Induction Of Settlement Research Articles

Complete genome sequence of Metabacillus sp. cB07, a bacterium inducing settlement and metamorphosis of coral larvae.

The settlement and metamorphosis of coral larvae are the bottleneck of coral recruitment. They are critical for the extension of coral population, which is the basis of the restoration of degraded coral reef ecosystem. In this study, we described the genomic characteristics of Metabacillus sp. cB07, which can efficiently induce larvae settlement and metamorphosis of coral Pocillopora damicornis. This function is first reported in the genus Metabacillus. Strain cB07 was isolated from the coral Porites pukoensis, and comprised one circular chromosome of 4,148,576bp (44.14mol%G+C content), containing 4148 protein coding sequences. To explore the potential mechanism of coral larvae settlement and metamorphosis induced by Metabacillus sp. cB07, we predicted that numerous genes related to the bacterial inductive ability. The genome of Metabacillus sp. cB07 will be helpful for further insights into the mechanism of bacterial induction of settlement and metamorphosis of coral larvae.

Reduced seawater pH alters marine biofilms with impacts for marine polychaete larval settlement

Ocean acidification (OA) can negatively affect early-life stages of marine organisms, with the key processes of larval settlement and metamorphosis potentially vulnerable to reduced seawater pH. Settlement success depends strongly on suitable substrates and environmental cues, with marine biofilms as key settlement inducers for a range of marine invertebrate larvae. This study experimentally investigated (1) how seawater pH determines growth and community composition of marine biofilms, and (2) whether marine biofilms developed under different pH conditions can alter settlement success in the New Zealand serpulid polychaete Galeolaria hystrix. Biofilms were developed under six pH(T) treatments (spanning from 7.0 to 8.1 [ambient]) in a flow-through system for up to 14 months. Biofilms of different ages (7, 10 and 14 months) were used to assay successful settlement of competent G. hystrix larvae reared under ambient conditions. Biofilm microbiomes were characterized through amplicon sequencing of the small subunit ribosomal rRNA gene (16S and 18S). Biofilm community composition was stable over time within each pH treatment and biofilm age did not affect larval settlement selectivity. Seawater pH treatment strongly influenced biofilm community composition, as well as subsequent settlement success when biofilms were presented to competent Galeolaria larvae. Exposure to biofilms incubated under OA-treatments caused a decrease in larval settlement of up to 40% compared to the ambient treatments. We observed a decrease in settlement on biofilms relative to ambient pH for slides incubated at pH 7.9 and 7.7. This trend was reversed at pH 7.4, resulting in high settlement, comparable to ambient biofilms. Settlement decreased on biofilms from pH 7.2, and no settlement was observed on biofilms from pH 7.0. For the first time, we show that long-term incubation of marine biofilms under a wide range of reduced seawater pH treatments can alter marine biofilms in such a way that settlement success in marine invertebrates can be compromised.

Settlement of larvae from four families of corals in response to a crustose coralline alga and its biochemical morphogens

Healthy benthic substrates that induce coral larvae to settle are necessary for coral recovery. Yet, the biochemical cues required to induce coral settlement have not been identified for many taxa. Here we tested the ability of the crustose coralline alga (CCA) Porolithon onkodes to induce attachment and metamorphosis, collectively termed settlement, of larvae from 15 ecologically important coral species from the families Acroporidae, Merulinidae, Poritidae, and Diploastreidae. Live CCA fragments, ethanol extracts, and hot aqueous extracts of P. onkodes induced settlement (> 10%) for 11, 7, and 6 coral species, respectively. Live CCA fragments were the most effective inducer, achieving over 50% settlement for nine species. The strongest settlement responses were observed in Acropora spp.; the only non-acroporid species that settled over 50% were Diploastrea heliopora, Goniastrea retiformis, and Dipsastraea pallida. Larval settlement was reduced in treatments with chemical extracts compared with live CCA, although high settlement (> 50%) was reported for six acroporid species in response to ethanol extracts of CCA. All experimental treatments failed (< 10%) to induce settlement in Montipora aequituberculata, Mycedium elephantotus, and Porites cylindrica. Individual species responded heterogeneously to all treatments, suggesting that none of the cues represent a universal settlement inducer. These results challenge the commonly-held notion that CCA ubiquitously induces coral settlement, and emphasize the critical need to assess additional cues to identify natural settlement inducers for a broad range of coral taxa.

