Most of the structures described above for Monorhaphis basal spicules were observed and described by Schulze [9] at the level possible for that time. Recently, Müller et al. [25] and Wang et al. [34,35,36, 38, 39, with references] presented descriptions of the same structures and proposed a model for spicule morphogenesis. We compare and contrast our examination of the structures of these spicules with those in earlier studies.
Organic material associated with basal Monorhaphis spicules
Our observations of organic material occurring on the surface of the Monorhaphis spicules conforms with earlier reports by Schulze [9]. He described “der faserigen Nadelscheide” [fibrous sheath of spicule] in smaller body spicules [9: Pl. XL, fig. 7] and “Netz der faserigen Nadelscheide” [net of fibrous sheath of spicule] in the mature basal spicule [9: Pl. XL fig. 8]. The collagen nature of this net was recognized by Ehrlich et al. [63, 64] who also demonstrated that it is hydroxylated fibrillary collagen [65]. The fact that it can be easily detached from the basal spicule surface, that small body spicules occur between the net (having fibrous structure—Figs. 12d, 13b) and the basal spicule surface (Figs. 11b, 12b, 13a), and that there is no direct relation between the meshes of the net and the tubercles and ridges on the spicule surface, suggest that the net is not directly participating in the process of basal spicule biomineralization, which must instead be controlled by the sclerosyncytium (multinucleate scleroblast masses) [42]. In our opinion, the net is better considered as a structural element of the sponge body keeping it connected with the basal spicule.
Apart from the exterior collagen net that covers the spicule surface, organic substances permeate the spicule silica (Fig. 14b) and our analysis indicates that these are proteins which were already reported– silicatein and/or galectin [2, 28], or collagen [3, 64, 65]. The ability to precipitate silica by much more simple synthetic minicollagens has been demonstrated [66].
Spicule structure
The nature of the axial filament of the studied spicules was beyond the scope of the present study, but there are several recent attempts to understand this in spicules of Monorhaphis and other sponges using X-ray based approaches with respect to silicateins [47,48,49,50]. However, no experimental evidence was presented in these publications for the presence of silicateins in the samples under study, but it was assumed, and there was no reported attempt to demineralize the spicule with the aim of isolating and characterizing the silicateins. Thus, the nature and origin of the axial filament in Monorhaphis remains to be determined.
All earlier papers described very regularly layered structures of the basal spicules in transverse section [9, 17, 30, 38, 39, 48, with references] composed mainly of PG layers (Figs. 2, 3, 5). At the nanoscale the silica of the PG layers has a granular structure [25, 36, Fig. 14a] what is typical of other hexactinellids as well [14]. The material that builds the spicules is always amorphous opaline silica and differs slightly in structure between the outermost AL, which is more condensed than the rest of the spicule. This could be explained by a smaller amount of structural water in the amorphous silica of the AL than in the rest of the spicule (figs. 16, 17).
Our studies of the immature spicule, which is characterized by the presence exclusively of PG layers (this corresponds to a growth stage illustrated by Schulze [9] when the sponge body has enveloped a major part of a spicule) lead us to conclude that they do not follow the growth model proposed by Wang et al. [24,25,26,27,28,29,30,31,32,33,34,35,36, 38, 39]. These authors used comitalia (smaller body spicules) of Monorhaphis as the basis of their reasoning, not a large basal spicule, but didn’t show that the structure of these spicules is the same as a basal spicule. In addition, they used an artificially broken spicule and the resultant detachment pattern of the apical spicule layers [34]. They reported step-like structures of broken layers (with steps facing downward, toward the spicule center) that were interpreted as mimicking the spicule growth pattern [34: fig. 5a, p. 277]. This result subsequently influenced interpretation and modelling of Monorhaphis spicule growth. They suggested that growth in spicule length proceeds by cones being added one above another from the apex of the spicule, which later extend downward (Fig. 18 left). Thus, new layers are added by a downward extension of the cones, and by engulfing already existing spicule layers (Fig. 18 left). Our observations of the young basal spicule clearly indicate that growth is fundamentally different, as discussed below (Figs. 18 right and 19).
The natural surface of the young spicule and its well preserved apex showed the presence of more or less natural step-like faces directed toward the spicule tips (not the center) and we suggest these correspond to growth lines. These lines or steps are more pronounced and more densely spaced near the lower tip of the spicule, and less well-pronounced and more widely spaced at the apex. This indicates that growth or extension of the spicule is proceeding in the direction of the tips (Fig. 18 right), not from the upper tip toward the center. Moreover, it suggests that growth of different layers (PG, TL and AL) may proceed simultaneously in different locations along the spicule. In other words, formation of one layer does not proceed over the whole spicule length and surface immediately, but initially is spatially limited. Internal layers may lengthen closer to the upper tip, while thickening of the spicules is achieved by progressive growth of more external layers. We also observed that the faces of growth lines in the center of the spicule are in opposite directions in the young spicule (Fig. 2c, h). Those in the upper portion of the spicule show step-like faces directed upward (Fig. 1a, b), while those from the lower part are directed downward (Fig. 2d–f), thus, the initial growth of the spicule must be bidirectional, as could be expected from what we know about smaller hexactinellid monaxial spicules (diactines).
