Robert M. Woollacott

Effects of duration of larval swimming period on early colony development in Bugula stolonifera (Bryozoa: Cheilostomata)

Laboratory experiments with larvae of the cheilostome bryozoan Bugula stolonifera Ryland, 1960 assessed the time to settlement in the presence of a constantly available polystryrene substrate, the development of competence for metamorphosis, and the effects of the duration of swimming period on early colony development. Sexually mature colonies of B. stolonifera were collected on 11 and 18 September 1987; 2 and 18 August, 1988; and 6, 12, 19, and 26 September 1988, from Eel Pond (Woods Hole, Massachusetts, USA) and were maintained at 20°C. In the presence of a constantly available substrate, cumulative percent settlement curves were sigmoid, with 75% of larvae settled in 3.2±0.5 h. Typically, 50% of the larvae settled in less than 3 h and 95% settled in 6.1±1.2 h. The number of settled individuals that developed feeding ancestrulae by 3 d and the number that developed first-feeding autozooids by 6 d was assessed as a function of duration of larval swimming. Individuals which were kept swimming for 8 and 10 h after hatching developed significantly more slowly to the ancestrula and autozooid stages in 13 out of 14 experiments than did larvae that swam 2 or 6 h. This is the first report for any bryozoan that prolongation of the larval free-swimming period affects the rate of colony development.

Effect of microbial films on settlement of bryozoan larvae (Bugula simplex, B. stolonifera and B. turrita)

The influence of microbial films on larval settlement was studied for sympatric species of cellularioid cheilostome bryozoans (Bugula simplex, B. stolonifera, and B. turrita). Settlement experiments were conducted in the laboratory in 1981 with and without a choice of filmed or unfilmed substrates. When given a choice of substrates, the larvae of all three bryozoan species significantly preferred the filmed substrate. When not given a choice of substrates, both B. simplex and B. turrita larvae tended not to settle on the unfilmed substrate, while the filmed and unfilmed substrates were equally attractive to B. stolonifera. Microbial films enhanced the settlement of all three species.

A simplified placenta‐like system for the transport of extraembryonic nutrients during embryogenesis of Bugula neritina (bryozoa)

Embryos of the marine cheilo‐ctenostome bryozoan Bugula neritina undergo a marked increase in volume (about 500‐fold) during embryogenesis while being retained in a brood chamber. Previous morphological studies indicate that shortly after transfer of the zygote to the brood chamber, the epithelium of the maternally‐derived portion of the brood chamber, the ooecial vesicle, differentiates in regions adjacent to the embryonary space from a squamous to a columnar form suggesting that the parent is involved as a source of extraembryonic nutrients required for the extensive growth of the embryo.

Association of bacteria with bryozoan larvae

Seven species of marine bryozoans were examined for the presence of bacteria associated with the larvae. In three species (Bugula neritina, B. pacifica, and B. simplex), rod-shaped bacteria were consistently observed in the pallial sinus of the larvae, independent of geographic location, year, or season. Larvae of closely related bryozoans (B. stolonifera, B. turrita, Scrupocellaria bertholetti, and Tricellaria occidentalis) occurring sympatrically with those containing bacteria lacked this apparent symbiosis. Those bryozoans examined that possessed the bacteria-larva association are among the most frequently encountered fouling bryozoans.

Structure and swimming behavior of the larva of Haliclona tubifera (Porifera: Demospongiae)

