Exposure to copper sulfate impairs survival, post-embryonic midgut development and reproduction in Aedes aegypti.

Aedes aegypti is a vector of several global human viruses responsible for high human morbidity and mortality. The method to prevent the transmission of vector-borne viruses is mainly based on the control of the insect vector using insecticides. Among these chemicals, copper sulfate is a compound widely used in agriculture with the potential to be used as an alternative to control these insects. This study evaluated the effects of the exposure of A. aegypti larvae to copper sulfate on survival, midgut morphology, blood-feeding and fecundity. The exposure to CuSO4 decreased the survival of A. aegypti during the immature phase. Adults obtained from exposed larvae had their lifespan decreased at all tested concentrations. The exposure to CuSO4 impaired the development in the transition from larvae to pupae and from pupae to adult. The number of eggs laid by females developed from larvae treated with CuSO4 was significantly lower than in control. In addition, the egg hatching rates were also negatively affected. The midguts of treated larvae and pupae showed epithelial disorganization. The number of cleaved caspase-3 cells increased in the midgut of exposed pupae compared to control. Moreover, there was a reduction in proliferating cells in treated larvae and pupae compared to the control. In conclusion, the results reveal that CuSO4 exposure has insecticidal activity against A. aegypti, which may be related to the impairment of the midgut metamorphosis and reduced proliferation of stem cells, with the consequent impairment of female mosquito fertility and fecundity.

Avian ceca are indispensable for hindgut enteric nervous system development.

The enteric nervous system (ENS), which is derived from enteric neural crest cells (ENCCs), represents the neuronal innervation of the intestine. Compromised ENCC migration can lead to Hirschsprung disease, which is characterized by an aganglionic distal bowel. During the craniocaudal migration of ENCCs along the gut, we find that their proliferation is greatest as the ENCC wavefront passes through the ceca, a pair of pouches at the midgut-hindgut junction in avian intestine. Removal of the ceca leads to hindgut aganglionosis, suggesting that they are required for ENS development. Comparative transcriptome profiling of the cecal buds compared with the interceca region shows that the non-canonical Wnt signaling pathway is preferentially expressed within the ceca. Specifically, WNT11 is highly expressed, as confirmed by RNA in situ hybridization, leading us to hypothesize that cecal expression of WNT11 is important for ENCC colonization of the hindgut. Organ cultures using embryonic day 6 avian intestine show that WNT11 inhibits enteric neuronal differentiation. These results reveal an essential role for the ceca during hindgut ENS formation and highlight an important function for non-canonical Wnt signaling in regulating ENCC differentiation.

Onset of nutrient consumption during early life stage digestive system development of two tuna species (Thunnus orientalis and Thunnus albacares).

To specify the timing of exogenous nutrient consumption in the larvae of two commercially important tuna species, the Pacific bluefin tuna (PBF) Thunnus orientalis and the yellowfin tuna (YFT) Thunnus albacares, the gene expressions of peptide transporter 1 (PEPT1) were examined. The mRNA expressions of PEPT1 first occurred at 2 days post hatching (dph) in PBF larvae and 3 dph for the YFT, and PEPT1 was found to only be expressed in the intestinal tract. The histological changes of the digestive tract of the YFT larvae were observed and compared to PBF larvae from a previous study. The intestines were developed at the hatching day for both species. It was found that the developmental timing of internal organs differed between the species, with the YFT showing an approximately one-day delay. The major organs such as liver, pancreas and gall bladder that excrete digestive enzymes appeared at 1 dph for PBF and 2 dph for YFT. The development of external morphological features was similar to organ development timings, with mouth-opening and first feeding starting at 2 dph for PBF, and 3 dph for YFT. Growth during the first month is rapid and variable for both species, ranging from 1.06 to 1.56 mm/d. Our findings provide new information about the early onset of feeding and larval development for the two species which would contribute to future aquaculture.

Expression of Piwi, MMP, TIMP, and Sox during Gut Regeneration in Holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirotida).

Mesodermal cells of holothurian Eupentacta fraudatrix can transdifferentiate into enterocytes during the regeneration of the digestive system. In this study, we investigated the expression of several genes involved in gut regeneration in E. fraudatrix. Moreover, the localization of progenitor cells of coelomocytes, juvenile cells, and their participation in the formation of the luminal epithelium of the digestive tube were studied. It was shown that Piwi-positive cells were not involved in the formation of the luminal epithelium of the digestive tube. Ef-72 kDa type IV collagenase and Ef-MMP16 had an individual expression profile and possibly different functions. The Ef-tensilin3 gene exhibited the highest expression and indicates its potential role in regeneration. Ef-Sox9/10 and Ef-Sox17 in E. fraudatrix may participate in the mechanism of transdifferentiation of coelomic epithelial cells. Their transcripts mark the cells that plunge into the connective tissue of the gut anlage and give rise to enterocytes. Ef-Sox9/10 probably controls the switching of mesodermal cells to the enterocyte phenotype, while Ef-Sox17 may be involved in the regulation of the initial stages of transdifferentiation.

