Major muscle systems in the larval caenogastropod,Ilyanassa obsoleta,display different patterns of development

This study describes the anatomical and developmental aspects of muscular development from the early embryo to competent larval stage in the gastropod Ilyanassa obsoleta. Staining of F‐actin revealed differential spatial and temporal patterns of several muscles. In particular, two major muscles, the larval retractor and pedal retractor muscles originate independently and display distinct developmental patterns similar to observations in other gastropod species. Additionally, together with the larval retractor muscle, the accessory larval muscle developed in the embryo at the trochophore stage. Therefore, both these muscles develop prior to ontogenetic torsion. The pedal retractor muscle marked the most abundant growth in the mid veliger stage. Also during the middle stage, the metapodial retractor muscle and opercular retractor muscle grew concurrently with development of the foot. We show evidence that juvenile muscles, such as the buccal mass muscle and siphon muscle develop initially during the late veliger stage. Collectively, these findings substantiate that larval myogenesis involves a complex sequence of events that appear evolutionary conserved within the gastropods, and set the stage for future studies using this model species to address issues concerning the evolution and eventual fates of larval musculature in molluscs. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Evolution of the molluscan body plan: the case of the anterior adductor muscle of bivalves

The separated shell plates with the rearranged musculature (adductor muscle) is a novelty for bivalves. Despite its importance in the bivalve bodyplan, the development of the anterior adductor muscle remains unresolved. In this study, we investigate the myogenesis of the bivalve species Septifer virgatus to reveal the developmental origin of the larval muscles in bivalves, focusing on the anterior adductor muscle. We observed that larval retractor muscles are differentiated from the ectomesoderm in bivalves, and that the anterior adductor muscles are derived from primordial larval retractor muscles via segregation of the myoblast during the veliger larval stage. Through the comparative study of myogenesis in bivalves and its related taxa, gastropods, we found that both species possess myoblasts that emerge bilaterally and later meet dorsally. We hypothesize that these myoblasts, which are a major component of the main larval retractor in limpets, are homologous to the anterior adductor muscle in bivalves. These observations imply that the anterior adductor muscle of bivalves evolved as a novel muscle by modifying the attachment sites of an existing muscle.     

Neuromuscular development in Patellogastropoda (Mollusca: Gastropoda) and its importance for reconstructing ancestral gastropod bodyplan features

Within Gastropoda, limpets (Patellogastropoda) are considered the most basal branching taxon and its representatives are thus crucial for research into evolutionary questions. Here, we describe the development of the neuromuscular system in Lottia cf. kogamogai. In trochophore larvae, first serotonin‐like immunoreactivity (lir) appears in the apical organ and in the prototroch nerve ring. The arrangement and number of serotonin‐lir cells in the apical organ (three flask‐shaped, two round cells) are strikingly similar to those in putatively derived gastropods. First, FMRFamide‐lir appears in veliger larvae in the Anlagen of the future adult nervous system including the cerebral and pedal ganglia. As in other gastropods, the larvae of this limpet show one main and one accessory retractor as well as a pedal retractor and a prototroch muscle ring. Of these, only the pedal retractor persists until after metamorphosis and is part of the adult shell musculature. We found a hitherto undescribed, paired muscle that inserts at the base of the foot and runs towards the base of the tentacles. An apical organ with flask‐shaped cells, one main and one accessory retractor muscle is commonly found among gastropod larvae and thus might have been part of the last common ancestor.     

Out-of-the tropics or trans-tropical dispersal? The origins of the disjunct distribution of the gooseneck barnacle Pollicipes elegans

