Microbial diversity in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by fluorescence in situ hybridization (FISH)

We have employed electronmicroscopical methods (SEM, TEM) to document the microbial community associated with the marine sponge Aplysina cavernicola (formerly Verongia cavernicola, class Demospongiae). Five dominant bacterial types were identified, three of which resemble the morphotypes originally described by Vacelet (1975). One bacterial type possesses morphological properties that are characteristic of the genus Planctomyces. In addition, morphologically uniform bacteria which reside inside the nuclei of host cells were observed. Using in situ hybridization with fluorescently labelled rRNA probes directed against known bacterial groups, the phylogenetic affiliation of the mesohyl bacteria was assessed. It could be shown that the vast majority of mesohyl bacteria belongs to the domain Bacteria with a low GC content. Among the Bacteria, the delta-Proteobacteria were most abundant, followed by the gamma-Proteobacteria and representatives of the Bacteroides cluster. Clusters of Gram-positive bacteria with a high GC content were also found consistently in low amounts. No hybridization signal was obtained with probes specific to the domain Archaea, to the alpha- and beta-Proteobacteria and to the Cytophaga/Flavobacterium cluster. This study describes for the first time the application of the “top-to-bottom approach” using 16S rRNA probes and in situ hybridization to assess the microbial diversity in Aplysina sponges. Received: 18 December 1998 / Accepted: 12 March 1999     

Characterizing the resident, fermentative microbial consortium in the hindgut of the temperate-zone herbivorous fish, Hermosilla azurea (Teleostei: Kyphosidae)

The zebraperch, Hermosilla azurea Jenkins and Evermann, a warm-temperate marine fish species with a strictly macroalgal diet, has a relatively long digestive tract with an enlarged hindgut and an associated blind caecum (HC). In zebraperch sampled off Santa Catalina Island, California (33°19′42′′N; 118°18′37′′W) in years 1995 through 2001, direct cell counts, gut epithelium assessment of bacterial attachment, and short-chain fatty acid (SCFA) analyses verified that the zebraperch HC possesses a dense and morphologically diverse, fermentative microbiota. Bacterial cell counts and morphological diversity were significantly higher in HC contents compared to anterior gut regions, suggesting that microbial populations were growing along the digestive tract. Similarly, electron micrographs of the HC epithelium revealed attached microbes, further supporting the possibility that these organisms constitute resident microbiota. Five different SCFAs were detected in all three regions of the digestive tract, but levels were up to three times greater in HC contents. Acetate was consistently the prevailing SCFA in all gut regions. Sequence analysis of bacterial 16S rDNA was used to identify predominant bacterial groups in HC contents. Of the seven main bacterial types identified, Enterovibrio spp. were the dominant bacteria in HC contents followed by species of Bacteroides,Faecalibacterium, and Desulfovibrio. Taken together, our findings show that the zebraperch HC harbors a consortium of microbes that appears to assist in the breakdown of algal polysaccharides in the herbivorous diet of the fish.     

Heavy metal contamination and physiological variability in the Brazilian mangrove crabs Ucides cordatus and Callinectes danae (Crustacea: Decapoda)

 Physiological studies were made on the crabs Ucides cordatus (L.) and Callinectes danae sampled from populations living in “polluted” mangroves on the southeast littoral of Brazil. Analysis of Cu, Cd, Zn, and Fe of sediments and crab tissues showed interspecific differences in tissue concentrations, and significantly higher levels of Cu, Cd, and Zn in “polluted” populations compared to “unpolluted” crabs living in uncontaminated mangrove in the same geographical area. Individuals of both species from the polluted site showed significantly greater capacities for regulating blood osmotic concentrations at low salinity (9‰). However, U. cordatus showed a reduced hypo-regulatory ability in 34‰S. Differences in ionoregulation were also seen. “Polluted”C. danae showed significantly higher Na/ K-ATPase levels in posterior gills compared to “unpolluted” crabs. Oxygen consumption rates (M˙ _O2) were elevated in U. cordatus, but depressed in C. danae from the “polluted” population. Individuals of both species from this site showed significantly lower O:N ratios, mainly because of an increased net efflux of ammonia. Adenylate energy charge (AEC) values of muscle and hepatopancreas in “unpolluted” and “polluted” populations of both species were not significantly different. These physiological differences are discussed in relation to the known acute physiological and metabolic effects of heavy metals in crustaceans, and interpretated in the light of possible adaptive changes following long-term exposure to contamination. Received: 6 August 1999 / Accepted: 22 June 2000     

