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     

Impact of conditions of cultivation and adsorption on antimicrobial activity of marine bacteria

The potency of free-living and animal-associated marine bacteria to produce antimicrobial substances has been studied in 491 strains isolated from northern and southern parts of the Pacific Ocean. A total of 26% (126 out of 491) of the strains examined produced antimicrobial compounds against 11 test bacterial strains (TBS) including the fish pathogens Aeromonas hydrophila and Vibrio anquillarum. Antimicrobial substances (AS) produced by marine bacteria were especially active against Staphylococcus epidermidis, Proteus vulgaris, Enterococcus faecalis, and Candida albicans. Twelve strains, isolated from different sources, were chosen as promising candidates, producing a number of AS. Production of AS varied within 24 to 72 h, increasing in a culture medium based on natural sea water with Br-ions, and after attachment to polymeric surfaces. In order to study the influence of adsorption, selected strains with a high potential for antimicrobial production were cultivated on polymeric surfaces with different hydrophobicities and chemical functionalities. These parameters of the surface hydrophobicity (measured by means of water contact angles) and chemical functionality of the surfaces were manipulated using the photo- and thermochemistry of a polymeric system (diazo-naphto-quinone/novolak) commonly used as a photoresistant material in semiconducto-manufacturing. The highest antimicrobial activities occurred on hydrophilic surfaces (standard exposed photoresistant films), whereas the number of attached cells was greater on hydrophobic surfaces, characterized as unexposed resistant films. These results suggest that the chemical nature of induced hydrophilicity may also be a major factor in controlling antimicrobial activity of adsorbed bacteria. Received: 5 March 1997 / Accepted: 24 August 1997     

Role of bacteria in larval settlement and metamorphosis of the polychaete Hydroides elegans

The serpulid polychaete Hydroides elegans Haswell, 1883 is an early colonist of new substrata in Pearl Harbor, Oahu, Hawaii. When metamorphically competent, larvae of H. elegans will settle rapidly upon an acceptably biofilmed surface, but not on a clean surface. In this study we found the ability of larvae to respond selectively to inductive surfaces to be retained for at least 3 wk. Of a series of bacterial strains isolated from Hawaiian marine biofilms, 13 induced larval settlement, 11 gave moderate or mixed results, and 10 others did not stimulate the settlement of H. elegans. The amount of settlement induced by monospecific strains was rarely as great as with natural, multispecies films. Most of the isolated bacteria were motile Gram-negative rods, but Gram-positive strains were also present, and diverse metabolic types were represented in the study. Biofilms killed by treatment with heat, ultraviolet radiation or chemical fixatives were no longer inductive. Soluble, dialyzable, heat-stable bacterial products induced settlement and metamorphosis more slowly. The range of bacteria producing an inductive signal suggests either that there are multiple cues, or that the cue is common to many bacteria. Chemical signals characteristic of early microbial biofilms may indicate freshly available substrata with optimal potential for the growth and survival of H. elegans. Received: 30 January 1998 / Accepted: 12 September 1998     

Thraustochytrid protists as a component of marine microbial films

Although ubiquitous in the marine environment, the presence and importance of thraustochytrid fungoid protists in primary film formation of freshly immersed surfaces in the sea have not been investigated. We isolated thraustochytrids from surfaces of glass, aluminium, mild steel and fibre glass panels immersed in sea water for 1 to 4 d, but not from those of copper and cupro-nickel. Microscopic examination of the primary film on glass surfaces revealed the presence of thraustochytrids. Thraustochytrids grew to varying population densities on surfaces of glass, aluminium and fibre glass in the laboratory. Scanning electron microscopy of the growth of Ulkenia profunda Gaertner, isolated from the primary film, on surfaces of glass and aluminium suggested that cells of the thraustochytrid attached directly to the surfaces, without producing copious extracellular polysaccharides. The presence of ectoplasmic net elements was not a prerequisite for attachment of cells to surfaces. Cell surface hydrophobicity of the thraustochytrid, as estimated by the MATH assay, might play an important role in adhesion. The presence of thraustochytrid cells on a polystyrene surface markedly induced settlement of barnacle larvae (Balanus amphitrite), as compared to barnacle extract and a control. Received: 26 July 1999 / Accepted: 21 January 2000     