Early developmental stages of Haliclona tubifera are retained by the parent and larvae are released on illumination of dark‐adapted colonies. Larvae are parenchymellae without provision for feeding on particulate matter and without identifiable sensory and neuronal elements. Larvae, however, are strongly photonegative on release and remain so throughout the duration of larval existence. The anterior and lateral epidermal fields are composed predominantly of elongate flagellate cells which form a pseudostratified columnar epithelium. Interspersed among elongate cells are globular flagellate cells which, based on ultrastructural criteria, are thought to be secretory in function. The posterior surface is not flagellated and is composed of a cuboidal epithelium. A 5–7 cells wide ring of epidermal cells with flagella 50 μm in length forms a prominent flagellar band separating the posterior from lateral fields. Measurements of swimming speeds in the horizontal plane indicate that H. tubifera larvae swim at speeds intermediate between Halichondria melanadocia, which have a posterior tuft of 25 μm flagella and Haliclona sp, with a posterior ring 75 μm flagella. Morphometric analysis of larval length, larval diameter, and flagellar band diameter shows significant correlations in all pair‐wise tests. Band diameter varies less than larval length or width suggesting that it is a conserved trait. A negative allometry exists between band diameter and larval length. As larval length increases elongate flagellar cells of the lateral and anterior surfaces become relatively more important for locomotion. A prominent cup‐like sheath of subepidermal cells separates the epidermis from the central region of the larval interior. These cells are not associated with collagen as are cells similar in appearance and location in several other haplosclerid larvae. Their function remains unknown. Spicules occur in the posterior region. As in the adult, these spicules are hastate oxeas, but they are significantly different in length, width, and length/width from those of the adult. Their function is unknown, but several obvious potential functions in both larval and postlarval life exist and await further study. Archeocytes are the dominant cell type of the central region. Bacteria are found inter‐ and intracellularly. Based on morphological data, they appear to be of a single type, pleomorphic rods without thick walls, but further study utilizing isolates in culture is needed to establish their diversity and identity. Intracellular bacteria are associated with a cell type provisionally designated a bacteriocyte. Three stages of bacteriocytes are described based on anatomical criteria, presence/absence of bacteria, and what appear to be stages in degradation of bacteria. The significance of this association and of the bacteriocyte cell type remain to be explored.

Spermatozoa of Ciona intestinalis and analysis of ascidian fertilization

The complex array of vestments which surrounds the ascidian oocyte indicates that spermatozoa of these urochordates may be highly modified to effect penetration through the egg‐envelopes and to achieve successful fusion with the female gamete. Examination of the spermatozoa of Ciona intestinalis reveals, however, that they lack an acrosomal vesicle and do not possess detectable amounts of actin. Furthermore, the spermatozoa do not undergo observable morphological alterations when exposed to ammoniated seawater, a treatment known to produce aggregation and an acrosome reaction in other organisms. Ciona spermatozoa do contain significant quantities of proteases. It is hypothesized that the localization of these enzymes may be related to the ridge‐like surface ornamentation which is observed as a component of the head region of Ciona spermatozoa.

Serotonin and Dopamine Have Opposite Effects on Phototaxis in Larvae of the Bryozoan Bugula neritina

Adult colonies of the bryozoan Bugula neritina release short-term anenteric larvae that initially are strongly photopositive. Over the course of several hours larvae lose their initial photopositivity and either become photonegative or alternate between positive and negative phototaxis. We report that newly released photopositive larvae rapidly become photonegative upon exposure to 10-6-10-5 M serotonin or its metabolic precursor, 5-hydroxytryptophan. This behavior was not observed in two congeners of B. neritina, nor in larvae of three other species of bryozoans and seven species from four additional phyla. Antibodies to serotonin label cells in the region of the equatorial nerve-muscle ring and in two tracts extending from the apical disc to this ring. In a separate series of experiments, larvae treated with dopamine (10-7-10-5 M) significantly prolonged their photopositive period. This effect was also obtained with the D2 dopamine receptor agonist, quinpirole (10-6-10-5 M). HPLC analysis determined that newly released photopositive larvae contained 0.120 pmol dopamine/ {mu}g protein. These findings implicate serotonin and dopamine as important neurochemical regulators of phototaxis in larvae of B. neritina.

