Phenotypic variability in the Caribbean Orange Icing sponge Mycale laevis (Demospongiae: Poecilosclerida)

Sponge species may present several morphotypes, but sponges that are morphologically similar can be separate species. We investigated morphological variation in Mycale laevis, a common Caribbean reef sponge. Four morphotypes of M. laevis have been observed (1) orange, semi-cryptic, (2) orange, massive, (3) white, semi-cryptic, and (4) white, massive. Samples of M. laevis were collected from Key Largo, Florida, the Bahamas Islands, and Bocas del Toro, Panama. Fragments of the 18S and 28S rRNA ribosomal genes were sequenced and subjected to phylogentic analyses together with sequences obtained for 11 other Mycale species and additional sequences retrieved from GenBank. Phylogenetic analyses confirmed that the genus Mycale is monophyletic within the Order Poecilosclerida, although the subgenus Aegogropila is polyphyletic and the subgenus Mycale is paraphyletic. All 4 morphotypes formed a monophyletic group within Mycale, and no genetic differences were observed among them. Spicule lengths did not differ among the 4 morphotypes, but the dominant megasclere in samples collected from Florida and the Bahamas was the strongyle, while those from Panama had subtylostyles. Our data suggest that the 4 morphotypes constitute a single species, but further studies would be necessary to determine whether skeletal variability is due to phentotypic or genotypic plasticity.     

Svenzea zeai, a Caribbean reef sponge with a giant larva, and Scopalina ruetzleri: a comparative fine-structural approach to classification (Demospongiae, Halichondrida, Dictyonellidae)

Abstract. Svenzea zeai , abundant on many deep Caribbean fore-reef habitats but of uncertain systematic position within the Demospongiae, is closely examined histologically and cytologically for evidence of its phylogenetic relationship beyond the traditional analysis of gross morphology and skeletal structure. We document that S. zeai is a bacteriosponge containing substantial quantities of unicellular photosynthetic and autotrophic microbes; that the most abundant cell type is an unusual cell with refractile granules that only few species share and whose composition and function are still enigmatic; and that it produces the largest—by a factor of 3—embryos and larvae recorded in the phylum Porifera. A combination of characters such as the granular cells, ciliary pattern, and aspects of larval shape and behavior are comparable with those of Scopalina ruetzleri , family Dictyonellidae, a prominent member of the Caribbean mangrove community. These results support our earlier decision to establish Svenzea as a new genus in Dictyonellidae to accommodate its unprecedented skeletal structure, styles in isodictyal reticulation.     

Metamorphosis of a sesarmid river crab, Armases roberti: stimulation by adult odours versus inhibition by salinity stress

In an experimental laboratory study with the megalopa stage of Armases roberti, a freshwater-inhabiting species of crab from the Caribbean region, we evaluated the combined, potentially antagonistic effects of odours from conspecific adults and of stepwise salinity reductions (simulating upstream migration, reaching within 1 week conditions of 2‰ or freshwater). Neither of these treatments affected the rate of survival, but the duration of development to metamorphosis was significantly (by about 25%) shortened, when odours from conspecific adult crabs were present, regardless of the salinity conditions. Our results indicate that the metamorphosis-stimulating effect of chemical cues from an adult population of A. roberti is far stronger than the potentially retarding effect of increasing hypoosmotic stress. This suggests that the final phase of larval development, including the processes of settlement and metamorphosis, occurs in this species in freshwater habitats, where conspecific populations live.     

Novel protein from larval sponge cells,ilborin, is related to energy turnover and calcium binding and is conserved among marine invertebrates

Sponges (phylum Porifera) are early-branching animals, whose outwardly simple body plan is underlain by a complex genetic repertoire. The transition from a mobile larva to an attached filter-feeding organism occurs by metamorphosis, a process accompanied by a radical change of the body plan and cell transdifferentiation. The continuity between larval cells and adult tissues is still obscure. In a previous study, we have produced polyclonal antibodies against the major protein of the flagellated cells covering the larva of the sponge Halisarca dujardini, used them to trace the fate of these cells and shown that the larval flagellated cells transdifferentiate into the choanocytes. In the present work, we identified the sequence of this novel protein, which we named ilborin. A search in the open databases showed that multiple orthologues of the newly identified protein are present in sponges, cnidarians, flatworms, ctenophores and echinoderms, but none of them has been described yet. Ilborin has two conserved domains: triosephosphate isomerase-barrel, which has enzymatic activity against macroergic compounds, and canonical EF-hand, which binds calcium. mRNA of ilborin is expressed in the larval flagellated cells. We suggest that the new protein is involved in the calcium-mediated regulation of energy metabolism, whose activation precedes metamorphosis.     

