Induction of larval settlement and metamorphosis of the sea urchin Strongylocentrotus intermedius by glycoglycerolipids from the green alga Ulvella lens

In aquaculture centers of the northern region of Japan, "Nami-ita" (waved polycarbonate plates), on which the green alga Ulvella lens Crouan frat. (Chaetophoraceae: Chaetophorales) was cultured, are used to promote larval settlement and metamorphosis of the sea urchin species Strongylocentrotus intermedius (A. Agassiz) and S. nudus (A. Agassiz). We investigated chemical inducer(s) for larval settlement and metamorphosis of these sea urchins with extracts of U. lens. Bioassay-guided separation of the methanol extract using a combination of column and thin-layer chromatography led to the isolation of several active compounds, the chemical structures of which were determined by spectral and chemical methods. These active compounds were identified as glycoglycerolipids, all comprising several molecular species: sulfoquinovosyl monoacylglycerols (SQMGs), sulfoquinovosyl diacylglycerols (SQDGs), monogalactosyl monoacylglycerols (MGMGs), monogalactosyl diacylglycerols (MGDGs), digalactosyl monoacylglycerols (DGMGs) and digalactosyl diacylglycerols (DGDGs). Among these glycolipids, SQMGs, MGMGs, MGDGs and DGMGs induced larval metamorphosis of the sea urchin S. intermedius. SQMGs and MGDGs induced larval metamorphosis at a concentration of 5 µg ml^-1, whereas SQDGs and DGDGs only induced larval settlement. These glycoglycerolipids are new congeners of chemical inducers to settlement and metamorphosis of planktonic larvae of sea urchins. The findings would provide a better understanding of larval settlement and metamorphosis in sea urchins.     

Dissolved histamine: a potential habitat marker promoting settlement and metamorphosis in sea urchin larvae

Many species of marine invertebrate larvae settle and metamorphose in response to chemicals produced by organisms associated with the adult habitat, and histamine is a cue for larvae of the sea urchin Holopneustes purpurascens. This study investigated the effect of histamine on larval metamorphosis of six sea urchin species. Histamine induced metamorphosis in larvae of three lecithotrophic species (H. purpurascens, Holopneustes inflatus and Heliocidaris erythrogramma) and in one planktotrophic species (Centrostephanus rodgersii). Direct comparisons of metamorphic rates of lecithotrophic and planktotrophic larvae in assays cannot be made due to different proportions of larvae being competent. Histamine (10 μM) induced metamorphosis in 95% of larvae of H. purpurascens and H. inflatus after 1 h, while the coralline alga Amphiroa anceps induced metamorphosis in 40–50% of these larvae. Histamine (10 μM) and A. anceps induced 40 and 80% metamorphosis, respectively, in the larvae of H. erythrogramma after 24 h. Histamine (10 μM) and the coralline alga Corallina sp. induced 30 and 70% metamorphosis, respectively, in the larvae of C. rodgersii after 24 h. No metamorphosis of any larval species occurred in seawater controls. Larvae of two planktotrophic species (Tripneustes gratilla and Heliocidaris tuberculata) did not metamorphose in response to histamine. Seagrasses, the host plants of H. inflatus, induced rapid metamorphosis in larvae of the two Holopneustes species, and several algae induced metamorphosis in C. rodgersii larvae. Histamine leaching from algae and seagrasses may act as a habitat marker and metamorphic cue for larvae of several ecologically important sea urchin species.     

Effects of temperature and salinity acclimation of adults on larval survival,physiology, and early development of Lytechinus variegatus (Echinodermata: Echinoidea)

Larval survival and developmental rates of Lytechinus variegatus (Lamarck) were determined as a function of temperature and salinity in two experiments by: (1) directly transferring fertilized eggs to 35, 30, 27.5, 25, 20, 15, and 10S seawater at 18 and 23°C, and (2) acclimation of adult sea urchins to the conditions described above for 1 to 4 wk prior to spawning. Developmental rates and percent survival of larvae prior to metamorphosis decreased at salinities below 35 (Q₁₀ values for metamorphosis=0.380 to 0.384). Temperature and salinity significantly (P<0.05) affected metabolic rates of L. variegatus plutei. These results show that L. variegatus larvae are stenohaline when compared to larvae of other echinoderm species. LC₅₀ values (S), developmental rates, and survival to metamorphosis indicate that acclimation of adult sea urchins to lower salinity prior to spawing and fertilization does not enhance development or survival of embryos exposed to low salinity.     

