Remarks on the life cycle
The only available information concerning the life cycle of Bicellariella ciliata comes from the paper of Eggleston  who studied bryozoan reproductive seasonality around the Isle of Man, Irish Sea. In this area two generations per year are produced in this species, including an overwintering and a summer generation. The overwintering colonies reproduce in spring and die in August, whereas their descendants – summer colonies – reproduce in autumn and die off in November. Thus, the lifespan of one colony does not exceed one year.
Distal parts of the branches in fertile colonies show a zonation reflecting subsequent states of embryonic development in the ovicells and the polypide recycling in the fertile zooids . The partition includes a zone at the very distal tip of each branch with young zooids without ooecia, a zone below containing zooids with ovicells and eggs, next zone of zooids with degenerating polypides and early embryos inside the brood chambers, a zone of zooids with degenerated polypides seen as “brown bodies” and late embryos or larvae in the ovicells, and a zone with regenerating polypides and empty ovicells. Below this sequence is repeated, starting from zooids with regenerated polypides, empty ovicells and eggs and so on.
The observations of Eggleston  show that in B. ciliata some zooids are able to produce more than one larva per reproductive season in the Irish Sea. Our material (large overwintering colonies collected in June) from the Skagerrak region, between the Baltic and the North Sea also demonstrated a similar zonation (Figure 4E) although polypide degeneration starts in zooids with middle-aged or late embryos. Nevertheless, a repeated production of embryos was not detected. Whereas reproducing zooids with eggs and embryos of different age were present at the tips of the branches, the zonation described above was never observed in more proximal parts of the colony where zooids had empty ovicells. In these parts of the colony we found only two zooids with a brown body and a young oocytic doublet. Therefore, it is likely that zooids reproduce only once per season in the Skagerrak region and polypides degenerate without further regeneration. Seasonal observations are necessary to prove the above suggestion. Functional polypides, however, were only seen on the tips of the branches and only a single brown body per zooid was detected in our material.
The initial and final position of the ovary has been subject to debate in bugulids. Claparède  was the first who observed that early female cells (prospective ovary) in Bugula avicularia originate at the early polypide bud that have no funicular cords yet. In fully-formed zooids the female gonad is situated on the proximal part of the funiculus near the caecum in this species. These observations are in accordance with those of Huxley , p. 191 who studied the same species and found a “single … ovum” being “attached to the funiculus … close to the stomach” in young zooids without ovicells. In older zooids the ovary does not change its position. In contrast, it is placed on the basal wall of the cystid and is not directly connected to the funiculus in Bugula flabellata and B. plumosa. In the latter species the formation of the female gonad on the cystid wall was also described by Salensky . However, Dyrynda and King  state that the ovary is suspended by funicular cords in a position immediately proximal to the gut of the developing polypide.
Vigelius  described the earliest recognizable ovaries on the basal wall in B. calathus. He also noticed that in older zooids it loses its contact with the cystid wall and is freely suspended in the zooidal coelom or connected to the funicular strand. It is quite possible that Vigelius observed ovulation in this case.
According to the observations of Calvet  the position of the ovary varies in B. simplex. In support of Claparède , Calvet found early ovaries located near the developing polypide in young zooidal buds. The definite female gonad is either suspended to the funicular strands in the zooidal cavity or attached to the peritoneal lining of the zooidal wall or stomach. Calvet  noticed that he saw two ovaries in some zooids, and this was supported by Schultz  and Borg  in Electra crustulenta. Our finding of two young doublets of female cells in the developing polypide bud indirectly supports these observations.
In Bicellariella ciliata, Nitsche  considered that there was no special ovary in this species (and also in two species of Bugula studied), and that 2–3 oocytes are “budded” from the internal surface of the “endocyst” (epithelial lining of the cystid wall) being surrounded by a thin membrane (follicle cells). In contrast, Joliet  mainly found the ovary to develop within the funiculus in B. ciliata and some other species. He described two different kinds of eggs, one developing in association with the funiculus and another (“parietal”) on the body wall. However, Joliet assumed that the parietal eggs originate in connection with the funicular strands passing through interzooidal pores.
