Gregarious and Nongregarious Larval Settlement in the Aggregation-Forming Annelid Ficopomatus enigmaticus.

Earlier studies have shown that biofilms can mediate the larval settlement of the polychaete Hydroides elegans and that changes in the bacterial community structure and density of biofilms often alter the larval settlement response. However, the chemical cues that mediate this response remain unknown. In this study, both successional changes in the bacterial community structure and the chemical profiles of subtidal biofilms are described and related to the larval settlement response. Multispecies biofilms were developed on polystyrene Petri dishes and granite rock in the subtidal zone over a period of 20 days. The effects of the substratum and age on the bacterial community structure and chemical profiles of the biofilms were evaluated with two molecular methods (microarray (PhyloChip) and denaturing gradient gel electrophoresis) and with gas chromatography-mass spectrometry, respectively. Both age and substratum altered the bacterial community structures and chemical profiles of the biofilms. Age had a greater effect in shaping the bacterial community structure than did the substratum. In contrast, the type of substratum more strongly affected the chemical profile. Extracts of biofilms of different ages, which developed on different substrata, were tested for the settlement of H. elegans larvae. The extracts induced larval settlement in a biofilm-age-dependent manner, and extracts originating from different substrata of the same age showed no differences in larval settlement. Our results suggest that the larval settlement response cannot be predicted by the overall chemical composition of the biofilm alone.

Regulatory effects of mussel ( Aulacomya maoriana Iredale 1915) larval settlement by neuroactive compounds, amino acids and bacterial biofilms

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 365:103-113 (2008) - DOI: https://doi.org/10.3354/meps07503 A hierarchy of settlement cues influences larval behaviour in a coral reef sponge Piers Ettinger-Epstein1,2, Steve Whalan1,2,*, Christopher N. Battershill2,3, Rocky de Nys1,2 1School of Marine and Tropical Biology, and 2AIMS@JCU, Sir George Fisher Building, James Cook University, Townsville, Queensland, 4811, Australia 3Australian Institute of Marine Science PMB 3, Townsville MC, Queensland 4810, Australia *Corresponding author. Email: stephen.whalan@jcu.edu.au ABSTRACT: For sessile marine invertebrates, processes contributing to larval release, dispersal, and settlement in favourable habitats are central to patterns of distribution and community structure. We quantified larval release patterns, phototactic behaviour, and settlement in response to environmental cues for the coral reef sponge Luffariella variabilis. Individual sponges released up to 830 larvae d–1. Larvae displayed phototactic behaviours by swimming upwards after release for brief periods (40 min), after which time most larvae (>95%) exhibited negative phototaxis. Light played a role in determining numbers and rates of larval settlement. Light levels of 56 µmol s–1 m–2 reduced the rate of settlement and inhibited larval settlement by 60% compared to dark controls. However, at lower light levels (0.7 to 0.34 µmol s–1 m–2), both time to settlement and numbers of larvae settling were consistent with settlement in dark controls. Larval settlement increased in the presence of other larvae, with >95% of larvae settling when placed in treatments with 50 individuals, compared to 50% settlement for treatments containing only 1 individual. The gregarious settlement of L. variabilis larvae was associated with conspecific larval settlement cues. Settlement in ‘conditioned’ water from which 200 larvae had previously settled and subsequently been removed was 80%, compared to 20% in controls. Our study unequivocally demonstrates that a conspecific cue not related to adults or other biotic or abiotic factors induces settlement in larvae. Our observations, the preference of larvae to settle in response to low light levels and of settlement increased by gregariousness, correspond with the cryptic and clumped distribution of L. variabilis in the field. KEY WORDS: Porifera · Light · Gregariousness · Larval settlement · Settlement cues Full text in pdf format PreviousNextCite this article as: Ettinger-Epstein P, Whalan S, Battershill CN, de Nys R (2008) A hierarchy of settlement cues influences larval behaviour in a coral reef sponge. Mar Ecol Prog Ser 365:103-113. https://doi.org/10.3354/meps07503 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 365. Online publication date: August 18, 2008 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2008 Inter-Research.