The chemical cue tetrabromopyrrole from a biofilm bacterium induces settlement of multiple Caribbean corals

Microbial biofilms induce larval settlement for some invertebrates, including corals; however, the chemical cues involved have rarely been identified. Here, we demonstrate the role of microbial biofilms in inducing larval settlement with the Caribbean coral Porites astreoides and report the first instance of a chemical cue isolated from a marine biofilm bacterium that induces complete settlement (attachment and metamorphosis) of Caribbean coral larvae. Larvae settled in response to natural biofilms, and the response was eliminated when biofilms were treated with antibiotics. A similar settlement response was elicited by monospecific biofilms of a single bacterial strain, Pseudoalteromonas sp. PS5, isolated from the surface biofilm of a crustose coralline alga. The activity of Pseudoalteromonas sp. PS5 was attributed to the production of a single compound, tetrabromopyrrole (TBP), which has been shown previously to induce metamorphosis without attachment in Pacific acroporid corals. In addition to inducing settlement of brooded larvae (P. astreoides), TBP also induced larval settlement for two broadcast-spawning species, Orbicella (formerly Montastraea) franksi and Acropora palmata, indicating that this compound may have widespread importance among Caribbean coral species.     

Fluorescence of coral larvae predicts their settlement response to crustose coralline algae and reflects stress

Multi-coloured homologues of the green fluorescent protein generate some of the most striking visual phenomena in the ocean. Despite their natural prominence in reef-building corals and widespread use in biotechnology, their biological role remains obscure. Here, we experimented with larvae of Acropora millepora to determine what can be learned about a coral larva or recruit from its fluorescent colour. We performed 12 crosses between seven A. millepora colonies representing differing fluorescence phenotypes, the larvae of which were exposed to a natural settlement cue (crustose coralline algae) and heat-light stress. Parental effects explained 18 per cent of variation in colour and 47 per cent of variation in settlement. The colour of the larval family emerged as a predictor of the settlement success: redder families were significantly less responsive to the provided settlement cue (p = 0.006). This relationship was owing to a correlation between parental effects on settlement and colour (r(2) = 0.587, p = 0.045). We also observed pronounced (16%) decline in settlement rate, as well as subtle (2%), but a statistically significant decrease in red fluorescence, as a consequence of heat-light stress exposure. Variation in settlement propensity in A. millepora is largely owing to additive genetic effects, and is thought to reflect variation in dispersal potential. Our results suggest an optical signature to discriminate between long- and short-range dispersing genotypes, as well as to evaluate stress. Further research in this direction may lead to the development of field applications to trace changes in coral life history and physiology caused by global warming.     

Isolation,characterization, and in vitro culture of larval and adult epidermal cells of the frogXenopus laevis

Summary Methods for the isolation and in vitro culture of larval and adultXenopus laevis epidermal cells have been developed. Epidermal cells of stage 52–54 tadpoles and adult epidermal cells were enzymatically dissociated and purified (98%) by Percoll-density centrifugation and unit-gravity sedimentation. Both cell types attached on fibronectin-coated dishes and proliferated for 1 wk when the proper medium was used. There were four significant differences between larval and adult cells: a) Adult cells had a greater buoyant density than larval cells. b) Keratin synthesis patterns were markedly different. c) A combination of medium F12 and Eagle's minimum essential medium was optimal for growth of larval cells whereas MCDB151 medium was optimal for adult cells. d) Adult cells needed fetal bovine serum (>5%) whereas larval cells grew without fetal bovine serum. In contrast to these differences, larval and adult cells had two similar properties: a) Insulin had a potent effect on the growth of both cells, and b) The optimal Ca⁺⁺ concentration for cell growth was quite low for both cell types; 0,1 mM for larval cells and below 0.05 mM for adult cells. These results suggest that low Ca⁺⁺ levels are essential for both cornifying (adult) and uncornifying (larval) amphibian keratinocytes. The culture techniques described herein for larval and adult epidermal cells provide a new in vitro model for analyzing development of the epidermis during amphibian metamorphosis. This study was supported by grant (HD 24438) from the National Institutes of Health, Bethesda, MD.     

