Loss and gain of the juvenile rudiment and metamorphic competence during starvation and feeding of bryozoan larvae

SUMMARY In many animals, larval structures and juvenile rudiments develop independently. One advantage of this independence is that juvenile rudiments can be expended as a nutrient reserve or for energy conservation. When bryozoan cyphonautes larvae were starved, structures required for settlement and metamorphosis shrank. When the larvae were again fed, these structures grew back. Starvation reduced the size of both the internal sac, a rudiment of postlarval juvenile structures, and the pyriform organ, which functions in sensing and crawling on the substratum at settlement. In contrast, starvation affected neither the size of the larval shell nor the lengths of the ciliary bands used in swimming and feeding. Starved larvae that had reduced the pyriform organ and internal sac did not metamorphose in response to stimuli from a laminarian alga. The laminarian alga did stimulate metamorphosis of the same larvae after renewed feeding, when the larvae had regrown these structures. Thus starved larvae expended body parts needed for settlement and metamorphosis when food was scarce while retaining structures for feeding, swimming, and defense. Starved larvae thereby retained the capacity to regrow structures needed for settlement and metamorphosis when they again encountered food. Advantages from expendable juvenile rudiments may enhance selection for their being developmentally distinct from structures for larval swimming and feeding.     

Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis

In ascidians, the events of metamorphosis transform the non-feeding, mobile tadpole larva into a filter-feeding, fixed juvenile, and the process involves rearrangements of cells, two organs and physiological changes. Differential screening was used to isolate two genes that are not expressed in swimming larvae but are expressed immediately after the initiation of metamorphosis in Ciona intestinalis. One of the genes, Ci-meta1, encodes a polypeptide with a putative secretion signal sequence, 6 epidermal growth factor (EGF)-like repeats and 13 calcium-binding EGF-like repeats. The gene begins to be expressed immediately after the beginning of metamorphosis in the adhesive organ and is likely to be associated with the signal response for metamorphosis. Another gene named Ci-meta2 encodes a protein with a putative secretion signal and three thrombospondin type-1 repeats. Ci-meta2 gene expression begins at the larval stage and is upregulated in the metamorphosing juveniles. Ci-meta2 expression is found in three regions; the adhesive organ which is also associated with settlement, the neck region between the trunk and the tail of the larva which is associated with tail resorption, and dorsal regions of the trunk which correspond to the location of the siphon primordium. This gene may be involved in the dynamic arrangement of cells during ascidian metamorphosis.     

Energetics of larval swimming and metamorphosis in four species of Bugula (Bryozoa)

The amount of energy available to larvae during swimming, location of a suitable recruitment site, and metamorphosis influences the length of time they can spend in the plankton. Energetic parameters such as swimming speed, oxygen consumption during swimming and metamorphosis, and elemental carbon and nitrogen content were measured for larvae of four species of bryozoans, Bugula neritina, B. simplex, B. stolonifera, and B. turrita. The larvae of these species are aplanktotrophic with a short free-swimming phase ranging from less than one hour to a maximum of about 36 hours. There is about a fivefold difference in larval volume among the four species, which scales linearly with elemental carbon content and, presumably, with the amount of endogenous reserves available for swimming and metamorphosis. Mean larval swimming speeds (in centimeters per second) were similar among species. Specific metabolic rate and larval size were inversely related. For larvae of a given species, respiration rates remained similar for swimming and metamorphosis; however, because metamorphosis lasts about twice as long as a maximal larval swimming phase, it was more energetically demanding. Larger larvae expended more energy to complete metamorphosis than did smaller larvae, but in terms of the percentage of larval energy reserves consumed, swimming and metamorphosis were more "expensive" for smaller larvae. A comparison of the energy expended during larval swimming calculated on the basis of oxygen consumption and on the basis of elemental carbon decrease suggests that larvae of Bugula spp. may not use significant amounts of dissolved organic material (DOM) to supplement their endogenous energy reserves.     