The health and survival of benthic marine communities depends upon an ecological and developmental transition from pelagic larva to benthic adult in most invertebrate species, and this is orchestrated by the environment. The majority of larvae are induced to settle by biochemical cues and many species have long been known to preferentially settle in the dark. Combined, these data suggest that larval responses to light and biochemical cues may be ontogenetically linked, but this is yet to be explored at the molecular level. My PhD research explores the nature and perception of environmental cues that regulate larval settlement by using parallel behavioural and transcriptomic assays on individual larvae of the sponge Amphimedon queenslandica, a representative of an early-branching animal lineage.Tracking the vertical position of A. queenslandica larvae over time revealed that larvae of this sponge descend to the benthos at twilight, by which time they are competent to respond to biochemical cues, consistent with them naturally settling in the dark. Larval settlement assays under three different light regimes (natural day-night, constant dark or constant light) were complimented with transcriptomic assays on individual larvae to identify candidate molecular pathways underlying the different settlement responses that were observed. Constant light prevents larval settlement in response to biochemical cues, likely via actively repressing chemostransduction; this is consistent with the sustained upregulation of a photosensory cryptochrome and two putative inactivators of G-protein signalling in the constant light only. These transcriptome data also suggest that the photo- and chemosensory systems may be hierarchically integrated into ontogeny to regulate larval settlement via nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) signalling in this sponge.Larval responses to biochemical settlement cues also change with age, at both behavioural and transcriptional levels. The majority of larvae settled in response to biochemical cues when induced at 5 and 8 hours post emergence (hpe) from the adult sponge. Genome-wide gene expression spanning five time points through larval ontogeny identified candidate gene co-expression modules for underlying larval ability to respond to biochemical cues. Identification of transcription factors, with known functional roles in animal development, within these candidate co-expression modules suggests a potential mechanism for regulating the global developmental rates of larvae.To explore how larvae respond to different biochemical cues at the behavioural and molecular levels, larvae were presented with two different species of coralline algae (CA) at five time points through larval development (1.5, 3, 5 and 8 hpe). Despite significant differences in larval settlement responses to these two species of CA at 3 and 5 hpe, there was no difference in larval transcriptional profiles at 2 hours post induction (hpi). These data suggest that a small number of genes may have a large effect on settlement in this sponge and that components of the GPCR-NO signalling pathway may account for different behavioural responses to biochemical cues. We also identified carryover effects of inductive cue on the expression of a small suite of genes, including two components of the GPCR-NO pathway, in benthic post-larvae at 2 hpi. These data also provide another line of evidence to support a central role for the GPCR-NO pathway in mediating larval settlement in this sponge that belongs to one of the earliest branching of the extant animal lineages. The research presented in this dissertation are consistent with a conserved role for GPCR-NO signalling in regulating larval settlement and metamorphosis throughout the animal kingdom.

Bugula neritina is a sessile marine bryozoan with a pelagic larval stage. Larvae frequently settle on boat hulls, facilitating the introduction of B. neritina to bays and estuaries worldwide. Adrenergic agonists, such as the vertebrate hormone noradrenaline, inhibit larval settlement in a variety of marine invertebrate species, including B. neritina. Light also inhibits B. neritina larval settlement, yet the underlying mechanisms by which light and adrenergic compounds exert their effects on larvae are not well understood. Octopamine is considered the invertebrate analog of noradrenaline, and may be an endogenous hormone involved in larval settlement pathways. I observed the effects of the adrenergic agonist noradrenaline and the adrenergic antagonist phentolamine on larval settlement, and found that high concentrations of noradrenaline increased larval mortality, inhibited larval attachment, and increased larval swimming behavior. High concentrations of phentolamine also increased larval mortality, but increased larval attachment and decreased larval swimming behavior. I used fluorescent labeling and microscopy to localize sensory system components, and found that larvae possess adrenergic-like receptors, as well as tyrosine hydroxylase-like and octopamine-like immunoreactivity. I also exposed larvae to phentolamine in both dark and light conditions, and found that light significantly inhibited larval attachment, but phentolamine blocked those inhibitory effects. These results suggest that B. neritina larvae possess adrenergic-like receptors, which serve as the binding sites for noradrenaline and phentolamine. These are likely octopamine receptors, and octopamine may be one endogenous compound involved in controlling larval phototaxis and settlement behavior. Light may increase octopamine production, thereby stimulating cilial activity, extending swimming behavior, and preventing larvae from attaching to a substrate. This research sheds light on previously unknown sensory mechanisms in B. neritina larvae, and may aid in the development of new biofouling control strategies.

