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.     

Induction of larval settlement and metamorphosis in the donkey-ear abalone,Haliotis asinina Linnaeus,by chemical cues

The effects of the chemical inducers, gamma-aminobutyric acid (GABA) and potassium chloride (KCl), on the larval settlement and metamorphosis of the donkey-ear abalone, Haliotis asinina, was investigated. H. asinina larvae (5–6 h post-hatch) were exposed to a range of GABA (0.125–2.00 μM) and KCl (1.00–12.00 mM) concentrations for 72 h. Results of the dose response experiments showed that settlement and metamorphosis vary according to the dose levels of the inducer compounds. Under controlled laboratory conditions, 0.45–0.50 μM and 6.0 mM seemed to be the optima for GABA and KCl, respectively, as these concentrations elicited the greatest number of postlarvae that metamorphosed, settled or survived. However, GABA generally promoted better attachment and metamorphic response as well as survival than KCl in H. asinina postlarvae.     

Degradation of gamma-aminobutyric acid (GABA) by marine microorganisms

Abstract By degrading the settlement inducer gamma-aminobutyric acid (GABA), bacteria may affect the larval settlement of sedentary marine invertebrates. Nearly one third of bacterial isolates from surfaces suitable for abalone ( Haliotis ) settlement were able to grow on GABA as sole carbon source. Compared with similar compounds, GABA was a good source of carbon, nitrogen and energy, and it was utilized concomitantly with glucose. GABA-metabolizing enzymes were constitutive in Pseudomonas fluorescens and inducible in Aeromonas hydrophila . High-affinity ( K _m: 20–50 μ M) and low-affinity ( K _m: 7–9 mM) uptake systems were produced in response to low and high GABA concentrations, respectively, in the growth medium. Within the ecologically significant temperature range (12–24°C), specific GABA degradation rates varied 2.5-fold in young cells of P. fluorescens . This organism los its ability to degrade GABA during the stationary phase. The results suggest that marine bacteria have the potential to affect invertebrate larval settlement by removing GABA from the settlement habitat.     

Improvements to a passive trap for quantifying barnacle larval supply to semi-exposed rocky shores

Planktonic cyprid larvae of the intertidal barnacle, Semibalanus balanoides (L.) and the infralittoral/sublittoral Balanus crenatus Brug., are readily captured in small, chambered, passive traps deployed on rocky shores. Several structural developments of a previous trap design improved urea (killing solution) retention and capture of cyprids of both species. The design adopted for assessment of the larval supply/settlement relationship of the focal species, S. balanoides, included additional internal baffles and the replacement of the cylindrical trap opening with a coned aperture. Three sizes of cone aperture area were compared, with the intermediate size (1 cm²) being optimal for measuring larval supply over the full range of wave action on local shores. Most daily catches included larvae that were recognizable as having been dead before they were trapped, and had recently passed through the guts of grazing limpets (Patella vulgata L.). One site showed that ∼32% of trapped S. balanoides were so-called ‘faecal’ cyprids. An inability to distinguish these in trap samples would lead to a significant over-estimation of larval supply. Preliminary data indicated that S. balanoides larval supply to the intertidal is enhanced by onshore winds (or perhaps increased wave turbulence), whereas B. crenatus supply was positively correlated with offshore winds (or decreased turbulence). A clear ‘supply/settlement’ relationship for S. balanoides at six sites along ∼40 km of coastline was derived from 1 cm² coned trap catches and daily counts of settlement on ceramic tiles. There were no instances of disproportionately high settlement in relation to larval supply, but at five sites there were days when settlement apparently failed. The identification and explanation of settlement success and failure is fundamental to understanding the importance of larval supply in structuring marine benthic populations and communities.     

