On nitric oxide signaling,metamorphosis, and the evolution of biphasic life cycles

Summary Complex life cycles are ancient and widely distributed, particularly so in the marine environment. Generally, the marine biphasic life cycle consists of pre‐reproductive stages that exist in the plankton for various periods of time before settling and transforming into a benthic reproductive stage. Pre‐reproductive stages are frequently phenotypically distinct from the reproductive stage, and the life cycle transition (metamorphosis) linking the larval and juvenile stages varies in extent of change but is usually rapid. Selection of suitable adult sites apparently involves the capacity to retain the larval state after metamorphic competence is reached. Thus two perennial and related questions arise: How are environmentally dependent rapid transitions between two differentiated functional life history stages regulated (a physiological issue) and how does biphasy arise (a developmental issue)? Two species of solitary ascidian, a sea urchin and a gastropod, share a nitric oxide (NO)‐dependent signaling pathway as a repressive regulator of metamorphosis. NO also regulates life history transitions among several simple eukaryotes. We review the unique properties of inhibitory NO signaling and propose that (a) NO is an ancient and widely used regulator of biphasic life histories, (b) the evolution of biphasy in the metazoa involved repression of juvenile development, (c) functional reasons why NO‐based signaling is well suited as an inhibitory regulator of metamorphosis after competence is reached, and (d) signaling pathways that regulate metamorphosis of extant marine animals may have participated in the evolution of larvae.     

Appendage regeneration in anamniotes utilizes genes active during larval-metamorphic stages that have been lost or altered in amniotes: The case for studying lizard tail regeneration

This review elaborates the idea that organ regeneration derives from specific evolutionary histories of vertebrates. Regenerative ability depends on genomic regulation of genes specific to the life-cycles that have differentially evolved in anamniotes and amniotes. In aquatic environments, where fish and amphibians live, one or multiple metamorphic transitions occur before the adult stage is reached. Each transition involves the destruction and remodeling of larval organs that are replaced with adult organs. After organ injury or loss in adult anamniotes, regeneration uses similar genes and developmental process than those operating during larval growth and metamorphosis. Therefore, the broad presence of regenerative capability across anamniotes is possible because generating new organs is included in their life history at metamorphic stages. Soft hyaluronate-rich regenerative blastemas grow in submersed or in hydrated environments, that is, essential conditions for regeneration, like during development. In adult anamniotes, the ability to regenerate different organs decreases in comparison to larval stages and becomes limited during aging. Comparisons of genes activated during metamorphosis and regeneration in anamniotes identify key genes unique to these processes, and include thyroid, wnt and non-coding RNAs developmental pathways. In the terrestrial environment, some genes or developmental pathways for metamorphic transitions were lost during amniote evolution, determining loss of regeneration. Among amniotes, the formation of soft and hydrated blastemas only occurs in lizards, a morphogenetic process that evolved favoring their survival through tail autotomy, leading to a massive although imperfect regeneration of the tail. Deciphering genes activity during lizard tail regeneration would address future attempts to recreate in other amniotes regenerative blastemas that grow into variably completed organs.     

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.     

Metamorphic Competence, a Major Adaptive Convergence in Marine Invertebrate Larvae

Larvae from diverse marine-invertebrate phyla are able to respondrapidly to environmental cues to settlement and to undergo veryrapid metamorphic morphogenesis because they share the developmentaltrait of metamorphic competence. The competent state, characteristicof larvae as diverse as those of cnidarian planulae, molluscanveligers, and barnacle cyprids, is one in which nearly all requisitejuvenile characters are present in the larva prior to settlement.Thus metamorphosis, in response to more or less specific environmentalcues (inducers), is mainly restricted to loss of larva-specificstructures and physiological processes. Competent larvae oftwo "model marine invertebrates" studied in the authors' laboratory,the serpulid polychaete Hydroides elegans and the nudibranchPhestilla sibogae, complete metamorphosis in about 12 and 20hr, respectively. Furthermore, little or no de novo gene actionappears to be required during the metamorphic induction processin these species. Contrasting greatly with the slow, hormonallyregulated metamorphic transitions of vertebrates and insects,competence and consequent rapid metamorphosis in marine invertebratelarvae are conjectured to have arisen in diverse phylogeneticclades because they allow larvae to continue to swim and feedin the planktonic realm while simultaneously permitting extremelyfast morphological transition from larval locomotory and feedingmodes to a different set of such modes that are adaptive tolife on the sea bottom.     