Larval metamorphosis of the sea urchins,<Emphasis Type="Italic"> Pseudocentrotus depressus</Emphasis> and<Emphasis Type="Italic"> Anthocidaris crassispina</Emphasis> in response to microbial films

In Japan, mass-production of sea urchin juveniles involves the culture of periphytic diatom films on plastic plates in 5- to 15-tonne tanks for the induction of larval metamorphosis. This study focused on the larval response of sea urchins, Pseudocentrotus depressus and Anthocidaris crassispina, to natural microbial films in the sea and diatom-based films formed in the tanks. The effect of diatoms and bacteria on larval metamorphosis was also examined using laboratory-cultured diatom-based films in the presence of germanium dioxide and antibiotics during film culture. Furthermore, the nature of the cue of the cultured diatom-based film was also investigated. Results showed that P. depressus and A. crassispina metamorphosed both on natural microbial films and diatom-based films in a tank. In the sea, the metamorphosis (%) of P. depressus increased gradually in accordance with the immersion period of film formed on glass slides, whereas the larval metamorphosis of A. crassispina had a bell-shaped response curve. In the tank, although the diatom-based films showed a low inducing activity for larval metamorphosis of A. crassispina, the metamorphosis of P. depressus larvae increased linearly in accordance with the diatom density. These results suggest that diatom-based films could promote the larval metamorphosis of P. depressus, but are less important in A. crassispina. In a simultaneous larval assay (May), P. depressus showed a higher percentage of metamorphosis than A. crassispina. We concluded that the former is more sensitive to diatom-based film than the latter and that this is due to differences in their natural habitats. For laboratory-cultured diatom-based film, both species of sea urchins showed a similar response, in which reduction in diatom and bacteria density resulted in a decrease in the original inducing activity. There seems to be a synergistic effect between diatom and bacteria in inducing larval metamorphosis. Films subjected to treatment with 0.1 N HCl were no longer inductive for either sea urchin, while those films treated with 40^°C heat or EtOH (5% and 10% EtOH) showed a significant reduction in the inducing activity. Thus the surface-associated cues may be highly susceptible to the above treatments.Communicated by T. Ikeda, Hakodate     

Larval settlement and metamorphosis of the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> in response to biofilms

Biofilms were allowed to develop on glass slips immersed 1.0–1.5 m below the sea surface in Tachibana Bay, Nagasaki, Japan, for different periods of time from November 2003 to January 2005. The effects of age, immersion month, dry weight, bacterial and diatom densities of these biofilms on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Furthermore, biofilms were subjected to various treatments to investigate the nature of the settlement and metamorphosis cue in the biofilm. Pediveliger larvae of the mussel settled and metamorphosed in response to biofilms. Settlement and metamorphosis to the post-larval stage significantly increased with the biofilm age. In addition, the biofilm activity varied depending on the immersion month (season), e.g., for biofilms with the same age, those immersed between June and August had higher activities than those immersed between November and March. The activity of the biofilm also positively correlated with the dry weight, bacterial and diatom densities. These three quantitative parameters of the biofilm were significantly affected by the film age but were not affected by the immersion month, suggesting that other parameters (e.g., community structures, extracellular products) also affected the inductive activity of the biofilm. The fixative agents (formalin and glutaraldehyde), heat, ethanol, ultraviolet irradiation and antibiotics treatments of the biofilm resulted in significant reduction or loss of its inductive activity. The survival of bacterial cells in the treated films where activities were either reduced or lost also decreased significantly. No settlement and metamorphosis were obtained when larvae were exposed to the conditioned water of the biofilm. Thus, larvae of M. galloprovincialis settled and metamorphosed in response to a cue produced by living bacteria in the biofilm. The cue may be a bacterial extracellular product which was susceptible to the above treatments.     

Larval metamorphosis of the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> in response to <Emphasis Type="Italic">Alteromonas</Emphasis> sp. 1: evidence for two chemical cues?