Identification of the antifungal peptide-containing symbiont of the marine sponge Theonella swinhoei as a novel δ-proteobacterium, “Candidatus Entotheonella palauensis”

 The Palauan sponge Theonella swinhoei (class Demospongiae, order Lithistida, family Theonellidae) harbors filamentous bacterial symbionts that contain theopalauamide, an antifungal, bicyclic glycopeptide. In this study, the filamentous symbionts were shown to be novel bacteria belonging to the δ-subdivision of proteobacteria. The 16S rRNA gene sequence was determined using a combination of denaturing gradient-gel electrophoresis (DGGE) and specific polymerase chain-reaction (PCR) primers, and its source was confirmed by in situ hybridization. In a series of culture experiments, the filamentous bacteria were propagated in a mixed culture on agar plates. Related 16S rRNA gene sequences were isolated from related sponges with slightly different chemistry. The taxonomic status “Candidatus Entotheonella palauensis” is proposed for the theopalauamide-containing filamentous bacteria from T. swinhoei. Received: 12 June 1999 / Accepted: 22 January 2000     

Microstructure of settlement-marks in the otoliths of tropical reef fishes

The morphology and ultrastructure of the otolith settlement-mark was examined in 44 tropical reef-fish species spanning nine families. A classification scheme based on similar otolith characteristics is presented. Three major categories are identified based on changes in increment width and optical qualities of the settlement-mark. Of the 44 species examined, 39 possessed “abrupt” settlement-marks (Type I) characterised by a rapid decrease in increment width (up to 50% reduction) over settlement. Type I settlement-marks were found in all nine families examined. The 39 species spanned the whole range of possible larval durations (Pomacentrus moluccensis, 15 d ± 0 SE; Naso hexacanthus, 91.2 d ± 2.97 SE). Four of the 44 species possessed “zonal” settlement-marks (Type II), featuring a band of increments that are wider than pre-settlement increments. Species in this category are the labrids Corisaygula, Thalassoma bifasciatum, T. lunare and an unidentified acanthurid (Acanthurus sp. 2). One species of acanthurid (N. brevirostris) possessed a “gradual” settlement-mark (Type III), manifest as a gradual decrease in increment width during the settlement period. A possible fourth type was identified from the literature. Gnatholepis thompsoni and Coryphopterus glaucofraenum possessed a settlement-mark with increment widths that increased post-settlement. Available data suggest a poor relationship between the structure of the settlement-mark and the magnitude of metamorphosis (previously reported as internal and external morphological change). Evidence suggests that the increment profile over early development and the increment transitions associated with the settlement event are taxon-specific and may enable late-larval stage fishes to be identified to species level. Received: 21 May 1997 / Accepted: 3 February 1999     

Echolocation behavior of the bat <Emphasis Type="Italic">Vespertilio murinus</Emphasis> reveals the border between the habitat types “edge” and “open space”

We test the hypothesis that echolocation behavior can be used to find the border between bat habitats. Assuming that bats react to background targets in “edge space” but not in “open space”, we determined the border between these two habitat types for commuting individuals of the parti-colored bat Vespertilio murinus. We recorded sequences of bats’ echolocation signals while they flew parallel to the walls of large buildings and to the ground and determined the signals’ average bandwidth, duration, and pulse interval. These parameters varied systematically with the estimated horizontal and vertical distances between the bats and the background. A distinct effect of horizontal distance to the background on echolocation behavior was found for horizontal distances of less than 6 m, thus indicating the border between edge and open space. Only a few bats flew at vertical distances below 5 m. However, enough passages at vertical distances of 5 m and above indicated that the vertical border is somewhere below a distance of 5 m. Within edge space, V. murinus reacted to the background by reducing signal duration, increasing bandwidth at closer distances, and often emitting one signal per wing beat. In open space, signal parameters did not vary as a function of distance to the background. There, V. murinus emitted the longest signals with the narrowest bandwidth and often made one or two wing beats without emitting a pulse. With our data we support with statistical methods the hypothesis that echolocation behavior reveals the border between the habitat types “edge” and “open space”.     