The gastropod nervous system in metamorphosis

Many gastropods, including the sea hare Aplysia californica, undergo metamorphosis in passing from the larval to the juvenile phases of their life cycle. During metamorphosis, the gastropod nervous system is affected by both progressive and regressive neuronal events. In addition to this metamorphic reorganization, the nervous system appears to be centrally involved in initiating metamorphosis. We propose that gastropods not only possess temporally distinct neuronal adaptations for the specific needs of the larval and juvenile phases, but also another transient neuronal adaptation specialized to subserve the metamorphic episode.     

Metamorphosis and fish vision

Many species of fish exhibit metamorphosis in which dramatic external transformations occur as a consequence of coordinated changes in gene expression within an organism. Because postembryonic development and change appears to be the rule rather than the exception in teleost fish species, we view metamorphosis as one of many developmental strategies in fish which have continued plasticity as a common theme. Metamorphic changes are manifested in the visual system by modification of photoreceptor peak sensitivity rod photoreceptor cell addition, and retinal reorganization. These changes correspond to significant changes in the natural habitat of the animal and in its visual capabilities as demonstrated behaviorally. Thyroxine is the main metamorphic hormone as has also been found in amphibia. The sequence of metamorphic events occur in all teleosts, but they are compressed in time in direct developing animals suggesting that such animals might prove useful for understanding the evolution of metamorphosis in fish. It seems likely that rod photoreceptors may have evolved in conjunction with the change from larval to juvenile stage through metamorphosis in indirect developing fishes. During evolution, the contraction and/or loss of the larval stage has resulted in earlier appearance of rod photoreceptors during development although they always arise later than cone photoreceptors. This ontogenetic developmental sequence supports Walls's (1942) proposal that cones are phylogenetically older than rods and suggests that rods may have evolved several times.     

Metamorphosis and fish vision

Many species of fish exhibit metamorphosis in which dramatic external transformations occur as a consequence of coordinated changes in gene expression within an organism. Because postembryonic development and change appears to be the rule rather than the exception in teleost fish species, we view metamorphosis as one of many developmental strategies in fish which have continued plasticity as a common theme. Metamorphic changes are manifested in the visual system by modification of photoreceptor peak sensitivity, rod photoreceptor cell addition, and retinal reorganization. These changes correspond to significant changes in the natural habitat of the animal and in its visual capabilities as demonstrated behaviorally. Thyroxine is the main metamorphic hormone as has also been found in amphibia. The sequence of metamorphic events occur in all teleosts, but they are compressed in time in direct developing animals suggesting that such animals might prove useful for understanding the evolution of metamorphosis in fish. It seems likely that rod photoreceptors may have evolved in conjunction with the change from larval to juvenile stage through metamorphosis in indirect developing fishes. During evolution, the contraction and/or loss of the larval stage has resulted in earlier appearance of rod photoreceptors during development although they always arise later than cone photoreceptors. This ontogenetic developmental sequence supports Walls's (1942) proposal that cones are phylogenetically older than rods and suggests that rods may have evolved several times.     

The genetic covariance between life cycle stages separated by metamorphosis

Metamorphosis is common in animals, yet the genetic associations between life cycle stages are poorly understood. Given the radical changes that occur at metamorphosis, selection may differ before and after metamorphosis, and the extent that genetic associations between pre- and post-metamorphic traits constrain evolutionary change is a subject of considerable interest. In some instances, metamorphosis may allow the genetic decoupling of life cycle stages, whereas in others, metamorphosis could allow complementary responses to selection across the life cycle. Using a diallel breeding design, we measured viability at four ontogenetic stages (embryo, larval, juvenile and adult viability), in the ascidian Ciona intestinalis and examined the orientation of additive genetic variation with respect to the metamorphic boundary. We found support for one eigenvector of G (g_obsmax), which contrasted larval viability against embryo viability and juvenile viability. Target matrix rotation confirmed that while g_obsmax shows genetic associations can extend beyond metamorphosis, there is still considerable scope for decoupled phenotypic evolution. Therefore, although genetic associations across metamorphosis could limit that range of phenotypes that are attainable, traits on either side of the metamorphic boundary are capable of some independent evolutionary change in response to the divergent conditions encountered during each life cycle stage.     

