Exoskeletons of Bougainvilliidae and other Hydroidolina (Cnidaria, Hydrozoa): structure and composition

The exoskeleton is an important source of characters for the taxonomy of Hydroidolina. It originates as epidermal secretions and, among other functions, protects the coenosarc of the polypoid stage. However, comparative studies on the exoskeletal tissue origin, development, chemical, and structural characteristics, as well as its evolution and homology, are few and fragmented. This study compares the structure and composition of the exoskeleton and underlying coenosarc in members of “Anthoathecata” and some Leptothecata, but does so mainly in bougainvilliid polyps histological analyses. We also studied the development of the exoskeleton under experimental conditions. We identified three types of glandular epidermal cells related to the origin of the exoskeleton and the secretion of its polysaccharides component. The exoskeleton of the species studied is either bilayered (perisarc and exosarc, especially in bougainvilliids) or corneous (perisarc). The exoskeleton varies in chemical composition, structural rigidity, thickness, extension, and coverage in the different regions of the colony. In bilayered exoskeletons, the exosarc is produced first and appears to be a key step in the formation of the rigid exoskeleton. The exoskeleton contains anchoring structures such as desmocytes and “perisarc extensions.”

The glandular epithelial cells of Hydroidolina are responsible for secreting compounds (e.g., structural proteins and enzymes, phenols, polysaccharides) that are associated with the development of the exoskeleton (Knight, 1970;Kossevitch, Herrmann & Berking, 2001;Böttger et al., 2012;Hwang et al., 2013;Mendoza-Becerril et al., 2016). These compounds have been identified in other organisms; for example, in the chitin in fungi, the chitin can have different morphological expression or it cannot be expressed (Wagner, 1994). The chitin system of the exoskeleton can be split into molecular matrix (MM) and molecular synthesis (MSS) (Wagner, 1994). The MM is the extracellular substance containing the molecules, and is located at the outer surface of the epithelium, while the MSS is the biosynthetic apparatus that produces the genetically encoded molecules (Wagner, 1994).
The aim of this study is to use histological and histochemical observations to analyzed and compare the structure and composition of the coenosarc and exoskeleton in polyps of five families of ''Anthoathecata'' and three families of Leptothecata, focusing on variable exoskeleton of the poorly known Bougainvilliidae Lütken, 1850 (Hydroidolina, ''Anthoathecata''). Additionally, we investigated the formation of the exoskeleton under different experimental conditions for five species of Bougainvilliidae, Pandeidae, and Oceaniidae, to verify the presence of MM and understand its cellular origin, morphology and variation in chemical compositional.

RESULTS
The analysis of the longitudinal sections revealed the presence of different patterns of organization. For the hydroids, there are three morphologically distinct patterns, viz., the basal hydrorhiza (formed by stolons), median hydrocaulus (=stem, stalk), and distal hydranth (Figs. 1A-1B). On the other hand, early stages of colonial development (in culture conditions) had five different patterns of organization, viz., the free stolon/branch, hydrorhiza, side-branch, stolonal hydranth/developing polyp, and terminal hydranth (Fig. 1C). The epidermal layer is thin in the region of the tentacles and gonophores. A thin, acellular mesoglea underlies the polyp epidermis and is thinner in the tentacles. The gastrodermis is a thick layer and contains some cells that most likely correspond to zooxanthellae (appears to be Symbiodinium), observed in only some species (e.g., Halecium bermudense) (Fig. 2).
We provide below histological/histochemical description of the exoskeleton in Bougainvilliidae and other members of Hydroidolina, as well as of the epidermal cells that may be associated with the exoskeleton and anchoring structures (Tables 3-6).

Exoskeleton organization in Bougainvilliidae Lütken, 1850 and other "Anthoathecata"
Staining was variable among the different glandular cells. In general the bougainvilliid hydroids present three types of epidermal glandular cells: vacuolated glandular cells (PAS-positive cells), highly granulated glandular cells (HgBpB and NYS-positive cells), and mucous glandular cells (PAS, HgBpB, and AB-positive cells). In other ''Anthoathecata,'' such as Eudendrium carneum (Eudendriidae) we observed vacuolated glandular cells with affinity for TB and PAS and mucous glandular cells, positively for H, PAS and AB, while Turritopsis sp. (Oceaniidae) glandular cells with affinity for PAS, HgBpB and NYS. In the hydranth of Leuckartiara cf. octona (Pandeidae), we observed cells with thin granular film apically and granules. In some ''Anthoathecata,'' Turritopsis sp. and L. cf. octona i-cells are commonly found grouped at the base of the hydrocaulus epidermis.