Myogenesis is currently investigated in a number of invertebrate taxa using combined techniques, including fluorescence labeling, confocal microscopy, and 3D imaging, in order to understand anatomical and functional issues and to contribute to evolutionary questions. Although developmental studies on the gross morphology of bivalves have been extensively pursued, organogenesis including muscle development has been scarcely investigated so far. The present study describes in detail myogenesis in the scallop Nodipecten nodosus (Linnaeus, 1758) during larval and postmetamorphic stages by means of light, electron, and confocal microscopy. The veliger muscle system consists of an anterior adductor muscle, as well as four branched pairs of striated velum retractors and two pairs of striated ventral larval retractors. The pediveliger stage exhibits a considerably elaborated musculature comprising the velum retractors, the future adult foot retractor, mantle (pallial) muscles, and the anterior and posterior adductors, both composed of smooth and striated portions. During metamorphosis, all larval retractors together with the anterior adductor degenerate, resulting in the adult monomyarian condition, whereby the posterior adductor retains both myofiber types. Three muscle groups, i.e., the posterior adductor, foot retractor, and pallial muscles, have their origin prior to metamorphosis and are subsequently remodeled. Our data suggest a dimyarian condition (i.e., the presence of an anterior and a posterior adductor in the adult) as the basal condition for pectinids. Comparative analysis of myogenesis across Bivalvia strongly argues for ontogenetic and evolutionary independence of larval retractors from the adult musculature, as well as a complex set of larval retractor muscles in the last common bivalve ancestor.     

Development of the musculature in the limpet Patella (Mollusca, Patellogastropoda)

 Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered. Received: 23 June 1998 / Accepted: 25 November 1998     

Torsion in Patella caerulea (Mollusca, Patellogastropoda): ontogenetic process, timing, and mechanisms

Abstract. Torsion is a process in gastropod ontogenesis where the visceral body portion rotates 180° relative to the head/foot region. We investigated this process in the limpet Patella caerulea by using light microscopy of living larvae, as well as scanning electron microscopy (SEM) of larvae fixed during the torsion process. The completion of the 180° twist takes considerably less time in larvae of Patella caerulea than previously described for other basal gastropod species. At a rearing temperature of 20–22°C, individuals complete ontogenetic torsion in ?2 h. Furthermore, the whole process is monophasic, i.e., carried out at a constant speed, without any evidence of distinct ‘fast” or ‘slow” phases. Both larval shell muscles—the main and the accessory larval retractor—are already fully contractile before the onset of torsion. During the torsion process both retractors perform cramp‐like contractions at ～30 s intervals, which are followed by hydraulic movements of the foot. However, retraction into the embryonic shell occurs only after torsion is completed. The formation of the larval operculum is entirely in‐dependent from ontogenetic torsion and starts before the onset of rotation, as does the mineralization of the embryonic shell. The reported variability regarding the timing (mono‐ versus biphasic; duration) of torsion in basal gastropod species precludes any attempt to interpret these data phylogenetically. The present findings indicate that the torsion process in Patella caerulea, and probably generally in basal gastropods, is primarily caused by contraction of the larval shell muscles in combination with hydraulic activities. In contrast, the adult shell musculature, which is independently formed after torsion is completed, does not contribute to ontogenetic torsion in any way. Thus, fossil data relying on muscle scars of adult shell muscles alone appear inappropriate to prove torted or untorted conditions in early Paleozoic univalved molluses. Therefore, we argue that paleontological studies dealing with gastropod phylogeny require data other than those based on fossilized attachment sites of adult shell muscles.     

Developmental dynamics of myogenesis in the shipworm <Emphasis Type="Italic">Lyrodus pedicellatus</Emphasis> (Mollusca: Bivalvia)

Background

The shipworm Lyrodus pedicellatus is a wood-boring bivalve with an unusual vermiform body. Although its larvae are brooded, they retain the general appearance of a typical bivalve veliger-type larva. Here, we describe myogenesis of L. pedicellatus revealed by filamentous actin labelling and discuss the data in a comparative framework in order to test for homologous structures that might be part of the bivalve (larval) muscular ground pattern.

Results

Five major muscle systems were identified: a velum retractor, foot retractor, larval retractor, a distinct mantle musculature and an adductor system. For a short period of larval life, an additional ventral larval retractor is present. Early in development, a velum muscle ring and an oral velum musculature emerge. In late stages the lateral and dorsal mantle musculature, paired finger-shaped muscles, an accessory adductor and a pedal plexus are formed. Similar to other bivalve larvae, L. pedicellatus exhibits three velum retractor muscles, but in contrast to other species, one of them disappears in early stages of L. pedicellatus. The remaining two velum retractors are considerably remodelled during late larval development and are most likely incorporated into the elaborate mantle musculature of the adult.