Aggregations of the sessile, serpulid tubeworm Hydroides elegans are commonly encountered on hard surfaces in Pearl Harbor, Hawai'i, and a bio-organic film has been found to be one requisite for larval settlement in this species. To determine if additional preor postsettlement factors influence larval distribution, we recorded settlement frequency, location, and survival on biofilmed settlement plates, either unoccupied as controls, or occupied by live juvenile or adult worms, tubes of dead adult worms, or plastic mimics of adult worm tubes. Early post-settlement mortality was extremely low in all trials (<1.0 %), and thus not responsible for structuring populations of H. elegans. The hypothesis that larvae settle preferentially on or near conspecific individuals (i.e., gregariously) was falsified: larvae did not settle faster (laboratory) or in greater numbers (field) on surfaces already occupied by H. elegans or their tubes. In the laboratory, settling larvae of H. elegans did not actively select crevices created where adult worm tubes or worm-tube mimics intersected with the substratum; in the field, however, there was significantly more settlement in tube crevices than expected by chance. Because this pattern appeared only in moving water, it probably results from hydrodynamics. In flowing water, larvae are entrained in eddies shed on the leeward side of roughness elements, such as the tubes of adult worms or their mimics, and attach there if these sites are otherwise acceptable (i.e., biofilmed). A similar pattern of enhanced settlement in crevices adjacent to tubes occurred on the plates with juvenile worms on the last day of field trial 1, when the diameter of their tubes (0.6 mm) was large enough to create eddies capable of entraining conspecific larvae. Dense aggregations of H. elegans found on hard surfaces in bays and estuaries most likely result from passive deposition of larvae in crevices beside tubes of conspecific individuals, followed by selective attachment in these locations if the bio-organic film is acceptable. Additional key words: larvae, settlement, active choice, passive deposition, fouling organism Many sessile marine invertebrates occur in non-random distributions (reviews: Meadows & Campbell 1972; Crisp 1976, 1984; Pawlik 1992). Clumped distributions of conspecific individuals may result from gregarious behavior by larvae at the time of settlement (e.g., Wilson 1968, 1970; Wethey 1984; Raimondi 1988), larval response to exogenous metamorphic inducers (e.g., Harrington 1921; Hadfield 1977; Morse & Morse 1984; Morse et al. 1988), or an active preference by larvae for specific surface topographies, such as crevices or elevations (e.g., Chabot & Bourget 1988; LeTourneux & Bourget 1988; Walters & Wethey a Author for correspondence. Present address: Department of Biology, University of Central Florida, 4000 Central Florida Blvd., Orlando, FL 32816-2368, USA. E-mail: ljwalter @pegasus.cc.ucf.edu 1991). Alternatively, clumped distributions may arise when passive entrainment by water currents deposits larvae near conspecific individuals (e.g., Wethey 1986; Butman 1987; Havenhand & Svane 1991), or when differential post-settlement mortality leaves individuals in only a few locations after widespread settlement (e.g., Keough & Downes 1982; Woodin 1985; Walters 1992a). If aggregations result from active larval choice, to what cues are larvae responding? Only a single cue, or two or more cues may be necessary for larval settlement. If the cue is associated with a specific organism, such as a food source, then settlement will be significantly reduced, or even absent, if the organism is not present (e.g., Morse & Morse 1984; Hadfield & Scheuer 1985; Morse et al. 1988). If the cue is associated with a bio-organic film, then larvae will not This content downloaded from 157.55.39.153 on Mon, 19 Sep 2016 04:45:07 UTC All use subject to http://about.jstor.org/terms Causes of settlement patterns in a serpulid polychaete settle on an unfilmed surface (e.g., Maki et al. 1989; Hadfield et al. 1994; Keough & Raimondi 1995). If larvae actively respond to topographic complexity created by conspecific individuals, then settlement on or near mimics will be similar to settlement around conspecifics (Wethey 1986; Walters 1992b). If larval settlement is gregarious, then the larvae must be responding to a chemical signal unique to their species (e.g., Burke 1986; Pawlik 1992). Such a compound may be surface bound (e.g., Wilson 1968, 1970; Crisp & Meadows 1962, 1963; Larman & Gabbott 1975) or waterborne (e.g., Svane et al. 1987; Tamburri et al. 1992). If surface bound, then cues may be attached to surfaces such as sand grains (e.g., Wilson 1968, 1970; Highsmith 1982; Jensen & Morse 1984; Pearce & Scheibling 1990) or sediments (Suer & Phillips 1983); alternatively, settlement cues may be associated with the source animals