Bacterial isolates from multi-species biofilms were identified by 16S rDNA gene sequences and investigated for their inductive effects as monospecific biofilms on larval metamorphosis of Mytilus galloprovincialis. Alteromonas sp. 1 biofilm was found to have inductive activity, which increased with increasing cell density. The cue(s) of Alteromonas sp. 1 biofilm responsible for inducing larval metamorphosis was further investigated. Treatment of the biofilm with formalin, ethanol, heat or ultraviolet irradiation resulted in a significant reduction in the inductive activity of Alteromonas sp. 1, and the crude extract of surface-bound products of the biofilm showed no activity. These results indicated that if the cue was a surface-bound chemical cue, it was unstable, or susceptible to the treatments or the extraction process. On the other hand, the inductive activity of treated biofilms had a linear regression to the cell survival of bacteria, indicating a metabolically active biofilm was a requirement for larval metamorphosis. Conditioned water of the biofilm did not induce larvae to metamorphose. However, larval crawling behavior in the conditioned water was the same as that in the biofilm prior to larval metamorphosis, and significantly different to larval behavior in seawater. This indicated that a potential or partial waterborne cue existed, but remained inactive when alone. A synergistic effect of the conditioned water with formalin-fixed Alteromonas sp. 1 biofilm resulted in a significant increase in larval metamorphosis. Heat treatment and fractionation of the conditioned water demonstrated that the waterborne cue was heat-stable and <3,000 Da in molecular weight. Platinum-coating, Lentil Lectin and Wheat Germ Agglutinin treatments of the formalin-fixed biofilm significantly reduced its synergistic effect with the conditioned water, suggesting that a surface-bound cue was present on the biofilm and that the cue might be associated with the bacterial exopolysaccharide or glycoprotein. Evidence presented here suggests that two chemical cues derived from bacteria act synergistically on larval metamorphosis of Mytilus galloprovincialis.     

Growth and metamorphosis of Aplysia oculifera larvae in laboratory culture

Eggs of Aplysia oculifera (Adams and Reeve, 1850) were incubated in the laboratory. They hatched 8 to 9 d after spawning. Shell length (SL) of the hatched larvae was 102±2 m. Larvae were fed on the unicellular algae Isochrysis galbana in a concentration of 10⁴ cell ml^-1, and after 45 to 60 d grew to a maximum SL of 385±11 m. Larvae survived up to 330 d. A total of 12 species of algae from the natural habitat of A. oculifera were examined as metamorphosis inducers. Red algae Dasia sp., Jania sp., Hypnea sp. and Liagora sp. induced metamorphosis in 66.7±21.2, 28.3±17.7, 26.0±18.5 and 4.0±8.0% of the larvae, respectively. Green algae Enteromorpha intestinalis and Ulva sp. induced metamorphosis in 37.0±11.0 and 9.0±10.4% of the larvae, respectively. Cladophora sp. and Codium dichotomum, and the brown algae Padina pavonia, Colpomenia sinuosa, Hydroclathrus clathratus and Cystoseira sp. did not induce metamorphosis. There was no significant difference in the rate of metamorphosis between young (2 to 4 mo) and old (6 to 8 mo) larvae. Postmetamorphic juveniles grew and developed only when fed with E. intestinalis. They grew to a body length of>8 mm in 50 d. Postmetamorphic juveniles did not survive on other algae. The longevity of the planktonic A. oculifera larvae supports the hypothesis that the larvae can exist in the plankton and survive for several months until the next recruitment. The advantage of non-specificity in metamorphosis induction is discussed.     

Correlating gene expression with larval competence,and the effect of age and parentage on metamorphosis in the tropical abalone<Emphasis Type="Italic"> Haliotis asinina</Emphasis>