Conclusions

To our knowledge, this is the first account of any larval retractor system that might contribute to the adult bodyplan of a (conchiferan) mollusk. A comparative analysis shows that a pedal plexus, adductors, a larval velum ring, velum retractors and a ventral larval retractor are commonly found among bivalve larvae, and thus most likely belong to the ground pattern of the bivalve larval musculature.

     

Muscle development in Antalis entalis (Mollusca,Scaphopoda) and its significance for scaphopod relationships

We applied fluorescence staining of F-actin, confocal laser scanning microscopy, as well as bright-field light microscopy, SEM, and TEM to examine myogenesis in larval and early juvenile stages of the tusk-shell, Antalis entalis. Myogenesis follows a strict bilaterally symmetrical pattern without special larval muscle systems. The paired cephalic and foot retractors appear synchronously in the early trochophore-like larva. In late larvae, both retractors form additional fibers that project into the anterior region, thus enabling retraction of the larval prototroch. These fibers, together with the prototroch, disappear during metamorphosis. The anlagen of the putative foot musculature, mantle retractors, and buccal musculature are formed in late larval stages. The cephalic captacula and their musculature are of postmetamorphic origin. Development of the foot musculature is dramatically pronounced after metamorphosis and results in a dense muscular grid consisting of outer ring, intermediate diagonal, and inner longitudinal fibers. This is in accordance with the proposed function of the foot as a burrowing organ based on muscle-antagonistic activity. The existence of a distinct pair of cephalic retractors, which is also found in basal gastropods and cephalopods, as well as new data on scaphopod shell morphogenesis and recent cladistic analyses, indicate that the Scaphopoda may be more closely related to the Gastropoda and Cephalopoda than to the Bivalvia.     

Development and metamorphic loss of the musculature in larvae of the nudibranch Phestilla sibogae: A functional ontogeny

Developmental programmes for many marine invertebrates include the assembly of muscular systems appropriate to the functions of swimming and feeding in pelagic larvae. Upon metamorphosis, that musculature is often radically re-organized to meet very different demands of post-larval life. To investigate the development and fate of musculature in the nudibranch Phestilla sibogae, embryos, larvae and metamorphosing stages were fixed, labelled with phalloidin and examined with confocal microscopy. The resultant images revealed the sequential development of both large retractor muscles and numerous finer muscles that allow the larva to manipulate the velum, foot and operculum. Observations of living specimens at the same stages as those fixed for microscopy revealed the actions of the muscles as they developed. During metamorphosis, muscles with shell attachments disintegrate as the larva transforms into a shell-less juvenile. Notably, the massive velar, pedal and opercular retractor muscles disappear during metamorphosis in a sequence that corresponds to their loss of function. Other muscles, however, that appear to be important to the embryo and free-swimming larva persist into juvenile life. The comprehensive and detailed observations of the musculature presented here provide a solid foundation for comparisons with other species with different phylogenies and life histories.     

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 of the marine gastropod Phestilla Sibogae Bergh (nudibranchia: aeolidacea). I. Light and electron microscopic analysis of larval and metamorphic stages

The structure and fate of transitory larval organs (velum, shell, operculum, retractor muscles, part of the epidermis) of Phestilla sibogae Bergh were studied before, during, and after metamorphosis with both light and electron microscopy to elucidate the morphology of these organs and the mechanisms by which they are lost.Loss of the velar lobes is the first morphological sign of metamorphosis, and involves selective dissociation and subsequent ingestion of the ciliated velar cells; the remaining aggregate of supportive cells is apparently incorporated into cephalic epidermis. Attachment of the larval body to shell and operculum is primarily at sites of retractor muscle insertions; once the velum is gone, the attachment between shell and larval body is lost and the shell is cast off as the visceral organs exit through the shell aperture. Merger of visceral and cephalopedal elements results in flattening of the postlarval body and reorientation of internal organs. Simultaneously, a rapid spreading of epipodial epidermis over the lateral, dorsal, and posterior sides of the body produces the definitive integument. The squamous cells which comprise the larval perivisceral epidermis are pushed ahead of the definitive epidermis and are seen shortly after the shell is cast as a constricted aggregate of cells on the posterior end of the body. Autolysis of the left and right retractor muscles begins during metamorphosis and no trace of them is left after 24 to 48 h. The metapodial mucous glands which hypertrophy before metamorphosis are also lost within 48 h following exit of the post larva from the shell. Metamorphosis produces a detorsion caused in part by muscular action and in part by continuing growth and development.     

