Interspecific variation in metamorphic competence in marine invertebrates: the significance for comparative investigations into the timing of metamorphosis

Metamorphosis in marine invertebrate larvae is a dynamic, environmentallydependent process that integrates ontogeny with habitat selection.The capacity of many marine invertebrate larvae to survive andmaintain metamorphic competence in the absence of environmentalcues has been hypothesized to be an adaptive convergence (Hadfieldand others 2001). A survey of the literature reveals that asingle generalized hypothesis about metamorphic competence asan adaptive convergence is not sufficient to account for interspecificvariation in this character. In an attempt to capture this variation,we discuss the "desperate larva hypothesis" and propose twoadditional hypotheses called the "variable retention hypothesis"and the "death before dishonor hypothesis." To validate theseadditional hypotheses we collected data on taxa from the publishedliterature and performed a contingency analysis to detect correlationsbetween spontaneous metamorphosis, habitat specificity and/orlarval life-history mode, three characters relevant to environmentallyinduced settlement and metamorphosis. In order to account forphylogenetic bias in these correlations, we also constructeda phylogeny of these taxa and again performed a character-correlationanalysis. Both these tests suggest that juvenile habitat specificityis correlated to the capacity of individuals to retain the competentlarval state in the absence of substrate cues and thereforevalidate the existence of more than one hypothesis about metamorphiccompetence. We provide new data from the sea urchin Lytechinuspictus that suggest that nitric oxide (NO) and thyroxine hormonesignaling interact to determine the probability of settlementin response to a settlement cue. Similarly, we provide evidencethat thyroxine signaling in the sand dollar Dendraster excentricusincreases spontaneous metamorphosis in the absence of cues fromadult conspecifics in a manner that is independent of larvalage.     

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

Background

A metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus.

Results

Our results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae.

Conclusions

We conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom - metamorphosis.     

LARVAL GROWTH AND THE INDUCTION OF METAMORPHOSIS OF A TROPICAL SPONGE-EATING NUDIBRANCH

The Hawaiian dorid nudibranch Hypselodoris infucata (Rppell& Leuckart, 1828) was reared to metamorphosis in the laboratory.Egg diameter averaged 88 µm and veligers hatched, afterabout 5 days at 25°C, with a coiled shell averaging 149µm in length. Larvae fed on phytoplankton grew to a maximumshell length of 306 µm in about 15 days. Larval growthwas affected by both quality and quantity of the algal diet.Some larvae were competent to metamorphose 16 days post-hatchingat 26°C, and competence could be retained for at least oneweek. Metamorphosis is complete within 24 hours of exposureto an inducer. At least three species of sponge, Halichondriacoerulea, Sig-modocia sp., and Tedania macrodactyla, in additionto the nudibranch's adult prey species, Dysidea sp., inducedmetamorphosis of competent larvae. Primary films grown on glassslides also induced metamorphosis in some larvae, as did 20µM excess potassium ions in seawater. These data suggestthat nonspecific induction of metamorphosis occurs in this specieseven though the adult nudibranchs are highly prey specific.This implies that in the field, H. infucata find Dysidea sp.after settlement and metamorphosis Present address: Dept of Genetics and Development, College ofPhysicians and Surgeons of Columbia Univeisity, 701 West 168thStreet, NewYork, 10032, USA (Received 22 June 1987; accepted 3 March 1988)     

Chemical induction of settlement and metamorphosis of Capitella capitata Sp. I (Polychaeta) larvae by juvenile hormone-active compounds

Summary

The influence of juvenile hormone (JH)-active chemicals on the settlement and metamorphosis of metatrochophore larvae of the polychaete annelid Capitella sp. I of the Capitella complex has been investigated. These studies demonstrate that JH-active chemicals are able to induce settlement and metamorphosis of Capitella larvae, and that these effects may possibly be mediated by protein kinase C induction. Evidence for the presence of JH-active compounds in marine sediments is also presented, suggesting that these chemicals may serve a natural role as chemical cues for settlement and metamorphosis for Capitella larvae in the marine environment.     