Interactions between shear rates and biofilms affecting exploratory behaviour by cyprids ofElminius modestus (Cirripedia)

The behaviour ofElrninius modestur Darwin cyprids suggests interaction between surface bacterial films acclimated to contrasting shear regimes and the range of shear rates over which observations were made. Exploratory behaviour is encouraged by filmed surfaces rather than biologically clean surfaces. Increasing shear rates encourage attachment to all surfaces, but cyprids detach from high-shear-acclimated films less often than from either low-shear-acclimated films, or clean surfaces. Furthermore, numbers of attaching cyprids are consistently lower on low-shear-acclimated films than on high-shear films.     

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.     

High recovery of culturable bacteria from the surfaces of marine algae

The culturability of heterotrophic marine bacteria obtained from the surfaces of two species of marine algae (Lobophora variegata andHalimeda copiosa) was assessed by comparing total DAPI-stained cell counts to colony-forming bacterial counts on two agar media. The colony-forming bacterial counts on a low-nutrient medium (LN) consisting of seawater and agar were significantly greater for both algal species than counts obtained on a high-nutrient medium (HN) similar in composition to that typically used for the isolation of heterotrophic marine bacteria. On average, 14 and 58%, respectively, of the total bacteria fromL. variegata andH. copiosa were culturable on LN. These recovery rates far exceed those typically reported for marine bacteria. Of 119 LN strains obtained in pure culture, 55% failed to grow on HN. The yeast extract component of HN (1.5 gl^-1) was responsible for the majority of the observed inhibition, suggesting that this nutrient can be highly toxic to marine bacteria. Eighty-nine percent of the strains inhibited by HN were capable of growth when the nutrients in this medium were diluted by a factor of 100 with seawater. Of 66 epiphytic strains, 30 (45%) initially inhibited by HN showed the ability to adapt to this medium after a period of laboratory handling. The initial inability of low-nutrient-adapted bacteria to grow on high-nutrient media may be due to nutrient shock. The results presented here indicate that the culturability of specific populations of marine bacteria can be dramatically improved by the use of low-nutrient media. Further, the importance of developing new medium formulations that eliminate traditional nutrients, some of which are clearly toxic to bacteria, is demonstrated.     

Spatial and temporal patterns of reproduction,larval settlement,and recruitment of the compound ascidian Aplidium stellatum

Spatial and temporal variation in recruitment of the compound ascidian Aplidium stellatum was examined on a shallow-water limestone outcropping in the Northeastern Gulf of Mexico from 1983 to 1985. Fifty-two percent of the recruits appeared on vertical surfaces, which were rare at this site. In the laboratory, tadpole larvae of A. stellatum consistently settled on vertical vs horizontal surfaces in a 2 to 1 ratio, regardless of the area of vertical surface offered. This settlement response was insufficient to account entirely for the field recruitment pattern, suggesting greater mortality of newly-settled individuals or larvae on horizontal vs vertical surfaces. Fifty-six percent of the variation in recruitment over 21 months could be explained by variation in the percentage of zooids brooding larvae in adult colonies. Over 28 consecutive months, the percentage of zooids brooding larvae was positively correlated with egg counts of the previous month (r ²=0.75), which in turn were weakly correlated with monthly average water temperature (r ²=0.36). These results suggest that larval production contributed substantially to temporal variation in recruitment of A. stellatum. The short larval life of tadpoles of A. stellatum and the relative isolation of the population were probably responsible for the correlation between recruitment and larval production.     

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     

Influence of substratum heterogeneity and settled barnacle density on the settlement of cypris larvae