Midgut transcriptome assessment of the cockroach-hunting wasp Ampulex compressa (Apoidea: Ampulicidae).

The emerald jewel wasp Ampulex compressa (Hymenoptera: Ampulicidae) is a solitary wasp that is widely known for its specialized hunting of cockroaches as larvae provision. Adult wasps mainly feed on pollen and nectar, while their larvae feed on the cockroachs' body, first as ecto- and later as endoparsitoids. Little is known about the expression of digestive, detoxification and stress-response-related genes in the midgut of A. compressa, or about its transcriptional versatility between life stages. To identify gut-biased genes related to digestion, detoxification, and stress response, we explored the midgut transcriptome of lab-reared A. compressa, for both adults and larvae, by focusing on the top 100 significantly up- and down-regulated genes. From the top 100 significantly differentially expressed genes (DEGs), we identified 39 and 36 DEGs putatively related to digestion and detoxification in the adult wasps and larvae, respectively. The two carbohydrases alpha-glucosidase (containing an alpha-amylase domain) and glycosyl hydrolase family 31, as well as the two proteinases chymotrypsin and trypsin, revealed the highest gene diversity. We identified six significant DEGs related to detoxification, which comprise glutathione S-transferase, cytochrome P450s and UDP-glucuronosyltransferase. The gene expression levels that were significantly expressed in both life stages vary strongly between life stages, as found in genes encoding for chymotrypsin and trypsin or glycosyl hydrolases family 31. The number of genes related to alpha-glucosidase, glycosyl hydrolase family 31, and cytochrome P450s was found to be similar across nine reference hymenopteran species, except for the identified glycosyl hydrolase family 31 gene, which was absent in all reference bee species. Phylogenetic analyses of the latter candidate genes revealed that they cluster together with their homologous genes found in the reference hymenopteran species. These identified candidate genes provide a basis for future comparative genomic and proteomic studies on (ontogenetic) dietary transitions in Hymenoptera.

Ontogeny and development of the gastrointestinal system in Indian walking catfish (Clarias magur) during its early development.

Ontogeny of the digestive tract and its accessory organs and their further development in the Indian walking catfish (Clarias magur) were examined in larvae, starting from the day of hatching until 35 days post-hatching (dph) reared at 28-29 °C. Many organs at their primordial stage were seen on the day of hatching. These include opened oral cavity with monolayered epithelial lining and very few newly emerging taste buds and goblet cells, primordial pharyngeal teeth on slightly stratified epithelia of the pharyngeal plate, stomach anlage with some degree of the mucosal fold, and a few newly forming gastric glands embedded under its mucosa, primordial anterior and posterior intestine with the smooth mucosal surface, anal opening, and primordial liver and pancreas. At 1 dph, the stomach appeared to be bilobed with the first evidence of food particle in it, and the intestine had some initial folding. On the day of hatching, goblet cells appeared in all lengths of the gut, but not densely, except in the stomach; on it, they appeared at 2 dph. Pancreatic zymogen granules also appeared on this day. Supranuclear vesicles first appeared on 4-5 dph (7.9 ± 0.5-8.6 ± 0.8 mm TL), and they continue to exist until 35 dph. The developmental sequence in this fish confirmed it as an altricial species with some major histomorphological events after the onset of feeding; these include-the appearance of fully developed-pharyngeal teeth at 4 dph and onwards, pyloric sphincter, anterior to posterior intestinal sphincter at 6 dph, and the continuous development of buccopharyngeal cavity and stomach in their shape, size, and functionality until the completion of metamorphosis. Overall, the information on gastrointestinal development in the early life stage of C. magur will be useful for understanding its larval digestive physiology, and this, in turn, will help in designing effective larval feed for growth and survival.

Molecular Aspects of Regeneration Mechanisms in Holothurians.

