The importance of life-history stage and individual variation in the allorecognition system of a marine sponge

In marine invertebrates with complex life cycles, it may often be the case that trade-offs and behaviors differ between adult and larval stages. In this study, I examined the effects of life-history stage on allorecognition system function in the sponge, Haliclona sp. For sedentary marine invertebrates, allorecognition systems allow individuals to distinguish between genetically similar and distinct tissue they may encounter and are thought to reduce costly tissue fusion with individuals other than self or kin. Although it was found that sessile adults fused preferentially with self-tissue and exhibited a functioning allorecognition system, free-swimming larvae fused equally with sibling and non-sibling larvae resulting in swimming chimeras capable of successful metamorphosis, suggesting a stage-activated allorecognition system. In addition, adult sponges differed significantly in the propensity of their larvae to fuse suggesting variation in parental strategies. Analysis of larval swimming behavior indicated that larvae aggregate and are capable of increasing their encounters with other larvae and perhaps their probability of fusing in nature. The pursuit of fusion at this motile stage, along with evidence of a functioning adult allorecognition system, suggests that larvae may not express a recognition system, or that factors other than relatedness such as benefits to larval or adult chimeras, are involved in larval fusion and a stage-activated allorecognition system. In addition, this study demonstrates the presence of variation among individuals in the allorecognition system's ontogeny in the sponge Haliclona sp.     

Morphological chimeras of larvae and adults in a hydrozoan--insights into the control of pattern formation and morphogenesis

In the marine hydroid Hydractinia echinata, metamorphosis transforms the spindle-shaped larva into a primary polyp. It bears a hypostome with a ring of tentacles at its apical end, a gastric region in the middle and stolons at the base. In nature, metamorphosis is induced in response to external stimuli provided by bacteria. These stimuli can be replaced by artificial inducers, one of which is heat shock. Among heat shock treated stages are those undergoing complete metamorphosis but also specimens forming chimeras of different developmental stages. In the chimeric larvae, the posterior is transformed into the apical hypostome of the adult polyp while the anterior part of the larva persists as larval tissue. After transverse sectioning, these stage chimeras regenerate the missing body parts with respect to the nature of the tissue at the wound surface. This shows that the decision to make larva or polyp morphology depends not on the majority of the tissue in the original body section, but on stage specificity within the regenerating animal part. Single cells can escape from this general rule, since RFamide nerve cells which usually differentiate in polyp tissue appear in regenerated larval tails of sectioned stage chimeras. The results indicate that the pattern-forming system of the larva and of the adult have features in common. The primary signals controlling patterning along the anterior-posterior axis in larvae and the apical-basal axis in polyps arethus likelyto be the same while the interpretation of these primary signals by the individual cells changes during metamorphosis.     

The colonial urochordate Botryllus schlosseri: from stem cells and natural tissue transplantation to issues in evolutionary ecology

The urochordates, whose stem groups may have included the direct predecessors of the chordate line, serve as an excellent model group of organisms for a variety of scientific disciplines. One taxon, the botryllid ascidian, has emerged as the model system for studying allorecognition; this work has concentrated on the cosmopolitan species Botryllus schlosseri. Studies analyzing self-nonself recognition in this colonial marine organism point to three levels of allorecognition, each associated with different outcomes. The first level controls natural allogeneic rejections and fusions, in which blood-shared chimeras are formed. The second level leads to morphological resorption of partners within chimeras while the third allows the development of somatic and germ cell parasitic events. Recent studies on multi-chimeric entities formed in allogeneic fusions reveal evolutionary links between allorecognition, stem cell biology and ecology. Thus, the Botryllus system generates perspectives from different biological disciplines to yield a unique life history portrait.     

The evolution of metamorphosis in temnospondyls

Temnospondyls, possible relatives of extant amphibians and crudely similar to recent salamanders, are known from larval, neotenic and metamorphosed stages. Here, ontogenetic data of various temnospondyl taxa are analysed in order to recognize metamorphosis. Here, metamorphosis is strictly defined as a shift from an aquatic to a terrestrial existence. Following a check-list of criteria, the most likely metamorphosis-induced changes are proved in three temnospondyl lineages: eryopids, zatrachydids and dissorophoids. In a few other, unrelated taxa, terrestrial adults are known but no larval or metamorphosing forms. The distribution of metamorphosis among the Temnospondyli does not strictly correlate with phylogeny, which highlights the widespread occurrence of neoteny. In each group, characteristic patterns of metamorphosis are described and compared. Among temnospondyls, dissorophoids had the most intensive type of metamorphosis, characterized by a condensed ontogeny and a relatively small body size. The result was a distinct transformed morphotype with far-reaching terrestrial adaptations.     