On the Atlantic coast of Canada, Semibalanus balanoides (L.) is widely distributed in the mid-intertidal zone, whereas in the Gulf of St. Lawrence, this species is mostly limited to crevices. We tested the hypothesis of regional differences in microhabitat selection by barnacle larvae at settlement in 1984 and 1985 at St. Andrews, New Brunswick, Canada. Since larvae settle in microhabitats already colonized by adults, the relative influence of settled barnacle density and of different scales of substratum heterogeneity on settlement were evaluated experimentally at Capucins, Québec, (Gulf of St. Lawrence) and at St. Andrews, New Brunswick (Atlantic coast). On a large scale (>10 cm deep crevices) of heterogeneity, results show that, in the Gulf, cypris larvae settled nearly exclusively (93%) in natural crevices rather than on adjacent horizontal surfaces. On the Atlantic coast, settlement was more important outside than inside of crevices, when the substrata were either natural or artificial. This result is unique and contrasts sharply with all known reports on barnacle settlement in relation to surface contour. The influence of barnacle density on settlement was greater than that of large scale heterogeneity. On a small scale (<1.5 cm deep cracks), the presence of conspecifics had a stronger effect on settlement than heterogeneity in both regions. Field observations showed a relationship between larval settlement density and percentage of adult cover. Settlement increased up to 22 or 30% (Gulf and Atlantic coast) of adult cover and decreased afterwards. The results confirm the hypothesis of larval selection for cryptic habitats in the Gulf and the opposite behaviour (preferences for horizontal surfaces) on the Atlantic coast. This microhabitat selection is apparent at large scales of heterogeneity, whereas at small scales, the presence of conspecifics is the predominant factor.Contribution to the programme of GIROQ (Groupe interuniversitaire de recherches océanographiques du Québec)     

Association of bacteria with larvae of the gutless protobranch bivalve Solemya reidi (Cryptodonta: Solemyidae)

Rod-shaped bacteria were consistently observed by transmission electron microscopy in the locomotory test of larvae and in the perivisceral cavity of post-larvae of Solemya reidi, a gutless protobranch bivalve known to possess intracellular chemoautotrophic bacterial symbionts in the adult gill. Bacteria develop within granular vesicles in the larval test, where they either remain to be ingested at metamorphosis, or are released into the space separating the test and embryo, to be subsequently ingested through the larval mouth. In either case, bacteria lie within the perivisceral cavity following metamorphosis. Bacteria were not seen either in or on gametes or in gills of juveniles. It is hypothesized that these bacteria represent a transmission stage of the gill symbionts present in adult S. reidi and are not evident in gametes or gills of juveniles due to cryptic packaging within granular vesicles. Perpetuation of this symbiosis would therefore be assured through vertical transmission, as is typical of other marine invertebrate-bacteria endosymbioses.Harbor Branch Oceanographic Institution Contribution No. 602     

A finite element method to solve a population dynamics stage-structured model of intertidal barnacles

Many marine organisms are fixed or highly sedentary as adults but the adult population may be strongly dependent on the oceanic transport of planktonic larvae. In order to assess interactions between oceanographic and biological processes that determine the population dynamics of marine organisms with a sessile adult phase restricted to the coastline and a planktonic larval phase, we present a stage-structured finite element model for the barnacle Balanus glandula that inhabits the rocky intertidal zone of central California, USA.     

High-frequency observations of early-stage larval abundance: do storms trigger synchronous larval release in <Emphasis Type="Italic">Semibalanus balanoides</Emphasis>?

The acorn barnacle, Semibalanus balanoides, is thought to release larvae in response to phytoplankton blooms, but there is evidence that another, unidentified cue for release may exist. We conducted high-frequency sampling in Little Harbor, Massachusetts, USA, to determine whether early-stage larval abundance was related to several environmental variables, and to characterize vertical distributions of the larvae. Larval concentrations peaked at 2.52 and 1.02 individuals l⁻¹ during two storms. Larvae were more abundant near the surface than near the bottom. We suggest the hypothesis that turbid conditions and upward-swimming behavior may protect newly-released larvae from predation and cannibalism. Future studies should test this hypothesis with barnacles and other invertebrates.     

Antimicrobial activity of extracts of Caribbean gorgonian corals

Extracts of 39 species of Caribbean gorgonians were tested for antimicrobial activity against 15 strains of marine bacteria. The bacteria consisted of three opportunistic pathogens, Vibrio parahaemolyticus, Leucothrix mucor, and Aerococcus viridans, and 12 strains isolated from either healthy or decayed gorgonians. Overall, only 15% (79 out of 544) of the tests resulted in antibacterial activity with 33% (13 out of 39) of the gorgonians inhibiting only one bacterial strain and 23% (9 out of 39) showing no activity. The extracts of four Pseudopterogorgia species showed relatively high levels of activity, inhibiting 43 to 86% of the bacterial strains. The potency of the active Pseudopterogorgia species was variable, however, and three additional Pseudopterogorgia species were inactive against all bacterial strains. With the exception of one sensitive strain, Vibrio species were resistant to gorgonian metabolites. Our results indicate that organic extracts of most Caribbean gorgonians do not possess potent, broad-spectrum antibacterial activity inhibitory to the growth of opportunistic marine pathogens and bacteria associated with healthy and decayed gorgonian surfaces. These findings suggest that the inhibition of bacterial growth is not the primary ecological function of gorgonian secondary metabolites and that bacteria may not be important selective agents in the evolution of gorgonian secondary chemistry.     