Holothurians, or sea cucumbers, belong to the phylum Echinodermata. They show good regenerative abilities. The present review provides an analysis of available data on the molecular aspects of regeneration mechanisms in holothurians. The genes and signaling pathways activated during the asexual reproduction and the formation of the anterior and posterior parts of the body, as well as the molecular mechanisms that provide regeneration of the nervous and digestive systems, are considered here. Damage causes a strong stress response, the signs of which are recorded even at late regeneration stages. In holothurian tissues, the concentrations of reactive oxygen species and antioxidant enzymes increase. Furthermore, the cellular and humoral components of the immune system are activated. Extracellular matrix remodeling and Wnt signaling play a major role in the regeneration in holothurians. All available morphological and molecular data show that the dedifferentiation of specialized cells in the remnant of the organ and the epithelial morphogenesis constitute the basis of regeneration in holothurians. However, depending on the type of damage, the mechanisms of regeneration may differ significantly in the spatial organization of regeneration process, the involvement of different cell types, and the depth of reprogramming of their genome (dedifferentiation or transdifferentiation).

Drosophila melanogaster sex peptide regulates mated female midgut morphology and physiology.

Drosophila melanogaster females experience a large shift in energy homeostasis after mating to compensate for nutrient investment in egg production. To cope with this change in metabolism, mated females undergo widespread physiological and behavioral changes, including increased food intake and altered digestive processes. The mechanisms by which the female digestive system responds to mating remain poorly characterized. Here, we demonstrate that the seminal fluid protein Sex Peptide (SP) is a key modulator of female post-mating midgut growth and gene expression. SP is both necessary and sufficient to trigger post-mating midgut growth in females under normal nutrient conditions, and likely acting via its receptor, Sex Peptide Receptor (SPR). Moreover, SP is responsible for almost the totality of midgut transcriptomic changes following mating, including up-regulation of protein and lipid metabolism genes and down-regulation of carbohydrate metabolism genes. These changes in metabolism may help supply the female with the nutrients required to sustain egg production. Thus, we report a role for SP in altering female physiology to enhance reproductive output: Namely, SP triggers the switch from virgin to mated midgut state.

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Anatomy and histology of the alimentary canal of larvae and adults of Chrysoperla externa (Hagen, 1861) (Neuroptera: Chrysopidae).

The larvae of the lacewing Chrysoperla externa are important predators with the potential to be used in the biological control in agriculture. Although some studies provide important data on the gut morphology in lacewings, they are limited to few species. This study describes the anatomy and histology of the alimentary canal in the predatory larvae and herbivorous adult of C. externa. In larvae, the crop is the larger part of the foregut and it is connected to the midgut by the stomodeal valve. The midgut is an enlarged sac-like organ. At the mid-hindgut transition, there are eight Malpighian tubules. The hindgut is a non-functional vestigial region in the larvae. In adults, the crop has a diverticulum associated with large tracheal trunks, a conic proventriculus with sclerotized lips followed by an elongated tubular midgut. Histological analyses of larval and adult midgut show the presence of a single-layered epithelium with columnar cells with well-developed brush border, nests of regenerative cells, and a peritrophic matrix lining the midgut lumen. The hindgut in adults has an epithelium with cubic cells lined by a thin cuticular intima and rectal pads in the rectum. These data are discussed in comparison with the digestive tract in other Chrysopidae.

RYK-mediated filopodial pathfinding facilitates midgut elongation.

Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a-/- phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.

Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, ß-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas ß-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.

Aedes cadherin receptor that mediates Bacillus thuringiensis Cry11A toxicity is essential for mosquito development.

Aedes cadherin (AaeCad, AAEL024535) has been characterized as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) Cry11A toxins. However, its role in development is still unknown. In this study, we modified the cadherin gene using ZFN and TALEN. Even though we obtained heterozygous deletions, no homozygous mutants were viable. Because ZFN and TALEN have lower off-targets than CRISPR/Cas9, we conclude the cadherin gene is essential for Aedes development. In contrast, in lepidopteran insects loss of a homologous cadherin does not appear to be lethal, since homozygous mutants are viable. To analyze the role of AaeCad in vivo, we tagged this protein with EGFP using CRISPR-Cas9-mediated homologous recombination and obtained a homozygous AaeCad-EGFP line. Addition of Aedes Rad51 mRNA enhanced the rate of recombination. We then examined AaeCad protein expression in most tissues and protein dynamics during mosquito development. We observe that AaeCad is expressed in larval and adult midgut-specific manner and its expression pattern changed during the mosquito development. Confocal images showed AaeCad has high expression in larval caecae and posterior midgut, and also in adult midgut. Expression of AaeCad is observed primarily in the apical membranes of epithelial cells, and not in cell-cell junctions. The expression pattern observed suggests AaeCad does not appear to play a role in these junctions. However, we cannot exclude its role beyond cell-cell adhesion in the midgut. We also observed that Cry11A bound to the apical side of larval gastric caecae and posterior midgut cells exactly where AaeCad-EGFP was expressed. Their co-localization suggests that AaeCad is indeed a receptor for the Cry11A toxin. Using this mosquito line we also observed that low doses of Cry11A toxin caused the cells to slough off membranes, which likely represents a defense mechanism, to limit cell damage from Cry11A toxin pores formed in the cell membrane.