Effects of sediments on larval settlement of abalone Haliotis diversicolor

The effects of deposited sediments on the settlement of the abalone Haliotis diversicolor Reeve were examined through both field observations and a laboratory experiment. Occurrences of newly settled post-larvae (shell length < 500 μm) and the amount of suspended and deposited sediments were monitored at two stations (Stns 1 and 2), which experienced different sediment deposition conditions, located at Nagai on the coast of Sagami Bay, Japan. Quantities of suspended sediments at the two stations varied considerably during the survey period, but no significant difference was detected between the stations. Mean volume densities of sediments deposited on cobbles were significantly higher at Stn 2 than at Stn 1. Densities of newly settled post-larvae from the 2001 to 2004 cohorts were significantly higher at Stn 1. A laboratory experiment was conducted to assess the effects of sediment quantity and quality on larval settlement. Two substances with different physical properties, kaolin and clamshell powder, were used as sediments. Larvae were subjected to four different sediment treatments with crustose coralline algae (CCA) substrates; thin and thick treatments for both kaolin and clamshell powder. Negative (without CCA) and positive (with CCA) controls without sediments were also established. The rate of metamorphosis decreased as sediment thickness increased in both the kaolin and clamshell powder treatments. Larvae in the kaolin treatments appeared to be trapped by the kaolin, and most could not metamorphose successfully. There were no trapped larvae in the clamshell powder treatments. The results indicate that the quantity and physical properties of sediments deposited on substrata affect the settlement and behaviour of larval abalone. Experimental results suggest that the lower densities of newly settled post-larvae observed at Stn 2 may have been a result of larger quantities of deposited sediments, which reduced the availability of suitable substrate for larval settlement.     

Application of real-time PCR for simultaneous identification and quantification of larval abalone

A paucity of direct studies of marine invertebrate larval dispersal motivated the development of a high-throughput method for identification and quantification of pinto abalone (Haliotis kamtschatkana) larvae in seawater. DNA extracted from sample retentate provided template to screen for species-specific cytochrome oxidase I (COI) mitochondrial DNA sequence via quantitative PCR (QPCR) technology. Primers and a dual-labeled probe were designed and used to identify and quantify DNA from the target species in blind tests of unknown samples alongside a standard template quantity series. Quantity estimates derived from QPCR standard curves were verified via direct enumeration of larvae using light microscopy. Multiplex reactions containing an internal positive control minimized underestimation of quantity and false negatives via partial or full PCR inhibition, respectively. Planned controlled field release and collection experiments to examine larval dispersion patterns via sampling over short and long postrelease times anticipate similar QPCR assays for other marine invertebrate species to aid investigations of larval dispersal in the marine environment.     

Sediment-associated cues for larval settlement of the infaunal spionid polychaetes Polydora cornuta and Streblospio benedicti

Do patchy distribution patterns of infaunal polychaetes result from active site selection of larvae influenced by sediment-associated microbial cues? This hypothesis was tested with still-water laboratory settlement assays revealing the acceptance or rejection of polychaete larvae to qualitatively different sediments. Laboratory brood cultures of the spionid polychaetes Polydora cornuta and Streblospio benedicti yielded a sufficient number of larvae with planktotrophic development for bioassays. High settlement rates (75-95%) of test larvae were observed in response to natural sediment. Sterilization of natural sediment significantly decreased settlement of P. cornuta (25-55%) while combustion of sediment significantly decreased the settlement rate in both species (5-50%). Differences in settlement responses to sediments treated by sterilization or combustion most likely resulted from a variety of factors such as modified sediment fabric, grain size distribution and quantity of adsorbed organic matter. To experimentally address the potential role of microorganisms and microbial metabolites as mediators of larval settlement, ashed sediment was inoculated with viable microorganisms obtained from natural sediment. In both polychaete species, this treatment significantly increased larval settlement in comparison to the control of ashed sediment indicating that larval settlement was at least partially mediated by the presence of microorganisms associated with sediment.     