Effects of algal and larval densities on development and survival of asteroid larvae

Effects of larval and algal culture density and diet composition on development and survival of temperate asteroid larvae were studied in the laboratory at Santa Cruz, California, USA, during summer and fall of 1990. Larvae of Asterina miniata were reared at two densities, 0.5 or 1.0 ml^-1, and fed one or two species of cultured phytoflagellates — Dunaliella tertiolecta alone or mixed with Rhodomonas sp. — at three concentrations of 5x10², 5x10³, and 5x10⁴ total cells ml^-1. Algal concentration strongly influenced larval development; however, larval density also had a marked effect. Development progressed further with increasing algal concentration. Larval growth and differentiation were sometimes uncoupled; i.e., growth measures were directly related to food level, while differentiation indicators were less so. At the lowest food level, growth was negative and differentiation was arrested at early precompetent stages; these larvae never formed juvenile rudiments or brachiolar attachment structures. Development times of larvae given more food ranged from 26 to 50 d and depended directly on food availability. Development time to metamorphosis at the highest food concentration was similar for siblings fed D. tertiolecta alone or mixed with Rhodomonas sp. In contrast, when food level was an order of magnitude lower, larvae fed the algal mixture metamorphosed significantly earlier than larvae fed the unialgal diet. This suggests interactive effects of food quantity and food quality. Survival was little affected by larval or food density, except at the lowest ration. Feeding experiments in well-controlled laboratory conditions are useful to predict and compare the physiological or developmental scope of response of larvae to defined environmental factors; however, results from such studies should not be extrapolated to predict rates and processes of larval development in nature.     

Implications of life history for genetic structure and migration rates of southern African coastal invertebrates: planktonic,abbreviated and direct development

The amount of genetic structure in marine invertebrates is often thought to be negatively correlated with larval duration. However, larval retention may increase genetic structure in species with long-lived planktonic larvae, and rafting provides a means of dispersal for species that lack a larval dispersal phase. We compared genetic structure, demographic histories and levels of gene flow of regional lineages (in most cases defined by biogeographic region) of five southern African coastal invertebrates with three main types of larval development: (1) dispersal by long-lived planktonic larvae (mudprawn Upogebia africana and brown mussel Perna perna), (2) abbreviated larval development (crown crab Hymenosoma orbiculare) and (3) direct development (estuarine isopod Exosphaeroma hylecoetes and estuarine cumacean Iphinoe truncata). We hypothesized that H. orbiculare, having abbreviated larval development, would employ a strategy of larval retention, resulting in genetic structure comparable to that of the direct developers rather than the planktonic dispersers. However, regional population structure was significantly lower in all species with planktonic larvae, including H. orbiculare, than in the direct developers. Moreover, nested clade analysis identified demographic histories resulting from low levels of gene flow (isolation by distance and allopatric fragmentation) in the direct developers only, and migration rates were significantly higher in all three species having planktonic larvae than in the direct developers. We conclude that the amount of genetic structure within marine biogeographic regions strongly depends on the presence or absence of free-swimming larvae. Whether such larvae are primarily exported or retained, whether they have long or short larval duration, and whether or not they are capable of active dispersal seems to have little effect on connectivity among populations.     

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.     