Our data are in accordance with the opinion that the early female cells originate from the polypide bud and that the final position of the female gonad may vary in the same species. Although the earliest ovary that we found was in contact with both, the developing polypide and the cystid wall, the zone of contact with the former was much larger than with the latter. Also, we observed a few instances when collapsed ovaries after ovulation did not touch the cystid wall and were only connected with the polypide gut. However, fully-formed ovaries were normally located on the zooidal wall. The reason why some authors described the ovary to originate from the cystid wall in Bugulidae might be that the space between this wall and the polypide bud is rather limited. Thus, the female cells appearing on the polypide bud quickly come into contact with the zooidal wall.
Follicle provisioning of the oocyte
In bryozoans oogenesis is of the alimentary type as in most invertebrates [54–56]. In most brooding cheilostomes and in particular in Bicellariella ciliata it combines both, follicular and nutrimentary types of oocyte provisioning, the latter provided by a nurse-cell. In contrast, in non-brooding cheilostomes and all ctenostomes (both belonging to the class Gymnolaemata) oogenesis is of exclusively follicular type. For instance, in the ctenostome Bowerbankia gracilis follicle cells are transformed from squamous to cuboidal suggesting a higher metabolic activity . Furthermore, well-developed cisternae of RER and numerous vesicles in the follicle cells suggest synthesis and transport of nutrients to the developing oocyte. Besides, numerous coated endocytotic pits occurring at the oolemma are supposed to indicate uptake of the presumptive heterosynthesized yolk, whereas the increase of the RER with flocculent material and the occurrence of annulate lamellae in the oocyte are supposed to point to an autosynthetic mode of yolk formation. Eckelbarger , p. 199 termed this mode as “mixed yolk synthesis”.
Size increase and strong development of the synthetic apparatus are also characteristic of the follicular cells in Bugula flabellata and Bicellariella ciliata (our data). In the latter species large numbers of ergastoplasmic vesicles bearing flocculent material appear in the cytoplasm of the follicle cells. Being close to the cell membrane facing the oocyte these ergastoplasmic vesicles probably play a major role in delivering proteins that are subsequently accumulated as protein yolk platelets within the ooplasm of the developing oocyte. The onset of vitellogenesis in the oocytes is noticeable by the yolk platelets and microvilli formation in both species. Thus, obtaining the heterosynthesized yolk by pinocytosis is a common strategy to accumulate nutrients in the developing egg. In contrast, the oolemma is reported as being not microvillous in the cheilostome Celleporella hyalina. Instead, microvilli develop on the surface of the partially ovulated oocyte that is exposed to the visceral coelom of the maternal zooid.
Besides, in B. flabellata MVB-s frequently occuring in the ooplasm are considered as membrane loci for autosynthesized yolk platelets . Some autosynthetic activity seems to be present in both the oocyte and the nurse cell in Bicellariella ciliata since cisternae of the RER are scattered throughout their cytoplasm. MVB-s were also detected in ripe oocytes.
Nutrimentary provisioning of the oocyte
Similar to most of the brooding cheilostomes reviewed in [14, 24, 30] oocytes develop in doublets with a nurse cell in B. ciliata. In general, nurse cells play a trophic role as well as in determination of the oocyte polarity . As described in the cheilostomes Chartella papyracea and Bugula flabellata by Dyrynda and King  the nurse cell with its very large nucleus serves as an additional source of RNA. This RNA is supplied to the oocyte (also as ribosomes) and thus supports and, possibly, accelerates its development. Also, in B. flabellata an endoplasmatic reticulum is highly developed in the nurse cell, and cisternae of vesicular endoplasmatic reticulum added by occasional yolk granules are abundant in B. ciliata too. Similarly, in both species the nurse cells have very large nuclei with folded nuclear envelopes and nucleoli. Despite the absence of obvious free ribosomes within the cytoplasm, such an appearance points to a high level of RNA synthesis. Additionally, the nucleoplasm of the nurse cell associated with the mid-stage oocyte differs to that associated with a late-stage oocyte. A slightly higher proportion of euchromatin within the nucleoplasm of the former suggests a higher synthetic activity. Microvilli formation during oogenesis in B. ciliata possibly indicates an uptake of the nutritive material by the nurse cell. Dyrynda and King  detected limited pinocytosis by the nurse cells in both, C. papyracea and B. flabellata, although the fate of the material is unknown. In conclusion, all these data argue in favour for the suggestion that trophic and accessory functions are characteristic of the nurse cells that provide yolk, its precursors and RNA for its sibling see also .