Developmental and Tissue Patterns of the Basal Expression of Chicken Avian ß-Defensins.

Defensins are a class of antimicrobial peptides in vertebrates that function as the first line of innate immunity with potent antimicrobial and immunomodulatory activities. Fourteen defensins, namely, avian ß-defensin 1 to 14 (AvBD1-14), have been identified in chickens. Before characterizing the role of AvBDs in innate immunity during the early development of chickens, we collected tissue segments from the liver, spleen, and gastrointestinal (GI) tract including the esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, and colon from broilers at days 1, 3, 7, 14, and 28. After RNA isolation and reverse transcription, we determined the expression levels of the 14 AvBD genes in these tissues during the first 28 days after hatching by real-time PCR. The results suggested the AvBDs were widely expressed in the chicken liver, spleen, and gastrointestinal (GI) tract. Interestingly, we did not detect AvBD11 expressed in the GI tract, even in the liver and spleen. Additionally, AvBDs were differentially expressed in the chicken GI tract. AvBD5 and AvBD14 were expressed most abundantly in the proximal GI tract, especially the esophagus and crop. Moreover, AvBD5, AvBD7, AvBD9, and AvBD14 were expressed in an inverted-V pattern with the peak being the observed expression at days 3, 7, or 14 in the chicken spleen, esophagus, duodenum, and cecum. Other AvBDs presented biphasic or inverted-V expression patterns in different tissues. The expression levels of all detected AvBDs were strengthened after hatching rather than decreasing steadily. Therefore, AvBDs were found to be expressed widely in the chicken liver, spleen, and GI tract and their expression levels were primarily up regulated during the early development of chicken, implying the potential essential roles of AvBDs in early innate defense and infection resistance of chickens.

Action of Bacillus thuringiensis (Bacillales: Bacillaceae) in the midgut of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae).

The relevance of Bacillus thuringiensis in pest control strategies have been increasing in recent decades. This entomopathogenic microorganism is considered safe, and its action is highly selective to target organisms. Its toxic effect is attributed to the toxins synthesized during sporulation. The nature of the produced toxins depends on the bacterial variety. Some varieties of B. thuringiensis can synthesize from one to eight distinct toxins, which have specific effects on different orders of susceptible insects. The sugarcane borer, Diatraea saccharalis, is a holometabolous lepidopteran that causes severe damage to sugar cane cultures during its larval phase. Therefore, to evaluate B. thuringiensis serovar Aizawai GC-91 efficiency and its effects on the midgut of first instar D. saccharalis larvae, we reared the insects with contaminated artificial diet for 144 h. Larvae mortality during this period revealed that 5, 10, and 20 g/L concentrations were the most efficient, resulting in 100% mortality of treated larvae, with LC50 and LC90 estimated at 0.307 and 1.330 g/L, respectively. Furthermore, histological and ultrastructural analyses revealed damage to midgut cells. Within the first 24 h of treatment, the midgut of infected insects presented peritrophic membrane degeneration, cytoplasmic vacuolization, and cellular hypertrophy; spherites and calcium granules were also observed. At 48 h of treatment, the digestive system had collapsed, with microvilli degeneration, basement membrane and muscular fiber bundles disruption, and cellular lysis. These results confirm the toxicity of the B. thuringiensis serovar Aizawai GC-91 on D. saccharalis larvae and its potential use as a biocontroller against this pest species.

A non-bilaterian perspective on the development and evolution of animal digestive systems.

Digestive systems and extracellular digestion are key animal features, but their emergence during early animal evolution is currently poorly understood. As the last common ancestor of non-bilaterian animal groups (sponges, ctenophores, placozoans and cnidarians) dates back to the beginning of animal life, their study and comparison provides important insights into the early evolution of digestive systems and functions. Here, I have compiled an overview of the development and cell biology of digestive tissues in non-bilaterian animals. I will highlight the fundamental differences between extracellular and intracellular digestive processes, and how these are distributed among animals. Cnidarians (e.g. sea anemones, corals, jellyfish), the phylogenetic outgroup of bilaterians (e.g. vertebrates, flies, annelids), occupy a key position to reconstruct the evolution of bilaterian gut evolution. A major focus will therefore lie on the development and cell biology of digestive tissues in cnidarians, especially sea anemones, and how they compare to bilaterian gut tissues. In that context, I will also review how a recent study on the gastrula fate map of the sea anemone Nematostella vectensis challenges our long-standing conceptions on the evolution of cnidarian and bilaterian germ layers and guts.