themselves (e.g., Crisp & Meadows 1962, 1963; Larman & Gabbott 1975). Settlement cues may be produced by conspecific animals during only a restricted part of their lives (e.g., cyprids of Semibalanus balanoides respond only to conspecific cyprids, Wethey 1984), throughout their lives, or even longer than the life span of an individual if the cues are associated with animal secretions, such as calcareous or membranous tubes (Wilson 1970). Among the Polychaeta, active larval choice at settlement results in monospecific stands of sessile forms in both the Sabellariidae and the Serpulidae (e.g., Wilson 1968, 1970; Scheltema et al. 1981; Jensen & Morse 1984). Approximately one third of sabellariid species build reefs (Pawlik & Chia 1991). Secretions used to cement sand grains into tubes have been documented to promote larval settlement in many of these reef-building species (e.g., Wilson 1968, 1970, 1974; Jensen & Morse 1984; Pawlik 1986), while tube cements were not effective in promoting larval settlement in species that did not build reefs (Wilson 1977; Pawlik 1988; Pawlik & Chia 1991). Among serpulids, active preferences at settlement for microbial films, specific substratum, and conspecific individuals have been reported. DeSilva (1962) found that Spirorbis tridentatus preferentially settled on stones with bio-organic films. Kirchman et al. (1982) also found that bacteria were responsible for settlement of Janua (Dexiospira) brasiliensis. Larvae of J. brasiliensis rarely settled on the diatom Nitzchia, and individual strains of marine bacteria varied in their capacity to induce settlement. Settlement of Hydroides elegans increased linearly on glass slides with microbial films of increasing age until films were 3 d old (Hadfield et al. 1994). Likewise, Scheltema et al. (1981) and Toonen & Pawlik (1994) found microbial films to be a requisite for settlement of H. dianthus. Knight-Jones (1951), Gee (1964), Williams (1964), Stebbing (1972), MacKay & Doyle (1978), and Al-Ogily (1985) have all found that preferences for specific macroalgae led to aggregations of Spirorbis spp. These preferences may be associated exclusively with the algae, bacteria on algal surfaces, or both (Fenical 1993). Conspecific adults on the surface of the alga significantly increased settlement of Spirorbis borealis and S. pagastecheri (Knight-Jones 1951). Crisp (1977) found that tubes of conspecific animals enhanced settlement of the reef-building serpulid Pomatoleios kraussii in laboratory trials, and Scheltema et al. (1981) reported that, in still-water, laboratory choice experiments, larvae of the tubeworm Hydroides dianthus settled almost exclusively on biofilmed surfaces occupied by previously settled individuals of the same species (4-133 resident individuals on 1.5 X 1.5 cm slate settlement surfaces). In no-choice, still-water, laboratory experiments, Toonen & Pawlik (1994) found that a small proportion of larvae of H. dianthus readily settled on biofilmed surfaces, while the majority settled only if conspecific animals were also present on the substratum. However, Butman (pers. comm.) did not find any evidence for gregariousness in this species in still-water trials when test plates had been soaked in a slurry of ground adults. Likewise, in 17-19 d recruitment studies conducted in the field, Mullineaux & Garland (1993) saw no evidence of gregariousness in H. dianthus. Here, we consider the possibility that larvae of the ubiquitous, sessile, tube-building polychaete Hydroides elegans (HASWELL 1883) require conspecific individuals for settlement induction, in addition to a microbial film. Although not a reef-building species, it may completely cover hard surfaces submerged in many tropical and warm temperate regions including Australia (Allen 1953; Wisely 1958), India (Sasikumar et al. 1989), Japan (Arakawa & Kubota 1973), and Hawai'i (Edmondson 1944), and it has been suggested that high densities of this species in habitats where space is not limited are the result of gregarious larval behavior (e.g., Hurlbut 1991). To explore the hypothesis that larvae of H. elegans settle in response to conspecific individuals, we ran laboratory and field experiments designed to keep the ages of bio-organic films similar, while separating the importance of (1) post-settlement mortality, (2) gregarious larval settlement behavior, (3) active larval choice for certain topographic features, and (4) hydrodynamics. The importance of an exogenous inducer associated with specific living organisms, other than those making up the biofilms, was not considered because larvae of H. elegans settle on many living and 103 This content downloaded from 157.55.39.153 on Mon, 19 Sep 2016 04:45:07 UTC All use subject to http://about.jstor.org/terms Walters, Hadfield, & del Carmen non-living substrata (Edmondson 1944; Vasishtha et al. 1995; Walters, Smith, & Hadfield, unpubl.).