The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp. in the inhibition of larval settlement

It has been suggested that bacteria associated with soft-bodied organisms are suggested to produce bioactive compounds against the attachment of invertebrate larvae and bacteria onto the surface of these organisms. Our recent study has demonstrated that epibiotic bacteria from the surface of the soft coral Dendronephthya sp. (Coelenterata: Octocoralia, Alcyonacea) inhibit the growth of bacteria commonly found in marine natural biofilms. In the present study, the effect of 11 epibiotic bacteria isolated from the surface of Dendronephthya sp. on larval settlement of the tubeworms Hydroides elegans was examined using laboratory bioassay. Among 11 bacterial isolates, 2 strains (18%) inhibited the larval settlement of H. elegans (Haswell), 4 strains (36%) were “inductive” to larvae and the remaining 5 strains (46%) were “non-inductive”. There was no correlation between the antifouling activities of bacterial isolates and their phylogenetic origin, i.e. closely related bacterial strains showed different effects on larval settlement of H. elegans. When all “inductive”, “non-inductive” and “inhibitive” bacterial isolates were mixed in a 1:1:1 ratio, the effect of the resultant multispecies film on larval settlement became “inhibitive”. Waterborne compounds of Vibrio sp. and an unidentified α-Proteobacterium, which suppressed the settlement of H. elegans and Bugula neritina (L.) larvae, were further investigated using size fractionation and bioassay-guided enzymatic analysis. It was found that antilarval settlement compounds from these bacteria were heat-stable polysaccharides with a molecular weight >100 kDa. The results indicate that the bacteria associated with the soft coral Dendronephthya sp. may contribute to the antifouling mechanisms of the soft-bodied organisms by producing compounds that are against bacterial growth and settlement of macrofoulers on the surface of their host.     

A Glycoprotein in Shells of Conspecifics Induces Larval Settlement of the Pacific Oyster Crassostrea gigas

Settlement of larvae of Crassostrea gigas on shell chips (SC) prepared from shells of 11 different species of mollusks was investigated. Furthermore, the settlement inducing compound in the shell of C. gigas was extracted and subjected to various treatments to characterize the chemical cue. C. gigas larvae settled on SC of all species tested except on Patinopecten yessoensis and Atrina pinnata. In SC of species that induced C. gigas larvae to settle, settlement was proportionate to the amount of SC supplied to the larvae. When compared to C. gigas SC, all species except Crassostrea nippona showed lower settlement inducing activities, suggesting that the cue may be more abundant or in a more available form to the larvae in shells of conspecific and C. nippona than in other species. The settlement inducing activity of C. gigas SC remained intact after antibiotic treatment. Extraction of C. gigas SC with diethyl ether (Et₂O-ex), ethanol (EtOH-ex), and water (Aq-ex) did not induce larval settlement of C. gigas larvae. However, extraction of C. gigas SC with 2N of hydrochloric acid (HCl-ex) induced larval settlement that was at the same level as the SC. The settlement inducing compound in the HCl-ex was stable at 100°C but was destroyed or degraded after pepsin, trypsin, PNGase F and trifluoromethanesulfonic acid treatments. This chemical cue eluted between the molecular mass range of 45 and 150 kDa after gel filtration and revealed a major band at 55 kDa on the SDS-PAGE gel after staining with Stains-all. Thus, a 55 kDa glycoprotein component in the organic matrix of C. gigas shells is hypothesized to be the chemical basis of larval settlement on conspecifics.     

Effects of GABA and epinephrine on the settlement and metamorphosis of the larvae of four species of bivalve molluscs

Competent larvae of different marine bivalve species were treated with GABA and epinephrine at different concentrations and times of exposure to test the ability of the drugs to induce settlement and metamorphosis. GABA induced both settlement and metamorphosis in the mussel Mytilus galloprovincialis, the clams Venerupis pullastra and Ruditapes philippinarum and the oyster Ostrea edulis. Maximum induction of settlement (>39%) was achieved after exposure of V. pullastra larvae to 10⁻⁴ M GABA; this concentration of GABA also induced the highest percentages of metamorphosis in the four species studied. Epinephrine was identified as an active inducer of settlement and metamorphosis in bivalve molluscs. Exposure to 10⁻⁵ M epinephrine induced significant levels of settlement in Mytilus, Venerupis and Ostrea. In contrast, epinephrine failed to induce settlement behaviour in Ruditapes. Maximum induction of metamorphosis was produced by 10⁻⁵ M epinephrine in mussels, clams and oysters; Ruditapes showed the highest percentage of metamorphosis (>78%). This is the first report in which the involvement of GABA in the settlement and metamorphosis of bivalve molluscan larvae is demonstrated. It was also recognised that epinephrine plays a role not only in inducing metamorphosis but also in initiating settlement.     