Waterborne chemical compounds in tropical macroalgae: positive and negative cues for larval settlement

Settlement sites of marine invertebrate larvae are frequently influenced by positive or negative cues, many of which are chemical in nature. Following from the observation that many shallow-water, Hawai'ian marine macroalgae are free of fouling by sessile invertebrates, we predicted that the algae are chemically protected and dependent on either surface-bound or continuously released soluble compounds to deter settling invertebrate larvae. To address the importance of waterborne algal compounds, we experimentally determined whether larvae of two of Hawai'i's dominant hard-surface fouling organisms, the polychaete tube worm Hydroides elegans and the bryozoan Bugula neritina, would settle in the presence of waters conditioned by 12 species of common Hawai'ian macroalgae (representing the Phaeophyta, Chlorophyta, Rhodophyta and Cyanophyta). The results included a full spectrum of biological responses by each larval species to waterborne algal compounds. Larval responses to conditioned water were consistent for each algal species, but the outcomes were not predictable based on the taxonomic relationships of the algae. For example, among the species of Phaeophyta examined, different conditioned waters were: (1) toxic, (2) inhibited settlement, (3) simulated settlement, or (4) had no effect, compared to larvae in control dishes containing filtered seawater. Additionally, larval responses to aged (24 h) conditioned waters could not be predicted from the results of assays run with conditioned waters utilized immediately after preparation. Finally, settlement by larvae of one species did not predict outcomes of tests for the other species. Four of 12 shallow-reef Hawai'ian macroalgae tested released compounds into surrounding waters that immediately killed or inhibited settlement by both H. elegans and B. neritina (toxic: Dictyota sandvicensis; inhibitory: Halimeda discoidea, Sphacelaria tribuloides, Ulva reticulata); the remaining 8 algal species prevented settlement by one of these fouling organisms but for the other had no effect or, in some cases, even stimulated settlement     

Growth,dispersal, and identification of planktonic labrid and pomacentrid reef-fish larvae in the eastern Pacific Ocean

Planktonic larvae of six genera of labrid and pomacentrid reef fishes were captured in march 1985 in the eastern Pacific Ocean several hundred kilometers from the nearest reefs. The larvae were identified to genus by fin-ray counts as well as by comparison of their larval otolith morphology with that of known species. The larval otolith morphologies of known species were derived from measurements of the larval otolith embedded within the otoliths of settled juveniles (as delineated by the daily otolith-increment marks corresponding to the late larval period). The body morphology and melanophore patterns of the eastern Pacific labird and pomacentrid larvae closely matched those of congeneric larvae described from other oceans. Growth rates of larvae less than about 70 d old were similar between taxa (from 0.13 to 0.19 mm d^-1). After about 70 d in the plankton, labrid larvae grew much more slowly (0.06 mm d^-1 in Xyrichtys sp.). Labrid larvae had long larval durations (up to 131 d in Xyrichtys sp.), while the larval lives of the pomacentrids appeared to be shorter and much less variable. Larvae of many different ages occurred within the same water mass, and young cohorts of larvae appeared continuously over the sampling period. Some larvae were as young as 21 d, indicating that reef-fish larvae are capable of rapid long-distance dispersal (at least 18 km d^-1).     

An experimental evaluation of transgenerational isotope labelling in a coral reef grouper

Transgenerational isotope labelling (TRAIL) using enriched stable isotopes provides a novel means of mass-marking marine fish larvae and estimating larval dispersal. The technique, therefore, provides a new way of addressing questions about demographic population connectivity and larval export from no-take marine protected areas. However, successful field applications must be preceded by larval rearing studies that validate the geochemical marking technique, determine appropriate concentrations and demonstrate that larvae are not adversely affected. Here, we test whether injection of enriched stable barium isotopes (¹³⁵Ba and ¹³⁷Ba) at two dose rates produces unequivocal marks on the otoliths of the coral reef grouper Epinephelus fuscoguttatus. We also assess potential negative effects on reproductive performance, egg size, condition and larval growth due to injection of adult female fish. The injection of barium isotopes at both 0.5 and 2.0 mg Ba/kg body weight into the body cavities of gravid female fish was 100% successful in the geochemical tagging of the otoliths of larvae from the first spawning after injection. The low-dose rate produced no negative effects on eggs or larvae. However, the higher dose rate of 2 mg Ba/kg produced small reductions in yolk sac area, oil globule area, standard length and head depth of pre-feeding larvae. Given the success of the 0.5 mg Ba/kg dose rate, it is clearly possible to produce a reliable mark and keep the concentration below any level that could affect larval growth or survival. Hence, enriched Ba isotope injections will provide an effective means of mass-marking grouper larvae.