Behavior and development of larvae in the sponge Haliclona amboinensis

Sponges play important roles in marine ecosystems by contributing to habitat complexity and benthopelagic coupling of nutrients. Yet, the reproduction and settlement behaviors of diverse sponge species are not well understood. Here, we examined the brooding demosponge Haliclona amboinensis, which is common on shallow reefs in Bolinao, northwestern Philippines. Gravid sponges were found between the months of May and August, coinciding with warmer sea surface temperature. Sponges released parenchymella larvae from brood chambers in the mid‐morning, suggesting that light and temperature may serve as cues to initiate hatching. Larvae immediately swam toward the surface upon emergence and migrated to the bottom of the tanks 1–2 hr after release. The presence of light and crustose coralline algae induced high larval settlement. Metamorphosis proceeded rapidly in vitro, with larval cells spreading laterally on the substrate. The osculum was first visible at 3 days after settlement. The short pelagic duration of larvae in H. amboinensis promotes local recruitment and may be important for the maintenance of sponge populations in the face of disturbances.     

A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus

Abstract

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.     

Symbiosis of Mycale (Mycale) vansoesti sp. nov. (Porifera, Demospongiae) with a coralline alga from North Sulawesi (Indonesia)

Abstract. The symbiotic association between the new sponge species Mycale vansoesti and the coralline alga Amphiroa sp. from the Bunaken Marine Park (North Sulawesi, Indonesia) is described. The alga completely pervades the sponge. The color of the sponge ectosome is white, both on the external surface and on the atrial wall, but where the alga is present the sponge takes on the light pink color of the alga. The sponge spicular complement is characterized by mycalostyles, anisochelae of two types, sigmas (often "C" shaped), and extremely abundant toxas organized in bundles forming toxadragma. In the association, the sponge shows very low silicate value, and consequently the alga represents the main skeleton of the sponge. On the other hand, the sponge affects the morphology of the alga, leading to a cylindrical shape, with thalli running parallel to the sponge surface. This association seems to be obligate for the sponge, as we found no sponges of this species living in isolation.     

Sponge paraphyly and the origin of Metazoa

In order to allow critical evaluation of the interrelationships between the three sponge classes, and to resolve the question of mono‐ or paraphyly of sponges (Porifera), we used the polymerase chain reaction (PCR) to amplify almost the entire nucleic acid sequence of the 18S rDNA from several hexactinellid, demosponge and calcareous sponge species. The amplification products were cloned, sequenced and then aligned with previously reported sequences from other sponges and nonsponge metazoans and variously distant outgroups, and trees were constructed using both neighbour‐joining and maximum parsimony methods. Our results suggest that sponges are paraphyletic, the Calcarea being more related to monophyletic Eumetazoa than to the siliceous sponges (Demospongiae, Hexactinellida). These results have important implications for our understanding of metazoan origins, because they suggest that the common ancestor of Metazoa was a sponge. They also have consequences for basal metazoan classification, implying that the phylum Porifera should be abandoned. Our results support the upgrading of the calcareous sponge class to the phylum level.     

The larval stages of the sea urchin, Strongylocentrotus purpuratus

The adult body plan of Strongylocentrotus purpuratus is established within the imaginal rudiment during the larval stages. To facilitate the study of these stages, we have defined a larval staging scheme, which consists of seven stages: Stage I, four-arm stage; Stage II, eight-arm stage; Stage III, vestibular invagination stage; Stage IV, rudiment initiation stage; Stage V, pentagonal disc stage; Stage VI, advanced rudiment stage; and Stage VI, tube-foot protrusion stage. Each stage is characterized by significant morphological features observed for the first time at that stage. This scheme is intended as a guide for determining the degree of larval development, and for identifying larval and adult structures. Larval anatomy was visualized using light and confocal microscopy as required on living material, whole mount fixed specimens, and serial sections. Antibody staining to localize specific gene products was also used. Detailed analysis of these data has furthered our understanding of the morphogenesis of the rudiment, and has suggested provocative questions regarding the molecular basis for these events. We intend this work to be of use to investigators studying gene expression and morphogenesis in postembryonic larvae.     

Discrimination of conspecific-built chimneys from human-built ones by the burrowing crayfish, Fallicambarus fodiens (Decapoda, Cambaridae)

Abstract. The vast majority of tested juvenile crayfish of Fallicambarus fodiens consistently preferred conspecific-built mud chimneys over similar-looking human-built chimneys. When the chimneys were surrounded by transparent acetate sheets, the crayfish no longer discriminated between conspecific-built and human-built chimneys. This suggests that visual cues were not important in allowing the crayfish to discriminate between the two chimney types. In the absence of chimneys, significantly more crayfish showed an initial preference for mud saturated with water that had contained conspecifics, over similar mud saturated with distilled water. This indicates that chemical cues play a role in allowing these crayfish to discriminate between the conspecific-conditioned mud and the control wet mud. The preference of F. fodiens individuals for conspecific-built chimneys appears to be based, at least in part, on chemical cues, possibly in addition to tactile cues.     

