Peptidergic and serotonergic immunoreactivity in the metamorphosing ophiopluteus of Ophiactis resiliens (Echinodermata, Ophiuroidea)

Abstract. Antibodies against the echinoderm-specific neuropeptide S1 and against 5HT were used to examine the fate of the larval nervous system during metamorphosis in the ophiuroid Ophiactis resiliens . In contrast to most echinoderms, the onset of peptidergic and serotonergic expression was delayed to the advanced ophiopluteus stage, in particular for 5HT. In advanced ophioplutei, peptidergic immunoreactivity was located in simple fibres associated with the ciliated bands, a stomach nerve ring, and cells along the antero-lateral arms. 5HT immunoreactivity was concentrated in 2 oral ganglia in the adoral projections, located at the posterior rim of the mouth. Clusters of 5HT-positive cells were also found along the antero-lateral arms. The ophiopluteus lacked a serotonergic (or peptidergic) anterior ganglion. In echinoids, holothuroids, and crinoids, anterior ganglia are thought to have a sensory role in settlement and metamorphosis. Given that ophioplutei metamorphose in the plankton and that larval structures degenerate before settlement, the absence of apical ganglia correlates with the lack of a functional role for larval structures in substrate selection and settlement. Although most of the larval nervous system degenerated during metamorphosis, the adoral projections and associated oral ganglia appeared to be incorporated into the juvenile mouth, suggesting a potential role for larval neurons in contributing to oral neuronal structures in the adult. S1-positive neurons and fibres in the rudiment developed de novo and in parallel with development of the epineural canal. This structure gives rise to the primordia of the adult circumoral nerve ring and radial nerves, indicating that differentiation of the adult nervous system begins in the early stages of metamorphosis.     

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death

The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis.     

Anatomy of the serotonergic nervous system of an entoproct creeping-type larva and its phylogenetic implications

Abstract. Although the internal phyletic relationships of Spiralia (and Lophotrochozoa) remain unresolved, recent progress has been made due to molecular phylogenetic analyses as well as developmental studies of crucial taxa such as Mollusca, Sipuncula, or Annelida. Despite this progress, the phylogenetic position of a number of phyla, such as Entoprocta, remains problematic, mainly due to their unique morphology, their aberrant mode of development, and their exclusion in most large-scale phylogenetic analyses. In order to extend the morphological dataset of this enigmatic taxon, we herein describe the anatomy of the serotonergic nervous system of the creeping-type larva of Loxosomella murmanica . The apical organ is very complex and comprises six to eight centrally positioned flask cells and eight bipolar peripheral cells. In addition, a prototroch nerve ring, an anterior nerve loop, a paired buccal nerve, and an oral nerve ring are found. Moreover, the larva of L. murmanica has one pair of pedal and one pair of lateral longitudinal nerve cords and thus expresses a tetraneurous condition. Several paired serotonergic perikarya, which form contact with the pedal nerve cords but not with the lateral ones, are found along the anterior–posterior axis. The combination of a complex larval serotonergic apical organ and (adult) tetraneury, comprising one pair of ventral and one pair of more dorsally situated lateral longitudinal nerve cords without ganglia, has so far only been reported for basal molluscs and may be diagnostic for a mollusc–entoproct clade. In addition, the larva of Loxosomella expresses a mosaic of certain neural features that are also found in other larval or adult Spiralia, e.g., a prototroch nerve ring, an anterior nerve loop, and a buccal nervous system.     

