Induction of larval settlement by KCI in three species of Bugula (Bryozoa)

Elevation of the K+ concentration in seawater (added as KC1) induces larval settlement and metamorphosis in Bugula simplex, B. stolonifera, and B. turrita. All three of these bryozoan species have similar bell-shaped dose-response curves: 5 mM excess K+ is sufficient to increase settlement and metamorphosis significantly over seawater controls in all the species and optimal responses to excess K concentrations occur ations at 10-25 mM, 5-10 mM, and 10-15 mM for B. simplex, B. stolonifera, and B. turrita, respectively. Percent settlement in all three species is decreased at levels greater than 25 mniM excess KC1. Similar bell-shaped curves have been observed for KCl-induced settlement of larvae from other, but not all, invertebrate groups. Larvae of B. turrita do not settle at a significantly higher level than seawater controls when exposed to excess K+ concentrations >25 mM even in the presence of a second favorable artificial inducer, filmed chicken eggshell membrane, indicating that K+ at high concentrations interferes with the normal settlement process. Furthermore, pulse exposures for 5 min, 15 min, and 30 min to 10 mM excess K+ are ineffective at inducing settlement over levels in seawater controls. When larvae of B. turrita were induced to settle by 10 mM excess K+, completion of metamorphosis was not significantly different from that of controls lacking excess KC1 (86% and 91%, respectively). However, if larvae induced by 10 mM excess K+ are not removed from the 10 mM excess K+ after settlement, the success ratio of metamorphosis is significantly lower than in controls (72% and 91%, respectively). Our results confirm previous studies demonstrating that settlement of bryozoan larvae can be induced by excess K+. Bryozoan larvae examined to date become responsive around 5-10 mM excess K+. These data suggest that shared underlying features exist in the mechanism triggering settlement and metamorphosis in bryozoans and that excess K+ is a useful experimental tool for inducing settlement of bryozoan larvae. Additional key words: metamorphosis, Cheilostomata, Bugula simplex, B. stolonifera, B. turrita Many benthic marine invertebrates possess a larval stage in their life cycle. The larval stage is concluded with metamorphosis to the juvenile stage, a process often mediated by cues from the environment (Crisp 1974; Scheltema 1974; Chia & Rice 1978; Chia 1989). The isolation and characterization of such cues triggering this transformation have motivated much research (for a review see Pawlik 1992; Gibson & Chia 1994). However, with few exceptions, the identification of natural inducers has remained elusive. As a result of this difficulty, researchers have used various artificial means for triggering settlement and/or metamorphosis in order to gain insight into these processes. Organic compounds are one such category of inducers (e.g., Morse et al. 1979; Baloun & Morse 1984; Pawlik 1986, 1990; Rittschof et al. 1986; Yool et al. 1986; Pennington & Hadfield 1989; Tegtmeyer & Rittschof 1989; Zimmer-Faust & Tamburi 1994). In addition to organic compounds, certain inorganic cations such as K+ are effective in initiating settlement and/or metamorphosis of many invertebrates (Pawlik 1992; Pechenik & Gee 1993; Pearce & Scheibling 1994). K+, however, is not a universal trigger, as elevated K+ apparently does not affect settlement and metamorphosis in anthozoans (Morse et al. 1988), some bivalves (Eyster & Pechenik 1987; Gustafson et al. 1991), and ascidians (Grave & Nicoll 1939). Indeed, elevated levels of K+ apparently inhibit settlement of barnacle cyprids (Rittschof et al. 1986) and some ascidian tadpoles (Lynch 1961). Present data do reveal an overall phylogenetic pattern to the KC1 response. For example, in molluscs, although all gastropod larvae examined metamorphose in response to elevated K+ levels, larvae of most bivalves do not (an exception is Saccostrea commercialis [Nell & Holiday 1986]). Similarly, in cnidarians, settlement can be triggered by K+ in hydrozoans, but not in anthozoans. The goal of the present study is to assess the efficacy of K+ as an artificial inducer of settlement and metamorphosis in the cheilostomes Bugula simplex, B. stolonifera, and B. turrita. Effective inducers of settlement are important agents in studies investigating larval energetics, metamorphic competence, and effects of larval swimming duration on subsequent juvenile success, as these types of studies depend on the ability to induce metamorphosis reliably. Excess potassium ions potentially offer one such experimental tool. Settlement of bryozoan larvae is tightly coupled to metamorphosis in that settlement is irreversible and is marked by eversion of the metasomal (internal) sac, the first morphogenetic movement of metamorphosis in larvae of Bugula spp. and other bryozoans (Zimmer & Woollacott 1977). Thus, settlement in bryozoans is not exclusively a behavioral change associated with substratum exploration; instead, it is a clearly defined process whereby larvae permanently attach to the substratum. We considered a larva settled only after attachment to the substratum via eversion of its metasomal sac. Eiben (1976) observed that KCI induced larval settlement in the ctenostome Bowerbankia gracilis, as did Lynch (1947) with the cheilostome Bugula neritina, and Stricker (1989) with the cheilostome Membranipora membranacea. In contrast, Lynch (1949) reported that larvae of B. simplex (as B. flabellata RYLAND 1958) from Woods Hole, Massachusetts), were inhibited by concentrations of excess K+ greater than 100 mM. In the present study, we: (1) re-examine, at lower concentrations, the effectiveness of excess K+ in triggering settlement of larvae of B. simplex from Woods Hole; (2) extend these investigations to larvae of its sympatric congeners B. stolonifera and B. turrita; (3) assess the effect of elevated concentrations of K+ on inhibiting settlement in the presence of a second artificial inducer; (4) examine the effectiveness of pulse vs. continuous exposure of larvae of B. turrita to excess K+; and (5) determine the effect of exposure to excess K+ on the ability of individuals to complete metamorphosis.