Ovicell formation and structure
Although the pioneering research of Nitsche  studied different aspects of the sexual reproduction of Bicellariella ciliata, a number of important points were either overlooked or left without an answer. One of them is the origin of the ooecium. Despite the Nitsche’s description of the ovicell structure and formation being very precise and detailed, our study showed that the ooecium in this species is not formed by the maternal zooid as was generally assumed [30, 36, 59, 60], but see . Instead its long basal tube-like part derives from the next distal zooid, hence being confluent with the visceral coelomic cavity of the latter. Although formed from the distal zooid, the anlage of the ooecium is not positioned distally in respect to the anlage of the ooecial vesicle that is produced by the maternal zooid. This is explained by a lateral (not a proximal) orientation of the ovicell opening and the corresponding positioning of two ovicell parts. The same situation is known for the ovicells of Bugula neritina. Similar long tube-like basal parts of the ooecium were also described in Bugula pacifica by Nielsen  and a number of species from the bugulid genus Cornucopina by Harmer .
Similar to Bugula neritina the basal part of the ooecium has a transverse septum with a pore plugged by the non-specialized epithelial cells in B. ciliata. Because of this septum and the cell plug, the ooecium in B. neritina was concluded to be a heterozooid (kenozooid) [14, 23, 61–65]. In contrast, Ostrovsky and Schäfer  argued that the ooecium cannot be considered as a heterozooid in cheilostomes since there is not a special pore-cell complex in the pore connecting the coelomic cavities of the ooecium-producing zooid and the ooecium itself see also . The results of our study support this statement and show that the ooecium is not a heterozooid in B. ciliata. Instead, the ooecium is an outgrowth of the next distal zooid. The absence of a pore-cell complex does not allow considering the ooecium as a special polymorph. The same holds true for Bugula neritina.
Although simplified, Nitsche’s  scheme of the muscular system of the ooecial vesicle precisely shows a large vertical retractor and a group of thin radiating depressors between it and the distal wall of the vesicle. Also, the retractor muscle was described and illustrated by Hincks  in its contracted and retracted state in this species. In Bugula the position of the retractor is generally similar, although Vigelius  illustrated two such muscles in the ooecial vesicle of B. calathus – one vertical and one oblique. In B. neritina the retractor is vertical  and in Bicellariella ciliata it is tilted distally. The position and attachment points of the depressor muscles are variable. For instance, in B. neritina they are described to cross the cavity of the vesicle, being proximally attached to the upper and lower parts of the ooecial vesicle . In B. calathus they are arranged in a bundle being proximally attached near one of the retractors and distally in the centre of the embryophore. In Bicellariella ciliata it is a meshwork of 2–3 thin interconnected fibres running parallel on each side of the retractor muscle and insert at different sites on the hypertrophied epithelium of the ooecial vesicle.
Every brooding episode is accompanied by hypertrophy of the embryophore that collapses after larval release in cheilostome bryozoans. Beside the temporal hypertrophy of the epithelium of the ooecial vesicle that provides nutrients to the embryo, there is a considerable volume increase of the embryo during brooding. These two processes occur simultaneously.
Our ultrastructural data agree well with the results of Wollacott and Zimmer  who studied Bugula neritina, but we also obtained new data. The appearance and ultrastructure of the prospective nutritive epithelium of the ooecial vesicle changes as soon as a zygote is transferred into the brooding cavity. The thin epithelial lining first consisting of squamous cells with few mitochondria, ribosomes and cisternae of the endoplasmic reticulum, transforms into a highly active ‘tissue’ involved in nutrient transport, production and delivery. Cells become cuboidal in shape and bear various organelles such as RER, mitochondria and Golgi-bodies in high numbers. The numerous cisternae of the RER are clear evidence of a high protein synthesis. Synthesized proteins are subsequently modified by the Golgi-bodies that form numerous secretory vesicles that have been found at the apical half of the epithelial cells close to the cell membrane and especially at the areas of foldings formation in between the cells. As described by Woollacott and Zimmer , these secretory vesicles are most probably the primary transport vehicles of nutritive material within the hypertrophied epithelium towards the brood chamber. The aforementioned vesicular bodies are most probably the result of the fusion of these secretory vesicles.