Cytoenzymatic investigation of intracellular digestion in the symbiont-bearing hydrothermal bivalve <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis>

Invertebrates harbouring endosymbiotic chemoautotrophic bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. Bathymodiolids are dominants of the biomass at geochemically distinct vent sites of the Mid Atlantic Ridge (MAR) and thus are good candidates to study biological processes in response to site-specific conditions. To satisfy their nutritional requirements, these organisms depend to varying extent on two types of chemoautotrophic symbionts and on filterfeeding. The quantitative relationships of the nutritional modes are poorly understood. Using enzyme cytochemistry, electron microscopy and X-ray microanalysis, the structural and functional aspects of the cellular equipment necessary for lysosomal digestion was studied. We provide evidence for the following: (1) the basis of intracellular digestion of symbionts in Bathymodiolus azoricus from two geochemically distinct vent sites was not mainly in the large lysosomal bodies as previously thought (based on the membranous content resembling bacteria); (2) senescent bacteria are autolysed, possibly by bacterial acid phosphatase, that is more likely a cell cycling of the symbionts rather than an active lysosomal digestion by the host; (3) the consistent absence of hydrolases may indicate the improper use of the name “lysosome” for large vesicles at the base of the gill bacteriocytes (4) nutrient transfer in B. azoricus, therefore, may more likely be accomplished through leaking of metabolites from the symbiont to the host, not excluding lysosomal resorption of dead bacteria as an auxiliary strategy for organic molecule transfer; (5) evidence is provided for microvillar transfer of substances from the seawater that may indicate filter-feeding, in non-symbiotic ciliated gill cells of mussels from Lucky Strike; (6) two types of lysosomal vesicles can be distinguished in digestive cells based on their enzymatic content and their elemental composition.     

Spawning and development in <Emphasis Type="Italic">Osedax</Emphasis> boneworms (Siboglinidae,Annelida)

We report observations on spawning and early development in bone-eating worms of the genus Osedax. Individual females of Osedax rubiplumus were observed at 1820 m depth freely spawning hundreds of oocytes, and females of an undescribed species, Osedax “orange collar”, were observed spawning in laboratory aquaria. Cytological and molecular analysis of the spawned oocytes of two Osedax species revealed no evidence for the bacterial endosymbionts that the female worms require for their nutrition, suggesting that the bacteria must be acquired later from the environment, as they are in other siboglinids. Individual O. “orange collar” females released an average of 335 (±130) eggs per day, but the number of oocytes spawned per day varied greatly, suggesting that not all the females spawned daily. Fertilization rates of the spawned oocytes varied from 0 to 100%, though most females showed nearly 100% fertilization rates. Oocytes spawned in the laboratory at 4–6°C were negatively buoyant. If fertilized, these oocytes extruded polar bodies and then after at least four hours cleaved unequally. Subsequent cleavages occurred in a spiral pattern at roughly 2-h intervals, resulting in free-swimming trochophore larvae after 24 h. These lecithotrophic trochophores swam for 9–16 days before settling with several hooked chaetae, similar to those of dwarf Osedax males. The larval life span of the Osedax species studied in the laboratory appears to be shorter than in closely related Vestimentifera. Osedax rubiplumus, on the other hand, has much larger oocytes and so may have greater dispersal potential than these other Osedax species. The high fecundity and apparently continuous reproduction of Osedax boneworms permits the opportunistic exploitation of sunken vertebrate bones. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Linking abundance and diversity of sponge-associated microbial communities to metabolic differences in host sponges

Many sponge species contain large and diverse communities of microorganisms. Some of these microbes are suggested to be in a mutualistic interaction with their host sponges, but there is little evidence to support these hypotheses. Stable nitrogen isotope ratios of sponges in the Key Largo, Florida (USA) area grouped sponges into species with relatively low δ¹⁵N ratios and species with relatively high δ¹⁵N ratios. Using samples collected in June 2002 from Three Sisters Reef and Conch Reef in the Key Largo, Florida area, transmission electron microscopy (TEM) and denaturing gradient gel electrophoresis were performed on tissues of the sponges Ircinia felix and Aplysina cauliformis, which are in the low δ¹⁵N group, and on tissue of the sponge Niphates erecta, which is in the high δ¹⁵N group. Results showed that I. felix and A. cauliformis have large and diverse microbial communities, while N. erecta has a low biomass of one bacterial strain. As the low δ¹⁵N ratios indicated a microbial input of nitrogen, these results suggested that I. felix and A. cauliformis were receiving nitrogen from their associated microbial community, while N. erecta was obtaining nitrogen solely from external sources. Sequence analysis of the microbial communities showed a diversity of metabolic capabilities among the microbes of the low δ¹⁵N group, which are lacking in the high δ¹⁵N group, further supporting metabolic differences between the two groups. This research provides support for hypotheses of mutualisms between sponges and their associated microbial communities.     