Molecular and cellular changes in skin and muscle during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) are accompanied by changes in deiodinases expression

Flatfish metamorphosis is the most dramatic post-natal developmental event in teleosts. Thyroid hormones (TH), thyroxine (T4) and 3,3??-5??-triiodothyronine (T3) are the necessary and sufficient factors that induce and regulate flatfish metamorphosis. Most of the cellular and molecular action of TH is directed through the binding of T3 to thyroid nuclear receptors bound to promoters with consequent changes in the expression of target genes. The conversion of T4 to T3 and nuclear availability of T3 depends on the expression and activity of a family of 3 selenocysteine deiodinases that activate T4 into T3 or degrade T4 and T3. We have investigated the role of deiodinases in skin and muscle metamorphic changes in halibut. We show that, both at the whole body level and at the cellular level in muscle and skin of the Atlantic halibut (Hippoglossus hippoglossus) during metamorphosis, the coordination between activating (D2) and deactivating (D3) deiodinases expression is strongly correlated with the developmental TH-driven changes. The expression pattern of D2 and D3 in cells of both skin and muscle indicate that TH are necessary for the maintenance of larval metamorphic development and juvenile cell types in these tissues. No break in symmetry occurs in the expression of deiodinases and in metamorphic developmental changes occurring both in trunk skin and muscle. The findings that two of the major tissues in both larvae and juveniles maintain their symmetry throughout metamorphosis suggest that the asymmetric changes occurring during flatfish metamorphosis are restricted to the eye and head region.     

Evolution of larval form in ophiuroids: insights from the metamorphic phenotype of Ophiothrix (Echinodermata: Ophiuroidea)

Comparison of development through metamorphosis in Ophiothrix species provided insights into the evolutionary relationships between Type I (ophiopluteus only) and Type II (ophiopluteus and vitellaria) patterns of development in the Ophiuroidea. As typical of Type I developers, the six inner larval arms in Ophiothrix spongicola were fully resorbed at metamorphosis and no remnants of ciliated epithelia were retained. The postero-lateral arms function as locomotory organs for the developing juvenile and were discarded at settlement. In contrast, in O. ciliaris the epithelia of the inner arms were transformed into ciliated ridges, similar to those seen in vitellariae and the postero-lateral arms were resorbed rather than being discarded. Larval arm resorption in O. ciliaris is similar to that in Type II developers. The metamorphic phenotype of O. ciliaris provides a link between Type I and II development. The presence of two types of metamorphosis in congeneric ophiuroids and the variable metamorphic phenotype of O. ciliaris was unexpected. It appears that closely related ophiuroids and individual species may have the capacity to metamorphose using either Type I or Type II pathways. Although the phylogenetic distribution of metamorphic phenotypes indicates that Type II development may be the ancestral state, comparative morphology suggests that a developmental dichotomy based on larval arm resorption may not be appropriate for the Ophiuroidea. Until metamorphosis is characterized for more taxa, the ancestral developmental mode for the Ophiuroidea will remain a matter of conjecture.     

Chiton integument: Metamorphic changes in Mopalia muscosa (Mollusca,Polyplacophora)

Summary The larval integument and juvenile girdle integument of Mopalia muscosa (Mollusca: Polyplacophora) were studied by light microscopy. Within 24 h of settlement, eight distinctive changes occur that characterize metamorphosis: loss of the functional prototroch and apical tuft, secretion of a cuticle over the mantle field followed by the secretion of calcareous shell plates and the extrusion of spicules into the cuticle, a 20% decrease in length, secretion of chitinous hairs and the incorporation of the lateral ciliated bands into the pallial grooves. Similar changes which were often not recognized as metamorphic have been reported for other species. Evidence for metamorphosis being a common developmental feature of chitons is presented.     