Microvilli are commonly known to be a sign of active transmembrane transport. Because of the absence of pinocytotic channels, coated vesicles associated with the area of microvilli (foldings) formation and the presence of numerous larger vesicles binding to the apical cell membrane, Woollacott and Zimmer  considered them as zone of active secretion in Bugula neritina. Our data confirm this hypothesis. Foldings formed in between epithelial cells and associated organelles together with flocculent material appearing in the brooding cavity clearly indicate exocytosis in B. ciliata. The presence of few sites of endocytosis outside the zones of foldings could indicate a transport of waste products from the embryo. This means that the membrane material added to the apical cell membrane of the embryophore by exocytosis excels the amount of membrane material that is ‘retrieved’ at the same time. Multivesicular bodies (MVB) are commonly known to play a role similar to autophagy and are most likely involved in uptake and degradation of membrane material derived from endocytotic processes as well as of excessive surface membrane material . The increase in the size of MVB-s as well as their increasing occurrence inside the embryophoral cells coincides with the enlargement of the areas of foldings formation during the gestation of the embryo.
The cuticle bordering the hypertrophied epithelium adjacent to the embryo is much thinner when compared to the areas of the ooecial vesicle not facing the brooding cavity. No pores or channels were recorded which suggests that the nutritive material is passing the cuticle in a dissolved state. As taken for B. neritina by Woollacott and Zimmer  an osmotic gradient can be the driving force moving the dissolved nutritive matter across the cuticle. Dissolved nutritive matter passing the cuticle and entering the brooding cavity appears as an electron-dense flocculent material. In Bugula stolonifera ( as B. avicularia) the hypertrophied epithelium of the ooecial vesicle has been described to produce an “albuminous liquid” that nourishes the embryo . Using light microscopy, Marcus could not see the nutritive material, but the proposed “albuminous liquid” nicely correlates with “flocculent material” discovered in B. ciliata. Also Woollacott and Zimmer  report that pinocytotic channels and vesicles contain some material that is of similar electron-density with that between apical infoldings in the growing embryo of B. neritina.
In early developmental stages the flocculent material is mostly recognized in the fluid of the brooding cavity. Although loosely arranged, the fertilization envelope acts as some kind of a barrier, passing only small amounts of nutrients to the embryo (Figure 8B,C). Consequently, the early stages of embryonic development are predominantly supported by the reserves accumulated in the egg. In this time the outer membranes of the embryonic cells show low activity or differentiation. The formation of pinocytotic vesicles containing similar dense flocculent material that occurs in the brood cavity, is an evidence of the beginning of endocytosis by the embryo. It has been predominantly recorded in embryonic cells that are opposed to the embryophore. Also, the flocculent material is mostly found in the area between embryophore and embryo. Further, membranous infoldings of the embryonic cells start to develop. In contrast to B. neritina they are formed all over the embryo and are not restricted to the area adjacent to the embryophore. Therefore, an uptake of nutritive material occurs around the entire embryonic surface in B. ciliata.
There are numerous single-membrane limited bodies of various size and shape found in the embryonic cells facing the embryophore. These single membrane bodies seem to be filled with the same flocculent material and appear to be involved in processing or accumulating nutrients. The supposed canalized transport of the endocytotic vesicles towards these bodies also points to this suggestion.
The fact that the fertilization envelope gradually collapses as well as the number of infoldings on the surface of the embryonic cells and of the endocytotic vesicles increases indicates that the embryonic development shifted primarily to an external source. Despite the high endocytotic activity, numerous yolk inclusions of two types stored during oogenesis still occur in large number in later embryonic stages, being mainly accumulated in the centre of the late embryo. In general, it is assumed that these stored nutritive resources are used during larval life and metamorphosis to differentiate into the ancestrula of the colony [14, 68]. Finally, the growing embryo occupies the entire brooding cavity in close contact to the embryophore. According to the ‘definition’ of a placenta which involves always both, an embryonic and maternal part see, for instance, , a placenta-like system is established only from this time on in B. ciliata.
The funicular strands in contact with the basal parts of the epithelial cells of the embryophore most probably function as pathways for nutrient transport . They connect the hypertrophied epithelium with a feeding polypide. When the latter degenerates the nutrients can descend from two sources: (1) from the degenerated polypide of the maternal zooid that transforms into a brown body, and (2) from neighboring feeding zooids, since the maternal zooid is interconnected to them via several pore plates each .