The interpretation of the growth of the Monorhaphis basal spicule proposed in this study is also supported from a biological viewpoint, because Monorhaphis, as for all hexactinellid sponges, is syncytial in nature, and thus silica may be deposited in any location on the spicule under the body cover. Conversely the model of Wang et al. [34,35,36, 38, 39] suggests that growth occurs only at the upper tip of the basal spicule (and thus also of the sponge). Our observations are in contrast to the “cone in cone structure” model [34,35,36, 38, 39] and in agreement with Schulze’s [9] observations. He illustrated specimens with the upper portion of the spicule protruding from the sponge body. Such a situation is not possible in the model by Wang et al. [34,35,36, 38, 39], but entirely plausible in the light of our observations, hence our new proposed growth model.
The simultaneous lengthwise growth of the spicules in both directions characterizes only the early stages of spicule development when it is nearly completely covered by a sponge body (Fig. 16). When the lower tip is well inserted into sediment, the sponge body is “climbing up” the spicule [9]. This movement of the sponge body from the lower part of the spicule upwards is registered by showing step-like faces of more external layers directed toward the lower spicule tip (Figs. 2d–f and 18), while growth at the upper tip progresses upward.
Tuberculate layer (TL)
The tuberculate structure was previously illustrated and described [9: Pl. XLIV, figs. 1–3, 8]. It has also been described in several recent papers [25, 34], but there are some inconsistencies in these descriptions [25: fig. 3f]. It was reported that the surface of the basal spicule is “occasionally decorated by rectangular protrusions” [25: fig. 3f]. Some suggested [34: fig. 3c, d] that a collagen net with holes and tubercles [“hemi-spherical” protrusions (knobs)] and that the protrusions (= tubercles) fit into the holes of the collagen net and are of similar size. However, their observations were not on basal spicules but much smaller body spicules, that are smooth. Subsequently, we have demonstrated that there is no direct relationship between the net openings and the tubercles (Fig. 12). Additionally, the shape of the holes in the net do not correspond to the shape of the tubercles, disposition of the holes in the net is irregular and different from the disposition of the tubercles on the spicule surface (see Figs. 12, 13) which show high regularity. Thus, the suggestion that tubercles (= protrusion) “exist in the initial phase of silica formation” [34] has not been confirmed in this study, and neither has the suggestion that “At later stages, the protrusions seem to melt”. The growth of tubercles in size and change of shape is gradual and it is the second (the first being PG) phase of silica deposition. At later stages they are gradually covered by incipient AL (Fig. 6).
According to Wang et al. [39: p. 2050, explanation to fig. 2], the zone of smooth surface is followed by „zone of tiny coarse protrusions (tc)” or that “penultimate lamellae [is] cluttered with tiny bumps” [and]”a thin layer of a finely woven network (fn)” that under higher magnification is revealed as “layer riddled with depressions with elevated rims (fig. 2e, i)”. We have shown with SEM observation (Fig. 6e, f), “protrusions” pass gradually into fully developed protrusions/tubercles and those, in turn, pass gradually into (are covered by) the AL (Fig. 6b–d). The apparent depressions [39] are in fact an optical artefact caused by specific illumination of the surface with tubercles (see Fig. 10e). The same surface under oblique reflected light looks like it has depressions (optical illusion) and with SEM categorically shows tubercles (Fig. 17).
Annular layer (AL)
As demonstrated by our analyses, the AL layer is of mineral opaline composition, is similar to the rest of the spicule but differing in structure. It corresponds to the “Querriffle zone” [transverse hackle zone] of Schulze [9: p. 118] who also considered it to be mineral, and the “banded ribbon” zone of others [34, 35, 39], who first suggested that it is a “collagen net” [34: p. 273, fig. 3c, d, 35: fig. 5b]. We propose to call it the “annular layer”, because the name proposed earlier [39] is based partly on the presence of “bands” that are taphonomic or optical artefacts, not original features (see below). Wang et al. [35: fig. 3.10c] also marked and described the AL as “a solid fibrous (collagen) sheet (fi)” on a copy of the original figure from Schulze [9: Pl. XLIV, fig. 8a], where it is clearly described as “Querriffel ziegenden Lamellen” [(hackle with lamellae); fig. 8a, p. 119].
