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     

Translocation of fixed carbon from symbiotic bacteria to host tissues in the gutless bivalve Solemya reidi

Naturally-occurring lipophilic inducers of larval settlement and metamorphosis wer isolated and identified for Phragmatopoma californica, a gregarious tube worm from southern California. Organic solvent extraction of the sand/organic cement matrix of tubes diminished the inducing capacity of the tube matrix. The inducing capacity was restricted to a single, highly active, HPLC-purified fraction of the organic solvent extract. Chemical analysis of this fraction revealed a mixture of free fatty acids (FFAs), dominated by eicosapentaenoic acid (20:5, 20%), palmitic acid (16:0, 14%) and palmitoleic acid (16:1, 12%). In assays of the nine FFAs that each contributed 3% or more to the active fraction, only 16:1, 18:2, 20:4 and 20:5 induced larval settlement and metamorphosis, while the others were ineffective. The larval response was contact-dependent, highly specific, and concentration-dependent, with a significant response to 16:1 and 20:4 at as low as 10 g FFA spread onto 1 g of sand (surface area 36 cm²). Active FFAs were extracted at approximately 14 g g^-1 sand from the tube matrix, although the levels encountered by larvae in nature are believed to be higher.     

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.     

Effects of salinity and adult extract on settlement of the oligohaline barnacle Balanus subalbidus

Balanus subalbidus (Henry) has the most oligohaline distribution of three congeneric barnacles in Chesapeake Bay and tolerates prolonged exposure to fresh water. We studied larval settlement (i.e., permanent attachment and metamorphosis) of B. subaldius in the laboratory, over a 3 yr period, May 1989 to March 1992, under the following conditions: (1) across an array of salinities at 25°C in the presence and absence of settlement factor consisting of adult B. subalbidus extract; (2) in the presence of conspecific or congeneric settlement factors; and (3) cyprids which were, and were not, induced to delay metamorphosis were compared in their capacities to settle in a range of salinities. Discrepancies between salinity profiles of larval settlement in the laboratory and adult oligohaline distribution in the estuary were striking, and there was a significant interaction between salinity and settlement factor. Averaging results of four different batches of larvae, although peak settlement (87±9%) of B. subalbidus occurred at 2 ppt salinity in the presence of adult cue, substantial settlement also occurred at higher salinities: >70% at 5, 10 and 15 ppt; and 47% at 20 and 25 ppt. In addition, settlement in the absence of settlement factor was relatively high (>50%) and peaked at mid-salinity ranges (e.g. 56±10% at 15 ppt). Variation observed in settlement among larval batches reflected detailed differences in settlement between adjacent test salinities. No difference in settlement occurred between replicate aliquots of cyprids within a batch. Cyprids of B. subalbidus settled most abundantly in the presence of settlement factor extracted from conspecifics, followed in decreasing order by settlement factor extracted from B. improvisus and B. eburneus. Delay of metamorphosis produced by keeping B. subalbidus cyprids for 8 d at 5°C resulted in a decreased level of settlement, but settlement frequency patterns of delayed and non-delayed cyprids were indistinguishable relative to salinity. These results indicate that the oligohaline distribution of adult B. subalbidus is probably not determined by larval behavior at settlement. We suggest that pre-settlement behavior, resulting in larval retention in low saline waters, could be an important factor in determining distribution of this species.     

Changes in biochemical and fatty acid composition of the razor clam <Emphasis Type="Italic">Solen marginatus</Emphasis> (Solenidae: Bivalvia) during larval development

Aquaculture studies have revealed that polyunsaturated fatty acids are critical for maintaining substantial growth, survival and reproductive rates, and high food conversion efficiencies for a wide variety of marine and freshwater organisms. The aim of this study was to investigate the gross biochemical and fatty acid composition of both neutral and polar lipid compartments of the razor clam Solen marginatus throughout embryonic and larval development. High levels of stored reserves in S. marginatus eggs allow a short larval development, lasting only 8 days. The energy required for embryogenesis was obtained from stored proteins. During larval development from D-shaped veliger until settlement, protein, lipid, and carbohydrate reserves were indistinctly stored for metamorphosis. Although total lipids increased, fatty acids in both neutral and polar lipids decreased during embryonic development. The depots allow a short larval development in which settlement is reached with lower amounts of stored neutral and polar lipids than the contents found in the oocytes. Non-methylene-interrupted dienoic fatty acid levels were similar to those of some polyunsaturated fatty acids, with increasing percentages at the onset of metamorphosis. This study indicates that S. marginatus exhibits a different pattern in the use of gross biochemical and fatty acid reserves during larval development compared to other razor clam and bivalve species, mainly due to the large size of its eggs and the short larval development stage reported in this species.     