Flagellar basal apparatus and its utility in phylogenetic analyses of the porifera

A broadly based comparative study was initiated to assess components of the flagellar basal apparatus as a character set in phylogenetic analyses of poriferans. The flagellated (monociliated) epidermal cells of sponge larvae were selected for study. Taken together, they create a field of locomotory cells analogous to a multiciliated surface. Larvae of six species in four orders of the Demospongiae were examined by transmission electron microscopy. Results are compared with findings taken from the literature on larvae of five additional species of demosponges and four species of calcareans. Data were assembled on six components of the basal apparatus: (1) basal body, (2) basal foot, (3) accessory centriole, (4) transverse cytoskeletal system, (5) longitudinal cytoskeletal system, and (6) association with Golgi body. Where evidence permits assessment, all have Type II basal bodies. Basal feet are diverse and are subdivided into three categories based on structural complexity. The most anatomically intricate (Type III) is found only in larvae of Mycale spp. Accessory centrioles are present or absent depending on the species, but their occurrence is without overall taxonomic pattern. When present, accessory centrioles are oriented perpendicularly to the long axis of the basal body, but as ascertained from relationship to the anterior‐posterior axis of the larvae they are without consistent orientation with regard to the plane of effective beat of the flagellum. Transverse and longitudinal cytoskeletal systems are also diverse among larvae. The existence of cross‐striated rootlets is convincingly established only in larvae of calcareans, and such rootlets are present in larvae of all four calcareans studied to date. Three apparently new rootlet structures are described: lateral arms of the transverse cytoskeletal system from larvae of Aplysilla sp. and Haliclona tubifera; laminar sheets of the longitudinal system from larvae of Aplysilla sp. and M. cecilia; and paraxial rootlet in larvae of H. tubifera. A robust similarity in structure of the basal appartus is observed among the three species of halichondrids reported here for the first time. In comparison with the flagellar basal apparatus found in adults, those of larvae are more complex and more diverse. Review of studies on adult sponges that include information on the basal apparatus reveals the absence of a longitudinal rootlet system in all cases. Additionally, there exists a high degree of concordance between properties of the basal apparatus in the one sclerosponge and the one hexactinellid studied to date. These basal apparatus are also the simplest in construction of those found in sponges. Conversely, the basal apparatus of demosponges are varied. Although consistent presentation of the basal apparatus is evident in certain taxa, any discernable systematic pattern in their overall configuration remains obscure. Finally, we conclude that the flagellar basal apparatus of sponges is more similar to that found in choanoflagellates than it is to that observed in eumetazoans.