Testing the evolutionary constraints of metamorphosis: The ontogeny of head shape in Triturus newts

In vertebrates with complex, biphasic, life cycles, larvae have a distinct morphology and ecological preferences compared to metamorphosed juveniles and adults. In amphibians, abrupt and rapid metamorphic changes transform aquatic larvae to terrestrial juveniles. The main aim of this study is to test whether, relative to larval stages, metamorphosis (1) resets the pattern of variation between ontogenetic stages and species, (2) constrains intraspecific morphological variability, and (3) similar to the “hour‐glass” model reduces morphological disparity. We explore postembryonic ontogenetic trajectories of head shape (from hatching to completed metamorphosis) of two well‐defined, morphologically distinct Triturus newts species and their F1 hybrids. Variation in head shape is quantified and compared on two levels: dynamic (across ontogenetic stages) and static (at a particular stage). Our results show that the ontogenetic trajectories diverge early during development and continue to diverge throughout larval stages and metamorphosis. The high within‐group variance and the largest disparity level (between‐group variance) characterize the metamorphosed stage. Hence, our results indicate that metamorphosis does not canalize head shape variation generated during larval development and that metamorphosed phenotype is not more constrained relative to larval ones. Therefore, metamorphosis cannot be regarded as a developmental constraint, at least not for salamander head shape.     

Sequential predator effects across three life stages of the African tree frog, <Emphasis Type="Italic">Hyperolius spinigularis</Emphasis>

While theoretical studies of the timing of key switch points in complex life cycles such as hatching and metamorphosis have stressed the importance of considering multiple stages, most empirical work has focused on a single life stage. However, the relationship between the fitness components of different life stages may be complex. Ontogenetic switch points such as hatching and metamorphosis do not represent new beginnings—carryover effects across stages can arise when environmental effects on the density and/or traits of early ontogenetic stages subsequently alter mortality or growth in later stages. In this study, I examine the effects of egg- and larval-stage predators on larval performance, size at metamorphosis, and post-metamorphic predation in the African tree frog Hyperolius spinigularis. I monitored the density and survival of arboreal H. spinigularis clutches in the field to estimate how much egg-stage predation reduced the input of tadpoles into the pond. I then conducted experiments to determine: (1) how reductions in initial larval density due to egg predators affect larval survival and mass and age at metamorphosis in the presence and absence of aquatic larval predators, dragonfly larvae, and (2) how differences in mass or age at metamorphosis arising from predation in the embryonic and larval environments affect encounters with post-metamorphic predators, fishing spiders. Reduction in larval densities due to egg predation tended to increase per capita larval survival, decrease larval duration and increase mass at metamorphosis. Larval predators decreased larval survival and had density-dependent effects on larval duration and mass at metamorphosis. The combined effects of embryonic and larval-stage predators increased mass at metamorphosis of survivors by 91%. Larger mass at metamorphosis may have immediate fitness benefits, as larger metamorphs had higher survival in encounters with fishing spiders. Thus, the effects of predators early in ontogeny can alter predation risk even two life stages later.     

Effects of allogeneic contact on life-history traits of the colonial ascidian Botryllus schlosseri in Monterey Bay

The formation of chimeric colonies following allogeneic contact between benthic invertebrates may strongly affect colony fitness. Here we show that, in a field population of the colonial ascidian Botryllus schlosseri in Monterey Bay, California, more than 20% of all colonies occur in allogeneic contact with conspecifics. We experimentally assessed the effects of allogeneic contact on the following life-history traits under natural field conditions: growth, age and size at first reproduction, and egg production (fecundity). When compared with isolated colonies, and in some cohorts also with colonies that rejected allogeneic neighbors, colonies that fused with neighbors incurred reduced fitness in terms of most life-history traits measured. We propose that one of the benefits of precise allorecognition is that, in fused colonies, it limits the unit of selection to chimeric individuals composed of closely related kin.     