Larval growth and settlement of the European oyster (Ostrea edulis) as a function of food quality measured as fatty acid composition

Larval growth rate and settlement of the European flat oyster Ostrea edulis were experimentally studied as a function of the composition of dietary fatty acids. Diets differing in fatty acid composition were composed by mixtures of the microalgae Isochrysis galbana, Pavlova lutheri and Chaetoceros calcitrans. Fatty acid content in the tissue of the feeding larvae, analyzed by gas chromatography and mass spectrometry, reflected the composition in the diet. Larval growth rate was significantly correlated to the three omega-3 polyunsaturated fatty acids (PUFA) C18:3, C18:4 and C22:6, with minor differences for neutral and polar lipids. No relation between growth rate and the omega-3 PUFA C20:5 was detected, a PUFA often implied as essential for bivalves. It is suggested that naturally occurring variability in fatty acid composition may constrain larval growth. In settlement experiments in both still water and flume flow little substrate selectivity was found for some contrasting substrates. It is concluded that differences in dietary fatty acids may explain as much of settlement success as the variability of substrates. Received: 12 October 1998 / Accepted: 6 April 1999     

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.     

Larval settlement and metamorphosis of the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> on different macroalgae

Settlement of mussels is commonly associated with macroalgae. The effects of 19 macroalgal species on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Settlement and metamorphosis inducing activities of macroalgae Chlorodesmis fastigiata and Ceramium tenerrimum collected each month during the period between January 2005 and April 2006 were also investigated. Furthermore, C. fastigiata and C. tenerrimum were subjected to various treatments to investigate the roles of bacteria and diatoms on the algal surface in the induction of larval settlement and metamorphosis of M. galloprovincialis and the characteristics of the cues in these two macroalgae. Pediveliger larvae of M. galloprovincialis settled and metamorphosed in high percentages on Cladophora sp., Chlorodesmis fastigiata, Centroceras clavulatum, and Ceramium tenerrimum, all of which were filamentous in morphology. Macroalgae that were cylindrical, phylloid, flabellate, palmate and pinnate all showed low (<8%) percentages of post-larvae but four other filamentous macroalgae also had low mussel larval settlement, suggesting that chemical factors may also be involved. Seasonal variation had no effect on inductive activities of C. fastigiata and C. tenerrimum. Treatment of C. fastigiata and C. tenerrimum with formalin, ethanol and heat resulted in the significant decrease or loss of their inductive activities. Survival of algal cells within treated macroalgae also decreased significantly. Treatment of the two macroalgae with antibiotics and GeO₂ reduced the numbers of bacteria and diatoms on their surface but did not affect their inductive activities, indicating that the cue was produced by macroalgae and not by coexisting bacteria and diatoms. However, conditioned water and crude extracts of these two macroalgae did not induce larval settlement and metamorphosis. Thus, larvae of M. galloprovincialis settled and metamorphosed on specific filamentous macroalgae. The chemical cues produced by C. fastigiata and C. tenerrimum were susceptible to ethanol and heat treatments and were not recovered in the conditioned water nor in the extracts. The finding that inactive C. tenerrimum can be produced from culturing its apical segments provides a new tool to elucidate the chemical cue(s) from macroalgae through manipulation of their culture conditions.     