Genie Control of Axolotl Metamorphosis

SYNOPSIS. Neoteny in the Mexican axolotl, Ambystoma mexicanum,is caused by homozygosity for a single recessive gene. The dominantallele causing physical metamorphosis is found in the closelyrelated species, Ambystoma tigrinum, with which it can hybridize.Despite the failure of axolotls to undergo physical metamorphosis,they do undergo a cryptic metamorphosis. A larval-to-adult hemoglobinform change, serum protein changes and other physiological eventsusually associated with amphibian metamorphosis occur duringearly larval life at ages comparable to the age at which Ambystomatigrinum undergoes both the cryptic and external metamorphicevents. Axolotl cryptic metamorphosis can be induced precociouslyby immersion of the larvae in low concentrations of thyroxine;physical metamorphosis can be induced with higher thyroxineconcentrations. The site of action of the gene responsible foraxolotl neoteny has not been identified. A change in the sensitivityof external metamorphic processes to thyroxine, or reduced hormonalstimulation by the pituitary or hypothalamus may be responsible.A comparison of these functions in Ambystoma tigrinum and theaxolotl may identify the lesion.     

Studies on the factors influencing larval settlement in Balanus amphitrite and Mytilus galloprovincialis

Understanding of factors influencing settlement(attachment and metamorphosis) of marine invertebratelarvae is of great importance in aquaculture andcontrol of biofouling. The influence of two factors onsettlement of larvae was assessed from two separateinvestigations: 1, the influence of age (endogenousfactor) on cyprids of the barnacle Balanusamphitrite; and 2, the influence of a microbial film(exogenous factor) on pediveligers of the mussel Mytilus galloprovincialis.The settlement response of cypris larvae of B.amphitrite was found to be age-dependent. Oldercyprids responded more readily to settlement factorsthan newly molted ones. In M.galloprovincialis, competent pediveligers settled inresponse to a microbial filmed surface but not toan unfilmed surface. Moreover, a factor with MW of lessthan 5000 dalton, derived from culture medium of abacterial strain C1.1 (Pseudomonas-Alteromonasgroup), induced the settlement of M. galloprovincialis larvae.Thus, marine invertebrate larvae may require a periodof competence acquisition, during which they arepoorly responsive to settlement inducers. Uponacquisition of competence, larvae readily respondto external cues (e.g. microbial film, bacterialextracellular products).     

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     

Serotonin Plays an Early Role in the Metamorphosis of the HydrozoanPhialidium gregarium

Hydrozoan larvae normally metamorphose in response to an obligate external environmental cue. Application of certain artificial chemical stimuli will also induce metamorphosis. These chemicals and their inhibitors have been used to define and order some of the signal transduction events involved in this process. Results from this study show that exogenous application of serotonin (5-HT) will induce metamorphosis and that 5-HT immunoreactive cells are present in larvae when they are competent to metamorphose. The 5-HT inhibitors ketanserin, clozapine, and 5,7-DHT prevent metamorphosis from occurring as a response to a natural inducing stimulus. Additionally, 5-HT signaling occurs prior to both an influx of external Ca²⁺from seawater and activation of protein kinase C, two other steps in the metamorphic signal transduction pathway. The neuropeptide LWamide, previously shown to induce metamorphosis in a related hydrozoan,Hydractinia echinata,also induced metamorphosis inPhialidium.When larvae were cotreated with LWamide and the 5-HT antagonist ketanserin, settlement occurred but was not followed by polyp morphogenesis. These results are used to present a model for the action of 5-HT during metamorphosis inPhialidium gregarium.     