Despite the suggested transport to provide the embryophore with large amounts of materials for active synthesis, relatively few organelles were detected in those funicular cells that are in contact with the hypertrophied epithelium in B. ciliata. Besides mitochondria, numerous vesicles or vesicular reticulum were sometimes seen within some cells of the funicular cords. Thus, their role in nutrient transfer remains obscure.
In Bugula neritina, the funicular plexus that is in contact with the basal surface area of the hypertrophied epithelium enlarges during hypertrophy of the latter, therefore suggesting a more intense potential nutrient transport. Furthermore, funicular processes deeply penetrate in between the cells of the embryophore, and vesicular structures and mitochondria seem more numerous in the funicular cells . An increase of the funicular plexus in contact with the basal surface of the hypertrophied epithelium has also been detected in B. ciliata. However, it is most probably not the result of cell enlargement or proliferation. Instead, we suggest that embryonic growth and deformation of the ooecial vesicle are the source of the increased contact between epithelial and funicular cells in the narrowed cavity of the oocial vesicle. Besides, the funicular processes occuring in B. neritina are not present in B. ciliata.
Because of the formation of microlecithal oocytes and the great nutritional activity of the embryophore resulting in 500-fold increase of the embryo in B. neritina, its funicular plexus seems to require a stronger transport activity in comparison to that of B. ciliata (10-fold embryonic increase). Therefore, the larger degree of development and interconnection of the funicular and epithelial cells most probably reflects an increased demand for nutrients during embryogenesis in the former species. In B. ciliata the strong incongruence between the presumably high activity of the funicular cells and low number of the organelles in many cells remains enigmatic.
Matrotrophy is characteristic for three of five reproductive patterns that are known in cheilostomes [14, 21, 24]. A few species with pattern I produce numerous small microlecithal oocytes that after near/post-ovulatory coelomic fertilization are freely spawned into the water and further develop into long-living planktotrophic larvae. Four other patterns are characterized by an early intraovarian fertilization and development of short-living non-feeding larva. The most common is pattern II which involves the production of a few large macrolecithal oocytes followed by embryonic incubation either in external membranous sacs, skeletal chambers (ovicells) or in internal brooding sacs. In pattern III, the production of a few small micro- or mesolecithal eggs is followed by matrotrophic brooding. In contrast, in the pattern IV, extraembryonic nutrition (EEN) during incubation is associated with macrolecithal oogenesis. Pattern V is known only in the cheilostome family Epistomiidae and combines EEN with intracoelomic incubation (viviparity). Consequently patterns III, IV and V can be referred to as matrotrophic patterns.
Bicellariella ciliata has been known as a matrotrophic [23, 44]. Our data show that this species sexually reproduces in a way similar to the pattern III. It forms small eggs and employs matrotrophy via a placenta (placentotrophy). The most obvious difference is that its oocytes are of macrolecithal type. When defining pattern IV, Ostrovsky with co-authors  described it as having macrolecithal oogenesis and placental embryonic incubation. This combination of characters (of the patterns II and III) was inferred as an incipient matrotrophy illustrating an evolutionary step from non-matrotrophic to matrotrophic mode of parental provisioning [2, 70, 71].
Example of the pattern IV is Scrupocellaria ferox having large macrolecithal oocytes (about 140.0 μm, calculated from the data on its volume), embryophore and embryos that more than double in volume during brooding . Despite the mature oocyte also being macrolecithal in Bicellariella ciliata, it is not large as in S. ferox. Additionally, its diameter is considerably smaller than the incubating space of the ovicell (63.0 μm in oocyte vs. 150.0 μm in ovicell). Thus, it is comparable with the size of oligolecithal eggs in a number of Bugula species with pattern III, for instance, about 50.0 μm in B. calathus, and 36.0 μm in B. neritina. Subsequently, the embryonic increase is rather prominent (10-fold), and the same situation is characteristic of B. flabellata that has rather small (77.0 μm) macrolecithal eggs and 7.1-fold increase of embryo in volume . Thus, in comparison with the matrotrophic species possessing large macrolecithal oocytes B. flabellata and B. ciliata represent an example of the next evolutionary step from incipient to substantial matrotrophy, and the major attributes of this transition became a shift from large (comparable to a brooding cavity in volume) to small macrolecithal eggs supposedly accompanied by an increased level of EEN.