Relationship between larval and juvenile growth rates in two marine gastropods,Crepidula plana and C. fornicata

Previous studies on various marine mollusc species have shown that both larval and juvenile growth rates are substantially heritable, but few workers have examined the extent to which larval and juvenile growth rates covary. We examined the relationship between larval and juvenile growth rates in seven laboratory experiments conducted between 1986 and 1993, using the prosobranch gastropods Crepidula plana Say and C. fornicata (L.). In most experiments larvae were reared individually, measured twice nondestructively to determine larval grwoth rate, allowed or stimulated (daily 5-h exposure to 20 mM excess K⁺ in seawater) to metamophose, and then measured at least twice after metamorphosis to determine juvenile growth rates. Generally, there was no significant (p >0.10) relationship between larval and juvenile growth rates, suggesting that in these two species selection can act independently on the two stages of development. A positive correlation (p=0.007) between larval and juvenile growth rates was observed for C. fornicata in one experiment, but only for offspring from females maturing the most rapidly in laboratory culture. Even for these larvae, however, variation in larval growth rate explained<2% of the variation in juvenile growth rate, so that larval and juvenile growth rates are at most only weakly associated in this species.     

Patterns of formation and the ultrastructure of the larval skeleton of Pocillopora damicornis

The growth and development of the tissues and skeleton of settled larvae of the reef coral Pocillopora damicornis (Linnaeus), collected in December 1983 from Ko Phuket, Thailand, were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. The rate of development of larval skeletons was very variable, preventing the chronological sequencing of skeletal growth. However, four growth stages in the development of a complete larval skeleton from first settlement were identificd: Stage 1, deposition of the first elements of the basal plate upon settlement; Stage 2, completion of basal plate, and deposition of skeletal spines and ridges in positions corresponding to the septal cycles; Stage 3, formation of the corallite wall and septal and costal cycles; Stage 4, the complete larval skeleton which represented the maximum growth attained eight days after settlement. The configuration of the larval tissues, particularly the aboral ectoderm, mirrored the four developmental stages. The deposition of the larval skeleton was correlated with the metamorphosis of the aboral ectoderm from a columnar to a squamous morphology. The basal plate of the larval skeleton had two layers of crystals orientated perpendicular to each other. The architecture of the complete larval skeleton is described and compared to that of the adult skeleton of P. damicornis. The results are discussed with respect to previous concepts of the formation of the larval skeleton of scleractinian corals and coral calcification.     

Physiological and biochemical changes during lecithotrophic larval development and early juvenile growth in the northern stone crab,Lithodes maja (Decapoda: Anomura)

Larvae of the northern stone crab, Lithodes maja L., were reared in the laboratory from hatching to the second crab stage. complete larval development (at constant 9°C) lasted about 7 wk, invariably consisting of three pelagic zoeal stages and a semibenthic Megalopa; only two zoeal stages have been described in the literature. All larval stages are lecithotrophic. First feeding was consistently observed only after metamorphosis, in the first juvenile crab stage. In short intervals (every 1 to 5 d), developmental changes in biomass, B (expressed as: dry weight, W; carbon, C; nitrogen, N; hydrogen, H) and oxygen consumption (respiration, R) were measured in larvae and early juveniles; additionally, protein and carbohydrates were measured, but only in the zoeal stages and early Megalopa. Unusually high C contents (varying between 56 and 61% of W in eggs and freshly hatched Zoea I larvae from 12 different females) and high C:N weight ratios (8 to 11) indicate enhanced initial lipid stores, which are utilized as the major metabolic substrate during both embryonic and lecithotrophic larval development. Predominant degradation of lipids is shown indirectly; the C:N ratio decreased significantly, from 10 (at hatching) to 6 (at metamorphosis), while larval protein decreased only little, from ca. 55% of W (at hatching) to 48% (in the Megalopa). From hatching to metamorphosis, about 27% of the initially present W, 48% of C, 18% of N, and 52% of H were lost. This decrease in larval biomass can be described as an exponential function of development time. The major part of these losses were associated with metabolic energy requirements, while exuvial losses were comparably small. In each of the zoeal stages, only about 1 to 2% of late premoult (LPM) B was shed with the exuvia. The Megalopa, which produces a much thicker, calcified exoskeleton, lost 20% of LPM W, but only 5 to 8% of organic constituents (C, N, H). Much higher exuvial losses were measured in the Crab I stage (51% in W, 21% in C, 5% in N, and 7% in H). Maximum respiration was found in the actively swimming zoeal stages, a minimum in the predominantly benthic, mostly inactive Megalopa. The Crab I stage exhibits also a sluggish behaviour and low R, in spite of beginning food uptake and growth. Immediately after metamorphosis, the juvenile crab gained rapidly in W, in particular in its C fraction. A transitorily steep increase in the C:N ratio indicates a replenishment of partially depleted lipid stores, but also a rapid initial increase of inorganic C in the heavily calcified exoskeleton. Instantaneous rates of growth, assimilation, and net growth efficiency (K ₂) were high during the initial (postmoult) phase in the first juvenile crab stage (C-specific growth rate: 6% d^-1; K ₂:70%), but decreased towards zero values during laterstages of the moulting cycle; metabolism remained practically constant during the Crab I stage. Entirely lecithotrophic larval development from hatching to metamorphosis in L. maja is considered an adaptation to seasonally short and limited planktonic food production in subarctic regions of the northern Atlantic.     