The darker/grey and milky bands observed in our study on the surface of the AL under reflected light (but not with SEM; Fig. 10), correspond, without doubt, to the bands described earlier [39: p. 2051). It is suggested there that there is regularity in the width and distribution of these bands, and that there are two types: non-perforated and perforated [39: fig. 5b]. In contrast, our study found no regularity in the bands, and no perforated or non-perforated ones (Fig. 10). The AL is a solid silica layer with a granular structure and high organic content along its entire length. This can also be seen in figure 5a in Wang et al. [39].
These bands are not visible with SEM (Fig. 10b) and the difference in colour results from the fact that the lighter (milky) bands are not bound perfectly with the underlying TL; there is a micro-fissure between the AL and the TL surface, while the darker bands are tightly bound with the underlying TL. As a result of light refraction at the boundary between the AL and TL, when a fissure exists between the two layers, an impression of different colours of the bands appears. In the case of the darker bands, there is no light refraction at the boundary with the underlying TL because there is no fissure between the layers. Consequently they behave as an optically homogenous structure. The “depressions” or “perforations” [39: figs 2l, 5b–d, f, g] occurring in the lighter bands of the AL are an optical artefact corresponding to the tubercles (protrusions) of the underlying TL as seen through the silica of the AL. This taphonomic feature could occur after sponge death, i.e. during spicule collection, by a mechanical stress that can partly detach the AL from underlying TL surface. However, it cannot be excluded that this may have happened in earlier stages when the sponge was still alive, and it is due to differential stress caused by bending of the spicule in various directions when it was still anchored in the sediment.
An additional point that challenges interpretation of apparent depressions as locations of sclerocytes is the fact that hexactinellids only have sclerosyncytium [42], and we consider that this syncytial nature allows for formation of large siliceous structures such as the basal spicules of Monorhaphis, that would be not possible in cellular sponges [cf, 16] It is worth noting that the size of the megascleres in syncytial hexactinellids, are in general larger than the size of megascleres in demosponges, which are cellular.
A complex scenario of deposition of siliceous lamellae by supposed discrete sclerocytes located in the “depressions” on the spicule surface (despite the syncytial nature of hexactinellids) was developed [34, 35, 39] and claimed without evidence that there is “no reason to believe that all cells participating in lamella formation fuse to form syncytia” [39: p. 2053].
It was shown [35] that the axial cylinder contains proteinaceous material and called it the axial barrel, and its careful examination in transmitted light reveals that the axial cylinder is layered (however not regularly) (Fig. 5h, i), also shown by Schulze [9] and reaffirmed by Wang et al. [35]. The apparently homogenous structure of the axial cylinder, visible on a broken surface with SEM (Fig. 2k) shows a difference in organic content, resulting in different mechanical properties during the spicule breaking. Our observations demonstrate that there is a gradual transition from the “axial cylinder” toward the more external layered part of the spicule, that has lower organic content indicated by the absence of a brown hue (Fig. 5i).
Based on our observations and the discussion of ideas presented by Wang et al. [34,35,36, 38, 39] we propose a new model of Monorhaphis basal spicule morphogenesis.
The new model of basal Monorhaphis spicule structure and morphogenesis
After careful consideration of our observations and relevant literature, especially those of Schulze [9], we propose the following growth model for the basal spicule of M. chuni, which includes the various structural zones and their functional interpretations.
The early stages of spicule formation, when most of the spicule is covered by a soft organic body is rather rapid in relation to later growth stage, and increase in length proceeds in two directions, towards both tips of the spicule at unequal speed (Figs. 18, 19 middle). This is seen by the asymmetry of the spicule ends, the lower one being widely conical (see Fig. 2d–f), and the upper one being thinner and more narrow conical (Fig. 2a, b) suggesting faster growth toward the spicule apex. The direction of growth is maintained by the extension of the axial filament whose tip is not enclosed by silica during most of the process. It is not clear if it is ever enclosed by silica layer(s) in the final stage (Fig. 18 right: B) as has been observed for smaller spicules of Monorhaphis, and in demosponge spicules (Pisera unpublished).
The formation of axial filament and incipient silica layer in demosponges that are cellular and have discrete sclrocytes, is first intracellular after, during extracellular stage [17, 40, with references] axial filament is still growing for a limited time, but finally it is encased completely by several silica layers (Pisera unpublished observations). This terminates, in most cases, spicule elongation/enlargement (some exceptions to this scheme, such as desmas and large spicules of Thenea, that still are not well understood). However, hexactinellids are syncytial and have sclerosyncytium, and thus continuous growth (including axial filament growth that might be not encased by silica at all) may be maintained for a very long time, producing much larger spicules.