The effect of season on "biofilming";, as a cue for the settlement of marine invertebrate larvae, was investigated in a long-term field study during the years 1992-1994. The series of settlement experiments was conducted in a tidal rapid on the west coast of Scotland, and involved manipulations of artificial panels. Biofilming of substrata, whilst excluding larval settlement, was achieved by the enclosure of panels within tight-fitting (but removable) mesh screens so that the number of settlers on filmed and unfilmed substrata were counted in the initial absence of other incumbent post-larvae. Depending on larval species, the effects of biofilming were found to be either facilitatory or inhibitory. Significant within- and between-species seasonal differences in the settlement responses were detected, and a reversal of the effect of biofilming on larval settlement response, from inhibitory to facilitatory and vice versa, was noted with season in the case of some taxonomic groups and species (e.g. Tubulipora sp., Plagioecia sp., Electra pilosa (L.)). The present study emphasizes the need for extended field studies of larval responses to environmental cues, when the focus of interest is in drawing general inferences about naturally occurring behavioural patterns at settlement.

&lt;p&gt;Serpulids are a globally represented group of polychaetes and can be found in many habitats from the intertidal fringe to the subtidal environment and even in deep-sea ecosystems. These tube-dwelling worms are often described as pioneer species in new or disturbed habitats. Serpulids secrete a calcareous tube and often occur in aggregations. These patches can range from several centimetres to several metres in diameter and may even form reef systems. Accumulations of tube-dwelling worms provide a new habitat for other species and, therefore, serpulids are considered bioengineers. Serpulid aggregations are known to enhance biodiversity and species abundance and may increase water quality through their filter activity. Despite their ecological importance, their ecology and ontogeny have received little attention. Spirobranchus cariniferus, a New Zealand endemic intertidal serpulid, is a substantial contributor to intertidal ecosystems. For this and other Serpulidae, the link between larval development and larval settlement is missing. However, this connection is essential to understand recruitment and ecology of tube-dwelling worms. Therefore, in this thesis, I describe the ontogeny of S. cariniferus from larval development to recruitment and reproduction. In the first data chapter, I present my findings on the recruitment of S. cariniferus in the field. This serpulid settles aggregatively in the field but not necessarily in response to the presence of adult conspecifics, as has been previously reported. Abiotic factors such as sunlight or wave disturbance have a more substantial effect on recruitment rather than the occurrence of adult individuals of the same or a competing species. Additionally, this chapter provides support for the hypothesis that larvae of S. cariniferus may accumulate near the substrate before settlement. Many sessile marine invertebrate taxa occur in either aggregations or as solitary individuals, with potential benefits and disadvantages associated with each configuration. For S. cariniferus, solitary and aggregative individuals can be found in the same habitat. Therefore, the second data chapter compares growth and mortality for individuals living alone or in aggregation. While solitary and aggregative individuals elongate their tubes at a similar rate, further correlations of body to tube sizes lead to the conclusion that solitary worms focus more of their energy on tube length growth rather than body size increment compared to aggregative conspecifics. Mortality is highly variable but does not differ between both configurations. However, individuals living in a patch have a better ability to recover from damage to their tubes. In the last two decades, the idea that gonochorism is the general reproductive pattern for Serpulidae has been challenged, and instead it has been suggested by some that protandry is the more common trait. Therefore, with my third data chapter, I explore maturation and sex ratio of S. cariniferus and whether it changes for individuals living alone vs. in aggregation or based on size. While maturation depends on size, sex does not, and neither maturation nor sex ratio are dependent on whether individuals live in aggregation or not. Further, the ratio of females to males did not favour either sex consistently. For the first time in this species I found evidence of possible hermaphroditism. Through spawning trials and histological sections, I identified nine individuals which simultaneously contained oocytes and sperm cells. I suggest therefore, that S. cariniferus has alternating sexes rather than protandry as a reproductive strategy. In the fourth and final data chapter, I describe the metamorphosis and settlement behaviour of S. cariniferus larvae. For this serpulid species, settlement and metamorphosis are separate and distinct steps that involve both behavioural and morphological changes to the larvae. Further, this entire process can be quite prolonged (i.e. over several days), and at some points can be reversed. It is therefore very important that observations last longer than 24–48 hours, when studying serpulid settlement. As far as I am aware, this is the first study on a serpulid species to examine aggregative settlement in the field in relation to the presence of adult conspecifics and abiotic factors, and also to explicitly test for consequences of solitary vs. group living on growth and mortality. It is also the first to show evidence of hermaphroditism in this species. I hope my research and this thesis stimulates a more inclusive and holistic investigation of serpulids in the future. Larval development, settlement patterns and ontogeny need to be studied in detail if we want to understand the evolution, ecology, impacts and benefits of these and other sessile marine invertebrates.&lt;/p&gt;