Experimental investigation into the effects of suspended sediment on fertilisation, larval survival and settlement in a scleractinian coral

Laboratory and field experiments were used to determine whether high (≃100 mg l⁻¹), low (≃50 mg l⁻¹) and control (≃0 mg l⁻¹) levels of suspended sediment affected fertilisation, larval survival, and larval settlement in the scleractinian coral Acropora digitifera (Dana, 1846). Both high- and low-sediment treatments significantly decreased fertilisation, but post-fertilisation embryonic development was not inhibited by suspended sediments. Larval survival and larval settlement were significantly reduced in high- and low-sediment treatments. No difference was found between high- and low-sediment treatments in any of the three post-spawning processes investigated, suggesting that they are susceptible to sediment concentrations which are not exceptionally high even under natural conditions (>50 mg l⁻¹). The introduction of an additional stress in the form of high levels of suspended sediments coupled with naturally high variability in recruitment may have a considerable effect on the successful supply and settlement of coral larvae to a reef. Given that many coral communities are open reproductive systems, the consequences of disturbance events are not likely to be restricted to the impact area. Recruitment to a population may be reduced significantly in the presence of high levels of suspended sediments because of effects on larval survival and settlement. Recruitment of larvae to adjacent populations may also be affected due to a decreased fertilisation success and potential increases in mortality of larvae passing through the affected site. Received: 13 August 1998 / Accepted: 22 July 1999     

Measuring settlement intensity of echinoderms on coral reefs

A pilot study was conducted at Davies Reef on the central Great Barrier Reef between 20 November 1991 and 20 January 1992 to assess the suitability of settlement collectors for measuring larval settlement rates of echinoderms on coral reefs. The collectors were deployed for two months during summer when many echinoderm species are known to spawn. A total of 657 newly settled echinoderms were recovered from just 47 collectors, each having a volume of just 0.005 m³ but with a utilisable surface area of about 1.44 m², indicating that competent-to-settle larvae were very abundant during the sampling period. Echinoids (7.8 sampler^-1) and ophiuroids (5.6 sampler^-1) were the most abundant groups on the collectors. Asteroids (0.2 sampler^-1) and crinoids (0.3 sampler^-1 were less common. The asteroids were all identified to species and included the crown-of-thorns starfish Acanthaster planci, Choriaster granulatus and Culcita novaeguineae. The newly settled echinoids could not be distinguished from each other but included Echinometra mathaei and Mespilia globulus. The abundance of each of the five different classes of echinoderms on the samplers was correlated with their abundance on the natural substratum. Significant spatial variability was found in settlement rates of echinoids over 1000s of metres and ophiuroids over 100s of metres, but not over smaller spatial scales. It is concluded that the collectors can provide a useful tool for monitoring spatial and temporal variability in settlement rates of echinoderms on coral reefs and for testing hypotheses about patterns of larval dispersal.     