The non-geniculate crustose coralline alga (CCA) Mastophora pacifica can induce the metamorphosis of competent Haliotis asinina (Vetigastropoda) larvae. The ability to respond to this natural cue varies considerably with larval age, with a higher proportion of older larvae (e.g. 90 h) able to metamorphose in response to M. pacifica than younger larvae (e.g. 66 h). Here we document the variation in time to acquisition of competence within a larval age class. For example, after 18 h of exposure to M. pacifica, approximately 15 and 36% of 84 and 90-h-old H. asinina larvae had initiated metamorphosis, respectively. This age-dependent response to M. pacifica is also observed when different aged larvae are exposed to CCA for varying periods. A higher proportion of older larvae require shorter periods of exposure to CCA than younger larvae in order to initiate metamorphosis. In this experiment, as in the previous, a small proportion of young larvae were able to respond to brief periods of CCA exposure, suggesting that they had developed the same state of competency as the majority of their older counterparts. Comparisons of the proportions of larvae undergoing metamorphosis between families reveals that parentage also has a significant (P<0.05) affect on whether an individual will initiate metamorphosis at a given age. These familial differences are more pronounced when younger, largely pre-competent larvae (i.e. 66 h old) are exposed to M. pacifica, with proportions of larvae undergoing metamorphosis differing by as much as 10 fold between families. As these data suggest that variation in the rate of development of the competent state has a genetic basis, and as a first step towards identifying the molecular basis to this variation, we have identified numerous genes that are differentially expressed later in larval development using a differential display approach. Spatial expression analysis of these genes suggests that they may be directly involved in the acquisition of competence, or may play a functional role in the postlarva following metamorphosis.Communicated by M.S. Johnson, Crawley     

Induction of metamorphosis in larvae of the oyster Crassostrea gigas using neuroactive compounds

Chemical (neuroactive compounds at different concentrations and exposure times) and physical (water agitation, light) factors with potential effects on the metamorphosis of larvae of the oyster Crassostrea gigas (Thunberg) larvae have been studied. The neurotransmitters l-dihydroxyphenylalanine (DO), epinephrine (EP), norepinephrine (NE), and acetylcholine (AC) have been identified as very active inducers of metamorphosis, whilst serotonin (SE), dopamine (DA) and potassium (K) were less effective inducers. The -aminobutyric acid (GA) and ammonium (AM) were found ineffective at the concentrations tested. Exposure to 10^-4 M EP for 15 min was sufficient to promote >80% metamorphosis within 48 h, whereas NE required 2 h to exert comparable induction. Maximum induction by DO (>50%) was achieved after 2 h exposure to 10^-4 M. However, unlike EP and NE, DO was lethal at that concentration in the long term. Maximum induction by AC (30% metamorphosis) was achieved at a concentration of 10^-4 M. In contrast to other neurotransmitters, AC induced settlement behaviour, cementation and eventual metamorphosis, yielding postlarvae which were all attached to the substratum. EP and NE triggered the morphogenetic changes, by-passing settlement and leading to postlarvae not cemented to the substratum. DO induced mostly attached spat at low concentrations (10^-5 M) and unattached spat at high concentrations (10^-4 M), and a similar pattern was apparent for the weaker inducers SE and DA. Regarding physical factors, a highly reflectant surface significantly increased the percentage of attached spat obtained, compared to a dark bottom. No consistent effect of water current or light was detected on the production of unattached spat. The three different forms of induction are discussed in relation to different regulatory pathways of settlement and metamorphosis.     

Initiation of feeding and salinity tolerance in the pacific lamprey Lampetra tridentata

Changes in salinity tolerance were determined during metamorphosis in Lampetra tridentata. Lampreys in Phase 5 of metamorphosis were unable to withstand salinities>13.4S, while those in Phase 6 survived direct transfer to sea water (30S). This abrupt change in tolerance coincided with the opening of the foregut lumen. Parasitic feeding began at the end of Phase 7 of metamorphosis following the completion of tooth development.     

Evidence for a water-borne cue inducing metamorphosis in the dorid nudibranch mollusc Adalaria proxima (Gastropoda: Nudibranchia)

The dorid nudibranch Adalaria proxima (Alder & Hancock) is a specialist predator of the cheilostome bryozoan Electra pilosa (L.). Natural induction of metamorphosis of the pelagic lecithotrophic larva of A. proxima was assessed in response to solutions from sonicated prey tissue and (live) E. pilosa-conditioned seawater (Electra-CSW). We exploited the tendency of larvae to become entrapped (rafted) at the air-water interface in cultures to examine whether larvae require direct contact with the live prey for metamorphosis to proceed. Larvae metamorphosed when rafted above colonies of live E. pilosa, above plankton mesh bags isolating live E. pilosa, and in choline chloride controls; there was no metamorphosis of larvae that were rafted in filtered seawater controls. Entrapped veliger shells remained rafted throughout the experimental period in all cases. No metamorphosis occurred in treatments containing either the supernatants or pelleted particulates obtained from sonicated colonies of E. pilosa. Both one-colony and three-colony Electra-CSW induced metamorphosis of larvae. These data are at variance with previous results in showing that direct contact with the live prey is not necessary for metamorphosis to proceed. Furthermore, the fact that competent larvae metamorphosed in response to Electra-CSW in the absence of any other cue strongly suggests that the inductive cue is water-borne.     