&lt;p&gt;Serpulids are a globally represented group of polychaetes and can be found in many habitats from the intertidal fringe to the subtidal environment and even in deep-sea ecosystems. These tube-dwelling worms are often described as pioneer species in new or disturbed habitats. Serpulids secrete a calcareous tube and often occur in aggregations. These patches can range from several centimetres to several metres in diameter and may even form reef systems. Accumulations of tube-dwelling worms provide a new habitat for other species and, therefore, serpulids are considered bioengineers. Serpulid aggregations are known to enhance biodiversity and species abundance and may increase water quality through their filter activity. Despite their ecological importance, their ecology and ontogeny have received little attention. Spirobranchus cariniferus, a New Zealand endemic intertidal serpulid, is a substantial contributor to intertidal ecosystems. For this and other Serpulidae, the link between larval development and larval settlement is missing. However, this connection is essential to understand recruitment and ecology of tube-dwelling worms. Therefore, in this thesis, I describe the ontogeny of S. cariniferus from larval development to recruitment and reproduction. In the first data chapter, I present my findings on the recruitment of S. cariniferus in the field. This serpulid settles aggregatively in the field but not necessarily in response to the presence of adult conspecifics, as has been previously reported. Abiotic factors such as sunlight or wave disturbance have a more substantial effect on recruitment rather than the occurrence of adult individuals of the same or a competing species. Additionally, this chapter provides support for the hypothesis that larvae of S. cariniferus may accumulate near the substrate before settlement. Many sessile marine invertebrate taxa occur in either aggregations or as solitary individuals, with potential benefits and disadvantages associated with each configuration. For S. cariniferus, solitary and aggregative individuals can be found in the same habitat. Therefore, the second data chapter compares growth and mortality for individuals living alone or in aggregation. While solitary and aggregative individuals elongate their tubes at a similar rate, further correlations of body to tube sizes lead to the conclusion that solitary worms focus more of their energy on tube length growth rather than body size increment compared to aggregative conspecifics. Mortality is highly variable but does not differ between both configurations. However, individuals living in a patch have a better ability to recover from damage to their tubes. In the last two decades, the idea that gonochorism is the general reproductive pattern for Serpulidae has been challenged, and instead it has been suggested by some that protandry is the more common trait. Therefore, with my third data chapter, I explore maturation and sex ratio of S. cariniferus and whether it changes for individuals living alone vs. in aggregation or based on size. While maturation depends on size, sex does not, and neither maturation nor sex ratio are dependent on whether individuals live in aggregation or not. Further, the ratio of females to males did not favour either sex consistently. For the first time in this species I found evidence of possible hermaphroditism. Through spawning trials and histological sections, I identified nine individuals which simultaneously contained oocytes and sperm cells. I suggest therefore, that S. cariniferus has alternating sexes rather than protandry as a reproductive strategy. In the fourth and final data chapter, I describe the metamorphosis and settlement behaviour of S. cariniferus larvae. For this serpulid species, settlement and metamorphosis are separate and distinct steps that involve both behavioural and morphological changes to the larvae. Further, this entire process can be quite prolonged (i.e. over several days), and at some points can be reversed. It is therefore very important that observations last longer than 24–48 hours, when studying serpulid settlement. As far as I am aware, this is the first study on a serpulid species to examine aggregative settlement in the field in relation to the presence of adult conspecifics and abiotic factors, and also to explicitly test for consequences of solitary vs. group living on growth and mortality. It is also the first to show evidence of hermaphroditism in this species. I hope my research and this thesis stimulates a more inclusive and holistic investigation of serpulids in the future. Larval development, settlement patterns and ontogeny need to be studied in detail if we want to understand the evolution, ecology, impacts and benefits of these and other sessile marine invertebrates.&lt;/p&gt;