Characterization and expression of calmodulin gene during larval settlement and metamorphosis of the polychaete Hydroides elegans

The polychaete Hydroides elegans (Serpulidae, Lophotrochozoa) is a problematic marine fouling organism in most tropical and subtropical coastal environment. Competent larvae of H. elegans undergo the transition from the swimming larval stage to the sessile juvenile stage with substantial morphological, physiological, and behavior changes. This transition is often referred to as larval settlement and metamorphosis. In this study, we examined the possible involvement of calmodulin (CaM) - a multifunctional calcium metabolism regulator, in the larval settlement and metamorphosis of H. elegans. A full-length CaM cDNA was successfully cloned from H. elegans (He-CaM) and it contained an open reading frame of 450 bp, encoding 149 amino acid residues. It was highly expressed in 12h post-metamorphic juveniles, and remained high in adults. In situ hybridization conducted in competent larvae and juveniles revealed that He-CaM gene was continuously expressed in the putative growth zones, branchial rudiments, and collar region, suggesting that He-CaM might be involved in tissue differentiation and development. Our subsequent bioassay revealed that the CaM inhibitor W7 could effectively inhibit larval settlement and metamorphosis, and cause some morphological defects of unsettled larvae. In conclusion, our results revealed that CaM has important functions in the larval settlement and metamorphosis of H. elegans.     

Further characterization of genes expressed during Ciona intestinalis metamorphosis

Metamorphosis of ascidians is a dynamic event by which a nonfeeding, mobile tadpole larva is transformed into a filter-feeding, fixed juvenile. This process usually begins with the settlement of the larva and is followed by a series of coordinated morphogenetic movements that rearrange organs, tissues, and cells. To identify genes that are involved in the initiation of metamorphosis, we conducted differential screening between mRNAs of swimming larvae and those of juveniles in Ciona intestinalis. This screening permitted the isolation of cDNA clones for genes whose expression is upregulated during metamorphosis, and the characterization of four such genes (Ci-meta3, Ci-meta4, Ci-meta5 and Ci-meta6) is reported here. Ci-meta3 encodes a protein with a domain found in Sp1a and the RYanodine receptor. This gene is not expressed in early swimming larvae but is expressed in the endoderm region and part of the retractile tail region in metamorphosing juveniles. The predicted proteins encoded by Ci-meta4, Ci-meta5 and Ci-meta6 do not contain any known consensus motifs, nor do they show any similarity to known proteins. Ci-meta4 and Ci-meta5 are expressed weakly in mesenchyme cells of the early larva and strongly in the metamorphosing juvenile, while Ci-meta6 is expressed in the mesenchyme in the late larva. In addition, we characterized 53 independent cDNA clones whose expression was downregulated during the period from early swimming larvae to metamorphosing juveniles by taking advantage of the Ciona intestinalis cDNA project database and BLAST searches. The expression patterns of some of these clones were changed during the larval period.     

Presence of thyroid hormones in ascidian larvae and their involvement in metamorphosis

In this study we investigated the presence and localization of thyroxine in Ciona intestinalis larvae and its involvement in metamorphosis. To date, the mechanisms regulating the metamorphosis of ascidians remain largely unknown. In vivo treatment of swimming larvae with exogenous L-thyroxine and thiourea, and in vitro experiments utilizing high performance liquid chromatography, radioimmunoassay, and immunoperoxidase staining demonstrate the presence of thyroxine at the larval stage. This suggests that this hormone may participate in the control of metamorphosis and thus play a different role from that observed in adults.     

The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function

The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene (Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx "retinal" territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.     

Control of polytenic replication in dipteran larvae. I. Increased number of cycles in a mutant strain of Drosophila melanogaster

Previous studies have demonstrated that a consistent maximum number of polytenic replication cycles occurs in the salivary gland nuclei of a wild-type strain of D. melanogaster. Since that number is achieved within the larval period, the DNA synthesis of the prepupal period is believed to be that of propagation of the final cycle. Photometric determinations have been made, in this study, of the salivary gland nuclei of larvae and prepupae of the tu-h strain in which the larval period has been extended as a consequence of delay or failure of pupation. The DNA values indicate that a higher maximum number of polytenic replications is achieved in such nuclei. It is inferred, thereby, that initiation of polytenic replication is a function of the larval state and, since it is terminated by the intervention of metamorphosis, a hormonal dependence is suggested.     

Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.     

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml.Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR.To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml.On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.     

The use of computer-assisted motion analysis for quantitative studies of the behaviour of barnacle (Balanus amphitrite) larvae

The effects of larval density and age on pre-settlement swimming behaviour of Balanus amphitrite cyprid larvae were studied with the aid of computer-assisted motion analysis. Swimming behaviour was monitored in individual, in groups of 10-15 and in groups of 50-100 cyprids. There was a small, but significant effect of larval density on swimming speed and no effect on two other quantitative measures: rate of change of direction and net-to-gross displacement ratio. There was also small but significant variation in swimming speed between different batches of cyprids over the course of 2 years. Swimming behaviour of individual cyprid larvae was also monitored daily for 7 days, with the larvae maintained in the cold and dark between measurements to prevent settlement and metamorphosis. There were no significant behavioural differences observed over time indicating that larvae may be held in this manner experimentally without affecting these parameters.     