Structure and metamorphic remodeling of the larval nervous system and musculature of Phoronis pallida (Phoronida)

The structure of the larval nervous system and the musculature of Phoronis pallida were studied, as well as the remodeling of these systems at metamorphosis. The serotonergic portion of the apical ganglion is a U-shaped field of cell bodies that send projections into a central neuropil. The majority of the serotonergic cells are (at least) bipolar sensory cells, and a few are nonsensory cells. Catecholaminergic cell bodies border the apical ganglion. The second (hood) sense organ develops at competence and is composed of bipolar sensory cells that send projections into a secondary neuropil. Musculature of the competent larva includes circular and longitudinal muscle fibers of the body wall, as well as elevators and depressors of the tentacles and hood. The juvenile nervous system and musculature are developed prior to metamorphosis and are integrated with those of the larva. Components of the juvenile nervous system include a diffuse neural net of serotonergic cell bodies and fibers and longitudinal catecholaminergic fibers. The juvenile body wall musculature consists of longitudinal fibers that overlie circular muscle fibers, except in the cincture regions, where this pattern is reversed. Metamorphosis is initiated by the larval neuromuscular system but is completed by the juvenile neuromuscular system. During metamorphosis, the larval nervous system and the musculature undergo cell death, and the larval tentacles and gut are remodeled into the juvenile arrangement. Although the phoronid nervous system has often been described as deuterostome-like, these data show that several cytological aspects of the larval and juvenile neuromuscular systems also have protostome (lophotrochozoan) characteristics.     

A developmental study of serotonin-immunoreactive neurons in the larval central nervous system of the spider crabHyas araneus (Decapoda,Brachyura)

Larval development in crabs is characterized by a striking double metamorphosis in the course of which the animals change from a pelagic to a benthic life style. The larval central nervous system has to provide an adequate behavioural repertoire during this transition. Thus, processes of neuronal reorganization and refinement of the early larval nervous system could be expected to occur in the metamorphosing animal. In order to follow identified sets of neurons throughout metamorphosis, whole mount preparations of the brain and ventral nerve cord of laboratory reared spider crab larvae (Hyas araneus) were labelled with an antibody against the neurotransmitter serotonin. The system of serotonin-immunoreactive cell bodies, fibres and neuropils is well-developed in newly hatched larvae. Most immunoreative structures are located in the protocerebrum, with fewer in the suboesophaegeal ganglia, while the thoracic and abdominal ganglia initially comprise only a small number of serotonergic neurons and fibres. However, there are significant alterations in the staining pattern through larval development, some of which are correlated to metamorphic events. Accordingly, new serotonin-immunoreactive cells are added to the early larval set and the system of immunoreactive fibres is refined. These results are compared to the serotonergic innervation in other decapod crustaceans.     

Loss of sensory elements in the apical sensory organ during metamorphosis in the nudibranch Phestilla sibogae

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a water-borne chemical cue released by the adult nudibranch's prey, the coral Porites compressa. In competent larvae, the apical sensory organ (ASO) includes five serotonergic parampullary neurons; five ampullary neurons, the ampullae of which are filled with sensory cilia; and a basal neuropil. After sensing the coral cue, the ASO undergoes radical morphological changes: a deterioration of sensory elements in the ASO and serotonergic axons originating from them to innervate the velum. Three hours after metamorphic induction, the velar lobes are lost, the serotonergic axons begin to break apart, the five parampullary neurons begin to degenerate, and the five ampullary neurons retract away from the epidermal surface. The extent of deterioration evident by this time suggests that the parampullary and ampullary components of the ASO are no longer functional. By 10 h after metamorphic induction, labeling of the ciliary bundles in the ampullary neurons has disappeared, and it is likely that these cells have degenerated. The results presented here provide evidence that the sensory neurons of the ASO and probably the entire organ are solely larval structures that do not persist into the adult sensory-nervous system in P. sibogae.     

Neurogenesis in the mossy chiton,Mopalia muscosa (Gould) (Polyplacophora): evidence against molluscan metamerism