This study investigated the effect of UV-A and B (UVR) on larval settlement of the poly- chaete Hydroides elegans through their influence on biofilms. Multispecies biofilms were treated with 3 doses of UVR (10, 30 and 80 KJ m -2 ) at 2 environmentally realistic irradiance levels (4 and 2 W m -2 ) in the laboratory, and their ability to induce the settlement of H. elegans larvae was then exam- ined in both laboratory and field conditions. For the field experiment, only 10 and 80 KJ m -2 doses under 4 W m -2 were used. In addition, this study evaluated the effects of UVR on monospecies bacte- rial biofilms and then their ability to induce larval settlement in the laboratory. Results demonstrated that the ability of multispecies biofilms to trigger larval settlement could be compromised as a result of enhanced UVR exposure. Although larval settlement on multispecies biofilms treated with the low- est UV dose (at both irradiance levels) was at the same level as that of the control, the exposure of biofilms to the highest dose significantly reduced their larval settlement triggering ability. Further- more, UVR treatments decreased the percentage of metabolically active bacterial cells in mono- species biofilms; the effect increased with increasing UV dose. Larval settlement response to mono- species biofilms decreased with increasing UV dose, suggesting that the bacterial metabolic activity in biofilms is essential for the biofilms to have an inductive effect on larval settlement. This study sug- gests that enhanced UVR, which might occur due to ozone depletion, may have a significant effect on the larval settlement of H. elegans by affecting a biofilm's inductive cues.

BackgroundRecovery of degraded coral reefs is reliant upon the recruitment of coral larvae, yet the mechanisms behind coral larval settlement are not well understood, especially for non-acroporid species. Biofilms associated with reef substrates, such as coral rubble or crustose coralline algae, can induce coral larval settlement; however, the specific biochemical cues and the microorganisms that produce them remain largely unknown. Here, we assessed larval settlement responses in five non-acroporid broadcast-spawning coral species in the families Merulinidae, Lobophyllidae and Poritidae to biofilms developed in aquaria for either one or two months under light and dark treatments. Biofilms were characterised using 16S rRNA gene sequencing to identify the taxa associated with settlement induction and/or inhibition.ResultsWe show that light and biofilm age are critical factors in the development of settlement inducing biofilms, where different biofilm compositions impacted larval settlement behaviour. Further, we show that specific biofilm taxa were either positively or negatively correlated with coral settlement, indicating potential inducers or inhibitors. Although these taxa were generally specific to each coral species, we observed bacteria classified as Flavobacteriaceae, Rhodobacteraceae, Rhizobiaceae and Pirellulaceae to be consistently correlated with larval settlement across multiple coral species.ConclusionsOur work identifies novel microbial groups that significantly influence coral larval settlement, which can be targeted for the discovery of settlement-inducing metabolites for implementation in reef restoration programs. Furthermore, our results reinforce that the biofilm community on coral reef substrates plays a crucial role in influencing coral larval recruitment, thereby impacting the recovery of coral reefs.