Energetics of metamorphic climax in the pickerel frog (Lithobates palustris)

Anuran metamorphosis, the transition from aquatic larvae to terrestrial juveniles, is accompanied by significant morphological, physiological, and behavioral changes. Timing of metamorphosis and final size, which can influence adult fitness, may depend on sufficient energy accumulated during the larval period to support metamorphosis. However, only two species of anurans have been examined for energetic costs of metamorphosis, Rana tigrina and Anaxyrus terrestris. Based on these species, it has been hypothesized that differences in energy expenditure are related to duration of metamorphosis. To compare energetic costs of metamorphosis among species and examine this hypothesis, we quantified the total energy required for metamorphosis of Lithobates palustris tadpoles by measuring oxygen consumption rates over the duration of metamorphic climax using closed-circuit respirometry. Total energy costs for L. palustris were positively related to tadpole mass and duration of metamorphic climax. However, larger tadpoles completed metamorphosis more efficiently because they used proportionally less total energy for metamorphic climax than smaller counterparts. Costs were intermediate to R. tigrina, a larger species with similar metamorphic duration, and A. terrestris, a smaller species with shorter metamorphic climax. The results supported the hypothesis that amphibian species with more slowly developing tadpoles, such as ranids, require more absolute energy for metamorphosis in comparison to more rapidly developing species like bufonids.     

Induction of Settlement and Metamorphosis of the Veliger Larvae of the Nudibranch,Onchidoris bilamellata

Summary

Onchidoris bilamellata veligers were reared in the laboratory on a combination of phytoñagellates and diatoms. They attained metamorphic competence after a period of 28 to 32 days at 11°C, or 60 to 80 days at a temperature averaging 7.5° C.

Experimental evidence suggests that settlement is stimulated by a diffusible chemical emanating from living barnacles, whereas metamorphosis is induced by a chemical or mechanochemical cue, which is also associated with barnacles. Settlement and metamorphosis are considered to be separable events in O. bilamellata. The settlement response is reversible and can be repeated; it involves a characteristic behavioral repertoire including descent to the bottom, foot contortions and crawling on the pedal sole. Settlement occurs only in seawater that contains, or had previously contained, living barnacles. Metamorphosis is irreversible and involves the resorption of the velum, loss of the larval shell, and incorporation of the visceral mass into the cephalopedal mass. Metamorphosis is triggered only when physical contact with living or dead barnacles is made (dead barnacles refers to shell and tissue fragments which are only effective in inducing metamorphosis when they are used in combination with seawater that had previously contained living barnacles).

Settlement and metamorphosis in O. bilamellata is compared with that of other nudibranch species, and its unique settlement response is discussed.     

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.     

Differences in heat shock protein 70 expression during larval and early spat development in the Eastern oyster, Crassostrea virginica (Gmelin, 1791)

For a variety of species, changes in the expression of heat shock proteins (HSP) have been linked to key developmental changes, i.e., gametogenesis, embryogenesis, and metamorphosis. Many marine invertebrates are known to have a biphasic life cycle where pelagic larvae go through settlement and metamorphosis as they transition to the benthic life stage. A series of experiments were run to examine the expression of heat shock protein 70 (HSP 70) during larval and early spat (initial benthic phase) development in the Eastern oyster, Crassostrea virginica. In addition, the impact of thermal stress on HSP 70 expression during these early stages was studied. C. virginica larvae and spat expressed three HSP 70 isoforms, two constitutive, HSC 77 and HSC 72, and one inducible, HSP 69. We found differences in the expression of both the constitutive and inducible forms of HSP 70 among larval and early juvenile stages and in response to thermal stress. Low expression of HSP 69 during early larval and spat development may be associated with the susceptibility of these stages to environmental stress. Although developmental regulation of HSP 70 expression has been widely recognized, changes in its expression during settlement and metamorphosis of marine invertebrates are still unknown. The results of the current study demonstrated a reduction of HSP 70 expression during settlement and metamorphosis in the Eastern oyster, C. virginica.     