Influence of bacteria and diatoms in biofilms on metamorphosis of the marine slipper limpet <Emphasis Type="Italic">Crepidula onyx</Emphasis>

Larvae of the slipper limpet Crepidula onyx metamorphose in response to marine biofilms. In this study, we investigated how the percentage of larval metamorphosis in this species was affected by biofilms that differed in certain attributes. To manipulate bacterial and diatom cell densities and community composition, we developed biofilms in the laboratory (1) at different temperatures (16, 23 and 30°C) and salinities (20, 27 and 34‰), (2) with or without addition of antibiotics, and (3) in the light or in the dark. We also allowed biofilms to develop at three field sites with different prevailing environmental conditions so as to generate biofilms with different, but natural, attributes. Bacterial and diatom community composition in biofilms were determined using a DNA fingerprinting technique and microscopic examination, respectively. The effects of biofilms on metamorphosis were investigated in laboratory assays. The percentage of larval metamorphosis correlated with bacterial and diatom cell densities in only one of the three experiments conducted, but was substantially affected by differences in bacterial and diatom community composition in all three experiments. It also appears that metamorphosis of C. onyx depends on the simultaneous presence of both bacterial and diatom communities in biofilms.     

Development of the nasal olfactory organs in the larvae, settlement-stages and some adults of 14 species of Caribbean reef fishes (Labridae, Scaridae, Pomacentridae)

Larval fishes likely use a variety of settlement cues to locate and navigate toward the habitats they will inhabit as juveniles. Information about the morphology and state of development of the sensory organs of larval stages of fishes provides insight into their capabilities at the time of settlement. The peripheral olfactory organ of wild-caught late-stage larvae and early juveniles and some adults of 14 species of the Caribbean reef fishes wrasses (Labridae), parrot fishes (Scaridae) and damselfish (Pomacentridae) were examined using scanning electron microscopy and compared in terms of settlement specificity. Ages in days after hatching and days post-settlement were determined from the otoliths. Morphology of the nares and the olfactory epithelium are described for these species by stage. The separation of the anterior and posterior nares occurred before settlement in the labrids but in some specimens of scarids this separation was not complete by the time of settlement. Densities of ciliated and microvillous receptor cells and non-sensory ciliated epithelial cells were calculated. Densities of ciliated receptor cells ranged from 0.389 μm⁻² in a specimen of Thallasoma bifasciatum to 0.0057 μm⁻² in Bodianus rufus and of microvillous receptor cells from 0.038 μm⁻² in a Clepticus parrae juvenile to 0.266 μm⁻² in a juvenile Doratonotus megalepis. Densities of non-sensory cilia, also associated with high olfactory ability, were also high. The olfactory organ in wrasses is well developed prior to settlement and is comparable to that of adult fishes. The possible role of olfaction in larval schooling, reef cue detection and orientation toward habitat at settlement is discussed.

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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.     

Larval settlement in the serpulid polychaete Hydroides elegans in response to bacterial films: an investigation of the nature of putative larval settlement cue

Larval settlement in the marine polychaete Hydroides elegans (Haswell) is induced by certain bacteria in marine biofilms. The exact nature of the settlement cue that larvae of H. elegans receive from these bacteria remains unknown. In this study, we revealed some properties of the bacterially derived larval settlement cue by investigating the larval settlement inductive activity of two bacterial strains after various treatments. These two bacterial strains, Roseobacter sp. and an α-subclass Proteobacteria, are highly inductive to larval settlement of H. elegans. The larvae responded similarly to Roseobacter and Proteobacteria in all the larval settlement bioassays, suggesting that the larval settlement-inducing substances produced by these bacteria may share common characteristics. First of all, the larvae did not settle in the seawater conditioned by the bacteria attached as a film or by the bacteria that were freely suspended in seawater. The results suggest that the putative larval settlement cue is not released into seawater and, therefore, should be associated with the surface of the bacteria. Secondly, formaldehyde treatment entirely eliminated the larval settlement induction activity of the bacterial films, and streptomycin treatment reduced the percentage of larval settlement on the bacterial films in a concentration-dependent manner. Since both treatments can kill bacteria with little damage to the surface chemistry of bacterial cells, the decline in larval settlement is suggested be due to a reduction of the viable bacterial population in the bacterial films. In fact, the reduction of larval settlement in the streptomycin treatments coincided with the decrease in viable bacterial populations in broth cultures containing respective concentrations of streptomycin. These results suggest that the viability of Roseobacter and Proteobacteria is important to their settlement induction effect. Since the larval settlement induction activity of the bacterial strains appears to correlate with their viability, we suggest that the putative larval settlement cue is derived from a metabolic pathway in the bacteria and that the cue is exported to and concentrated at the extracellular polymer matrix of the bacterial cell, at which the larvae establish contact with the bacteria. The larval settlement cue may be highly susceptible to degradation so that a metabolically active bacterial film is needed to maintain the putative cue at a concentration that surpasses the threshold for induction of larval settlement. Received: 14 October 1998 / Accepted: 5 September 2000     