Parasite regulation of host insect metamorphosis: a new form of regulation in pseudoparasitized larvae ofTrichoplusia ni

Summary When eggs ofTrichoplusia ni (Lepidoptera) are stung by a parasitic wasp,Chelonus sp., the developing host larvae precociously initiate metamorphosis ten days later. Precocious initiation of metamorphosis occurs even in ‘pseudoparasitized’ stung hosts which contain no living parasites at the time of symptoms of host regulation by the parasite. In feeding, penultimate instar, pseudoparasitized hosts, the corpora allata activity, hemolymph juvenile hormone esterase activity, in vivo rates of juvenile hormone metabolism and changes in hemolymph protein composition all follow the pattern of the normal last instar. This and other evidence suggests the entire developmental pattern of the last larval instar is precociously expressed in penultimate instar, pseudoparasitized hosts. The cause of precocious expression of the developmental program leading to metamorphosis is a significant decrease in the critical size parameter that, in normal larvae, signals attainment of the last instar. The induction, in preultimate instar larvae, of the entire feeding stage developmental program leading to metamorphic commitment, using either biochemical, surgical or parasitic experimental probes, has not been previously reported. The results have important implications for the study of host-parasite endocrine interaction, of normal insect metamorphosis and even of human puberty.     

Variation in thyroid hormone action and tissue content underlies species differences in the timing of metamorphosis in desert frogs

Hormonal control of post-embryonic morphogenesis is well established, but it is not clear how differences in developmental endocrinology between species may underlie animal diversity. We studied this issue by comparing metamorphic thyroid hormone (TH) physiology and gonad development across spadefoot toad species divergent in metamorphic rate. Tissue TH content, in vitro tail tip sensitivity to TH, and rates of TH-induced tail tip shrinkage correlated with species differences in larval period duration. Gonad differentiation occurred before metamorphosis in species with long larval periods and after metamorphosis in the species with short larval periods. These differences in TH physiology and gonad development, informed by phylogeny and ecology of spadefoot metamorphosis, provide evidence that selection for the short larval periods in spadefoot toads acted via TH physiology and led to dramatic heterochronic shifts in metamorphic climax relative to gonad development.     

Carry-over effects of the larval environment on post-metamorphic performance in two hylid frogs

Life history theory and empirical studies suggest that large size or earlier metamorphosis are suitable proxies for increased lifetime fitness. Thus, across a gradient of larval habitat quality, individuals with similar phenotypes for these traits should exhibit similar post-metamorphic performance. Here we examine this paradigm by testing for differences in post-metamorphic growth and survival independent of metamorphic size in a temperate (spring peeper, Pseudacris crucifer) and tropical (red-eyed treefrog, Agalychnis callidryas) anuran reared under differing larval conditions. For spring peepers, increased food in the larval environment increased post-metamorphic growth efficiency more than predicted by metamorphic phenotype and led to increased mass. Similarly, red-eyed treefrogs reared at low larval density ended the experiment at a higher mass than predicted by metamorphic phenotype. These results show that larval environments can have delayed effects not captured by examining only metamorphic phenotype. These delayed effects for the larval environment link larval and juvenile life history stages and could be important in the population dynamics of organisms with complex life cycles.     

Interpreting Geographic Variation in Life-History Traits

The geographic variation in the length of the larval periodand the size at metamorphosis of the wood frog,Rana sylvatica,is examined for populations in the tundra of Canada, the mountainsof Virginia, and the lowlands of Maryland. We argue that theobserved differences in developmental plasticity, heriisbilitiesand genetic covariances of traits among localities result fromdifferential selection pressures in each environment, and arerelated to the physiological constraints inherent in developmentand to the degree of compromise between the timing and sizeat metamorphosis allowed in each environment. In Maryland populationsfitness has been maximized by evolutionary changes in size alone;body size in this population is canalized, has low heritabilityand is highly correlated with juvenile survival relative todevelopmental time. In Canada, minimum developmental time yieldsmaximum fitness; the length of the larval period in this populationis canalized and genetically monomorphic relative to body size.In contrast, fitness in the Virginia populations has been determinedby correlated and pleiotropic effects of genes on both developmentaltime and larval body size, and both traits are equally canalized,affect juvenile survivorship equally and display moderate heritabilities.These results stress the importance of interpreting variationin life-history traits relative to constraints inherent in developmentand those imposed by the environment. Heritability and survivorshipdata support the general notion that fitness traits should havelow levels of additive genetic variation, but also suggest thatantagonistic pleiotropy may act to preserve genetic variationin fitness traits under simultaneous selection, and cautionagainst inferring evolutionary importance of individual traitswithout considering the possible presence of pleiotropy.     

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.     

Changes in RNA,DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid‐based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post‐metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage‐specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post‐metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre‐metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre‐metamorphic larval phase. Growth was dominantly hypertrophical from the early‐ to mid‐metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.