Neurogenesis in the chiton Mopalia muscosa (Gould, 1846) was investigated by applying differential interference contrast microscopy, semithin serial sectioning combined with reconstruction techniques, as well as confocal laser scanning microscopy for the detection of fluorescence-conjugated antibodies against serotonin and FMRFamide. The ontogeny of serotonergic nervous structures starts with cells of the apical organ followed by those of the cerebral commissure, whereas the serotonergic prototroch innervation, pedal system, and the lateral cords develop later. In addition, there are eight symmetrically arranged serotonergic sensory cells in the dorsal pretrochal area of the larva. FMRFamide-positive neural elements include the cerebral commissure, specific "ampullary" sensory cells in the pretrochal region, as well as the larval lateral and pedal system. In the early juvenile the cerebral system no longer stains with either of the two antibodies and the pedal system lacks anti-FMRFamide immunoreactivity. Outgroup comparison with all other molluscan classes and related phyla suggests that the cord-like, nonganglionized cerebral system in the Polyplacophora is a reduced condition rather than a primitive molluscan condition. The immunosensitivity of the pedal commissures develops from posterior to anterior, suggesting independent serial repetition rather than annelid-like conditions and there is no trace of true segmentation during nervous system development. Polyplacophoran neurogenesis and all other available data on the subject contradict the idea of a segmented molluscan stem species.     

Programmed cell death in the apical ganglion during larval metamorphosis of the marine mollusc Ilyanassa obsoleta

The apical ganglion (AG) of larval caenogastropods, such as Ilyanassa obsoleta, houses a sensory organ, contains five serotonergic neurons, innervates the muscular and ciliary components of the velum, and sends neurites into a neuropil that lies atop the cerebral commissure. During metamorphosis, the AG is lost. This loss had been postulated to occur through some form of programmed cell death (PCD), but it is possible for cells within the AG to be respecified or to migrate into adjacent ganglia. Evidence from histological sections is supported by results from a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which indicate that cells of the AG degenerate by PCD. PCD occurs after metamorphic induction by serotonin or by inhibition of nitric oxide synthase (NOS) activity. Cellular degeneration and nuclear condensation and loss were observed within 12 h of metamorphic induction by NOS inhibition and occur before loss of the velar lobes, the ciliated tissue used for larval swimming and feeding. Velar disintegration happens more rapidly after metamorphic induction by serotonin than by 7-nitroindazole, a NOS inhibitor. Loss of the AG was complete by 72 h after induction. Spontaneous loss of the AG in older competent larvae may arise from a natural decrease in endogenous NOS activity, giving rise to the tendency of aging larvae to display spontaneous metamorphosis in culture.     

Fine structure of the doliolaria larva of the feather star Florometra serratissima (Echinodermata: Crinoidea), with special emphasis on the nervous system

The epidermis of the doliolaria larva of the Florometra serratissima is differentiated into distinct structures including an apical organ, adhesive pit, ganglion, ciliary bands, nerve plexus, and vestibular invagination. All these structures possess unique cell-types, suggesting that they are functionally specialized in the larva, except the vestibular invagination that becomes the postmetamorphic stomodeum. The epidermis also contains yellow cells, amoeboid-like cells, and secretory cells. The enteric sac, hydrocoel, axocoel, and somatocoels have differentiated but are probably not functional in the doliolaria stage. Mesenchymal cells, around the enteric sac and coeloms, appear to be actively secreting the endoskeleton and connective tissue fibers. The nervous system is composed of a nerve plexus, ganglion, and sensory receptor cells in the apical organ. The apical organ is a larval specialization of the anterior end; the ganglion is located in the base of the epidermis at the anterior dorsal end of the larva. The nerve plexus underlies most of the epidermis, although it is more prominent in the anterior region. Here, processes from sensory receptor cells of the apical organ, as well as those from nerve cells, contribute to the plexus. These processes contain one or a combination of organelles including vesicles, vacuoles, microtubules, and mitochondria. The configuration of glyoxylic acid-induced fluorescence, revealing catecholamine activity, correlates to the apical organ, nerve cells, and nerve plexus. Morphological evidence suggests that the nervous system may function in initiation and control of settlement, attachment, and metamorphosis. The crinoid larval nervous system is discussed and compared to that found in other larval echinoderms.     