The digestive system of xenacoelomorphs.

Interest in the study of Xenacoelomorpha has recently been revived due to realization of its key phylogenetic position as the putative sister group of the remaining Bilateria. Phylogenomic studies have attracted the attention of researchers interested in the evolution of animals and the origin of novelties. However, it is clear that a proper understanding of novelties can only be gained in the context of thorough descriptions of the anatomy of the different members of this phylum. A considerable literature, based mainly on conventional histological techniques, describes different aspects of xenacoelomorphs' tissue architecture. However, the focus has been somewhat uneven; some tissues, such as the neuro-muscular system, are relatively well described in most groups, whereas others, including the digestive system, are only poorly understood. Our lack of knowledge of the xenacoelomorph digestive system is exacerbated by the assumption that, at least in Acoela, which possess a syncytial gut, the digestive system is a derived and specialized tissue with little bearing on what is observed in other bilaterian animals. Here, we try to remedy this lack of attention by revisiting the different studies of the xenacoelomorph digestive system, and we discuss the diversity present in the light of new evolutionary knowledge.

Molecular and evolutionary aspects of the protochordate digestive system.

The digestive system is a functional unit consisting of an endodermal tubular structure (alimentary canal) and accessory organs that function in nutrition processing in most triploblastic animals. Various morphologies and apparatuses are formed depending on the phylogenetical relationship and food habits of the specific species. Nutrition processing and morphogenesis of the alimentary canal and accessory organs have both been investigated in vertebrates, mainly humans and mammals. When attempting to understand the evolutionary processes that led to the vertebrate digestive system, however, it is useful to examine other chordates, specifically protochordates, which share fundamental functional and morphogenetic molecules with vertebrates, which also possess non-duplicated genomes. In protochordates, basic anatomical and physiological studies have mainly described the characteristic traits of suspension feeders. Recent progress in genome sequencing has allowed researchers to comprehensively detail protochordate genes and has compared the genetic backgrounds among chordate nutrition processing and alimentary canal/accessory organ systems based on genomic information. Gene expression analyses have revealed spatiotemporal gene expression profiles in protochordate alimentary canals. Additionally, to investigate the basis of morphological diversity in the chordate alimentary canal and accessory organs, evolutionary developmental research has examined developmental transcription factors related to morphogenesis and anterior-posterior pattering of the alimentary canal and accessory organs. In this review, we summarize the current knowledge of molecules involved in nutrition processing and the development of the alimentary canal and accessory organs with innate immune and endocrine roles in protochordates and we explore the molecular basis for understanding the evolution of the chordate digestive system.

Sponge digestive system diversity and evolution: filter feeding to carnivory.

Sponges are an ancient basal life form, so understanding their evolution is key to understanding all metazoan evolution. Sponges have very unusual feeding mechanisms, with an intricate network of progressively optimized filtration units: from the simple choanocyte lining of a central cavity, or spongocoel, to more complex chambers and canals. Furthermore, in a single evolutionary event, a group of sponges transitioned to carnivory. This major evolutionary transition involved replacing the filter-feeding apparatus with mobile phagocytic cells that migrate collectively towards the trapped prey. Here, we focus on the diversity and evolution of sponge nutrition systems and the amazing adaptation to carnivory.

Ultrastructural differentiation of plasma membrane and cell junctions in the hindgut cells is synchronized with key developmental transitions in Porcellio scaber.

Differentiation of transporting epithelial cells during development of animal organisms includes remodelling of apical and basal plasma membranes to increase the available surface for transport and formation of occluding junctions, which maintain a paracellular diffusion barrier. This study provides a detailed ultrastructural analysis of apical and basal plasma membrane remodelling and cell junction formation in hindgut cells during late embryonic and early postembryonic development of the crustacean Porcellio scaber. Hindgut cells in late-stage embryos are columnar with flat apical and basal plasma membranes. In early-stage marsupial mancae the hindgut cells begin to acquire their characteristic dome shape, the first apical membrane folding is evident and the septate junctions expand considerably, all changes being probably associated with the onset of active feeding. In postmarsupial mancae the apical labyrinth is further elaborated and the septate junctions are expanded. This coincides with the transition to an external environment and food sources. First basal infoldings appear in the anterior chamber of early-stage marsupial mancae, but in the papillate region they are mostly formed in postmarsupial mancae. In molting late-stage marsupial mancae, the plasma membrane acquires a topology characteristic of cuticle-producing arthropod epithelia and the septate junctions are considerably reduced.