Pharmacological induction of larval settlement and metamorphosis in the blue mussel Mytilus edulis L

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10(-7)-10(-2) M as well as KCl at 10-40 mM K+ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) < 9% of the larvae settled after 48 h. Elevated K+ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10(-5) M and IBMX at 10(-6)-10(-4) M induced 41-83% larval settlement and ACH at 10(-7)-10(-5) M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemical, most of the larvae settled within 24 h. Compounds at concentrations of 10(-3)-10(-2) M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10(-6)-10(-4) M and L-DOPA at 10(-5) M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.     

Recruitment and metamorphosis of Haliotis larvae induced by molecules uniquely available at the surfaces of crustose red algae

Crustose red algae induce substratum-specific settlement, attachment and metamorphosis of the planktonic larvae of Haliotis rufescens Swainson (gastropod mollusc), upon direct contact by the larvae with any of a number of algal species tested. Larvae are not induced by contact with intact foliose red, brown or green macroalgae. Geniculate red algae are only slightly active. Larval settlement and metamorphosis are shown to be triggered by a class of chemical inducers associated with macromolecules and found in extracts of all species of crustose, geniculate, and foliose red algae tested; these inducers are not found in extracts of brown or green macroalgae. The substratum specificity of larval settlement and metamorphosis is shown to result from the unique availability of these inducers at the surfaces of the crustose red algae. Using a newly-developed improved method of purification based upon size-separation by gel-filtration, followed by ion-exchange chromatography over a diethylaminoethyl (DEAE)-acrylamide matrix, the principal inducer of Haliotis larval settlement and metamorphosis has been resolved from the red algal phycobiliproteins. Sensitivity of this inducer to reduction in molecular weight by digestion with trypsin demonstrates that this inducer is associated with protein.     

GABA is an inhibitory neurotransmitter in the neural circuit regulating metamorphosis in a marine snail

The marine mud snail, Tritia (=Ilyanassa) obsoleta, displays a biphasic life cycle. During the initial phase of early development, embryos hatch from benthic egg capsules to become weakly swimming veliger larvae. In the second phase, adult T. obsoleta are facultative carnivores and major agents of community disturbance. Metamorphosis is the irreversible developmental event that links these two life history stages. When physiologically competent, larvae can respond to appropriate environmental cues by settling onto their mudflat habitat and transforming themselves into miniature adult snails. Two neurotransmitters—serotonin and nitric oxide—have opposing effects on the metamorphic process in this species. In multiple other species of gastropod and bivalve molluscs, a third neurotransmitter, the classically inhibitory compound γ‐aminobutyric acid (GABA), can induce settlement or metamorphosis upon external application to competent larvae. In this situation, GABA is presumed to mimic the action of ligands from the juvenile environment that bind to larval chemosensory receptors and activate the metamorphic pathway. Results of our experiments contradict this commonly reported action of GABA on molluscan larvae. External application of GABA to competent larvae of T. obsoleta elicited no response, but instead attenuated the action of serotonin (5‐HT), a metamorphic inducer. Our investigations into the responses of larval T. obsoleta to multiple GABAergic reagents support our hypothesis that GABA functions internally as a neurotransmitter in the pathway that controls the initiation of metamorphosis. Our results also suggest that GABA acts directly on or downstream from serotonergic neurons to regulate the metamorphosis‐inducing effects of this neurotransmitter. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 736–753, 2018     