Settlement of larval blacklip abalone, Haliotis rubra, in response to green and red macroalgae

Surfaces from the habitat of adult Haliotis rubra were tested as inducers of larval settlement to determine the cues that larvae may respond to in the field. Settlement was high on the green algal species Ulva australis and Ulva compressa (Chlorophyta), the articulated coralline algae Amphiroa anceps and Corallina officinalis, and encrusting coralline algae (Rhodophyta). Biofilmed abiotic surfaces such as rocks, sand and shells did not induce settlement. Ulvella lens was also included as a control. Treatment of U. australis, A. anceps and C. officinalis with antibiotics to reduce bacterial films on the surface did not reduce the settlement response of H. rubra larvae. Similarly, treatment of these species and encrusting coralline algae with germanium dioxide to reduce diatom growth did not significantly reduce larval settlement. These results suggest that macroalgae, particularly green algal species, may play an important role in the recruitment of H. rubra larvae in the field and can be used to induce larval settlement in hatchery culture.     

Gill morphogenesis in the oyster Ostrea chilensis

Despite the importance of the gills in the acquisition of food by suspension-feeding bivalve mollusks, there is almost no information on gill organogenesis. By means of a series of stereoscan electron micrographs, this paper describes gill development in the Chilean oyster, Ostrea chilensis, from the brooded larval stages to 1-month-old spat. A single gill rudiment was observed on each side of the mantle at a shell length of 320 μm, and the rudiments increased in number and size until the end of the brooding period. During metamorphosis the gill filaments increased in number from 5 or 6 to between 7 and 9. The loss of the velum and the absence of functional gill filaments during metamorphosis are consistent with previous observations of weight loss during this critical period of the life history, because the newly settled juvenile lacks the ability to remove particles from suspension. The end of metamorphosis (100% of spat with dissoconch edge) was reached 36 h after larval settlement, when the gill filaments began to grow cilia, which increased in density and differentiated as the spat developed and acquired the capability of suspension-feeding, accounting for the increase in body weight previously recorded during this stage. The larval rudiments gave rise to the inner demibranchs. The outer demibranchs were observed 10 days after settlement, located between the inner demibranch and the mantle. In 1-month-old spat, the gill did not show differentiation between primary and secondary filaments, indicating that the heterorhabdic condition characteristic of adult oysters had yet to be attained. Received: 11 December 1998 / Accepted: 21 August 2000     

An energy budget for the free-swimming and metamorphosing larvae of Balanus balanoides (Crustacea: Cirripedia)

Analysis of biochemical components and measurements of oxygen consumption rates of cypris larvae of Balanus balanoides (L.) maintained in the laboratory at 10°C for up to 5 weeks after capture shows that lipid is the primary energy reserve, although later protein is utilised. Initially, the cyprids swim freely with an oxygen consumption rate of ca. 37×10^-3 l O₂ h^-1 cyprid^-1, but within a few days the rate falls to ca. 21×10^-3 l O₂ h^-1 cyprid^-1 when they cease swimming and explore the substratum. The cost of metamorphosis was calculated both from the loss of biochemical components and oxygen consumption rates during metamorphosis; the values were 2.8×10^-2 and 3.2×10^-2 cal cyprid^-1, respectively. A budget was collated from the data on respiration and biochemical composition, whereby the energy per cyprid was partitioned into that required for essential structural components (6.8×10^-2 cal), that needed for metamorphosis (3.0×10^-2 cal) and an excess available for swimming and exploring, which in the batches studied was about 5.0×10^-2 cal. This excess is mainly derived from the utilisation of lipid reserves and is used up usually 2 1/2 to 4 weeks after capture. During these measurements, samples of cyprids were taken at weekly intervals to test the rate of settlement and success of metamorphosis. The results showed that they lose their competence to metamorphose successfully approximately at the same time (3 to 4 weeks) that the energy supply for swimming and exploration is used up.