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     

Changing spatial patterns of DNA replication in the developing wing of Drosophila

Using an antibody against bromodeoxyuridine we have analyzed the distribution of S-phase nuclei in the wing disc of Drosophila as the larval disc transforms into the adult wing during metamorphosis. On the basis of the timing of replication three cell populations can be distinguished: the cells of the presumptive wing margin, the precursor cells of the longitudinal veins, and those of the intervein regions. In each of these populations the cell cycle is first arrested and later resumes at a specific time, so that at each developmental time point a characteristic spatial pattern of S-phase nuclei is seen. An interpretation of these changing patterns in terms of vein formation, compartments, and neural development is offered.     

DNA replication events during larval silk gland development in the silkworm, Bombyx mori

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.     

Short‐term larval food stress and associated compensatory growth reduce adult immune function in a damselfly

Abstract 1. In animals with a complex life cycle, larval stressors may carry over to the adult stage. Carry‐over effects not mediated through age and size at metamorphosis have rarely been studied. The present study focuses on the poorly documented immune costs of short‐term food stress both in the larval stage and after metamorphosis in the adult stage. 2. The present study quantified immune function [number of haemocytes, activity of prophenoloxidase (proPO) and phenoloxidase (PO)] in an experiment where larvae of the damselfly Lestes viridis were exposed to a transient starvation period. 3. Directly after starvation, immune variables were reduced in starved larvae. Levels of proPO and PO remained low after starvation, even after metamorphosis. In contrast, haemocyte numbers were fully compensated by the end of the larval stage, yet were lower in previously starved animals after metamorphosis. This can be explained as a cost of the observed compensatory growth after starvation. Focusing only on potential costs of larval stressors within the larval stage may therefore be misleading. 4. The here‐identified immunological cost in the adult stage of larval short‐term food stress and associated compensatory growth strongly indicates that physiological costs may explain hidden carry‐over effects bridging metamorphosis. This adds to the increasing awareness that the larval and adult stages in animals with a complex life cycle should be jointly studied, as trade‐offs may span metamorphosis.     

Lumichrome. A larval metamorphosis-inducing substance in the ascidian Hhalocynthia roretzi.

It has long been known that metamorphosis of ascidian larvae is induced by exposure to adult tunic extract or larval-conditioned seawater. However, such a natural 'inducer' has not been identified, probably due to its very low concentration in organisms. Here we have succeeded in isolating the same metamorphosis-inducing substance from the larvae, the larval-conditioned seawater, and the adult tunic of the ascidian Halocynthia roretzi. Structural analysis revealed that this substance was identical to lumichrome. Lumichrome was active toward H. roretzi larvae, but inactive toward another ascidian larvae, suggesting that lumichrome is species-specific. Riboflavin (vitamin B2), from which lumichrome might be derived from, was found to be inactive in induction of larval metamorphosis. In addition, it was demonstrated that lumichrome is localized predominantly in the basal region of the adhesive organ and the posterior part of the larval trunk. Thus, we propose that lumichrome functions as a natural inducer for larval metamorphosis in H. roretzi. This is the first natural metamorphosis-inducing substance to be identified in ascidians.     

Effect of changes in resource level on age and size at metamorphosis in Hyla squirella

Recent experiments suggest that timing of metamorphosis is fixed during development in some anurans, insects, and freshwater invertebrates. Yet, these experiments do not exclude a growth rate optimization model for the timing of metamorphosis. I manipulated food resources available to larvae of squirrel treefrogs (Hyla squirella) to determine if there is a loss of plasticity in duration of larval period during development and to critically test growth rate models for the timing of metamorphosis. Size-specific resource levels for individual tadpoles were switched from low to high or high to low at three developmental stages spaced throughout larval development. The effects of changes in resource availability on larval period and mass at metamorphosis were measured. Switching food levels after late limb bud development did not significantly affect larval period in comparison to constant food level treatments. Therefore, developmental rate in H. squirella is better described by a fixed developmental rate model, rather than a growth rate optimization model. The timing of fixation of developmental rate in H. squirella is similar to that found in other anuran species, suggesting a taxonomically widespread developmental constraint on the plasticity of larval period duration. Mass at metamorphosis was not significantly affected by the timing of changes in food levels; the amount of food available later in development determined the size at metamorphosis. Larval period and mass at metamorphosis were negatively correlated in only one of two experiments, which contrasts with the common assumption of a phenotypic trade-off between decreased larval period and increased mass at metamorphosis. Received: 19 August 1996 / Accepted: 20 June 1997