The ontogeny of functional design: metamorphosis of feeding behaviour in the tiger salamander (Ambystoma tigrinum)

The ontogeny of feeding behaviour was studied quantitatively in the tiger salamander, Ambystoma tigrinum , to elucidate the relative importance of morphological and environmental changes on form and function. High-speed films of prey capture provided data for a frame-by-frame analysis of seven kinematic parameters of feeding behaviour. By comparing underwater feeding of larval and metamorphosed individuals, the effect of morphological changes occurring at metamorphosis on the feeding kinematic pattern was determined. By comparing metamorphosed animals feeding in the water and on land, changes in feeding kinematics associated with the environmental transition (and thus the differing physical properties of water and air) were determined. Both univariate and multivariate analyses failed to demonstrate any differences between larval and metamorphosed aquatic feedings for seven kinematic variables. However, when individuals feed on land, a radical shift in hyoid kinematics was observed. In addition, multivariate analysis showed that terrestrial feedings differed from aquatic feedings in having longer duration head movements. The lack of a kinematic difference between larval and metamorphosed individuals feeding in the water indicates that the morphological changes occurring at metamorphosis do not impose any obligatory kinematic consequences. Rather, metamorphosed Ambystoma tigrinum acquire the ability to modulate their kinematic pattern depending on the environment.     

NK cell tolerance in mixed allogeneic chimeras

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c-->B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c--> B6 chimeras rejected a low dose of beta(2)-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and beta(2)-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.     

Paedogenesis in european newts (Triturus: salamandridae): cranial morphology during ontogeny

A cross-sectional analysis using different ontogenetic stages (larvae, juveniles, paedotypic, and metamorphic adults) of the smooth newt, Triturus vulgaris, and the alpine newt, T.alpestris, revealed a broad spectrum of perennibranchiation influences on cranial ontogeny in European newts, more pronounced than previously thought. These influences included marked variation in ossification levels, pronounced morphometric variability of many cranial elements, and considerable skull shape changes in the transition from larvae to the adult stage. In comparison with metamorphosed individuals, paedotypic newts had a higher level of variability in both individual cranial traits and cranial shape changes. Sexual size difference of the skull traits was mostly negligible, especially in comparison to the influence of paedogenesis. The main changes in cranial shape of the European newts occurred during metamorphosis. Cranial morphological organization in the majority of examined paedotypes corresponds to cranial organization at late larval stages prior to metamorphosis or at the onset of metamorphosis.     

The impact of metamorphosis on the cranial osteology of giant salamanders of the genus Dicamptodon

Pacific giant salamanders (Dicamptodon) rank among the largest terrestrial caudates. Their ontogeny produces two distinct morphs—larval‐neotenic and metamorphosed—which differ in many morphological traits. We identified changes that are initiated by metamorphosis (distinguishing transformed from neotenic specimens) and also recognized age‐related changes occurring irrespective of transformation. During metamorphosis, specimens remodel the palate, rearrange the vomerine dentition, expand the maxilla, broaden the cheek, foreshorten the posterior skull table and develop specific serrated suture patterns in the dermal bones. Instead, large larvae grow a robust pterygoid sutured with a fully ossified trapezoidal vomer and a short maxilla. Small larvae are readily distinguished by tooth count, morphology and arrangement from more advanced larvae. Age‐related features, irrespective of metamorphosis, include pedicellate teeth, morphological differentiation of parasphenoid, enlargement of the orbitosphenoid, distal expansion of columella, and loss of coronoid teeth.     

Cell proliferation and early differentiation during embryonic development and metamorphosis of Hydractinia echinata

The early embryonic development of Hydractinia lasts about 2.5 days until the developing planula larva acquires competence for metamorphosis. Most embryonic cells stop cycling on reaching the larval stage. In older larvae of Hydractinia, cells that are still proliferating occur exclusively in the endoderm in a typical distribution along the longitudinal axis. During metamorphosis, proliferation activity begins again. The number of S-phase cells has increased by the 9th hour after induction of metamorphosis. Proliferative activity starts in the middle gastric region and in basal parts of primary polyps. Tentacles and stolon tips are always free of replicating cells.     