The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina

Biofilms of marine bacteria and diatoms and their combinations were examined in laboratory choice assays to determine their effects on the attachment and successful metamorphosis of the larvae of the bryozoan Bugula neritina (Linnéus). The larval settlement in response to unfilmed surfaces, a natural biofilm (NBF) and adsorbed cells of three strains of bacteria, five strains of pennate diatoms and combinations of the two at different densities. Bacterial and diatom strains showed different effects on the larval settlement of B. neritina. Bacterial monospecific strains of an unidentified α-Proteobacterium and Vibrio sp. mediated the same percentage of settlement as a filtered seawater control. Biofilms of Pseudoalteromonas sp. caused significantly lower larval settlement. Larval settlement of B. neritina was negatively correlated with increasing densities of Pseudoalteromonas sp. The highest percentages of settlement were mediated by the biofilms of the diatom species Achnanthes sp., Amphora cofeaeformis, Amphora tenerrima, Nitzschia constricta and a 5-day-old natural biofilm, while the lowest settlement was found on a N. frustulum film. A three-way analysis of variance demonstrated that the density of bacteria and the presence of particular species of diatoms and bacteria in combined biofilms, significantly affected the settlement of B. neritina larvae. High settlement of larvae (50-90%) at all treatments indicated that B. neritina larvae are much more indiscriminate settlers than previously expected. Hence, using this species as a monitoring organism to trace ecologically relevant subtle changes of settlement cues in the natural environment should be carefully re-examined.     

Nature and role of newly described symbiotic associations between a sea anemone and gastropods at bathyal depths in the NW Atlantic

The hormathiid sea anemone Allantactis parasitica was found living as an epibiont on numerous species of gastropods at depths of 725-1100 m along the continental slope of eastern Canada. The proportion of bathyal gastropods hosting 1-6 sea anemones reached 72.5% in a single trawl. Although A. parasitica was occasionally found on other substrata (i.e. empty shells, pebbles), laboratory trials confirmed that they preferably associate with living gastropods. Settlement of planula larvae occurred significantly more often on the shells of live bathyal gastropods than on all other substrata present in the tanks. Juvenile sea anemones (∼ 1 mm diameter) readily moved from the mud or other inert substrata onto shells of burrowed bathyal gastropods. Conversely, larvae, juveniles and adults of A. parasitica never associated with any shallow-water gastropods when given the opportunity. Trials exposing predatory sea stars (Leptasterias polaris) from shallow and bathyal depths to bathyal gastropods (Buccinum undatum) with epibiotic A. parasitica, and to asymbiotic bathyal and shallow-water B. undatum, revealed adaptive behaviours in both prey and predator. Shallow-water gastropods (devoid of epibionts) reacted defensively to L. polaris, whereas bathyal gastropods relied mostly on their epibionts to protect them, thus falling prey to L. polaris when the epibionts were removed. L. polaris from bathyal depths typically ignored symbiotic gastropods, but they consistently preyed on asymbiotic ones, while L. polaris from shallow areas initially attempted to prey on all gastropods, but learned to avoid those harbouring sea anemones. Furthermore, living as epibionts afforded sea anemones a means to escape one of their own predators, the sea star Crossaster papposus. The mutualistic relationship between hormathiid sea anemones and bathyal gastropods from the NW Atlantic may have evolved in response to predation pressure.     

Influence of snail mucus trails and first snail meal on the behavior of malacophagous sciomyzid larvae

There has been considerable focus on the natural enemies of snails, particularly those of medical and veterinary significance. Much attention has focussed on members of the Family Sciomyzidae (Diptera), the majority of which feed on a range of mollusc species. However, little is known about the influence of first snail meal on subsequent prey choices, an important consideration in biocontrol. We examined neonate larval responses of Ilione albiseta to fresh and aged snail mucus trails of three snail species. Median neonate response rates to aged mucus trails for all three snail species tested were significantly (P < 0.001) weaker than for fresh mucus trails indicating a strategy which enhances the likelihood of reaching prey snail species without expending energy following “cold” trails. More than 78% of first instar larvae, fed on one snail species (Radix peregra or Stagnicola palustris) and subsequently offered a choice of these two snail species for the second snail meal, selected the snail species of the first snail meal suggesting that the first snail meal influences subsequent prey selection. However, the impact of the first snail meal on larval trail-following behavior is less clear-cut. While there may be some preference for the mucus trail of the snail species on which neonate larvae have fed, this does not exclude the larvae from following the mucus trails of other snail species. The results are discussed in the context of the potential use of I. albiseta as a biocontrol agent of vectors of snail borne diseases.     

