes, which have been pointed out as transitory stages of the same cell type in Echinodermata ( Eliseikina and Magarlamov, 2002; Matranga et al., 2005), were common in fresh and stained preparations in our study. For phagocytes, regardless of the stage, morphological features seem to be sufficient to identify this population, as observed here in Paracentrotus and other species elsewhere, such as Tripineuste gratilla , E. tribuloides , L. variegatus , E. lucunter , and A. lixula ( Work et al., 2020; Queiroz et al., 2021b). In the case of vibratile cells, the flagellum is very sensitive to handling/fixation, being easily lost during preparation. Nevertheless, we observed that even after losing the flagellum, vibratile cells can be promptly identified in cytological preparations by using the TB stain. They are the only cell type in sea urchins that displays β- metachromasia (purple color) after staining. This reaction was verified herein for both Paracentrotus species, and previously for E. tribuloides , E. lucunter , L. variegatus , and A. lixula ( Queiroz et al., 2021b) . An apparent purple coloration was also observed in TB-stained preparations in P. lividus ( Deveci et al., 2015) and Strongylocentrous purpuratus ( Holland et al., 1965).

Though crystal cells are considered typical coelomocytes in sea cucumbers (e.g. Hetzel, 1963), we provide the first record of a crystal cell in sea urchins. The crystalloid inside sea cucumber crystal cells dissolves under slight osmotic stress or staining procedures ( Hetzel, 1963; Queiroz et al., 2022). By contrast, the crystalloid in P. gaimardi cells was proven resistant to staining and SEM procedures in our study. This higher resistance suggests that the crystal present in the cells in P. gaimardi probably has a different composition from those found in holothuroids. Though they were already recorded in the echinoid Echinus esculentus ( Smith, 1981) , progenitor cells are commonly observed in holothurians and asteroids ( Smith, 1981; Vazzana et al., 2015). The progenitor cell observed in the present study was morphologically similar to progenitor cells observed in E. esculentus , in the sea star Marthasterias glacialis , and other holothurians ( Hetzel, 1963; Vazzana et al., 2015; Caulier et al., 2020; Andrade et al., 2021). A morphologically similar cell, named small cell, was described in P. lividus ( Deveci et al., 2015) , and fits the morphology of the progenitor cell observed here. However, crystal and progenitor cells may be widespread in this genus. They were very rare in the coelomic fluid, which may have hindered finding them in both species. Consequently, both species may have seven cell types, instead of six.

We observed that morphology, morphometry, and stain affinity were essential for the unequivocal identification of red, colorless, and granular spherulocytes in live and stained preparations. All spherulocytes of P. gaimardi and P. lividus were morphologically and/or cytochemically similar to those of E. tribuloides , E. lucunter , L. variegatus , Arbacia punctulata , and A. lixula in MT preparations ( Liebman, 1950; Queiroz and Custodio´, 2015; Queiroz et al., 2021b). Even analyzed through different methods, the morphology of living and stained red and colorless spherulocytes in Paracentrotus sea urchins were quite similar to those of T. gratilla ( Work et al., 2020) . Distinguishing colorless and granular spherulocytes in live preparations can be difficult since both are transparent spherule-filled coelomocytes. Still, accurate analysis of the shape and size of the spherules proved to be enough for identification. A similar situation may be seen in A. punctulata since “green and red threphocytes” have similar-sized spherules, while the “colorless threphocytes” spherules are large and irregular ( Liebman, 1950). By contrast, we had no problems in identifying the three different spherulocytes in MT-stained preparations. While red spherulocytes show either brownish spherules or an uncolored and empty cytoplasm (probably due to the removal of their content during slide preparation), colorless and granular spherulocytes are quite different since they stain blue and pink respectively (Cf. Fig. 3 ). These differences indicate the predominance of (proteo)glycans in the colorless spherulocytes, while the granular ones present a peptidic moiety. Although the specific chemical identity of these compounds needs further studies by more precise chemical methods, this clearly shows different physiological functions for these cell types. Colorless trephocytes of A. punctulata also stained blue in MT preparations ( Liebman, 1950). Additionally, the newly-described granular spherulocyte (Queiroz and Custodio´, 2015) may be easily identified in TB preparations because this spherulocyte is the only one that becomes grayish after staining (Cf. Fig. 1X inset and Supplementary Fig. 1 ).

We obtained detailed structural information for Paracentrotus coelomocytes through SEM analysis, confirming all morphological features observed in cytological preparations. Morphological aspects were quite specific for each cell type, and based on these characteristics, the coelomocytes could be divided into two main groups: non-spherulous and spherulous cells. Phagocytes, crystal, and progenitor cells fall within the first group. Crystal cells are easily identified due to the conspicuous crystalloid, while phagocytes and progenitor cells may be distinguished according to specific sizes and cytoplasmic complexity. The second group can be further divided into (1) cells with flattened or spread cytoplasm and a thicker nucleus; and (2) cells with thicker cytoplasm and depressed nucleus. Although vibratile cells and red spherulocytes share some characters with the first subgroup, the very spread cytoplasm of vibratile cells and the empty reticulated cytoplasm in red spherulocytes allow for accurate identification. Colorless and granular spherulocytes are in the second subgroup and can be distinguished according to their cytoplasmic spherules, which are large and heterogeneous in colorless spherulocytes, and smaller and homogeneous in granular spherulocytes.

Although the present work is focused on Paracentrotus , it certainly may be useful to identify coelomocytes in other sea urchins, in both live, stained, and SEM preparations, as observed for E. tribuloides , E. lucunter , L. variegatus , A. punctulata , A. lixula , and T. gratilla ( Liebman, 1950; Queiroz and Cust´odio, 2015; Work et al., 2020; Queiroz et al., 2021b). In the case of phagocytes, vibratile cells, progenitor cells, and crystal cells, they are different enough not to be confused. Still, the main characteristics of each type (e.g. cytoplasmic expansion of phagocytes, the flagellum of vibratile cells, the huge nucleus of progenitor cells, and the remarkable crystalloid of crystal cells) were seen in different types of preparation (Cf. Figs. 1 and 2 ). For spherulocytes, the situation is somewhat different. Except for some of our previous works (Queiroz and Cust´odio, 2015; Queiroz et al., 2021b), which detailed the granular spherulocyte, all other studies addressing sea urchin coelomocytes describe only red and colorless spherulocytes (e.g. Branco et al., 2014; Romero et al., 2016; Work et al., 2020). However, the present work provides a set of morphological, morphometric, and cytochemical characteristics that can be used to accurately differentiate red, colorless, and granular spherulocytes in Paracentrotus . Some of these features were already observed in other species (e.g. E. tribuloides , E. lucunter , L. variegatus , A. lixula , and A. punctulata – Liebman, 1950; Queiroz and Cust´odio, 2015; Queiroz et al., 2021b). We also confirm that cytocentrifugation may be quite useful to study echinoderm coelomocytes, as previously demonstrated by successful results in asteroids, holothuroids, and echinoids ( Taguchi et al., 2016; Queiroz and Cust´odio, 2015; Queiroz et al., 2021b; Queiroz et al., 2022).

Figures

Fig. 3. Different morphotypes (M1 to M3) of the spherulocytes of Paracentrotus sea urchins. A-L – Spherulocytes of P. gaimardi; M-Z – Spherulocytes of P. lividus. A-D and M P – Red spherulocyte; E-H and Q-T – Colorless spherulocyte; I-L and U-Z – Granular spherulocyte. A, E, I, M, Q, and U – Morphotype 1; B–C, F-G, J-K, N–O, R–S, V-X – Morphotype 2; D, H, L, P, T, Z – Morphotype 3. A-L and Q-Z – Mallory’s trichrome; M P – Toluidine blue. Scale: 10 μm. Arrow = nucleus. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1. Live and stained mature coelomocytes in Paracentrotus sea urchins. A-L – coelomocytes of P. gaimardi; M-X – coelomocytes of P. lividus. A, G, M, and S – Phagocytes; B, H, N, and T – Vibratile cell; C and I – Crystal cell; O and U – Progenitor cell; D, J, P, and V – Red spherulocyte; E, K, Q, and W – Colorless spherulocyte; F, L, R, and X – Granular spherulocyte. Columns A-F and M-R – Live cells; Columns G-L and S-X – Stained cells; A-B and D-F – Differential interference contrast; C and M-R – Light microscopy; G, J-L, S, V inset, W and X – Mallory’s trichrome; H–I, T-V, and X Inset – Toluidine blue. Black arrowhead =Flagellum; white arrowhead = crystalloid; Arrow =nucleus. Scale: A-X =10 μm; Insets =5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Scanning electron microscopy of mature coelomocytes in Paracentrotus sea urchins. A-F – coelomocytes of P. gaimardi; G-L – coelomocytes of P. lividus. A and G – Phagocyte; B and H – Vibratile cell; C – Crystal cell; I – Progenitor cell; D and J – Red spherulocyte; D inset – Typical mature Red spherulocyte; E and K – Colorless spherulocyte; F and L – Granular spherulocyte. Arrowhead = Flagellum; Asterisk = nucleus. Scale: A = 10 μm; B-L = 5 μm.