Sequential developmental programmes for retractor muscles of a caenogastropod: reappraisal of evolutionary homologues

Evolutionary changes in the development of shell-attached retractor muscles in gastropods are of fundamental importance to theories about the early evolution and subsequent diversification of this molluscan class. Development of the shell-attached retractor muscle (columellar muscle) in a caenogastropod has been studied at the ultrastructural level to test the hypothesis of homology with the post-torsional left retractor muscle (larval velar retractor) in vetigastropod larvae. The vetigastropod muscle has been implicated in the generation of ontogenetic torsion, a morphogenetic twist between body regions that is important to theories about early gastropod evolution. Two shell-attached retractor muscles develop sequentially in the caenogastropod, Polinices lewisii, which is a pattern that has been also identified in previous ultrastructural studies on a vetigastropod and several nudibranch gastropods. The pattern may be a basal and conserved characteristic of gastropods. I found that the first-formed retractor in larvae of P. lewisii is comparable to the larval velar retractor that exists at the time of ontogenetic torsion in the vetigastropod, Haliotis kamtschatkana. However, the post-metamorphic columellar muscle of P. lewisii is derived exclusively from part of the second-formed muscle, which is comparable to the second-formed pedal muscle system in the vetigastropod. I conclude that the post-metamorphic columellar muscle of P. lewisii, is not homologous to the larval velar retractor of the vetigastropod, H. kamtschatkana.     

Development of oral and branchial muscles in lancelet larvae of Branchiostoma japonicum

The perforated pharynx has generally been regarded as a shared characteristic of chordates. However, there still remains phylogenetic ambiguity between the cilia‐driven system in invertebrate chordates and the muscle‐driven system in vertebrates. Giant larvae of the genus Asymmetron were reported to develop an orobranchial musculature similar to that of vertebrates more than 100 years ago. This discovery might represent an evolutionary link for the chordate branchial system, but few investigations of the lancelet orobranchial musculature have been completed since. We studied staged larvae of a Japanese population of Branchiostoma japonicum to characterize the developmental property of the orobranchial musculature. The larval mouth and the unpaired primary gills develop well‐organized muscles. These muscles function only as obturators of the openings without antagonistic system. As the larval mouth enlarged posteriorly to the level of the ninth myomere, the oral musculature was fortified accordingly without segmental patterning. In contrast, the iterated branchial muscles coincided with the dorsal myomeric pattern before metamorphosis, but the pharynx was remodeled dynamically irrespective of the myomeric pattern during metamorphosis. The orobranchial musculature disappeared completely during metamorphosis, and adult muscles in the oral hood and velum, as well as on the pterygial coeloms developed independently. The lancelet orobranchial musculature is apparently a larval adaptation to prevent harmful intake. However, vestigial muscles appeared transiently with the secondary gill formation suggest a bilateral ancestral state of muscular gills, and a segmental pattern of developing branchial muscles without neural crest and placodal contributions is suggestive of a precursor of vertebrate branchiomeric pattern. J. Morphol. 275:465–477, 2014. © 2013 Wiley Periodicals, Inc.     

Ontogenetic torsion in two basal gastropods occurs without shell attachments for larval retractor muscles

Results of this study on two species of vetigastropods contradict the long-standing hypothesis, originally proposed by Garstang (1929), that the larval retractor muscles power the morphogenetic movement of ontogenetic torsion in all basal gastropods. In the trochid Calliostoma ligatum and the keyhole limpet Diodora aspera, the main and accessory larval retractor muscles failed to establish attachments onto the protoconch (larval shell) when the antibiotics streptomycin sulfate and penicillin G were added to cultures soon after fertilization. Defects in protoconch mineralization were also observed. Despite these abnormalities, developing larvae of these species accomplished complete or almost complete ontogenetic torsion, a process in which the head and foot rotate by 180 degrees relative to the protoconch and visceral mass. Analysis by using phalloidin-fluorophore conjugate and transmission electron microscopy showed that myofilaments differentiated within myocytes of the larval retractor muscles and adherens-like junctions formed between muscle and mantle epithelial cells in both normal and abnormal larvae. However, in abnormal larvae, apical microvilli of mantle cells that were connected to the base of the larval retractor muscles failed to associate with an extracellular matrix that normally anchors the microvilli to the mineralized protoconch. If morphogenesis among extant, basal gastropods preserves the original developmental alteration that created gastropod torsion, as proposed by Garstang (1929), then the alteration involved something other than the larval retractor muscles. Alternatively, the developmental process of torsion has evolved subsequent to its origin in at least some basal gastropod clades so that the original alteration is no longer preserved in these clades.     

Larval muscle contraction fails to produce torsion in a trochoidean gastropod.

The causes and effects of ontogenetic torsion in gastropods have been debated intensely for more than a century (1-19). Occurring rapidly and very early in development, torsion figures prominently in shaping both the larval and adult body plans. We show that mechanical explanations of the ontogenetic event that invoke contraction of larval retractor muscles are inadequate to explain the observed consequences in some gastropods. The classic mechanical explanation of Crofts (4, 5) and subsequent refinements of her explanation have been based on species with rigid larval shell properties (18, 19) that cannot be extrapolated to all gastropods. We present visual evidence of the lack of rigidity of the uncalcified larval shell in a basal trochid gastropod, Margarites pupillus (Gould), and provide photographic confirmation of our prediction that larval retractor muscle contraction is insufficient to produce more than local deformation or dimpling at the site of muscle insertion. These findings do not refute muscular contraction as a primary cause of ontogenetic torsion in gastropods that calcify their larval shells prior to the onset of torsion, nor do they refute the monophyly of torsion. They do, however, suggest that torsion may be a loosely constrained developmental process with multiple pathways to the more constrained end result (20, 21).     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

Molluscan muscle systems in development and evolution*

The evolutionary history of the various molluscan muscle systems reflects drastic modifications and reductions as well as true innovations. No less than eight main and independent muscle systems of the Mollusca are described and, based on the current understanding of molluscan phylogeny, their evolutionary histories are outlined.New data on the myogenesis of the Polyplacophora by means of fluorescence‐staining and image analysis by confocal laser scanning microscopy show that the pre‐oral region recapitulates a ‘worm‐grid’, and that the dorso‐ventral musculature passes a stage of multiple seriality as found in adult Solenogastres. Old and new data on bivalves and recent studies on primitive gastropods provide clear evidence that the larval musculature of both groups (and thus possibly of all conchiferans) is entirely independent from the adult condition. The growth of shell‐inserted muscles always necessitates substantial renewal of myocytes which is still poorly understood. Although very promising for phylogenetic purposes, the understanding of the developmental genetics of the various molluscan muscle systems is still in its infancy.     

Myogenesis in two polyclad platyhelminths with indirect development,Pseudoceros canadensis and Stylostomum sanjuania

SUMMARY Myogenesis of two representatives of Platyhelminthes, Stylostomum sanjuania and Pseudoceros canadensis, was followed from egg deposition until well‐differentiated free‐swimming larval stages, using F‐actin staining and confocal laserscanning microscopy. Zonulae adhaerentes are the only structures to stain before 50% of development between egg deposition and hatching in S. sanjuania, and before 67% of development in P. canadenis. Subsequently, irregular fibers appear in the embryo, followed by a helicoid muscle close to the apical pole. Three longitudinal muscle pairs form, of which the dorsal pair remains more pronounced than the others. Gradually, new muscles form by branching or from double‐stranded muscle zones adjacent to existing muscles. This results in an elaborate muscular bodywall that consists of a single helicoid muscle as well as multiple circular and longitudinal muscles. Diverse retractor muscles insert at the sphincter muscles around the stomodeum. The overall arrangement and formation mode of the larval musculature appears very similar in both species, although only P. canadensis has a primary circular muscle posterior to the helicoid muscle. Muscle formation in the apical region of the embryo precedes that at the abapical pole and the primary longitudinal muscles form slightly later than the primary circular muscles. Myogenesis and larval myoanatomy appears highly conserved among polyclad flatworms, but differs significantly from that of other trochozoan clades. Our data suggest that the larval muscular ground pattern of polyclad larvae comprises a bodywall consisting of a helicoid muscle, circular and longitudinal muscles, several retractor muscles, and sphincter muscles around the stomodeum.     

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