The exoskeleton of almost all species studied varies in thickness from region to region of the polyp as well as from species to species. The exoskeleton is divided into inner (=perisarc) and outer (=exosarc, as defined in Mendoza-Becerril et al., 2016) layers (Table 4), therefore     Notes.
-, not stained; <+, nearly unstained; +, weakly stained; ++, moderately stained; +++, intensely stained; x, not analyzed histologically; ø, without structure; *, structure not identified; ?, doubtful reaction. corresponding to the bilayered exoskeleton (cf. Mendoza-Becerril et al., 2016) (Tables 5 and  7). The inner layer is continuous from the hydrorhiza to the base of the hydranth (Fig. 3A), rigid and laminated, sometimes reticulated or with a gelatinous (''non-rigid'') appearance (Fig. 3B). When the inner layer extends over the hydranth, reaching the base of the whorl of tentacles (Fig. 3C), the base of the tentacles (Fig. 3D) or even entirely enveloping the tentacles (Fig. 3E) it is thin and gelatinous. This layer has an affinity for TB (with a blue staining) (Fig. 3F), eosin (pink) (Fig. 3G), PAS (Fig. 3H), HgBpB (Fig. 3I) and NYS (Fig. 3J), and the intensity of the staining varies throughout the polyp (Tables 2 and 3), suggesting a chemical composition of aminopolysaccharides (AP) associated with proteins. The inner layer of some species has an affinity for AB (suggesting presence of GAGs) at the region of the hydrocaulus, in others this layer has no affinity for PAS (suggesting absence of AP). The outer layer is usually thick and rugose, extending from the hydrorhiza to the hydranth, reaching the whorl of tentacles (Figs. 3A and 3C-3E) or covering up to the base of the tentacles (Fig. 3D). This layer has an affinity for TB (with a purple staining) (Fig.  3F), and is PAS-and AB-positive (Figs. 3H and 3K), suggesting a chemical composition of GAGs (Table 3). The outer layer is easily distinguished from the inner layer when treated with TB and AB techniques. However, it is difficult to distinguish both layers when the inner layer is thin or when the outer layer has no external material attached. The two layers may be connected by an anchoring system formed by extensions from the inner layer (''perisarc extensions''; Fig. 3F). The exosarc is frequently encrusted with thin organic and inorganic material (diatoms, mineral particles, bacterial film), therefore with a granular and rigid appearance (Figs. 3F-3I and 3K). Bilayered semi-transparent or opaque exoskeleton. Inner layer laminated, fairly thick (Table 5), continuous from hydrorhiza to hydranth base then ending abruptly, annulated in basal regions (hydrocaulus and side-branches) and throughout hydrocaulus at more or less regular intervals (Figs. 4A-4C). Outer layer not rigid, thin (Table 5), continuous from hydrorhiza to hydranth base (Fig. 4C).
x, not analyzed; •, approximate measures, difficult define boundaries layer; *, it was not possible to define the boundaries of the layer. The measures of perisarc thickness were obtained from the position of maximum perisarc thickness.

Exoskeleton organization in leptothecates
Hydroids of Clytiidae and Obeliidae with glandular cells, with affinity for TB, PAS, HgBpB and NYS (Table 3), abundant at hydrocaulus and gonophore base. I-cells rarely observed. Haleciid hydroids with vacuolated glandular cells with affinity for TB, PAS, and HgBpB, moreover with i-cells in the hydrocaulus.

Description of hydroidolinan exoskeleton under culture conditions
Polyps of Bougainvilliidae B. vestita, B. muscus and P. robusta, and Pandeidae L. cf. octona maintained in culture with both filtered and unfiltered seawater developed a bilayered exoskeleton. The inner layer of B. vestita (Fig. 21A) is thinner at apical hydranth region close to tentacular whorl and at tentacular base (Fig. 21A), weakly stained with HgBpB and NYS compared to hydrorhizal and hydrocauline regions. The hydrorhiza has a thin perisarc and thick exosarc (Fig. 21B). The exoskeleton of a new hydranth is not detectable under the stereomicroscope (Fig. 21C), requiring histological preparations for detection. On the contrary, in B. muscus the exoskeleton is clearly seen at low power magnification,  : dm, diatoms; ep, epidermis; gct, gastrovascular cavity; gs, secretory granules; gt, gastrodermis; inl, inner layer; ms, mesoglea; oul, outer layer. even in polyps 240 µm in height (Fig. 21D). B. vestita and B. muscus growing tips of the developing stolon and hydranth with 'non-rigid' inner layer (Figs. 21E and 21F), and outer layer encrusted with little external material. Inner layer of P. robusta is thinner, not rigid, with vertical divisions in some regions of hydrocaulus (Fig. 21G), and positive for HgBpB (Fig. 21H). Hydranth with thin exoskeleton (Fig. 22A) and developing polyps with granules in epidermal glandular cells (Figs. 22A and 22B). Outer layer of L. cf. octona with slightly  c, coenosarc; em, external material; ep, epidermis; es, exoskeleton; gc, glandular cells, gct, gastrovascular cavity; gt, gastrodermis; inl, inner layer; ms, mesoglea; oul, outer layer. different staining intensity with TB and AB compared to material developed under natural conditions. Exoskeleton at growing tips of the developing polyp of L. cf. octona with single ''non-rigid'' layer, encrusted with external material (Figs. 22C and 22D).
Polyps of Turritopsis sp. maintained in culture with unfiltered seawater developed the outer covering over exoskeleton but without encrusted material attached (Fig. 22E); polyps maintained in culture with filtered seawater did not developed an outer covering (developing polyp 2.1 µm) (Fig. 22F), and with affinity for PAS but not for AB, suggesting only AP present. Therefore, we assume this outer covering is not equivalent to the outer layer present in Bougainvilliidae. Polyps of Clytia sp. maintained in culture developed thin, AB-negative exoskeleton.

Features of epithelia and their cells
Epidermal I-cells at the base of the hydrocaulus of Turritopsis sp., L. cf. octona, H. bermudense can be differentiated into nematocysts or glandular cells, the latter participating in the production of different substances forming the exoskeleton. This indicates the importance of these cells for both cnidogenesis and skeletogenesis, a hypothesis to be tested in other hydrozoan taxa.
The three types of epidermal glandular cells (vacuolated, granulated and mucous) were more abundant in developing polyps in the majority of the studied species of Bougainvilliidae, but were not observed in polyps of D. conferta and P. disticha. Previous histochemical tests indicated that the gastrodermal glandular cells of the hypostome continually produce and contain GAGs (Syncoryne tenella (Wineera, 1972), accepted as Coryne eximia Allman, 1859(Schuchert, 2001; Hydra (Wood, 1979); MA Mendoza-Becerril, pers. obs., 2013); these substances may correspond to enzymes (Cowden, 1965). This is a different condition from the GAGs of the exoskeleton, which are not produced continuously based on our results and these are produced by epidermal mucous glandular cells.
Different enzymatic types and activities are specific for each function and region of the polyps, e.g., the enzyme acid phosphatase has been recorded in species of Leptothecata and its concentration has been associated with morphological variation of the species (Östman, 1982). Other important enzymes participating in exoskeleton formation have been recorded in several hydrozoans (Mendoza-Becerril et al., 2016). Chitin synthetase (Chs) is found in Hydractinia echinata (Mali et al., 2004). Chitinase is restricted to the gastrodermis of the hydrocaulus and absent in the epidermis and tentacles of Podocoryna carnea (accepted as Hydractinia carnea) and Hydra attenuata (accepted as Hydra circumcincta) (Klug et al., 1984). Phenoloxidase, produced in epidermal cells of Laomedea flexuosa, is involved in cross-linking of perisarc components (Knight, 1970;Kossevitch, Herrmann & Berking, 2001).