Larval settlement of the green sea urchin, Strongylocentrotus droebachiensis, in the southern Gulf of Maine

Abstract. Settlement timing and differential settlement for the larval stage of the green sea urchin, Strongylocentrotus droebachiensis , in the southern Gulf of Maine was studied during the summer of 1996. Settlement densities on astroturf panels were highest in June and early July (13 to 37 m ⁻² d⁻¹), and peaked in mid-June (199 m⁻² d⁻¹). Settlement was low to nonexistent from mid-July through August (0 to 2 m⁻² d⁻¹). During the peak in settlement, no selection for substrate type was observed. In the remainder of the settlement period, differential settlement occurred, with a preference for substrate covered with live coralline algae. Test diameter of newly settled urchins varied among the substrates, with urchins settling on live coralline algae having the largest test diameter (0.43 ± 0.01 mm). There were no differences in test diameter among the different weeks in which sampling was done. Sustained onshore winds occurred only during peak settlement, suggesting that wind drift currents may concentrate larvae and influence patterns of larval settlement.     

Vertical transmission of cyanobacterial symbionts in the marine sponge Chondrilla australiensis (Demospongiae)

The cyanobacterial symbionts of the marine sponge Chondrilla australiensis (Demospongiae) were examined using fluorescent microscopy and Transmission Electron Microscopy. Unicellular cyanobacteria with ultrastructure resembling Aphanocapsa feldmannii occur in the cortex and bacterial symbionts are located throughout the mesohyl. In C. australiensis, the developing eggs are distributed throughout the mesohyl and are surrounded by nurse cells attached to them by thin filaments. The nurse cells form cytoplasmic bridges with the eggs, apparently releasing their contents into the egg cytoplasm. The presence of cyanobacterial and bacterial symbionts inside developing eggs and nurse cells in 25% of female Chondrilla australiensiswas established using Transmission Electron Microscopy, suggesting that these symbionts are sometimes passed on to the next generation of sponges via the eggs.     

Effects of Adult Dreissena polymorpha on Settling Juveniles and Associated Macroinvertebrates

The impact of Dreissena polymorpha settlement on recruitment of juvenile mussels and density of other macroinvertebrates was studied in field experiments using blank concrete blocks and tiles (control), blocks and tiles with attached empty zebra mussel shells, and blocks and tiles with attached living mussels. On blocks, dominant invertebrate taxa showed colonization patterns coinciding with increased habitat complexity owing to zebra mussel settlement or the biodeposition of faeces and pseudofaeces. Adult and especially juvenile zebra mussels preferred blocks with empty shells to blank blocks and blocks with living mussels; this might possibly be caused by a chemical cue that induces gregarious settlement. Lower recruitment on blocks with attached living mussels compared to blocks with only shells could be the consequence of ingestion of larvae by adult mussels and of competition for food. On tiles, the sediments deposited and the organic content of the sediment were investigated. Sedimentation was significantly higher on shell‐only and live‐mussel tiles compared to blank tiles. Organic matter differed significantly between blank and live‐mussel tiles.     

The influence of temperature on growth rate and length of larval life of the gastropod, Crepidula plana Say

The relationship between rate of larval development and the potential to prolong larval life was examined for larvae of the marine prosobranch gastropod Crepidula plana Say. Larvae were maintained in clean glass dishes at constant temperatures ranging from 12–29°C and fed either Isochrysis galbana Parke (ISO) or a Tahitian strain of Isochrysis species (T-ISO). Under all conditions, larvae grew at constant rates, as determined by measurements of shell length and tissue biomass. Most larvae eventually underwent spontaneous metamorphosis. Regardless of temperature, faster growth was associated with a shorter planktonic stage prior to spontaneous metamorphosis. Within an experiment, higher temperatures generally accelerated growth rates and reduced the number of days from hatching to spontaneous metamorphosis. However, growth rates were independent of temperature for larvae fed ISO at 25 and 29°C and for larvae fed T-ISO at 20 and 25°C. Where growth rates were unaffected by temperature, time to spontaneous metamorphosis was similarly unaffected. Maximum durations of larval life at a given temperature were shorter for larvae of Crepidula plana than for those of the congener C. fornicata (L.), although both species grew at comparable rates. Interpretations of the ecological significance of these interspecific differences in delay capabilities will require additional data on adult distributions and larval dispersal patterns in the field.