Love at First Taste: Induction of Larval Settlement by Marine Microbes

Marine biofilms are composed of many species of bacteria, unicellular algae, and protozoa. Biofilms can induce, inhibit, or have no effect on settlement of larvae and spores of algae. In this review, we focus on induction of larval settlement by marine bacteria and unicellular eukaryotes and review publications from 2010 to September 2019. This review provides insights from meta-analysis on what is known about the effect of marine biofilms on larval settlement. Of great interest is the impact of different components of marine biofilms, such as bacteria and diatoms, extracellular polymeric substances, quorum sensing signals, unique inductive compounds, exoenzymes, and structural protein degradation products on larval settlement and metamorphosis. Molecular aspects of larval settlement and impact of climate change are reviewed and, finally, potential areas of future investigations are provided.

Disease Specific Bacterial Communities in a Coralline Algae of the Northwestern Mediterranean Sea: A Combined Culture Dependent and -Independent Approach.

Crustose coralline red algae (CCA) are important components of marine ecosystems thriving from tropical waters and up to the poles. They fulfill important ecological services including framework building and induction of larval settlement. Like other marine organisms, CCAs have not been spared by the increase in marine disease outbreaks. The white-band syndrome has been recently observed in corallines from the Mediterranean Sea indicating that the disease threat has extended from tropical to temperate waters. Here, we examined the microbiome and the pathobiome of healthy and diseased Neogoniolithon brassica-florida coralline algae in the Mediterranean Sea by combining culture-dependent and -independent approaches. The coralline white-band syndrome was associated with a distinct pathobiome compared to healthy tissues and showed similarities with the white-band syndrome described in the Caribbean Sea. A sequence related to the genus Hoeflea, order Rhizobiales, characterized the white-band disease pathobiome described by amplicon sequencing. No representative of this genus was isolated by culture. We, however, successfully isolated an abundant member of the healthy CCA microbiome, an Alphaproteobateria of the family Rhodobacteraceae. In conclusion, we did not identify a potential causative agent of the disease, but through the complementarity of culture dependent and independent approaches we characterized the healthy microbiome of the coralline and the possible opportunistic bacteria colonizing diseased tissues.

Inducers of settlement and metamorphosis of the shrimp Hippolyte inermis Leach in Posidonia oceanica

Larvae of the caridean shrimp Hippolyte inermis persist in the plankton of the Mediterranean up to about one month. Since they need to reach appropriate coastal areas for their recruitment in seagrass meadows, we hypothesized that leaves of Posidonia oceanica or, alternatively, algae present in their epiphytic biofilms, might be physically recognised as target substrates and trigger larval metamorphosis and settlement. Chemical cues could improve the finding of suitable habitats for settlement. Thus, the effects of leaves of P. oceanica and biofilms of the diatom Cocconeis scutellum parva, seasonally abundant in the leaf epiphytic stratum, were investigated along with the effect of volatile organic compounds (VOCs) extracted from the epiphytic diatom. The physical induction with P. oceanica accelerated larval settlement, stimulating an earlier and faster metamorphosis of larvae. C. scutellum parva produced a weaker effect on settlement; however, diatom’s VOCs had evident influence and accelerated metamorphosis and settlement. We concluded that such chemical cues as the VOCs produced by epiphytic diatoms, reinforce the effect of physical cues for the identification of suitable settlement locations for this shrimp.

Immuno-modulation of settlement cues in the barnacle, Amphibalanus amphitrite: significance of circulating haemocytes

Gregarious settlement in barnacles has been attributed to chemical cues originating from the associated microorganisms, lectins, and settlement-inducing protein complex from arthropodin, a glycoprotein present in their conspecifics/adults. In this study, we explored the influence of haemolymph haemocytes on the metamorphosis of barnacle, Amphibalanus amphitrite cyprids. The barnacle haemocytes were also characterized for the first time using flow cytometry. Cyprid metamorphosis was significantly higher in the presence of surface-bound haemocytes compared to water-borne cues from haemolymph or adult glycoprotein, a known settlement inducer. The flow cytometry-assisted haemocyte characterization, sorting and their subsequent microscopic evaluation indicated the presence of hyalinocytes and granulocytes. Tagging of these haemocytes with lectins revealed that a significant number of haemocytes had d-glucose/d-mannose and N-acetyl-d-galactosamine, which are important settlement cues. These glycoconjugates were also detected in the cyprid larva pointing out the origin of conspecific cues. The retention of these glycoconjugates in the adult haemocytes and their occurrence in adult leachants indicate pelago-benthic coupling in sessile barnacles wherein these glycoconjugates play a significant role in their gregarious settlement. Characterization of such a coupling process in other sessile benthic invertebrates will pave the way to the understanding of complexities in their population dynamics.

Index to Volume 235

Index to Volume 235

Sand Dollar Larvae Show Within-Population Variation in Their Settlement Induction by Turbulence.

Settlement-the generally irreversible transition from a planktonic phase to a benthic phase-is a critical stage in the life history of many shoreline organisms. It is reasonable to expect that larvae are under intense selection pressure to identify appropriate settlement habitat. Several decades of studies have focused mainly on local indicators that larvae use to identify suitable habitat, such as olfactory cues that indicate the presence of conspecifics or a favored food source. Our recent work has shown that the larvae of seashore-dwelling echinoids (sea urchins, sand dollars, and kin) can be primed to settle following a brief exposure to a broader-scale indicator of their approach to shore: an increase in fluid turbulence. Here we demonstrate that this priming shows within-population variation: the offspring of certain Pacific sand dollar (Dendraster excentricus) parents-both specific fathers and specific mothers, regardless of the other parent-are more responsive to turbulence than others. In particular, the observation of the effect correlating, in some cases, with specific fathers leads us to conclude that these behavioral differences are likely genetic and thus heritable. We also report that turbulence exposure causes larvae to temporarily sink to the bottom of a container of seawater and that larvae that respond in this way are also more likely to subsequently settle. We hypothesize a two-step scenario for the evolution of turbulence responsiveness at settlement and suggest that the evolutionary origin of these behaviors could be a driving force for population differentiation and speciation.

Resilience of Atlantic Slippersnail Crepidula fornicata Larvae in the Face of Severe Coastal Acidification

Globally, average oceanic pH is dropping, and it will continue to decline into the foreseeable future. This ocean acidification (OA) will exacerbate the natural fluctuations in pH that nearshore ecosystems currently experience daily, potentially pushing marine organisms to their physiological limits. Adults of Crepidula fornicata (the Atlantic slippersnail) have proven remarkably resilient to many environmental changes, which is perhaps not surprising considering that they are common intertidally, have a geographically large native range, and have been extremely successful at invading coastal water in many other parts of the world. However, the larvae of C. fornicata have been shown to be somewhat more vulnerable than adults to the effects of reduced pH. Research to date has focused on the physiological impacts of OA on C. fornicata larvae; few studies have explored shifts in gene expression resulting from changes in pH. In the present study, we examined the response of young (4-day old) C. fornicata larvae to two extreme OA treatments (pH 7.5 and 7.6) relative to pH 8.0, documenting both phenotypic and genome-wide gene expression responses. We found that rearing larvae at reduced pH had subtle influences on gene expression, predominantly involving downregulation of genes related to growth and metabolism, accompanied by significantly reduced shell growth rates only for larvae reared at pH 7.5. Additionally, 10-day old larvae that had been reared at the two lower pH levels were far less likely to metamorphose within six hours when exposed to inducer. However, all larvae eventually reached similarly high levels of metamorphosis 24 hours after settlement induction. Finally, there were no observed impacts of OA on larval mortality. Taken together, our results indicate that far future OA levels have observable, but not severe, impacts on C. fornicata larvae, which is consistent with the resilience of this invasive snail across rapidly changing nearshore ecosystems. We propose that future work should delve further into the physiological and transcriptomic responses of all life history stages to gain a more comprehensive understanding of how OA impacts the littoral gastropod C. fornicata.

Optimising microalgae diets in sea urchin Paracentrotus lividus larviculture to promote aquaculture diversification

The objective of this study was to optimise the rearing of the sea urchin Paracentrotus lividus (Lamarck, 1816) larvae through the choice of an optimal diet. The effects on larvae reared in oyster facilities and using different microalgae species were analysed.Four experimental diets were tested: 1) Isochrysis aff. galbana (Clone T-ISO), 2) Dunaliella tertiolecta, 3) Rhodomonas sp. and 4) a combined diet of these three species (1:1:1). The biometrics of larvae were carried out every two days. Post-larval survival was assessed when competence for settlement was achieved. Induction of settlement was carried out by contact between larvae and oyster shell particles. This method, adapted from oyster farming, was used for the first time in sea urchin culture. After 9 days post-settlement, metamorphosed juveniles were sampled and post-settlement survival was assessed. The biochemical composition (proteins, carbohydrates, lipids) of microalgae and larvae was measured.Age at competence was reached at 15 days post-fertilisation for all diets. Larval survival rate and growth in length and width were higher for larvae fed with Rhodomonas sp. These larvae also had the highest lipid content. Post-settlement survival was higher in larvae fed with the combined diet. Larvae in all treatments demonstrated normal growth and development, but Rhodomonas sp. presented nutritional advantages for sea urchin larvae: a more rapid development and a higher lipid content.The use of Rhodomonas sp., already widely used in oyster aquaculture, as a diet in Paracentrotus lividus culture could be a factor in enabling oyster farmers to diversify towards “echinoculture” and increase their revenue.

The peripheral nervous system of the ascidian tadpole larva: Types of neurons and their synaptic networks.

Physical and chemical cues from the environment are used to direct animal behavior through a complex network of connections originating in exteroceptors. In chordates, mechanosensory and chemosensory neurons of the peripheral nervous system (PNS) must signal to the motor circuits of the central nervous system (CNS) through a series of pathways that integrate and regulate the output to motor neurons (MN); ultimately these drive contraction of the tail and limb muscles. We used serial-section electron microscopy to reconstruct PNS neurons and their hitherto unknown synaptic networks in the tadpole larva of a sibling chordate, the ascidian, Ciona intestinalis. The larva has groups of neurons in its apical papillae, epidermal neurons in the rostral and apical trunk, caudal neurons in the dorsal and ventral epidermis, and a single tail tip neuron. The connectome reveals that the PNS input arises from scattered groups of these epidermal neurons, 54 in total, and has three main centers of integration in the CNS: in the anterior brain vesicle (which additionally receives input from photoreceptors of the ocellus), the motor ganglion (which contains five pairs of MN), and the tail, all of which in turn are themselves interconnected through important functional relay neurons. Some neurons have long collaterals that form autapses. Our study reveals interconnections with other sensory systems, and the exact inputs to the motor system required to regulate contractions in the tail that underlie larval swimming, or to the CNS to regulate substrate preference prior to the induction of larval settlement and metamorphosis.

Induction of settlement, growth and survival of juveniles of Paracentrotus lividus

The effect of different treatments on the settlement success, survival and growth of sea urchin juveniles has been evaluated. Two experiments were performed for assessing the ability of substrates or seawater to induce settlement. In the first test 500 competent larvae were inoculated in transparent plastic containers conditioned for ten days with: Tetraselmis marina, Cyilindrotheca closterium, Nitzschia sp., Maërl beds, 100μm filtered natural seawater and 1μm filtered and UV disinfected seawater. Maximum settlement values were obtained with C. closterium, Nitzschia sp. films and UV treated seawater; and significant differences were only detected between T. marina films and the other treatments. In the second test the following treatments were performed in order to investigate the effects of filtered seawater on settlement: 0.22μm filtered and autoclaved seawater (negative control), 1μm filtered and UV treated seawater and C. closterium (positive control). Settlement success was significantly higher for C. closterium than for autoclaved seawater. Survival and growth of juveniles were monitored for 32days on these treatments: T. marina, C. closterium, Nitzschia sp., Maërl beds and 100μm filtered natural seawater. Growth of juveniles could be adequately described by linear fitting and maximum growth rate was found for C. closterium film. There were no significant differences between treatments in terms of mortality at days 21 or 32. In general, the results showed that C. closterium biofilms provide good settlement, growth and survival of sea urchin juveniles which would allow improving general performance of these phases of culture compared to the other diets tested.

Coral larvae for restoration and research: a large-scale method for rearing Acropora millepora larvae, inducing settlement, and establishing symbiosis.

Here we describe an efficient and effective technique for rearing sexually-derived coral propagules from spawning through larval settlement and symbiont uptake with minimal impact on natural coral populations. We sought to maximize larval survival while minimizing expense and daily husbandry maintenance by experimentally determining optimized conditions and protocols for gamete fertilization, larval cultivation, induction of larval settlement by crustose coralline algae, and inoculation of newly settled juveniles with their dinoflagellate symbiont Symbiodinium. Larval rearing densities at or below 0.2 larvae mL−1 were found to maximize larval survival and settlement success in culture tanks while minimizing maintenance effort. Induction of larval settlement via the addition of a ground mixture of diverse crustose coralline algae (CCA) is recommended, given the challenging nature of in situ CCA identification and our finding that non settlement-inducing CCA assemblages do not inhibit larval settlement if suitable assemblages are present. Although order of magnitude differences in infectivity were found between common Great Barrier Reef Symbiodinium clades C and D, no significant differences in Symbiodinium uptake were observed between laboratory-cultured and wild-harvested symbionts in each case. The technique presented here for Acropora millepora can be adapted for research and restoration efforts in a wide range of broadcast spawning coral species.

Settlement patterns of the coral Acropora millepora on sediment-laden surfaces

Successful recruitment in corals is important for the sustenance of coral reefs, and is considered a demographic bottleneck in the recovery of reef populations following disturbance events. Yet several factors influence larval settlement behaviour, and here we quantified thresholds associated with light attenuation and accumulated sediments on settlement substrates. Sediments deposited on calcareous red algae (CRA) directly and indirectly impacted coral settlement patterns. Although not avoiding direct contact, Acropora millepora larvae were very reluctant to settle on surfaces layered with sediments, progressively shifting their settlement preference from upward to downward facing (sediment-free) surfaces under increasing levels of deposited sediment. When only upward-facing surfaces were presented, 10% of settlement was inhibited at thresholds from 0.9 to 16mgcm−2 (EC10), regardless of sediment type (carbonate and siliciclastic) or particle size (fine and coarse silt). These levels equate to a very thin (<150μm) veneer of sediment that occurs within background levels on reefs. Grooves within settlement surfaces slightly improved options for settlement on sediment-coated surfaces (EC10: 29mgcm−2), but were quickly infilled at higher deposited sediment levels. CRA that was temporarily smothered by sediment for 6d became bleached (53% surface area), and inhibited settlement at ~7mgcm−2 (EC10). A minor decrease in settlement was observed at high and very low light intensities when using suboptimal concentrations of a settlement inducer (CRA extract); however, no inhibition was observed when natural CRA surfaces along with more realistic diel-light patterns were applied. The low deposited sediment thresholds indicate that even a thin veneer of sediment can have consequences for larval settlement due to a reduction of optimal substrate. And while grooves and overhangs provide more settlement options in high deposition areas, recruits settling at these locations may be subject to ongoing stress from shading, competition, and sediment infilling.

Larval settlement and metamorphosis in a marine gastropod in response to multiple conspecific cues.

Larvae of the marine gastropod Crepidula fornicata must complete a transition from the plankton, where they are highly dispersed, to an aggregated group of benthic adults. Previous research has shown that selective settlement of larvae on conspecific adults is mediated by a water-borne chemical cue. However, variable experimental conditions have been used to study this cue, and standardization is needed in order to investigate factors that may have weak effects on settlement. In this study, we developed a time-course bioassay based on a full-factorial design with temporal blocking and statistical analysis of larval settlement rates in the lab. We tested this bioassay by examining settlement in the presence of an abiotic cue (KCl), and biotic cues (water conditioned with adult conspecifics and conspecific pedal mucus). Results confirmed settlement in the presence of both KCl and adult-conditioned water, and discovered the induction of settlement by pedal mucus. This optimized, standardized bioassay will be used in future experiments to characterize the complex process of larval settlement in C. fornicata, particularly to measure components of potentially small effect.

Draft Genome Sequence of Shewanella sp. Strain P1-14-1, a Bacterial Inducer of Settlement and Morphogenesis in Larvae of the Marine Hydroid Hydractinia echinata.

The assembly and annotation of the draft genome sequence of Shewanella sp. strain P1-14-1 are reported here to investigate the genes responsible for interkingdom interactions, secondary metabolite production, and microbial electrogenesis.

In situ changes of tropical crustose coralline algae along carbon dioxide gradients.

Crustose coralline algae (CCA) fulfill important ecosystem functions in coral reefs, including reef framework stabilization and induction of larval settlement. To investigate in situ the effects of high carbon dioxide on CCA communities, we deployed settlement tiles at three tropical volcanic CO2 seeps in Papua New Guinea along gradients spanning from 8.1 to 7.4 pH. After 5 and 13 months deployment, there was a steep transition from CCA presence to absence around pH 7.8 (660 μatm pCO2): 98% of tiles had CCA at pH > 7.8, whereas only 20% of tiles had CCA at pH ≤ 7.8. As pH declined from 8.0 to 7.8, the least and most sensitive CCA species lost 43% and 85% of cover, respectively. Communities on upward facing surfaces exposed to high light and high grazing pressure showed less steep losses than those on shaded surfaces with low grazing. Direct CO2 effects on early life stages were the main mechanisms determining CCA cover, rather than competitive interactions with other benthic groups. Importantly, declines were steepest at near-ambient pH, suggesting that CCA may have already declined in abundance due to the recent seawater pH decline of 0.1 units, and that future severe losses are likely with increasing ocean acidification.

Induction of Larval Settlement in the Reef Coral Porites astreoides by a Cultivated Marine Roseobacter Strain.

Successful larval settlement and recruitment by corals is critical for the survival of coral reef ecosystems. Several closely related strains of γ-proteobacteria have been identified as cues for coral larval settlement, but the inductive properties of other bacterial taxa naturally occurring in reef ecosystems have not yet been explored. In this study, we assayed bacterial strains representing taxonomic groups consistently detected in corals for their ability to influence larval settlement in the coral Porites astreoides. We identified one α-proteobacterial strain, Roseivivax sp. 46E8, which significantly increased larval settlement in P. astreoides. Logarithmic growth phase (log phase) cell cultures of Roseivivax sp. 46E8 and filtrates (0.22μm) from log phase Roseivivax sp. 46E8 cultures significantly increased settlement, suggesting that an extracellular settlement factor is produced during active growth phase. Filtrates from log phase cultures of two other bacterial isolates, Marinobacter sp. 46E3, and Cytophaga sp. 46B6, also significantly increased settlement, but the cell cultures themselves did not. Monospecific biofilms of the three strains did not result in significant increases in larval settlement. Organic and aqueous/methanol extracts of Roseivivax sp. 46E8 cultures did not affect larval settlement. Examination of filtrates from cell cultures showed that Roseivivax sp. 46E8 spontaneously generated virus-like particles in log and stationary phase growth. Though the mechanism of settlement enhancement by Roseivivax sp. 46E8 is not yet elucidated, our findings point to a new aspect of coral-Roseobacter interactions that should be further investigated, especially in naturally occurring, complex microbial biofilms on reef surfaces.

Inducers of settlement and moulting in post-larval spiny lobster.

The rapid and often remote location of suitable habitats used by migrating organisms is often critical to their subsequent recruitment, fitness and survival, and this includes in the marine environment. However, for the non-feeding post-larval stage of spiny lobsters, effective settlement cues for habitat selection are critical to their success but are poorly described. Therefore, the current study examined whether acoustic and substrate cues have the potential to shorten the time to moulting and affect their subsequent nutritional condition in the pueruli of the southern spiny lobster, Jasus edwardsii. Individuals moulted to first instar juveniles up to 38% faster when exposed to the underwater sound from two types of typical settlement habitat (coastal kelp- and urchin-dominated reefs) compared to those with no underwater sound. The settlement delay in the post-larvae without underwater sound also resulted in juveniles in poorer survival and nutritional condition as measured by their protein and lipid contents. In a separate experiment, post-larvae presented with seaweed and rock substrates were found to complete settlement and moult to juvenile by as much as 20% faster compared to those on the sand and control treatments. Overall, the results are the first to demonstrate that the pueruli of J. edwardsii have the ability to detect and respond to underwater sound, as well as determining that both acoustic and substrate cues play a role in modulating physiological development during settlement.