Our results corroborate the hypothesis that the coenosarc does not have a fixed composition of cell types. During development, different types of cells constantly migrate from specific areas of cell differentiation and proliferation to their final location (Chapman, 1974;Thomas & Edwards, 1991;Kosevich, 2013). Thus, cell action depends on a definite sequence of events, including cell multiplication, cell differentiation, and cell migration (see Kosevich, 2013;for Gonothyraea loveni). Also, the different epidermal glandular cells involved in exoskeletal development can change the type and secretion of one or several chemical components as the polyp grows (Kosevich, 2013).
We documented two types of structural exoskeleton, (a) the bilayered exoskeleton formed by an inner layer of perisarc surrounding the coenosarc and covered by an outer layer of exosarc in contact with the environment, and (b) a single coriaceous exoskeleton formed exclusively by the perisarc (Figs. 23C and 23D). The perisarc and exosarc vary in their chemical composition (AP or GAGs; Table 4), texture (Fig. 23D), thickness, extension and coverage of different regions of the colony (Fig. 23E). A thick exosarc is generally derived from the aggregation of extraneous inorganic (sand and mud grains) and organic materials (tests of radial centric and araphid pinnate diatoms) (Mendoza-Becerril et al., 2016). These extraneous materials lend a rigid granular appearance to the exoskeleton of Bougainvilliidae and Pandeidae (Fig. 23F).
All the species of Bougainvilliidae that we studied have a bilayered exoskeleton, with the possible exception of P. michaeli. For this species we found a discontinuous thin layer in its hydrorhiza and hydrocaulus. This thin layer could correspond to exosarc, contradicting the descriptions of P. michaeli stating that the ''pseudohydrotheca'' (=exosarc on the hydranth) is absent in this species (e.g. Schuchert, 2007).
Gonophores of the bougainvilliids studied (except P. michaeli) are completely enclosed by a bilayered exoskeleton, even in the species that have been described with an exoskeleton restricted to the gonophore pedicels, such as G. annulata (Nutting, 1901). As it is very thin and flexible, almost imperceptible in some cases when specimens are examined entire, the exoskeleton completely surrounding the gonophore is described in the literature as a filmy perisarc, loose filmy perisarc, or thin perisarc membrane (Schuchert, 2007).
Epidermal glandular cells of the colonies of Bougainvilliidae (B. vestita, B. muscus, and P. robusta) and Pandeidae (L. cf. octona), forming the molecular matrix (MM), apparently are differentiated at the developing border of the free stolons/branch, hydrorhiza, side-branch, stolonal and terminal hydranths. Developing extremities of growing polyps and hydranths of some bougainvilliids were covered by a ''non-rigid'' layer of GAGs, while regions near their origin were covered by a ''non-rigid'' exoskeleton formed by an exosarc, constituted predominantly by GAGs, and a perisarc with AP and proteins. This ''non-rigid'' layer may correspond to MM and to the cuticle described by Wineera (1972), possibly being involved in the process of formation of a rigid, bilayered exoskeleton (perisarc and/or exosarc). Particles and thin filaments present in the exoskeleton would serve to harden the structure.
The exosarc is produced first, and may be an important step in the formation of the rigid perisarc. The exosarc could interact with other molecules (e.g., AP, structural proteins) functioning as a single layer in developing polyps. This hypothesis is supported by the presence of AB-positive granules in the epidermal glandular cells at the base of the hydrocaulus of G. franciscana, and of TB-and PAS-positive granules in the skeletal outer layer and epidermal glandular cells of the developing hydranth of L. cf. octona. The MM with acid polysaccharides is an important element in the mineralization process in the stony coral Mycetophyllia reesi (Goldberg, 2001).
Variations in staining intensity suggest the presence of different concentrations of the chemical components, depending on the developmental stage of the polyps. The presence of GAGs in the perisarc of G. annulata, E. carneum, and C. gracilis indicates that acidic GAGs are trapped within the inner layer, maybe sclerotized in the presence of proteins.
Developing polyps have only an exosarc, suggesting that this is the first layer in the skeletal ontogenesis. Subsequently, epidermal cells differentiate, producing other specialized glandular cells and therefore changing the nature of the secreted compounds over time. Such changes may have prevented us from observing mucous glandular cells in some species, which would be capable of rapidly eliminating their secretions and developing into different cell types, as in Hydra pseudoligactis (accepted as Hydra canadensis), a species with epidermal cells that release acid GAGs (Burnett & Lambruschi, 1973).