Comparison of larval development and growth of the sea cucumberActinopyga echinites: Ovary-induced ova and DTT-induced ova

AdultActinopyga echinites (Jaeger) were collected from northern Taiwan in September 1989. Oocytes were induced to mature by bathing them in ovary juice (ovary-induced ova) or in 10^–2 M dithiothreitol (DTT-induced ova). The percentage of germinal vesicle breakdown (GVBD) increased from 0.4 to 6.4% in the former treatment and to 84% in the latter. After artificial fertilization, the embryos were cultured in seawater (35 S) at 25 to 28°C. Larvae were fed with the algaIsochysis aff.galbana at 10^4–5 cells/ml. Larvae from ovary-induced ova developed faster (18 d to the doliolaria stage) and grew to a larger size (1.13 mm length) than those from DTT-induced ova (20 d to the doliolaria stage and 0.62 mm in length). On the twelfth day, larvae from ovary-induced ova bear lipid spheres. The number of spheres is positively correlated with larval size. Lipid spheres may provide nutrient reserves for larvae during metamorphosis.     

The induction of larval settlement and metamorphosis of two sea urchins,Pseudocentrotus depressus and Anthocidaris crassispina,by free fatty acids extracted from the coralline red alga Corallina pilulifera

Lipophilic inducers of larval settlement and metamorphosis of Pseudocentrotus depressus and Anthocidaris crassispina, two commercially important sea urchin species in Japan, were isolated from the foliose coralline red alga Corallina pilulifera (collected in 1990 near Saga, Japan) and identified. Larval assays of the fractions obtained by silica gel column chromatography of the total lipids showed that non-polar groups of lipids were effective at inducing larval settlement and metamorphosis. The effective fractions were further subjected to gel filtration (Sephadex LH-20) and also to silica gel column chromatography, and the effective components isolated as single spots by thin-layer chromatography. The components at a concentration of ca. 0.4 mg paper^-1 (sample was adsorbed on a paper with 20 cm²) induced high rates of larval settlement of both P. depressus and A. crassispina. Chemical analyses of the components revealed a mixture of free fatty acids (FFAs), dominated by eicosapentaenoic acid (20:5, 41 to 50%), palmitic acid (16:0, 11 to 17%), arachidonic acid (20:4, 9 to 15%), and palmitoleic acid (16:1, 4 to 5%). In assays with the four standard FFAs, only 20:4 and 20:5 induced larval settlement and metamorphosis of the two species, while 16:0 and 16:1 were ineffective. The larvae underwent significant rates of settlement and metamorphosis in response to the two former FFAs at levels as low as 0.18 mg paper^-1. Amongst the free fatty acid components of the alga, 20:5 was isolated as the chemical inducer of larval settlement and metamorphosis of the sea urchins in the laboratory.     

A method for rearing Pecten maximus larvae in the laboratory

Development of the scallop Pecten maximus (L.) from egg to metamorphosis takes 33 to 38 days at 16°C. The shelled veliger first appears 2 days after fertilisation, and crawling pediveligers at around 2 days before metamorphosis. The pediveliger can attach temporarily to filamentous objects by means of a byssus thread and, even after metamorphosis, periods of attachment alternate with periods of crawling. The larvae are reared in polythene bins containing 30 l of full salinity sea water (pre-filtered through 0.2 filters), which is changed every 2 days. Mixtures of algal foods (Isochrysis galbana, Chaetoceros calcitrans, Pyramimonas obovata and Tetraselmis suecica), concentrated by centrifugation, are added at each change from the third day onwards to give a concentration of 50 cells/l. Larval density is maintained below 10/ml. Antibiotics are added at each change, but very careful sterilisation of all equipment is necessary to guard against the introduction of diseases.     