Variation in serotonergic and dopaminergic neuronal survival in the central nervous system of adult <Emphasis Type="Italic">Drosophila</Emphasis>

Loss of serotonergic and dopaminergic neurons may have serious implications for normal brain function. Drosophila models of neurodegenerative diseases utilize the short life-span and simple anatomy of the fly to characterize the molecular and genetic processes characteristic of each dysfunctional state. In fly embryonic and larval ventral nerve cords, serotonergic and dopaminergic neurons are positioned in a stereotypic pattern that is reorganized during metamorphosis. In this study, we examine the adult pattern of serotonergic and dopaminergic neurons within the adult fly ventral nerve cord. We find that the number of cells lost following metamorphosis is highly variable. Changes in cell number attributable to age are therefore likely to be highly masked by developmental variation. The source of this variation is probably apoptosis-based cell loss during pupal development.This work was supported by a Keck Scholars Award and NINDS R29 37322 to BGC and by the University of Virginia Medical Scientist Training Program to PAS.     

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.     

Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis

Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as 'non-self'. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose-dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult-type immune system.     

Subtype-specific neuronal remodeling during Drosophila metamorphosis

During metamorphosis in holometabolous insects, the nervous system undergoes dramatic remodeling as it transitions from its larval to its adult form. Many neurons are generated through post-embryonic neurogenesis to have adult-specific roles, but perhaps more striking is the dramatic remodeling that occurs to transition neurons from functioning in the larval to the adult nervous system. These neurons exhibit a remarkable degree of plasticity during this transition; many subsets undergo programmed cell death, others remodel their axonal and dendritic arbors extensively, whereas others undergo trans-differentiation to alter their terminal differentiation gene expression profiles. Yet other neurons appear to be developmentally frozen in an immature state throughout larval life, to be awakened at metamorphosis by a process we term temporally-tuned differentiation. These multiple forms of remodeling arise from subtype-specific responses to a single metamorphic trigger, ecdysone. Here, we discuss recent progress in Drosophila melanogaster that is shedding light on how subtype-specific programs of neuronal remodeling are generated during metamorphosis.     

Apical organs in echinoderm larvae: insights into larval evolution in the Ambulacraria

The anatomy and cellular organization of serotonergic neurons in the echinoderm apical organ exhibits class-specific features in dipleurula-type (auricularia, bipinnaria) and pluteus-type (ophiopluteus, echinopluteus) larvae. The apical organ forms in association with anterior ciliary structures. Apical organs in dipleurula-type larvae are more similar to each other than to those in either of the pluteus forms. In asteroid bipinnaria and holothuroid auricularia the apical organ spans ciliary band sectors that traverse the anterior-most end of the larvae. The asteroid apical organ also has prominent bilateral ganglia that connect with an apical network of neurites. The simple apical organ of the auricularia is similar to that in the hemichordate tornaria larva. Apical organs in pluteus forms differ markedly. The echinopluteus apical organ is a single structure on the oral hood between the larval arms comprised of two groups of cells joined by a commissure and its cell bodies do not reside in the ciliary band. Ophioplutei have a pair of lateral ganglia associated with the ciliary band of larval arms that may be the ophiuroid apical organ. Comparative anatomy of the serotonergic nervous systems in the dipleurula-type larvae of the Ambulacraria (Echinodermata+Hemichordata) suggests that the apical organ of this deuterostome clade originated as a simple bilaterally symmetric nerve plexus spanning ciliary band sectors at the anterior end of the larva. From this structure, the apical organ has been independently modified in association with the evolution of class-specific larval forms.     

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro

Summary We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T₃) at a concentration higher than 10⁻⁹ M to the medium could induce cell death of larval epithelial cells, but T₃ alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 μg/ml) and cortisol (0.5 μg/ml) besides T₃ were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.     