The ‘axial cylinder’ in Monorhaphis is formed next, and is already under the influence of the sclerosyncytium, not the axial filament. The axial cylinder occurs along the entire length of the spicule (Figs. 17, 18 right: A, B), hence its formation, as for the extension of the axial filament, must be continuous and simultaneous with the deposition of outer, layered silica in older parts of the spicule.
In the next step, PG layers begin to be deposited, which allows for an increase in spicule thickness. Some surface layers seem to be discontinuous initially. New layers begin to be formed in the central part of the spicule (Figs. 2c, h, 18) and are later extended toward the spicule tips. It appears that deposition of silica may be patchy, and that these patches are gradually fused into one continuous layer; each new layer transgressing over the earlier layer occurring below and near the tip (Fig. 18 right: A, B). The pattern of growth at the lower and apical tips differs. At the lower tip each successive (more external) layer has a reduced downward extent (layers are regressive in relation to underlying layers) (Fig. 2d–f). This may be because the sponge body is moving up the spicule (or the spicule is extruded downward), leaving the lower tip more and more exposed, and thus allowing for anchoring the sponge in the sediment (Fig. 18 left and 19 middle and right).
Additional growth results in formation of other morphologically and structurally various zones (Fig. 19) on the spicule surface. In the upper part of the spicule additional PG layers are formed (Fig. 19 left), while in the lower part two new structures appear: first tuberculation (Fig. 19 left: B, C) of the surface, and then (both in time and space), the AL (Fig. 19 left: D, E). The tubercles are formed by adding folded PG layers. Structurally TL is the same as a continuation of the PG. Tubercles are developed by progressive growth (down the spicule) in size and change in shape, by adding new, more folded layers above those beneath (Fig. 19 left: B, C).
After the tubercles reach their final size and shape, an incipient AL zone progressively starts to cover the TL (Fig. 19 left: D). The AL is structurally different from both the PG and TL by being granular and containing more organic material. The AL is always downward from the TL, and above fully formed tubercles. It appears that the AL is developed when the sponge body “climbs up” the spicule to be separated from the muddy sediment. There is no doubt that the AL is partly covered by sponge body (confirmed by direct observation) during its formation, but in lower parts is devoid of organic material even when the sponge is alive (Fig. 19 right), thus, no further growth in thickness takes place. During the later stages of spicule growth, the pattern of silica deposition differs in various zones and proceeds differently from the early stages of growth. Lengthwise spicule growth only takes place at the apical tip by the addition of new layers of PG which grow over previous layers (Fig. 18 right). This also occurs in young spicules which produce the PG. The growth at the lower part is arrested from the moment the AL is fully developed. In fact, the AL is deposited in an upward direction as is the TL zone. These layers are added above the TL and PG zones respectively as new layers (Fig. 19 left: B, D), and over each other during the course of the sponge “climbing” the spicule, thus adding to the thickness of the spicule. The result is that the sponge body moves up by extending the spicule, the TL surface is “moving up” the spicule by development of new incipient tubercles, and the AL is also “moving up” progressively covering fully developed tubercles.
The distribution of various morphological zones and structures on the spicule surface confirms that they are formed simultaneously on different parts of the spicule. The syncytial nature of hexactinellids is responsible for this pattern, as the syncytium may be functionally differentiated [42].
It is not clear why the AL and TL developed, but one can speculate that the outer AL is an adaptation to assist the sponge to remain associated with the spicule surface when “climbing up” during growth and it also prevents the sponge from sliding downwards on what would otherwise be a very smooth silica surface. The structure where elevations of the TL fit into depressions of the lower surface of the AL (Fig. 9b, c), can be compared to structural joints in woodworking called tenon and mortise or dovetail joints, used to connect two pieces of material to obtain a strong stable joint. Such joints help to keep the spicule layers together, stabilize and strengthen them and make the whole structure less flexible. However, the upper part of the spicule built only with PG and smooth layers, allows for bending of the spicule due to the possibility of movements along the layer’s surface. We hypothesise that the function of such a structure is to strengthen the spicule to prevent breakage (or bending) in the lower part and thus to assure sponge survival by keeping it above the muddy sediment. Some additional rigidity is achieved in the upper part of the spicule (covered with the soft body) by the presence of numerous other spicules – tautactins (of different size and diameter) that are located close to the basal spicule with their long rays tangential to the basal spicule.
Finally, one should stress that the basal spicule of Monorhaphis, with its unique features (including its large size and complex morphology), cannot be used as a general model of siliceous spicule formation, or even as a model for other smaller hexactinellid spicules (even other smaller body spicules of Monorhaphis), except perhaps for other basal hexactinellid spicules (e.g. spicules of Hyalonema which have a similar function and display similar surface structures; Pisera, unpublished data). However, the basic principle of increase in spicule length is the same in hexactinellid spicules (as well as in demosponge spicules), and is toward the spicule tips, not from the tips toward the spicule centre.