Combined effects of temperature and salinity on fed and starved larvae of the mediterranean mussel Mytilus galloprovincialis and the Japanese oyster Crassostrea gigas

The combined effects of temperature, salinity and nutrition on survival and growth of larvae of the Mediterranean mussel Mytilus galloprovincialis and the Japanese oyster Crassostrea gigas were studied over a period of 7 d in the laboratory. Ripe adults, collected in spring and summer 1987 from natural populations in the Bay of Arcachon, France, were induced to spawn. Larvae of both species were cultured at four temperatures (15°, 20°, 25° and 30°C), four salinities (20, 25, 30 and 35S) per temperature, and two levels of nutrition (fed and unfed) per temperature/salinity combination. The fed larvae received a mixed algal diet of 50 cells each of Isochrysis galbana and Chaetoceros calcitrans forma pumilum per microlitre. In both bivalve species, larvae survived over a wide range of temperature and salinity, with the exception of mussel larvae, which died at 30°C. Statistical analysis indicated that nutrition had the greatest effect on larval development, explaining 64 to 75% of the variance in growth of M. galloprovincialis and 54 to 70% in growth of Crassostrea gigas. Unfed mussel larvae displayed little growth. Compared with temperature, the effect of salinity was very slight. M. galloprovincialis larvae exhibited best growth at 20°C and 35S and C. gigas at 30°C and 30S.     

Einfluß von Benetzungsspannung und lonen auf die Substratbesiedlung und das Einsetzen der Metamorphose bei Bryozoenlarven (Bowerbankia gracilis)

Settlement and metamorphosis in larvae of Bowerbankia gracilis depend on the wettability of the substratum. The wettability of a solid can be characterized by it's specific contact angle, . Larvae settle on solids with angles >17°, but not on surfaces with high wettability properties. In an attempt to explain this phenomenon, we consider the larvae as a second liquid and their attachment as a second wetting. In such case, water and larvae would compete in wetting the substratum. Adhesion can be accounted for by assuming the wetting tension of solid to water to be lower than that of the larval surface (|_solid/water| < |_solid/larvae|). Adhesion would thus be favoured energetically. The rate of settlement and that of metamorphosis are demonstrated as depending on the degree of contact angle, , in an all-or-none fashion. Settlement of larvae on high-wetting solids is prevented by high capillary pressure accruing at the point of contact. Metamorphosis is initiated by contractions occurring after adhesion. Application of CsCl or KCl induces such contractions and metamorphosis in free-swimming larvae. MgCl₂ prevents the onset of metamorphosis but not adhesion. Two mechanisms are considered as possibly accounting for induction of metamorphosis: (1) mechanical stimulation by shearing the cilia at the point of contact, and (2) a chemical stimulation of the cell membrane after application of K⁺ or Cs⁺.     

Isolation and characterisation of zinc-binding proteins distinct from metallothionein from the eggs of the sea urchin Strongylocentrotus intermedius

Two low-molecular-weight zinc-binding proteins were purified from eggs of the sea urchin Strongylocentrotus intermedius (Echinodermata: Echinoidea) by gel-permeation and anion-exchange chromatography followed by high performance liquid chromatography. The characteristics of these proteins are distinct from those of metallothionein. Amino acid compositions show a low or intermediate content of cysteine and high amounts of acidic amino acids, glycine and histidine; they also contain methionine and aromatic residues. On the basis of these characteristics, the S. intermedius zinc-binding proteins appear similar to other metalloproteins isolated from both vertebrates and invertebrates. No typical metallothionein was detected in the extracts from S. intermedius eggs.     

Effect of Ca2+ on survival and development of metamorphosing bonefish (Albula sp.) leptocephali