Tissue-specific profile of DNA replication in the swimming larvae of Ciona intestinalis

The cell cycle is strictly regulated during development and its regulation is essential for organ formation and developmental timing. Here we observed the pattern of DNA replication in swimming larvae of an ascidian, Ciona intestinalis. Usually, Ciona swimming larvae obtain competence for metamorphosis at about 4-5 h after hatching, and these competent larvae initiate metamorphosis soon after they adhere to substrate with their papillae. In these larvae, three major tissues (epidermis, endoderm and mesenchyme) showed extensive DNA replication with distinct pattern and timing, suggesting tissue-specific cell cycle regulation. However, DNA replication did not continue in aged larvae which kept swimming for several days, suggesting that the cell cycle is arrested in these larvae at a certain time to prevent further growth of adult organ rudiments until the initiation of metamorphosis. Inhibition of the cell cycle by aphidicolin during the larval stage affects only the speed of metamorphosis, and not the formation of adult organ rudiments or the timing of the initiation of metamorphosis. However, after the completion of tail resorption, DNA replication is necessary for further metamorphic events. Our data showed that DNA synthesis in the larval trunk is not directly associated with the organization of adult organs, but it contributes to the speed of metamorphosis after settlement.     

Induction and regulation of metamorphosis in planktonic larvae:Phoronis mülleri (Tentaculata) as archetype

The larvae ofPhoronis mülleri are comprised of many diverse behavioural forms that can be manipulated experimentally to facilitate precise assertions about the induction of metamorphosis. Various parameters for inducing metamorphosis as exemplified inPhoronis, such as species-specific substrate, bacteria, the cations Rb⁺, Cs⁺ and Hg²⁺ and tensides, are considered, and their ecologic relevance to natural factors in the sea is demonstrated. Findings on metamorphosis in other marine larvae are summarized. The function of marine bacteria as “ecological ushers” is particularly emphasized.     

Expanding networks: Signaling components in and a hypothesis for the evolution of metamorphosis

Metamorphosis is a substantial morphological transition between2 multicellular phases in an organism's life cycle, often markingthe passage from a prereproductive to a reproductive life stage.It generally involves major physiological changes and a shiftin habitat and feeding mode, and can be subdivided into an extendedphase of substantial morphological change and/or remodeling,and a shorter-term phase (for example, marine invertebrate "settlement,"insect "adult eclosion," mushroom fruiting body emergence) wherethe actual habitat shift occurs. Disparate metamorphic taxadiffer substantially with respect to when the habitat shiftoccurs relative to the timing of the major events of morphogeneticchange. I will present comparative evidence across a broad taxonomicscope suggesting that longer-term processes (morphogenetic changes)are generally hormonally regulated, whereas nitric oxide (NO)repressive signaling often controls the habitat shift itself.Furthermore, new evidence from echinoids (sea urchins, sanddollars) indicates a direct connection between hormonal andNO signaling during metamorphosis. I incorporate 2 hypothesesfor the evolution of metamorphosis—one involving heterochrony,the other involving phenotypic integration and evolutionarilystable configurations (ESCs)—into a network model formetamorphosis in echinoderms (sea urchins, starfish, and theirkin). Early indications are that this core regulatory networkcan be acted upon by natural selection to suit the diverse ecologicalneeds of disparate metamorphic organisms, resulting in evolutionaryexpansions and contractions in the core network. I briefly speculateon the ways that exposure to xenobiotic pollutants and othercompounds might influence successful settlement of juvenilesin the wild. Indeed, environmentally regulated life historytransitions—such as settlement, metamorphosis, and reproductivematuration—may be developmental periods that are especiallysensitive to such pollutants.     