Induction of Metamorphosis in Larvae of a Sponge

Elevated exogenous KC1 and CsCl are effective artificial metamorphic triggers for many, but not all, invertebrate larvae. Neither taxonomic nor functional patterns in their effectiveness, however, are apparent. In our experiments, elevated exogenous KC1 promoted metamorphosis of larvae of the demosponge Aplysilla sp., but only when applied simultaneously with a biofilmed artificial substratum. Although the presence of this artificial substratum alone significantly enhanced settlement over seawater controls, its effectiveness was further augmented significantly in the presence of elevated KC1, indicating a synergistic interaction. Exposure of larvae to a 2-min pulse of 30 mM KC1 promoted metamorphosis as effectively as continuous exposure to 30 mM KC1. Moreover, 30 mM CsCl proved an effective trigger of metamorphosis both in the absence and presence of a biofilmed substratum. As with KC1, the CsCl data indicate the presence of a synergistic interaction between CsCl and the biofilmed substratum. This report provides preliminary insights into the induction of settlement and metamorphosis of sponges, metazoans which lack established sensory and neuronal elements, and it permits comparison with responses of eumetazoans. Additional key words: Porifera, Aplysilla, KC1, CsCl, settlement Sponges represent a grade in metazoan evolution for which the presence of neuronal cells and differentiated receptor cells is unconfirmed (Harrison & De Vos 1991). Additionally, sponge larvae are considered generally nonspecific in substratum preferences at settlement and metamorphosis (Bergquist 1978). The receptor site(s) and the internal pathway(s) of the metamorphic response in sponges remain unexplored. Elevated concentrations of exogenous K+ (as KC1) are effective in inducing larval settlement and metamorphosis in species of a wide range of marine invertebrates including a hydrozoan (Spindler & Muller 1972; Muller & Buchal 1973), a polychaete (Yool et al. 1986), at least 15 gastropods (Baloun & Morse 1984; Yool et al. 1986; Pechenik & Heyman 1987; Hubbard 1988; Davis et al. 1990; Todd et al. 1991; Inestrosa et al. 1993; Pechenik & Gee 1993; Campos et al. 1994; Gibson & Chia 1994; Yang & Wu 1995), a bivalve (Nell & Holliday 1986), a phoronid (Herrmann 1979), a brachiopod (Freeman 1993), 5 bryozoans (Eiben 1976; Stricker 1989; Wendt & Woollacott 1995), and 3 echinoderms (Cameron et al. 1989; Pearce & Scheibling 1994). Evidence indicates, however, that supplemental K+ is not a global inducer as aTo whom correspondence should be addressed. it is ineffective in stimulating settlement and metamorphosis in species of certain other marine invertebrates (at least at the concentrations tested) including 2 corals (Morse et al. 1988), 2 bivalves (Eyster & Pechenik 1987; Gustafson et al. 1991), 2 echinoderms (Rowley 1989; Johnson et al. 1991), and 2 ascidians (Grave & Nicoll 1940; Lynch 1961). In the barnacle Balanus amphitrite, supplemental K+ even inhibits settlement (Rittschof et al. 1986). Inspection of these data reveals little in the way of phylogenetic patterns in larval responses to K+. Only in 3 taxa (echinoderms, gastropods, and bryozoans) are responses of more than 3 species established. In gastropods and bryozoans, excess K+ was uniformly effective as an inducer of settlement and metamorphosis. In contrast, data on echinoderms (and smaller data sets on cnidarians and bivalves) indicate that larvae of some species within each group are affected by elevated K+ whereas others are not. Responses of larvae within deuterostome and protostome assemblages are also mixed. Finally, where response to elevated K+ varies within a clade, it does not correlate with overall differences in known features of developmental and larval biology. At present, an encompassing synthesis of K+ induction remains elusive from phylogenetic perspectives. To expand on this taxonomic baseline This content downloaded from 157.55.39.186 on Sun, 09 Oct 2016 06:18:30 UTC All use subject to http://about.jstor.org/terms Woollacott & Hadfield and to address the induction pathway in sponges, we examined responses of larvae of the demosponge Aplysilla sp. to elevated exogenous K+ and Cs+. Aplysilla sp. retains its early developmental stages and releases short-lived parenchymella larvae that are anenteric and, presumably, incapable of feeding on particulate matter (Woollacott & Hadfield 1989 [as Dendrilla cactus]; Woollacott & Pinto 1995). Most of the larval surface is comprised of a uniform field of flagellated (monociliated) cells. The anterior end (that facing forward during swimming) is cap-like and formed by larger flagellated cells. The posterior pole bears a tuft of long flagella. On release, larvae are initially strongly positively phototactic, but the strength of this response gradually diminishes during the swimming period.

Mechanisms of rapid morphogenetic movements in the metamorphosis of the bryozoan Bugula neritina (cheilostomata, cellularioidea). I. Attachment to the substratum

The larval morphology, settlement behavior, and the rapid morphogenetic movements that occur during the first 60 sec of metamorphosis of the cellularioid cheilostome bryozoan Bugula neritina have been examined and analyzed by light and electron microscopy. The larva attaches to the substratum at the onset of metamorphosis by the eversion of the internal sac. At the same time, the coronal cilia reverse their direction of beat, spreading an adhesive secreted by the neck region of the everting sac over the metamorphosing larva. During attachment, the larva goes through several configurations that coincide with the sequential contraction and relaxation of certain larval muscles. Histological and ultrastructural evidence indicates that the neck and wall regions of the internal sac are everted by the contraction of the muscles in the equatorial plane of the larva at the same time that the roof region in pulled toward the larval equator by the contraction of the axial muscles. The subsequent relaxation of the axial muscles allows the roof region to be everted by the antagonistic force generated by the sustained contraction of the equatorial musculature. After the roof region attaches to the substratum, the apical disc is temporarily retracted by a second contraction of the axial muscles. The apical disc subsequently reextends as the axial muscles relax just before coronal involution. A comparison of the ontogenetic sequence of rapid morphogenetic movements in the metamorphoses of cheilostome and ctenostome bryozoans indicates that cellularioid cheilostomes have undergone peramorphosis in the aspect of development.