Larval and adult brains

Apical organs are a well-known structure in almost all ciliated eumetazoan larvae, although their function is poorly known. A review of the literature indicates that this small ganglion is the "brain" of the early larva, and it seems probable that it represents the brain of the ancestral, holopelagic ancestor of all eumetazoans, the gastraea. This early brain is lost before or at metamorphosis in all groups. Protostomes (excluding phoronids and brachiopods) appear to have brains of dual origin. Their larvae develop a pair of cephalic ganglia at the episphere lateral to the apical organ, and these two ganglia become an important part of the adult brain. The episphere and the cerebral ganglia show Otx expression, whereas Hox gene expression has not been seen in this part of the brain. A ventral nervous system develops around the blastopore, which becomes divided into mouth and anus by fusion of the lateral blastopore lips. The circumblastoporal nerve ring becomes differentiated into a nerve ring around the mouth, becoming part of the adult brain, a pair of ventral nerve cords, in some cases differentiated into a chain of ganglia, and a ring around the anus. This part of the nervous system appears to be homologous with the oral nerve ring of cnidarians. This interpretation is supported by the expression of Hox genes around the cnidarian mouth and in the ventral nervous system of the protostomes. The development of phoronids, brachiopods, echinoderms, and enteropneusts does not lead to the formation of an episphere or to differentiation of cerebral ganglia. In general, a well-defined brain is lacking, and Hox genes are generally not expressed in the larval organs, although this has not been well studied.     

Neurometamorphosis of the ear in the gypsy moth,Lymantria dispar,and its homologue in the earless forest tent caterpillar moth,Malacosoma disstria

The adult gypsy moth, Lymantria dispar (Lymantriidae: Noctuoidea) has a pair of metathoracic tympanic ears that each contain a two-celled auditory chordotonal organ (CO). The earless forest tent caterpillar moth, Malacosoma disstria (Lasiocampidae: Bombycoidea), has a homologous pair of three-celled, nonauditory hindwing COs in their place. The purpose of our study was to determine whether the adult CO in both species arises from a preexisting larval organ or if it develops as a novel structure during metamorphosis. We describe the larval metathoracic nervous system of L. dispar and M. distria, and identify a three-celled chordotonal organ in the anatomically homologous site as the adult CO. If the larval CO is severed from the homologue of the adult auditory nerve (IIIN1b1) in L. dispar prior to metamorphosis, the adult develops an ear lacking an auditory organ. Axonal backfills of the larval IIIN1b1 nerve in both species reveal three chordotonal sensory neurons and one nonchordotonal multipolar cell. The axons of these cells project into tracts of the central nervous system putatively homologous with those of the auditory pathways in adult L. dispar. Following metamorphosis, M. disstria moths retain all four cells (three CO and one multipolar) while L. dispar adults possess two cells that service the auditory CO and one nonauditory, multipolar cell. We conclude that the larval IIIN1b1 CO is the precursor of both the auditory organ in L. dispar and the putative proprioceptor CO in M. disstria and represents the premetamorphic condition of these insects. The implications of our results in understanding the evolution of the ear in the Lepidoptera and insects in general are discussed. © 1996 John Wiley & Sons, Inc.     

Metamorphosis-related changes in the lateral line system of lampreys, <Emphasis Type="Italic">Petromyzon marinus</Emphasis>

Lamprey metamorphosis leads to considerable changes in morphology and behavior. We have recently reported that larval lampreys possess a functional lateral line system. Here we investigated metamorphic morphological changes in the lateral line system using light and electron microscopy. Functional modifications were studied by recording the trunk lateral line nerve activity of larvae and adults while stimulating neuromasts with approximately sinusoidal water motion. We found a general re-patterning of neuromasts on the head and trunk including an increase in numbers, redistribution within the pit lines, and shifts of the pit lines relative to external features. The trunk lateral line nerve response was qualitatively similar in adults and larvae. Both showed two neuronal populations responding to opposite directions of water flow. Magnitude of the response increased monotonically with stimulus amplitude. At low frequencies, the response lag relative to the stimulus maximum was approximately 220°, and the gain depended approximately linearly on frequency, confirming that superficial neuromasts are velocity detectors. Changes in phase lag with increasing stimulus frequency were steeper in larvae, suggesting slower afferent conductance. The response gain with frequency was smaller for adults, suggesting a narrower frequency discrimination range and decreased sensitivity. These changes may be adaptations for the active lifestyle of adult lampreys.