Larval settlement and metamorphosis in the slipper limpet Crepidula onyx (Sowerby) in response to conspecific cues and the cues from biofilm

In the marine environment, aggregated distribution in the genus Crepidula is a very common phenomenon. Works from Pechenik's group suggested that this is the result of gregarious settlement of larvae in response to cues associated with conspecific adults. In this study, we investigated the existence of larval metamorphic cues associated with adults of C. onyx, a slipper limpet introduced to Hong Kong from the U.S. in the 1970s, through a series of laboratory bioassays. The results showed that derived cues in adult C. onyx were waterborne and the waterborne cues were not derived from bacteria associated with the shell and soft body of the adult Crepidula. The natural biofilm also induced the larval metamorphosis of C. onyx. The cues from the biofilm were associated with the surface of the biofilm and were not waterborne. The aggregated distribution in nature of adult C. onyx may result from a selective larval settlement process. On a small scale in the water column near the conspecific adults, larvae of C. onyx initially detect the waterborne conspecific cues, which then lead to positive downward swimming or passive sinking. This activity increases the chances for larvae to make contact with the biofilm and to be exposed into the higher concentration of waterborne conspecific cues. This may eventually lead to the enhanced larval settlement pattern on or near the conspecific adults.     

Crustose coralline algae and a cnidarian neuropeptide trigger larval settlement in two coral reef sponges

In sessile marine invertebrates, larval settlement is fundamental to population maintenance and persistence. Cues contributing to the settlement choices and metamorphosis of larvae have important implications for the success of individuals and populations, but cues mediating larval settlement for many marine invertebrates are largely unknown. This study assessed larval settlement in two common Great Barrier Reef sponges, Coscinoderma matthewsi and Rhopaloeides odorabile, to cues that enhance settlement and metamorphosis in various species of scleractinian coral larvae. Methanol extracts of the crustose coralline algae (CCA), Porolithon onkodes, corresponding to a range of concentrations, were used to determine the settlement responses of sponge larvae. Cnidarian neuropeptides (GLW-amide neuropeptides) were also tested as a settlement cue. Settlement in both sponge species was approximately two-fold higher in response to live chips of CCA and optimum concentrations of CCA extract compared to 0.2 μm filtered sea water controls. Metamorphosis also increased when larvae were exposed to GLW-amide neuropeptides; R. odorabile mean metamorphosis reached 42.0±5.8% compared to 16.0±2.4% in seawater controls and in C. matthewsi mean metamorphosis reached 68.3±5.4% compared to 36.7±3.3% in seawater controls. These results demonstrate the contributing role chemosensory communication plays in the ability of sponge larvae to identify suitable habitat for successful recruitment. It also raises the possibility that larvae from distinct phyla may share signal transduction pathways involved in metamorphosis.     

Can benthic algae mediate larval behavior and settlement of the coral Acropora muricata?

The resilience of coral reefs relies significantly on the ability of corals to recover successfully in algal-dominated environments. Larval settlement is a critical but highly vulnerable stage in the early life history of corals. In this study, we analyzed how the presence of two upright fleshy algae, Sargassum mcclurei (SM) and Padina australis (PA), and one crustose coralline algae, Mesophyllum simulans (MS), affects the settlement of Acropora muricata larvae. Coral larvae were exposed to seawater flowing over these algae at two concentrations. Larval settlement and mortality were assessed daily through four variables related to their behavior: swimming, substratum testing, metamorphosis, and stresses. Temperature, dissolved oxygen, pH, algal growth, and photosynthetic efficiency were monitored throughout the experiment. Results showed that A. muricata larvae can settle successfully in the absence of external stimuli (63 ± 6 % of the larvae settled in control treatments). While algae such as MS may stimulate substrate testing and settlement of larvae in the first day after competency, they ultimately had a lower settlement rate than controls. Fleshy algae such as PA, and in a lesser measure SM, induced more metamorphosis than controls and seemed to eventually stimulate settlement. A diverse combination of signals and/or modifications of microenvironments by algae and their associated microbial communities may explain the pattern observed in coral settlement. Overall, this study contributes significantly to the knowledge of the interaction between coral and algae, which is critical for the resilience of the reefs.