Bonefish (Albula sp.) larvae (leptocephali) from the Gulf of California complete metamorphosis in ˜10 d in natural seawater (35‰S; Ca²⁺ conc = 10.5 mM). The increase in ossification that occurs near the end of the non-feeding metamorphic period, in addition to the ability of larvae to complete metamorphosis in dilute seawater (8‰ S) prompted the present study, where the effects of varying the external calcium ion concentration, [Ca²⁺]_e, of artificial seawater (ASW) on the survival, development and internal (whole-body) calcium ion content, (Ca²⁺)_i, of unfed metamorphosing larvae were investigated. Early-metamorphosing larvae placed in␣ASW, where [Ca²⁺]_e = 10.1 mM, survived for up to 10 d and developed normally without exogenous nutrients. In shorter-term experiments (4 to 5 d), no differences in survival were found for larvae in ASW with [Ca²⁺]_e rang-ing from 1.5 to 10.1 mM. However, in Ca²⁺-free ASW, most larvae died within 27 h and no larvae survived more than 42 h; the median lethal time (LT₅₀), and its 95% confidence limits, were 14.5 (10.0 to 20.9) h. High mortality (81% after 20 h) also occurred in 1.0 mM Ca²⁺ ASW, but 2 of 16 larvae tested survived for 96 h. The 96 h median tolerance limit (TL_M), corrected for control mortality, was 1.2 mM Ca²⁺. In natural seawater, larval (Ca²⁺)_i remained relatively constant ( = 0.419 mg larva⁻¹)␣in early- and intermediate-metamorphosing larvae, and then increased to a mean value of 0.739 mg larva⁻¹ in advanced larvae, indicating that Ca²⁺ was␣taken up from the medium at this stage; the increase in (Ca²⁺)_i corresponded to the period of ossification of the vertebral column. Internal (whole-body) magnesium ion content (Mg²⁺)_i showed no significant change during metamorphosis ( = 0.089 mg larva⁻¹). No significant differences in (Ca²⁺)_i were found in advanced larvae in natural seawater and those in ASW, with [Ca²⁺]_e ranging from 2.0 to 10.1 mM. However, clearing and staining revealed that ossification of the vertebral column had not yet occurred in advanced larvae from 2.0 to 10.1 mM Ca²⁺ ASW. Also, low [Ca²⁺]_e (1.0 to 2.0 mM) usually produced deformed larvae that swam erratically, at times showing “whirling” behavior. Received: 21 August 1996 / Accepted: 26 August 1996     

Sublethal effects of zinc and municipal effluents on larvae of the red abalone Haliotis rufescens

Experiments were conducted to develop a sensitive sublethal toxicity test protocol to determine the toxicity of municipal wastewater effluents to larvae of the red abalone Haliotis rufescens. In multiple tests, fertilized abalone embryos were exposed for 48 h to dilutions of a reference toxicant, zinc sulfate, and to dilutions of primary-and secondary-treated effluents. The resulting veliger larvae were examined microscopically for larval shell abnormalities. In a longer flowthrough experiment, abalone were exposed for the entire larval phase, from the two-cell stage through metamorphosis, to compare zinc effects on metamorphosis with zinc effects on short-term larval shell development. Dissolved oxygen, pH, salinity and temperature were measured daily in test solutions, and zinc concentrations were verified by chemical analysis. No observed effect concentrations (NOECs) for zinc were 39±2.1 g l^-1 in three 48 h exposures, and 19 g l^-1 for the 9 d exposure through metamorphosis. Median effect concentrations (EC₅₀s) were 68±6.9 g l^-1 in 48 h tests and 50 g l^-1 in the 9 d test. Abalone larvae were affected at lower concentrations of primary than of secondary effluent.     

Some observations on gametogenesis,larval development and substratum selection of the sea pen Ptilosarcus guerneyi

Ptilosarcus (Leioptilus) guerneyi (Gray) maintained in the laboratory, were observed to spawn in late March, 1972. Gametes, developed in the leaf proper, discharged through the mouths of feeding polyps and were fertilized externally in the sea water. The sea pen's eggs are 500 to 600 in diameter; a large female is capable of producing over 200,000 eggs in one season. A pear-shaped and free-swimming planula larva developed 4 days after fertilization, at a temperature of 12 °C. The larvae were ready to settle and metamorphose when 7 days old if favorable substratum was available, but would remain as planulae for at least 30 days if kept in glass dishes only. The 30-day-old larvae would metamorphose if a suitable substratum (coarse sand, for example) was presented. The larvae do not feed and, hence, development is lecithotrophic. Studies of histogenesis showed that metamorphosis greatly enhanced the rate of cellular differentiation. The high fecundity, lecithotrophic development, and the ability of substratum selection by the larvae explain the success of this species in maintaining a high-density population in many areas of sandy substratum in the shallow waters of Puget Sound (USA), despite the fact that it is preyed upon by 7 species of predators.