Population and reproductive biology of two sibling amphipod species from ascidians and sponges

 The population dynamics and reproductive biology of an ascidian- and a sponge-dwelling amphipod were examined. The two undescribed amphipod species, Leucothoe“ascidicola” and L.“spongicola”, are closely related to each other, and occur in ascidians and sponges, respectively, along the Florida Atlantic coast. L. “ascidicola” was abundant in solitary ascidians during fall 1997, disappeared during spring/early summer, and became abundant again in September 1998. During the time when L. “ascidicola” were absent from their hosts, a copepod became a frequent inhabitant of the ascidians but disappeared again when L.“ascidicola” returned to the ascidians in September 1998. The numbers of L.“spongicola” in sponges increased substantially during spring, when high reproductive activity was observed. Following this reproductive peak, both adult and juvenile amphipods apparently left the sponges, and during the summer amphipod numbers in the sponges were very low. Another small amphipod species, which often co-occurred with L.“spongicola”, showed less seasonal variation and was found in sponges throughout the whole study period. The percentage of ovigerous females per host unit was usually lower in the ascidian-dwelling than in the sponge-dwelling amphipods. In solitary ascidians, L.“ascidicola” amphipods usually occurred in groups of several adults, yet there never was more than one ovigerous female per ascidian. In contrast, several ovigerous L.“spongicola” females were found to cohabit in the same spongocoel. This suggests that intrasexual aggression may be stronger among reproductive amphipod females in the ascidians than in sponges. The size distributions of juvenile cohorts indicate that juvenile L.“ascidicola” remain for relatively long time periods in the parental ascidian, where they may reach sexual maturity. In contrast, in L.“spongicola”, only cohorts of very small juveniles could be identified, indicating that juveniles disperse shortly after emerging from the female's brood pouch. It is concluded that extended parental care is of very short duration or does not occur in the sponge-dwelling amphipod L. “spongicola”, possibly because fast-growing sponges with a highly branched spongocoel system do not allow long-lasting coexistence of parent-offspring groups. In contrast, the discrete character of the solitary ascidians may enhance the potential for exclusion of other species, resource monopolization by reproductive females, and furthermore for long-lasting extended parental care in the ascidian-dwelling amphipod. Groups of single parents together with cohorts of large juveniles are reported in the literature for amphipods and isopods from brachiopods, bivalves and ascidians, suggesting that these discrete biotic microhabitats may favor the evolution of extended parental care in peracarid crustaceans. Received: 30 July 1999 / Accepted: 8 May 2000     

Functional morphology and phylogeny of the rock-boring bivalves <Emphasis Type="Italic">Leiosolenus</Emphasis> and <Emphasis Type="Italic">Lithophaga</Emphasis> (Bivalvia: Mytilidae): a third functional clade

The functional morphology of the shell of rock-boring mytilids (especially Leiosolenus and Lithophaga) is analyzed and compared with that of several epifaunal and semi-infaunal mytilids. Semi-infaunal species are generally intermediate between epifaunal and rock-boring ones both in terms of shell form and the magnitude of forces pulling the shells against the substratum. A molecular phylogenetic analyses using 18s rDNA sequence data strongly suggests that Leiosolenus and Lithophaga are monophyletic genera but that the so-called Lithophaginae (or Leiosolenus plus Lithophaga) is a paraphyletic group. The common cylindrical shell form of rock-boring species, which is here called “lithophagiform” as a third functional mytilid clade, may be due to convergence, as is the case with ‘mytiliform’ and ‘modioliform’.     