In vitro bioassay of sheep gastrointestinal mucus for nematode paralysing activity mediated by substances with some properties characteristic of SRS-A

A bioassay is described for determining the inhibition of nematode larval migration from agar by substances exerting a paralysing action.In the assay, larval migration was completely inhibited by the anthelmintic levamisole (25 μg/ml) whereas biogenic amines, and prostaglandins E₁ and E₂ at 50 μg/ml, were without effect. Mucus from the gastrointestinal tract of sheep resistant to nematode infection inhibited larval migration by up to 93% whereas mucus from heavily infected sheep or sheep reared helminth free did not significantly inhibit larval migration. Mucus from sheep resistant to Trichostrongylus colubriformis inhibited the migration of larvae of other nematode species.The larval migration inhibitory (LMI) activity of mucus from resistant sheep was associated with components having some properties of slow reacting substance of anaphylaxis (SRS-A).Faecal samples from resistant sheep possessed significantly more LMI activity than faecal samples from heavily infected sheep or sheep reared helminth free. The level of LMI activity in the faeces of sheep undergoing challenge infection may be a useful indicator of the sheep's resistance status.The presence of the larval migration inhibitory activity in sheep mucus is discussed in relation to resistance to infection.     

Larval Chigger Mites Collected from Small Mammals in 3 Provinces,Korea

A total of 9,281 larval chigger mites were collected from small mammals captured at Hwaseong-gun, Gyeonggi-do (Province) (2,754 mites from 30 small mammals), Asan city, Chungcheongnam-do (3,358 mites from 48 mammals), and Jangseong-gun, Jeollanam-do (3,169 for 62 mammals) from April-November 2009 in the Republic of Korea (= Korea) and were identified to species. Leptotrombidium pallidum was the predominant species in Hwaseong (95.8%) and Asan (61.2%), while Leptotrombidium scutellare was the predominant species collected from Jangseong (80.1%). Overall, larval chigger mite indices decreased from April (27.3) to June (4.9), then increased in September (95.2) and to a high level in November (169.3). These data suggest that L. pallidum and L. scutellare are the primary vectors of scrub typhus throughout their range in Korea. While other species of larval chigger mites were also collected with some implications in the transmission of Orientia tsutsugamushi, they only accounted for 11.2% of all larval chigger mites collected from small mammals.     

Effect of rock type on the recruitment and early mortality of the barnacle Chthamalus montagui

Discontinuities in distribution of sessile species due to changes in available habitat have rarely been investigated. In the English Channel, soft sedimentary rocks including chalk could potentially present a barrier to eastward range extension of the southern barnacle Chthamalus montagui. To test the effect of rock type on recruitment and mortality, prepared settlement tiles of four calcareous rocks that form important platforms close to existing limits of distribution were fixed to the shore at a site in south-west England, known to have consistently high larval settlement. Prior to fixation, the roughness parameter “R_a” and Potential Settlement Sites (PSS) index was measured. Recruitment was monitored photographically on four occasions during the settlement season and subsequent mortality for 7 months after the settlement season. By the end of the settlement season, recruitment on the hard Kimmeridge Cementstone was significantly less than on the other three rock types and attributed to low surface roughness and fewest Potential Settlement Sites (PSS), yet only 28% of variance could be attributed to PSS. Post-recruitment mortality was more variable, however after 7 months there were significant differences between rock types. Simultaneous surveys of post-recruitment mortality in natural populations showed that although recruitment on chalk can potentially be high, survivorship was relatively low. Thus rock type could be influential in setting species geographical limits.