Larval settlement in the polychaete Hydroides elegans is mediated by marine biofilms, which are complex agglomerates of bacteria, diatoms, fungi and protozoa. The induction of H. ele- gans larval settlement by marine biofilms has been mainly attributed to bacteria and diatoms. In con- trast to bacteria-derived settlement cues, the nature and origin of diatom-derived settlement cues is poorly understood. In this study, we present the first investigation on the nature and origin of larval settlement cues produced by marine diatoms. Diatoms with inductive (i.e. Achnanthes sp. and Nitzschia constricta) and non-inductive (i.e. Amphora tenerrima and N. frustulum) effects on larval settlement of H. elegans were selected as model strains in this investigation. Larval settlement bio- assays with a choice between monospecies diatom films and unfilmed substratum revealed that the diatom-derived settlement cue was water-insoluble and associated with the film surface. There was a clear correlation between the surface coverage of diatoms in films and their inductive effect on larval settlement. In the case of Achnanthes sp., even the lowest surface coverage of 1.8% induced larval settlement significantly more than the control of filtered seawater. The interstitial distance between diatom cells at this low film density was within the body size range of H. elegans larvae (100 ± 30 µm). The inductive effect of diatom films on larval settlement prevailed even after heat treatments (121°C for 1 h) that completely killed the diatoms, as verified with the vital stain fluores- cein diacetate. These results suggest that the induction of larval settlement by diatoms is not related to their viability, and that, contrary to marine bacteria, diatom-induced larval settlement cues are composed of heat-stable surface components, such as capsular extracellular polysaccharides. These results not only demonstrate that different components of biofilms play inductive and inhibitive roles on larval settlement, but also that their relative space occupation deserves consideration regarding their roles as mediators of larval settlement.

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.

Spatial and temporal variation in larval settlement of reefbuilding corals in mariculture

BackgroundAggregated settlement of kin larvae in sessile marine invertebrates may result in a complex array of compatible and incompatible allogeneic responses within each assemblage. Each such aggregate can, therefore, be considered as a distinct self-organizing biological entity representing adaptations that have evolved to maximize the potential benefits of gregarious settlement. However, only sparse information exists on the selective forces and ecological consequences of allogeneic coalescence.ResultsWe studied the consequences of aggregated settlement of kin larvae of Stylophora pistillata (a Red Sea stony coral), under controlled laboratory settings. When spat came into contact, they either fused, establishing a chimera, or rejected one another. A one-year study on growth and survivorship of 544 settled S. pistillata genotypes revealed six types of biological entities: (1) Single genotypes (SG); (2) Bi-chimeras (BC); (3) Bi-rejecting genotypes (BR); (4) Tri-chimera entities (TC); (5) Three-rejecting genotypes (TR); and (6) Multi-partner entities (MP; consisting of 7.5 ± 2.6 partners). Analysis of allorecognition responses revealed an array of effector mechanisms: real tissue fusions, transitory fusions and six other histoincompatible reactions (borderline formation, sutures, overgrowth, bleaching, rejection, and partner death), disclosing unalike onsets of ontogeny and complex modes of appearance within each aggregate. Evaluations at the entity level revealed that MP entities were the largest, especially in the first two months (compared with SG: 571% in the first month and 162% in the seventh month). However, at the genotype level, the SG entities were the largest and the colonies with the highest-cost-per-genotype were the TR and the MP colonies. The cost was calculated as reduced average genotype size, from 27% and 12% in the first month to 67% and 64% in the seventh month, respectively. In general, MP exhibited the highest survivorship rate (85%, after one year) and SG the lowest (54%).ConclusionIn view of the above, we suggest that the driving force behind gregarious kin settlements in Stylophora pistillata stems from gained benefits associated with the immediate and long-term increase in total size of the MP entity, whereas survivorship rates did not draw a parallel link. Furthermore, the biological organization of MP entity exhibits, simultaneously, an intricate network of rejecting and fusible interactions in a single allogeneic intimate arena, where proposed benefits surpass costs incurred by discord among founders. Above results and documentations on gregarious settlement in other marine taxa bring us to suggest that the 'group level' of kin aggregates may serve as a ubiquitous legitimate selection entity in the evolution of a sessile mode of life in marine organisms.