Zooplankton: pre-southwest and northeast monsoons of 1993 to 1994, from the North Arabian Sea

Zooplankton samples from the North Arabian Sea Environment and Ecosystem Research (NASEER) cruises were analyzed to determine the basic taxonomic composition, biomass (standing stock) and the total and copepod numeric abundance; these characteristics are discussed with reference to the different monsoon periods. Cruises carried out during March 1993 and May 1994, categorized as pre-southwest monsoon periods, and a cruise in December 1994, categorized as a northeast monsoon period, are discussed in detail. The biomass of January 1992 versus August 1992 and August 1992 versus March 1993 differed significantly (F = 6.44, P≤ 0.05). Ranges of highest and lowest biomass from each cruise are also given. Distinct “high” and “low” production areas of statistically significant difference (F = 12.67, P≤ 0.05) were observed. The “high” and “low” production areas were mobile and followed the surface wind circulation patterns (wind reversal pattern) during the northeast and southwest monsoon periods. Overall zooplankton showed a patchy distribution. The overall zooplankton abundance and total copepod counts differed significantly between the Cruises 3 versus 4 and 4 versus 5 (F = 15.67, P≤ 0.05 and F = 34.39, P≤ 0.05, respectively). There was no significant difference (P≥ 0.05) in biomass, between eutrophic and oligotrophic stations, suggesting no difference between near shore and offshore waters. Thirty-eight taxonomic groups were identified from the samples, with copepods being the most dominant group, followed by chaetognaths and siphonophores. Copepods constitute an average of 52.50 to 74.93% of the total zooplankton count and reach maxima of 92.14% of the total zooplankton count at the outset of the southwest monsoon (March 1993) and 91.39% at the outset of the active northeast monsoon (December 1994). Received: 27 February 1998 / Accepted: 8 October 1999     

Fine structure of the “button setae” in the deep-sea pelagic copepods of the genus Euaugaptilus (Calanoida: Augaptilidae)

 Many species of the carnivorous copepod family Augaptilidae, a primarily meso- and bathypelagic group, possess specialized “buttons” on the setae of their maxilla and maxilliped, which have been thought to be a simple cuticular outgrowth. The fine structure of the button setae in three species of Euaugaptilus was examined by light- and electron microscopy from samples collected in the eastern Indian Ocean and the subtropical western North Pacific. The buttons are arranged in two rows along the inner surface of the setae. There are differences in the shape, size, and arrangement of buttons among the three species, Euaugaptiluslaticeps, E.longimanus, and E.magnus. The button setae have an elaborate internal structure consisting of “setal shaft”, “stalk”, “disc”, and “outer membrane”, with microtubule bundles in the setal lumen, but have no muscular tissues. The disc has only pin-point contact to the core of the stalk, but has connection over a wider area with the fibrous tissue surrounding the core, and the contact area between the stalk and the setal shaft is also small. These structures suggest that the disc will move freely on the apex of the core of the stalk and the stalk may be movable at its connection to the setal shaft. All these components of the button setae may function in concert to fit the surface of the discs firmly to the surface of a prey, and to absorb the energy generated by the struggling prey. Received: 30 November 1999 / Accepted: 19 May 2000     

Cryptic speciation in the recently discovered American cycliophoran <Emphasis Type="Italic">Symbion americanus</Emphasis>; genetic structure and population expansion

Symbion americanus was recently described as the second species in the phylum Cycliophora, living commensally on the American commercial lobster Homarus americanus. A previous genetic analysis of American and European populations of cycliophorans suggested that haplotype divergence in S. americanus was much greater than in its European counterpart S. pandora. This study examined the population structure and demographics of 169 individuals thought to belong to S. americanus collected from lobsters over 13 North American localities (Nova Scotia, Canada to Maryland, USA) between October 2003 and January 2006. Cytochrome c oxidase subunit I sequence data clearly suggested the presence of three cryptic lineages in a species complex, often co-occurring in the same lobster specimens. One of these lineages, named the “G” lineage, was represented by very few individuals and therefore was excluded from subsequent statistical analyses. The other two sympatric lineages, named the “T” and “C” lineages, showed different population structure and demography. Although limited geographic structure was found in the T lineage, the C lineage showed higher nucleotide and haplotype diversity values, as well as more variation between localities. The data also indicated that the T lineage underwent a recent population expansion, suggesting that the C and T lineages may have speciated in allopatry but a subsequent population expansion may have been responsible for their current sympatric distribution. Studies on the anatomy and ecology of the sympatric lineages of this species complex should provide further information on the identity of the holotype of S. americanus, which currently cannot be ascribed to any of the three cryptic lineages.     