The impact of larval predators and competitors on the morphology and fitness of juvenile treefrogs

Studies of phenotypic plasticity typically focus on traits in single ontogenetic stages. However, plastic responses can be induced in multiple ontogenetic stages and traits induced early in ontogeny may have lasting effects. We examined how gray treefrog larvae altered their morphology in four different larval environments and whether different larval environments affected the survival, growth, development, and morphology of juvenile frogs at metamorphosis. We then reared these juveniles in terrestrial environments under high and low intraspecific competition to determine whether the initial differences in traits at metamorphosis affected subsequent survival and growth, whether the initial phenotypic differences converged over time, and whether competition in the terrestrial environment induced further phenotypic changes. Larval and juvenile environments both affected treefrog traits. Larval predators induced relatively deep tail fins and short bodies, but there was no impact on larval development. In contrast, larval competitors induced relatively short tails and long bodies, reduced larval growth, and slowed larval development. At metamorphosis, larval predators had no effect on juvenile growth or relative morphology while larval competitors produced juveniles that were smaller and possessed relatively shorter limbs and shorter bodies. After 1 month of terrestrial competition among the juvenile frogs, the initial differences in juvenile morphology did not converge. There were no differences in growth due to larval treatment but there were differences in survival. Individuals that experienced low competition as tadpoles experienced near perfect survival as juvenile frogs but individuals that experienced high competition as tadpoles suffered an 18% decrease in survival as juvenile frogs. There were also morphological responses to juvenile competition, but these changes appear to be due, at least in part, to allometric effects. Collectively, these results demonstrate that larval environments can have profound impacts on the traits and fitness of organisms later in ontogeny.     

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

The barnacle, Balanus amphitrite, is one of the primary model organisms for rocky-shore ecology studies and biofouling research. This barnacle species has a complex life cycle during which the swimming nauplius molts six times and transforms into a cyprid stage. Cyprids must attach to a surface to metamorphose into a juvenile barnacle. To clarify the overall profile of protein expression during larval development and metamorphosis, 2-DE was used to compare the proteome of the nauplius, the swimming cyprid, the attached cyprid, and the metamorphosed cyprid. The proteome of the swimming cyprid was distinctly different from that of other life stages and had about 400 spots. The proteomes of the attached and metamorphosed cyprids were similar with respect to major proteins but had significantly lower numbers of spots compared to that of swimming larval stages. Obviously, synthesis of most proteins from swimming cyprids was switched off after attachment and metamorphosis. Our advanced MS analysis (MALDI-TOF/TOF MS/MS) allowed us to identify the proteins that were differentially and abundantly expressed in the swimming cyprid. These proteins included signal transduction proteins (adenylate cyclase and calmodulin) and juvenile hormone binding proteins. In summary, for the first time, we have analyzed the global protein expression pattern of fouling marine invertebrate larvae during metamorphosis. Our study provides new insights into the mechanisms of barnacle larval metamorphosis and also provides a foundation for exploring novel targets for antifouling treatments.     

Metamorphosis in the Urodelan amphibians: patterns, regulation mechanisms, and evolution

Patterns of metamorphosis and mechanisms of its regulation in primitive and advanced salamanders are compared. It is found that urodelan evolution was characterised by the following trends: 1) increase in the number of metamorphosing systems; 2) increase in the amplitude of metamorphic transformations of each particular system due to the progressive divergence of the larval and the adult morphology; 3) synchronization of metamorphic transformations and their concentration within a relatively short period of ontogeny; 4) increase in the role of the thyroid hormones (TH) in the regulation of metamorphosis. Structures that are induced by factors other than TH and develop independently of TH in primitive Urodela species acquire TH-dependence in phyletically more advanced salamanders. For instance, morphogenetic induction as a mechanism of ontogeny regulation is substituted by endocrine induction with TH as the inducing factor. The switch from morphogenetic to endocrine induction stimulates the following events: 1) optimization of ontogeny; 2) reduction of the metamorphosis duration; 3) formation of the dissociability of larval and post-metamorphic stages of ontogeny, which, in its turn, is a precondition for the swith to necrobiotic metamorphosis and to the direct development.     