The thin, discontinuous outer covering observed in the exoskeleton of athecate Turritopsis sp. and some leptothecates (O. dichotoma and O. sargassicola), with an affinity for AB, has adhered inorganic and/or organic external material. However, this covering is probably not part of the exoskeleton and not equivalent to the exosarc of bougainvilliids and some other members of ''Anthoathecata,'' because we have not observed a MM in the species with discontinuous outer covering throughout the polyp, and, at least in the case of Turritopsis sp., it does not develop when the polyp is maintained in filtered seawater. The outer covering is possibly formed by exogenous substances, particles or diatoms. For example, the diatoms secrete acid sugars in the form of uronic acids and sulfated sugars (Staats et al., 1999) and therefore positive to AB, thus our results indicate that leptothecate polyps are incapable of producing GAGs independently.
In most species we observed that the exoskeleton was laid down even when the colonies were maintained with filtered seawater, therefore suggesting its secretion is genetically encoded and innate (a putative MSS), and does not depend on age or environmental conditions. Exoskeleton thickness, especially of the exosarc, depends on the quantity and type of extraneous material (organic or inorganic) available under natural conditions (Rees, 1956;Schuchert, 2007;Mendoza-Becerril et al., 2016) and the exoskeletal morphology can be modified by environmental conditions (Murdock, 1976;Hughes, 1980) using any source of external material, even agglutinating particles egested by the polyp. Some species have a polymorphic expression of the exosarc in at least some species of Garveia. This was observed when comparing specimens from different environments. Similarly, it has been observed in other Hydroidolina (Rees, 1956;Murdock, 1976;Hughes, 1980). Different levels of contraction were observed in living and fixed polyps. A contracted hydranth of some species, such as B. muscus and G. franciscana, appears to be fully covered by the bilayered exoskeleton, even though their exoskeleton extends only from the hydrorhiza to the tentacular base of the hydranth; while other species, such as G. nutans and P. robusta, appear to be covered up to the whorl of tentacles, as delimited by a fold just below that, although the extended body clearly has free tentacles. Species such as B. vestita may have the hydranth completely covered by the exoskeleton even when fully extended, but, without a detailed analysis, may appear to have exoskeleton coverage similar to contracted hydranths of B. muscus or G. franciscana.
The thickness of the exoskeletal layer (Table 5) varies intraspecifically in some ''Filifera'' from different locations (e.g., Table 7 and G. franciscana, Vervoort, 1964). Consequently, this structural variation may lead to misidentifications of the species, especially when other diagnostic characters are absent (e.g., reproductive structures), and should be used cautiously as diagnostic for the taxonomy of groups such as Pandeidae and Bougainvilliidae (Millard, 1975;Calder, 1988). Nevertheless, the variation in thickness of the chitin-protein exoskeleton is considered a useful diagnostic character for some families of Leptothecata (e.g., Clytiidae, Cunha, Genzano & Marques, 2015).
Desmocytes (Fig. 23G) are specialized cells that are found along the upright hydrocauline coenosarc of the polyps and side branches of colonies of Bougainvilliidae, Eudendriidae, Pandeidae, and Haleciidae, among the species studied. They are characterized by a dense accumulation of chitin and protein filaments (Knight, 1970;Chapman, 1974), and therefore have a high affinity for PAS, HgBpB and NYS. These filaments aggregate into dense rods and reach the exoskeletal perisarc at the apical end of the desmocyte (Fig. 23G), and form rigid connections with the mesoglea at the basal end of the desmocyte (Fig. 23G).
In conclusion, our study added to the knowledge of the hydrozoan exoskeleton, but also left unanswered several questions on its structure and chemical composition: which specific components are present within the exoskeleton (e.g., glycoproteins, proteoglycans and hexuronic acids, more specifically, chondroitin sulfate and heparan sulfate)? What is the ratio of the different chemical components and what are their chemical interactions? What are the biomechanical properties related to the different types of exoskeletons and their biological consequences? Further investigations applying immunohistochemistry (e.g., to identify the type of GAGs), confocal microscopy (e.g., using congo red as a fluorescence marker for chitin), and transmission electron microscopy and X-ray diffraction may help to answer these questions.