Toxicity of the venom in three neotropical Crematogaster ants (Formicidae: Myrmicinae)

Bioassays were performed to investigate and compare the toxic properties of the contact venoms of three Neotropical Crematogaster species (C. sp. prox. abstinens, C. distans and C. brevispinosa rochai), whose main venom compounds are chemically different (long chain derivatives linked to an aldehyde or a primary acetate, and furanocembrenoid diterpenes, respectively). Different quantities of venom were topically applied on the bodies of three target insect species (workers of the termite Nasutitermes ephratae, workers of the ant Solenopsis sp., “media” and “major” workers of the ant Cephalotes pusillus). The toxicity of the venom greatly varied according to the Crematogaster species, the venom of C. b. rochai showing the highest toxic activity towards all target species. The sensitivity of the target species also greatly varied, the workers of N. ephratae showing a great sensitivity to the venoms, while the heavily armoured workers of the ant C. pusillus, particularly “major” ones, were resistant. Cuticle thickness was found to be a key factor in the resistance to the toxic effects of Crematogaster venom. Sensitivity of the target workers was also directly related to the amount of venom applied to their cuticle. Workers of C. distans were resistant to topical application of their own venom, while C. sp. prox. abstinens and C. b. rochai were not. In all cases, the venoms were slow acting, as several hours to one or even several days were needed to obtain a significant mortality. The main biological activities of the Crematogaster venom could be related to more immediate repellent properties, as recently shown with the European species C. scutellaris.     

Seasonal and interannual variations in size,biomass and chemical composition of the eggs of North Sea shrimp, <Emphasis Type="Italic">Crangon crangon</Emphasis> (Decapoda: Caridea)

In the shrimp Crangon crangon, an important fishery resource and key species in the southern North Sea, we studied temporal variations in size, biomass (dry weight, W) and chemical composition (C, N, protein and lipid) of eggs in an initial embryonic stage. Data from 2 years, 1996 and 2009, consistently revealed that egg size and biomass varied seasonally, with maxima at the beginning of the reproductive season (January), decreasing values throughout spring, minima in June–July, and a slight increase thereafter. This cyclic pattern explains why “Winter eggs” are on average larger and heavier than “summer eggs”. Using a modelling approach, we estimated the duration of oogenesis in relation to seasonally changing seawater temperatures. According to an additive model of multiple explanatory variables, the C content per newly laid egg showed in both years a highly significant negative relationship with day length (r2 = 0.38 and 0.40, respectively; P < 0.0001), a weak positive relationship with temperature (r2 = 0.08 and 0.09; P < 0.05), and a weak negative relationship with phytoplankton biomass (r2 = 0.11 and 0.12; P < 0.05) at the estimated time of beginning oogenesis. Phenotypic plasticity in initial egg size and biomass is interpreted as an adaptive reproductive trait that has evolved in regions with strong seasonality in plankton production and periods of larval food limitation. In contrast to biomass per egg, the percentage chemical composition remained similar throughout the reproductive period. Both the absolute and percentage values also showed significant interannual variations, which caution against generalizations based on short-term studies of reproductive traits of C. crangon and other species of shrimp.     

Morphological differences between females of different <Emphasis Type="Italic">Jaera</Emphasis> species (Crustacea: Asellota: Isopoda) in the White Sea: a possible solution to an old problem

It is well known that females of “Jaera albifrons” species complex cannot be determined on the basis of morphological analysis. In this work, an attempt to separate females of three Jaera species on the basis of morphometric characters, the length of so-called “coupling zones” (CZ), was made. Coupling zones are those parts of the partners’ bodies that contact during copulation. Female CZ is a region between the posterior edge of the pleotelson and the border between the fourth and the fifth thoracic segments, where openings of dorsal vaginas are situated. Male CZ extends from the anterior edge of the cephalon to the first abdominal segment carrying the copulative organ. Absolute and relative lengths of CZ were used. The latter was calculated as ratio of absolute length of CZ to body length. Two settlements of Jaera in the White Sea were studied for 3 years. Species composition of these populations was quite different (the first one was dominated by J. ischiosetosa and the second by J. albifrons and, 1 year by J. praehirsuta). Males of different species were shown to be distinct in terms of absolute and relative length of CZ. Frequency distributions of absolute and relative CZ length of females differed significantly in the two settlements. The experiments based on male choice in mixed samples were organized. They revealed that females chosen by males of different species differ significantly by CZ parameters.     

Inhibition and facilitation of bryozoan and ascidian settlement by natural multi-species biofilms: effects of film age and the roles of active and passive larval attachment

Sessile marine invertebrate larvae can recognize suitable settlement substrata by using various environmental cues, including organic/microbial biofilms. In laboratory choice assays, the effect of biofilms of varying ages on the settlement behaviour of two fouling organisms was assessed. The species included the arborescent cheilostome bryozoan Bugula flabellata (Thompson) and the solitary ascidian Ciona intestinalis (L.), both of which are characteristic of temperate sublittoral hard substratum assemblages in northwest Europe. Experiments were carried out using polystyrene petri dish substrata preconditioned with multispecies biofilms from natural laboratory-aquarium seawater for 1, 3, 6 or 12 d. Unfilmed (new, initially sterile) dishes were used as control substrata. Whereas the coronate larvae of B. flabellata generally were inhibited by biofilming, irrespective of film age, the settlement of tadpole larvae of C. intestinalis was facilitated on biofilmed substrata, and numbers of settled larvae generally increased with biofilm age: the highest mean numbers were counted on 12 d-old biofilms. In C. intestinalis, settlement and metamorphosis are processes which can be temporally separate and are possibly induced by different environmental cues. This study therefore distinguished between C. intestinalis larvae which were attached to the biofilm surface by the anterior, and those larvae entrapped by the biofilm but not settled in the conventional meaning of the term. As reported in previous studies, we did observe that such entrapped larvae could subsequently attach and develop successfully into sessile juveniles. Both the numbers of “attached” and “trapped” tadpoles increased with biofilm age. Assuming that “settlement” is essentially a process involving the active behavioural response of larvae to environmental cues, it seems that the facilitated attachment of C. intestinalis onto biofilmed substrata is due to the combined effect of active habitat selection and passive deposition/“entrapment” of larvae onto the “sticky” substratum. Received: 21 August 1996 / Accepted: 21 November 1996     

Evolutionary relationships of deep-sea hydrothermal vent and cold-water seep clams (Bivalvia: Vesicomyidae): results from the mitochondrial cytochrome oxidase subunit I

Phylogenetic relationships among vesicomyid clams (Bivalvia: Vesicomyidae) and their placement within the order Heterodonta were examined using mitochondrial encoded cytochrome oxidase subunit I (COI) DNA sequences. The presently analyzed vesicomyids represent a recent monophyletic radiation that probably occurred within the Cenozoic. Nucleotide phylogenetic analyses resolved discrete clades that were consistent with currently recognized species: Calyptogena magnifica, C. ponderosa, Ectenagena extenta, C. phaseoliformis, Vesicomya cordata, Calyptogena n. sp. (Gulf of Mexico), C. kaikoi, C. nautilei, C. solidissima and C. soyoae (Type-A). However, specimens variously identified as: V. gigas, C. kilmeri, C. pacifica, and V. lepta comprised two “species complexes”, each composed of multiple evolutionary lineages. Most taxa are limited to hydrothermal-vent or cold-seep habitats, but the “vent” versus “seep” clams do not constitute separate monophyletic groups. Current applications of the generic names Calyptogena, Ectenagena, and Vesicomya are not consistent with phylogenetic inferences. Received: 24 July 1997 / Accepted: 22 August 1997     

Phylogenetic analyses of potentially free-living <Emphasis Type="Italic">Symbiodinium</Emphasis> spp. isolated from coral reef sand in Okinawa,Japan

The existence of “free-living” Symbiodinium that can form symbioses with hosts is implied by the presence of hosts that produce Symbiodinium-free gametes and expulsion and/or expelled symbiotic algae from host. However, it is still unclear if potentially symbiotic Symbiodinium are found “free-living” in the coral reef environment. Sixteen Symbiodinium strains were established from samples taken from three sampling locations of coral reef sand in Okinawa, Japan. Phylogenetic analyses of the partial large subunit ribosomal DNA (28S-rDNA) and the internal transcribed spacer of ribosomal DNA (ITS-rDNA) conclusively showed that all 16 isolates belonged to Symbiodinium clade A sensu Rowan and Powers (1991). The lack of other Symbiodinium clades besides clade A in this study may be due to other clades not being readily culturable under culture conditions used here. The new isolates could be phylogenetically divided into four groups, though no sequences were identical to previously reported Symbiodinium. Two of the four groups were closely related to symbiotic Symbiodinium clade A isolated from a variety of host species. One isolate group formed a highly supported monophyly with Symbiodinium types that have previously been characterized as “free-living”. The remaining isolate group, although within clade A, was quite divergent from other clade A Symbiodinium. These results indicate that novel diversity of free-living Symbiodinium exists in coral sand.