Effects of developmental and growth history on metamorphosis in the gray treefrog, Hyla versicolor (Amphibia, Anura)

In ecological models, the timing of amphibian metamorphosis is dependent upon rate of larval growth, e.g., tadpoles that experience a decrease in growth rate can initiate metamorphosis early. Recent authors have suggested that this plasticity may be lost at some point during the larval period. We tested this hypothesis by exposing groups of tadpoles of the gray treefrog, Hyla versicolor, to different growth schedules. In endocrine models, metamorphosis is dependent on thyroxine levels and thyroxine is antagonized by prolactin (amphibian larval growth hormone), consistent with the idea that a rapidly growing tadpole can delay metamorphosis. Thus, we also manipulated the rate of development by supplementing or maintaining natural thyroxine levels for half of the tadpoles in each growth treatment. All tadpoles that received thyroxine supplements metamorphosed at the same time regardless of growth history. They also metamorphosed earlier than tadpoles not treated with thyroxine. Tadpoles not given thyroxine supplements metamorphosed at different times: those growing rapidly during day 15-34 metamorphosed earlier than tadpoles growing slowly. Growth rate before day 15 and after day 34 had no effect on metamorphic timing. The difference in larval period between these rapidly growing tadpoles and their sisters given thyroxine treatments was less than the same comparison for tadpoles that grew slowly during the same period. This apparent prolactin/thyroxine antagonism did not exist after day 34. These results are consistent with the hypothesis of a loss of plasticity in metamorphic timing.     

Plasticity in newt metamorphosis: the effect of predation at embryonic and larval stages

1. Some organisms under variable predator pressure show induced antipredator defences, whose development incurs costs and may be associated with changes to later performance. This may be of especial relevance to animals with complex life histories involving metamorphosis. 2. This study examines the effect of predation environment, experienced both during embryonic and larval stages, on palmate newt (Triturus helveticus) metamorphosis. Newt eggs were raised until hatching with or without exposure to chemical cues from brown trout (Salmo trutta), and larval development was monitored in the presence or absence of the cues. 3. Exposure to predator cues during the embryonic stage resulted in higher growth rates at the larval stage, reduced time to metamorphosis and size at metamorphosis. Metamorphs also had narrower heads and shorter forelimbs than those from predator‐free treatments. In contrast, exposure to predator cues during the larval stage did not affect metamorph characteristics. 4. These results indicate that developing embryos are sensitive to predator chemical cues and that the responses can extend to later stages. Reversion of induced defences when predation risk ceased was not detected. We discuss the possible adaptive significance of these responses.     

Contribution of ventral blood island mesoderm to hematopoiesis in postmetamorphic and metamorphosis-inhibited Xenopus laevis

In an effort to label very early erythrocyte and lymphocyte populations and to follow their fate in normally developing postmetamorphic frogs and goitrogen-treated permanent larvae, diploid (2N) and triploid (3N) ventral blood island (VBI) mesoderm was exchanged between neurula stage embryos (about 16-22 hr old). Beginning at 15 days of age, half of the 2N or 3N hosts were treated with sodium perchlorate to prevent thyroxine-induced developmental changes. At larval stages 55-59 (41-48 days) and at 1-2 months postmetamorphosis (110-120 days), the untreated control chimeras and age-matched perchlorate-treated chimeras were killed for analysis of the VBI contribution to blood, spleen, and thymus populations by flow cytometry. The data suggest that grafting of ventral blood island mesoderm is an effective way to label an early larval erythrocyte population that declines after metamorphosis. In perchlorate-blocked permanent larvae this early VBI-derived erythrocyte population persists. In contrast, grafting of VBI mesoderm was less useful as a method to label a larvally distinct lymphocyte population in the thymus and spleen. At the late larval stages that we examined, the proportion of VBI-derived cells in thymus and spleen was not different from that observed after metamorphosis. Inhibition of metamorphosis interfered with the thymocyte expansion that normally occurs after metamorphosis, but the proportion of VBI-derived cells in thymus and spleen was not affected. This suggests that lymphopoiesis occurring in late larval life and after metamorphosis uses a stable persisting population of VBI-derived stem cells as well as dorsally derived stem cells.     

A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene during molting and metamorphosis in Drosophila

In Drosophila, pulses of the steroid hormone ecdysone trigger larval molting and metamorphosis and coordinate aspects of embryonic development and adult reproduction. At each of these developmental stages, the ecdysone signal is thought to act through a heteromeric receptor composed of the EcR and USP nuclear receptor proteins. Mutations that inactivate all EcR protein isoforms (EcR-A, EcR-B1, and EcR-B2) are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heat shock promoter drives expression of an EcR cDNA, we have employed temperature-dependent rescue of EcR null mutants to determine EcR requirements at later stages of development. Our results show that EcR is required for hatching, at each larval molt, and for the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone-responsive genes is blocked and normal ecdysone responses of both imaginal and larval tissues are blocked at an early stage. These results show that EcR mediates ecdysone signaling at multiple developmental stages and implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis.