Amphioxus Larvae Research Articles

Asymmetric segregation of maternal mRNAs and germline-related determinants in Cephalochordate embryos: implications for the evolution of early patterning events in chordates.

How animal embryos determine their early cell fates is an important question in developmental biology. In various model animals asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification. Cephalochordates (amphioxus), which have three living genera (Asymmetron, Epigonichthys, Branchiostoma), are an early branching chordate lineage and thus occupy a key phylogenetic position for understanding the evolution of chordate developmental mechanisms. It has been shown that in the zygote of Brachiostoma amphioxus, which possess bilateral gonads flanking both sides of their trunk region, maternal transcripts of germline determinants form a compact granule. During early embryogenesis this granule is inherited by a single blastomere that subsequently gives rise to a cluster of cells displaying typical characteristics of primordial germ cells (PGC). These PGCs then come to lie in the tailbud region and proliferate during posterior elongation of the larva to join in the gonad anlagen at the ventral tip of the developing myomeres in amphioxus larvae. However, in Asymmetron and Epigonichthys amphioxus, whose gonads are present only on the right side of their body, nothing is known about their PGC development or the cellular/morphogenetic processes resulting in the asymmetric distribution of gonads. Using conserved germline determinants as markers, we show that similarly to Brachiostoma amphioxus, Asymmetron also employ a preformation mechanism to specify their PGCs, suggesting that this mechanism represents an ancient trait dating back to the common ancestor of Cephalochordates. Surprisingly, we found that Asymmetron PGCs are initially deposited on both sides of the body during early larval development; however, the left side PGCs cease to exist in young juveniles, suggesting that PGCs are eliminated from the left body side during larval development or following metamorphosis. This is reminiscent of the PGC development in the sea urchin embryo, and we discuss the implications of this observation for the evolution of developmental mechanisms.

Ancestral role of Pax6 in chordate brain regionalization.

The Pax6 gene is essential for eye and brain development across various animal species. Here, we investigate the function of Pax6 in the development of the anterior central nervous system (CNS) of the invertebrate chordate amphioxus using CRISPR/Cas9-induced genome editing. Specifically, we examined Pax6 mutants featuring a 6bp deletion encompassing two invariant amino acids in the conserved paired domain, hypothesized to impair Pax6 DNA-binding capacity and gene regulatory functions. Although this mutation did not result in gross morphological changes in amphioxus larvae, it demonstrated a reduced ability to activate Pax6-responsive reporter gene, suggesting a hypomorphic effect. Expression analysis in mutant larvae revealed changes in gene expression within the anterior CNS, supporting the conserved role of Pax6 gene in brain regionalization across chordates. Additionally, our findings lend support to the hypothesis of a zona limitans intrathalamica (ZLI)-like region in amphioxus, suggesting evolutionary continuity in brain patterning mechanisms. ZLI region, found in both hemichordates and vertebrates, functions as a key signaling center and serves as a restrictive boundary between major thalamic regions.

Hedgehog signaling controls mouth opening in the amphioxus

IntroductionThe left-sided position of the mouth in amphioxus larvae has fascinated researchers for a long time. Despite the fundamental importance of mouth development in the amphioxus, the molecular regulation of its development is almost unknown. In our previous study, we showed that Hh mutation in the amphioxus leads to no mouth opening, indicating a requirement of Hh signaling for amphioxus mouth formation. Nevertheless, since the Hh mutant also exhibits defects in early left-right (LR) patterning, it remains currently unknown whether the loss of mouth opening is affected directly by Hh deficiency or a secondary effect of its influence on LR establishment.ResultsWe demonstrated that knockout of the Smo gene, another key component of the Hh signaling pathway, in the amphioxus resulted in the absence of mouth opening, but caused no effects on LR asymmetry development. Upregulation of Hh signaling led to a dramatic increase in mouth size. The inability of Smo mutation to affect LR development is due to Smo’s high maternal expression in amphioxus eggs and cleavage-stage embryos. In Smo mutants, Pou4 and Pax2/5/8 expression at the primordial oral site is not altered before mouth opening.ConclusionsBased on these results and our previous study, we conclude that Hh signal is necessary for amphioxus mouth formation and that the Hh-mediated regulation of mouth development is specific to the mouth. Our data suggest that Hh signaling regulates mouth formation in the amphioxus in a similar way as that in vertebrates, indicating the conserved role of Hh signaling in mouth formation.

A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene.

Amphioxus is a promising model organism for understanding the origin and evolution of vertebrates due to its basal phylogenetic position among chordates. We here compared the mutation efficacy and mutation type of tail tips and gametes of amphioxus founders injected with Cas9 protein and six different sgRNAs targeting five distinct genes, and revealed a strong correlation for mutation efficacy and a mild correlation for mutation type among the two tissues. In addition, we also observed a positive relationship between gene insertions observed in tail tips and gametes of amphioxus founders injected with Tol2 transposase and two different transgenic constructs. Finally, we showed that amphioxus larvae which had their tail tips cut at the 3–4 gill-slit stage were able to recover within 6 days and developed a normal number of gonads at the adult stage, and that F0 larvae carry similar mutation efficacy and type in the posterior end to that in the tail tips after their metamorphosis. Together, these findings suggest a great potential for obtaining valid amphioxus founders with desired mutations and transgenes at as early as the early larval stage, which will certainly speed up the generation of amphioxus mutants and transgenes and make it more cost- and labor-effective.

Optical Clearing and Light Sheet Microscopy Imaging of Amphioxus.

Cephalochordates (amphioxi or lancelets) are representatives of the most basally divergent group of the chordate phylum. Studies of amphioxus development and anatomy hence provide a key insight into vertebrate evolution. More widespread use of amphioxus in the evo–devo field would be greatly facilitated by expanding the methodological toolbox available in this model system. For example, evo–devo research on amphioxus requires deep understanding of animal anatomy. Although conventional confocal microscopy can visualize transparent amphioxus embryos and early larvae, the imaging of later developmental stages is problematic because of the size and opaqueness of the animal. Here, we show that light sheet microscopy combined with tissue clearing methods enables exploration of large amphioxus specimens while keeping the surface and the internal structures intact. We took advantage of the phenomenon of autofluorescence of amphioxus larva to highlight anatomical details. In order to investigate molecular markers at the single-cell level, we performed antibody-based immunodetection of melanopsin and acetylated-α-tubulin to label rhabdomeric photoreceptors and the neuronal scaffold. Our approach that combines light sheet microscopy with the clearing protocol, autofluorescence properties of amphioxus, and antibody immunodetection allows visualizing anatomical structures and even individual cells in the 3D space of the entire animal body.

Formation of the initial kidney and mouth opening in larval amphioxus studied with serial blockface scanning electron microscopy (SBSEM)

BackgroundFor early larvae of amphioxus, Kaji et al. (Zool Lett 2:2, 2016) proposed that mesoderm cells are added to the rim of the forming mouth, giving it the quality of a coelomoduct without homology to the oral openings of other animals. They depended in part on non-serial transmission electron microscopic (TEM) sections and could not readily put fine structural details into a broader context. The present study of amphioxus larvae is based largely on serial blockface scanning electron microscopy (SBSEM), a technique revealing TEM-level details within an extensive anatomical volume that can be reconstructed in three dimensions.ResultsIn amphioxus larvae shortly before mouth formation, a population of compact mesoderm cells is present at the posterior extremity of the first left somite. As development continues, the more dorsal of these cells give rise to the initial kidney (Hatschek’s nephridium), while the more ventral cells become interposed between the ectoderm and endoderm in a localized region where the mouth will soon penetrate. SBSEM reveals that, after the mouth has opened, a majority of these mesoderm cells can still be detected, sandwiched between the ectoderm and endoderm; they are probably myoblasts destined to develop into the perioral muscles.ConclusionsSBSEM has provided the most accurate and detailed description to date of the tissues at the anterior end of amphioxus larvae. The present study supports the finding of Kaji et al. (2016) that the more dorsal of the cells in the posterior region of the first left somite give rise to the initial kidney. In contrast, the fate of the more ventral cells (called here the oral mesoderm) is less well understood. Although Kaji et al. (2016) implied that all of the oral mesoderm cells joined the rim of the forming mouth, SBSEM reveals that many of them are still present after mouth penetration. Even so, some of those cells go missing during mouth penetration and their fate is unknown. It cannot be ruled out that they were incorporated into the rim of the nascent mouth as proposed by Kaji et al. (2016). On the other hand, they might have degenerated or been shed from the larva during the morphogenetic interaction between the ectoderm and endoderm to form the mouth. The present SBSEM study, like Kaji et al. (2016), is based on static morphological data, and dynamic cell tracer experiments would be needed to decide among these possibilities.

Retinoic acid signaling and neurogenic niche regulation in the developing peripheral nervous system of the cephalochordate amphioxus.

The retinoic acid (RA) signaling pathway regulates axial patterning and neurogenesis in the developing central nervous system (CNS) of chordates, but little is known about its roles during peripheral nervous system (PNS) formation and about how these roles might have evolved. This study assesses the requirement of RA signaling for establishing a functional PNS in the cephalochordate amphioxus, the best available stand-in for the ancestral chordate condition. Pharmacological manipulation of RA signaling levels during embryogenesis reduces the ability of amphioxus larvae to respond to sensory stimulation and alters the number and distribution of ectodermal sensory neurons (ESNs) in a stage- and context-dependent manner. Using gene expression assays combined with immunohistochemistry, we show that this is because RA signaling specifically acts on a small population of soxb1c-expressing ESN progenitors, which form a neurogenic niche in the trunk ectoderm, to modulate ESN production during elongation of the larval body. Our findings reveal an important role for RA signaling in regulating neurogenic niche activity in the larval amphioxus PNS. Although only few studies have addressed this issue so far, comparable RA signaling functions have been reported for neurogenic niches in the CNS and in certain neurogenic placode derivatives of vertebrates. Accordingly, the here-described mechanism is likely a conserved feature of chordate embryonic and adult neural development.

The Bmp signaling pathway regulates development of left-right asymmetry in amphioxus

Establishment of asymmetry along the left-right (LR) body axis in vertebrates requires interplay between Nodal and Bmp signaling pathways. In the basal chordate amphioxus, the left-sided activity of the Nodal signaling has been attributed to the asymmetric morphogenesis of paraxial structures and pharyngeal organs, however the role of Bmp signaling in LR asymmetry establishment has not been addressed to date. Here, we show that Bmp signaling is necessary for the development of LR asymmetric morphogenesis of amphioxus larvae through regulation of Nodal signaling. Loss of Bmp signaling results in loss of the left-sided expression of Nodal, Gdf1/3, Lefty and Pitx and in gain of ectopic expression of Cerberus on the left side. As a consequence, the larvae display loss of the offset arrangement of axial structures, loss of the left-sided pharyngeal organs including the mouth, and ectopic development of the right-sided organs on the left side. Bmp inhibition thus phenocopies inhibition of Nodal signaling and results in the right isomerism. We conclude that Bmp and Nodal pathways act in concert to specify the left side and that Bmp signaling plays a fundamental role during LR development in amphioxus.

Locomotory control in amphioxus larvae: new insights from neurotransmitter data

Amphioxus larvae have a midbrain-level locomotory control center whose overall organization is known from serial TEM reconstructions. How it functions has been a puzzle, owing to uncertainty as to the transmitters used by each class of neurons, but this has recently become clearer. We summarize what is now known, and correct past misconceptions: The large paired neurons at the core of the control center are glutamatergic, and hence excitatory, the commissural neurons are GABAergic, hence probably inhibitory, and both motoneurons and ipsilateral projection neurons are cholinergic, suggesting that the latter, a class of interneurons, may be derived evolutionarily from the former. The data clarify some aspects of how fast and slow swimming are controlled and prevented from interfering with one another, but leave open the source of pacemaker activity, which could reside in the large paired neurons or circuits associated with them. A unusual type of non-synaptic junction links the fast and slow systems, but how these junctions function is open to interpretation, depending chiefly on whether they act to couple adjacent cells independent of cell type, or can have differential effects that vary with cell type. Some evolutionary implications are discussed.

CYP26 function is required for the tissue-specific modulation of retinoic acid signaling during amphioxus development.

During development, morphogens, such as retinoic acid (RA), act as mediators of intercellular communication systems to control patterning and cell fate specification processes. In vertebrates, the tightly regulated production and degradation of RA creates an anterior-posterior (A-P) morphogen gradient that is required for regional patterning of the embryo. RA catabolism in particular, mediated by members of the cytochrome P450 subfamily 26 (CYP26), has been highlighted as a key regulatory component for the formation of this gradient. RA-dependent developmental patterning is now widely recognized as a shared feature of all chordate groups (i.e. of vertebrates, tunicates, and cephalochordates). However, the evolutionary origin of the RA morphogen gradient still remains elusive. Thus, in the present study, we used pharmacological approaches to assess the roles of CYP26 enzymes in tissue-specific patterning processes in embryos and larvae of the cephalochordate amphioxus (Branchiostoma lanceolatum). Marker gene analyses revealed selective requirements for CYP26 activity in anterior endoderm, general ectoderm as well as central nervous system (CNS), but not in mesoderm. Furthermore, comparisons of the effects induced by CYP26 inhibition with those obtained by the pharmacological upregulation or downregulation of global RA signaling levels yielded evidence for a role of CYP26 in establishing an A-P RA gradient in the amphioxus embryo, important at least for patterning the CNS. Altogether, this work hence highlights the involvement of CYP26 in tissue-specific modulations of RA signaling activity in the amphioxus embryo and suggests that a RA morphogen gradient already functioned in the last common ancestor of all chordates.

Left-right asymmetry specification in amphioxus: review and prospects.

Extant bilaterally symmetrical animals usually show asymmetry in the arrangement of their inner organs. However, the exaggerated left-right (LR) asymmetry in amphioxus represents a true peculiarity among them. The amphioxus larva shows completely disparate fates of left and right body sides, so that organs associated with pharynx are either positioned exclusively on the left or on the right side. Moreover, segmented paraxial structures such as muscle blocks and their neuronal innervation show offset arrangement between the sides making it difficult to propose any explanation or adaptivity to larval and adult life. First LR asymmetries can be traced back to an early embryonic period when morphological asymmetries are preceded by molecular asymmetries driven by the action of the Nodal signaling pathway. This review sums up recent advances in understanding LR asymmetry specification in amphioxus and proposes upstream events that may regulate asymmetric Nodal signaling. These events include the presence of the vertebrate-like LR organizer and a cilia-driven fluid flow that may be involved in the breaking of bilateral symmetry. The upstream pathways comprising the ion flux, Delta/Notch, Wnt/β-catenin and Wnt/PCP are hypothesized to regulate both formation of the LR organizer and expression of the downstream Nodal signaling pathway genes. These suggestions are in line with what we know from vertebrate and ambulacrarian LR axis specification and are directly testable by experimental manipulations. Thanks to the phylogenetic position of amphioxus, the proposed mechanisms may be helpful in understanding the evolution of LR axis specification across deuterostomes.

The origin of dopaminergic systems in chordate brains: insights from amphioxus.

The basic anatomy of the central nervous system (CNS) is well conserved within the vertebrates and differs in significant ways from that of non-vertebrate chordates. Of the latter, amphioxus is of special interest, being the best available stand-in for the basal chordate condition. Immunohistochemical and gene expression studies on the developing CNS of amphioxus embryos and larvae are now sufficiently advanced that we can begin to assign specific neurotransmitter phenotypes to neurons identified by transmission electron microscopy (TEM), and then compare the distribution of cell types to that in vertebrate brains. Here, by monitoring tyrosine hydroxylase (TH) transcripts and protein, along with serial TEM, we identify a population of catecholamine-containing neurons in the anterior nerve cord of amphioxus larvae and describe their pattern of synaptic inputs and outputs. Inputs parallel those to the large paired neurons that control the larval escape response, suggesting that the TH+ system functions as an accessory excitatory and perhaps modulatory pathway in larval locomotion, with the added feature of recruiting an assortment of additional interneurons to the circuitry. The TH+ cells probably contain either L-DOPA or dopamine, and correspond closely with a cell population known from previous work on adult amphioxus to be dopaminergic. This population lies in a CNS domain now thought to comprise a combined vertebrate diencephalon plus mesencephalon, the implication being that dopaminergic nuclei in both of these brain regions could derive from a single dien-mesencephalic population in the last common ancestor of amphioxus and vertebrates.

An Animal Series of Embryological Models in Plaster by Meusel in Germany, circa 1900, probably Representing the Amphioxus

Twenty (20 cm high) models in plaster represented the beginning of the animal embryology. They were mounted on a wooden basis by a metallic wire. All the early models had a polar globule. The first stages of the segmentation were represented with successively : one, two, four, eight, and so on cells, then was a morula and a blastula stage with a cyst and then the gastrulation. At least, three models in a large size represented two hemi‐sections : one (33cm long) was probably an amphioxus larva with three layers dorsally and two ones ventrally ; the other one with ten big somites could be a drosophila sample ; and a frontal cross‐section with the beginning of the organogenesis. A label (L M M) on the basis corresponded to Meusel, a german craftsman which was active during two or three decades after 1900.These models could be the representation of the first embryological stages in the amphioxus or in a sea urchin which were largely studied in these times. Unfortunately the embryology even for its morphological aspects has made so much progress that these models have now only an historical interest and no more a teaching one.Support or Funding InformationBrieux‐Gillot Scientific Library, 48 rue Jacob, Paris 6th. France

Amphioxus tails: source and fate of larval fin rays and the metamorphic transition from an ectodermal to a predominantly mesodermal tail

It was previously discovered that tail fin rays of larval amphioxus are long ciliary rootlets in posterior epidermal cells. This work describes the heretofore unknown origin and fate of these organelles in the Florida amphioxus (Branchiostoma floridae). In late embryos, epidermal cells at the posterior end of the body increase in height, thus producing a tail fin. One ciliary rootlet in each cell elongates and also rotates through about 90°, soon becoming oriented parallel to the long axis of the cell and running continuously from the apical to the basal plasma membrane. During the subsequent growth of the larval tail, the rootlets and epidermal cells housing them reach lengths up to 120 μm. At metamorphosis, the rootlets become vacuolated and rapidly decrease in length along with the height of the tail epidermis. Contemporaneously, abundant extracellular dermal matrix accumulates in the sagittal plane of the body to produce a predominantly dermal tail fin. Throughout postmetamorphic life, the posterior epidermal cells, now without ciliary rootlets, thinly cover a largely dermal tail flange. Thus, the specialized morphology of the amphioxus tail fin is generated by two different cellular mechanisms, involving different cell populations (ectodermal and mesodermal), at different life-history stages.

Looking into eye evolution: amphioxus points the way

Looking into eye evolution: amphioxus points the way

Cloning, characterization and expression of tyrosinase-like gene in amphioxus Branchiostoma japonicum

Prophenoloxidase (tyrosinase) widely distributed in invertebrates and vertebrates, and plays a crucial role in the innate immune. In the present study, the full-length cDNA of a tyrosinase-like (designated AmphiTYR) was cloned from amphioxus Branchiostoma japonicum by PCR techniques. The full-length cDNA of AmphiTYR is 2314 bp, and its predicted open-reading frame codes for a protein of 544 amino acids with a predicted molecular mass of approximately 60.9 kDa and an isoelectric point of 5.65. It has a conserved putative copper-binding domain with six histidines in tyrosinase proteins. Six potential N-linked glycosylation sites and 14 conserved cysteine residues were also predicted to be present in B. japonicum tyrosinase. Homology analysis revealed that AmphiTYR was higher similar to vertebrates tyrosinases (32.5–40.5%) than to invertebrates phenoloxidase (6.4–25.4%). In the adult, AmphiTYR mRNA was expressed in the muscle, epidermis, notochord, ovary, hepatic caecum, pharynx and gill, but not in the neural tube, intestines and testis. During the different development stages from unfertilized egg to larvae of amphioxus, AmphiTYR expressed during all the amphioxus development. These results indicated that AmphiTYR gene not only play a pivotal role in innate immune but also play an important role during embryonic development of cephalochordate amphioxus.

A maternally established SoxB1/SoxF axis is a conserved feature of chordate germ layer patterning

Despite deep evolutionary roots in the metazoa, the gene regulatory network driving germ layer specification is surprisingly labile both between and within phyla. In Xenopus laevis, SoxB1- and SoxF-type transcription factors are intimately involved in germ-layer specification, in part through their regulation of Nodal signaling. However, it is unclear if X. laevis is representative of the ancestral vertebrate condition, as the precise roles of SoxF and SoxB1 in germ-layer specification vary among vertebrates, and there is no evidence that SoxF mediates germ-layer specification in any invertebrate. To better understand the evolution of germ-layer specification in the vertebrate lineage, we analyzed the expression of soxB1 and soxF genes in embryos and larvae of the basal vertebrate lamprey, and the basal chordate amphioxus. We find that both species maternally deposit soxB1 mRNA in the animal pole, soxF mRNA in the vegetal hemisphere, and zygotically express soxB1 and soxF throughout nascent ectoderm and mesendoderm, respectively. We also find that soxF is excluded from the vegetalmost blastomeres in lamprey and that, in contrast to vertebrates, amphioxus does not express soxF in the oral epithelium. In the context of recent work, our results suggest that a maternally established animal/vegetal Sox axis is a deeply conserved feature of chordate development that predates the role of Nodal in vertebrate germ-layer specification. Furthermore, exclusion of this axis from the vegetal pole in lamprey is consistent with the presence of an extraembryonic yolk mass, as has been previously proposed. Finally, conserved expression of SoxF in the forming mouth across the vertebrates, but not in amphioxus, lends support to the idea that the larval amphioxus mouth is nonhomologous to the vertebrate mouth.

Tail regression induced by elevated retinoic acid signaling in amphioxus larvae occurs by tissue remodeling, not cell death

The vitamin A derived morphogen retinoic acid (RA) is known to function in the regulation of tissue proliferation and differentiation. Here, we show that exogenous RA applied to late larvae of the invertebrate chordate amphioxus can reverse some differentiated states. Although treatment with the RA antagonist BMS009 has no obvious effect on late larvae of amphioxus, administration of excess RA alters the morphology of the posterior end of the body. The anus closes over, and gut contents accumulate in the hindgut. In addition, the larval tail fin regresses, although little apoptosis takes place. This fin normally consists of columnar epidermal cells, each characterized by a ciliary rootlet running all the way from an apical centriole to the base of the cell and likely contributing substantial cytoskeletal support. After a few days of RA treatment, the rootlet becomes disrupted, and the cell shape changes from columnar to cuboidal. Transmission electron microscopy (TEM) shows fragments of the rootlet in the basal cytoplasm of the cuboidal cell. A major component of the ciliary rootlet in amphioxus is the protein Rootletin, which is encoded by a single AmphiRootletin gene. This gene is highly expressed in the tail epithelial cells of control larvae, but becomes downregulated after about a day of RA treatment, and the breakup of the ciliary rootlet soon follows. The effect of excess RA on these epidermal cells of the larval tail in amphioxus is unlike posterior regression in developing zebrafish, where elevated RA signaling alters connective tissues of mesodermal origin. In contrast, however, the RA-induced closure of the amphioxus anus has parallels in the RA-induced caudal regression syndrome of mammals.

Active Metabolism of Thyroid Hormone During Metamorphosis of Amphioxus

Thyroid hormones (THs), and more precisely the 3,3',5-triiodo-l-thyronine (T(3)) acetic derivative 3,3',5-triiodothyroacetic acid (TRIAC), have been shown to activate metamorphosis in amphioxus. However, it remains unknown whether TRIAC is endogenously synthesized in amphioxus and more generally whether an active TH metabolism is regulating metamorphosis. Here we show that amphioxus naturally produces TRIAC from its precursors T(3) and l-thyroxine (T(4)), supporting its possible role as the active TH in amphioxus larvae. In addition, we show that blocking TH production inhibits metamorphosis and that this effect is compensated by exogenous T(3), suggesting that a peak of TH production is important for advancement of proper metamorphosis. Moreover, several amphioxus genes encoding proteins previously proposed to be involved in the TH signaling pathway display expression profiles correlated with metamorphosis. In particular, thyroid hormone receptor (TR) and deiodinases gene expressions are either up- or down-regulated during metamorphosis and by TH treatments. Overall, these results suggest that an active TH metabolism controls metamorphosis in amphioxus, and that endogenous TH production and metabolism as well as TH-regulated metamorphosis are ancestral in the chordate lineage.

The club‐shaped gland of amphioxus: export of secretion to the pharynx in pre‐metamorphic larvae and apoptosis during metamorphosis

AbstractIn amphioxus larvae, the club‐shaped gland is a tube connecting the pharyngeal lumen with the external environment. The functions of the gland and its fate during the larva‐to‐juvenile metamorphosis have long been controversial. Here we use a fixative including ruthenium red to preserve extracellular secretions (presumably glycoproteins) in late pre‐metamorphic larvae. This procedure reveals reddish, fibrogranular material in the lumen of the club‐shaped gland and in the pharynx adjacent to the gland's inner opening. This finding strengthens the idea that secretions of the club‐shaped gland are exported to the pharyngeal lumen to help form a mucous trap for capturing food particles entering the mouth. We also use the terminal desoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay to study apoptosis in the tissues of metamorphosing larvae. One of the earliest events of metamorphosis is the massive apoptotic destruction of the club‐shaped gland. Therefore, despite some previous opinions to the contrary, the cells of the gland do not survive to participate in the genesis of the definitive endostyle or any other post‐larval structures.