Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin.

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg's fertilization response.

Egg Case Protein 3: A Constituent of Black Widow Spider Tubuliform Silk.

Spider silk has outstanding mechanical properties, rivaling some of the best materials on the planet. Biochemical analyses of tubuliform silk have led to the identification of TuSp1, egg case protein 1, and egg case protein 2. TuSp1 belongs to the spidroin superfamily, containing a non-repetitive N- and C-terminal domain and internal block repeats. ECP1 and ECP2, which lack internal block repeats and sequence similarities to the highly conserved N- and C-terminal domains of spidroins, have cysteine-rich N-terminal domains. In this study, we performed an in-depth proteomic analysis of tubuliform glands, spinning dope, and egg sacs, which led to the identification of a novel molecular constituent of black widow tubuliform silk, referred to as egg case protein 3 or ECP3. Analysis of the translated ECP3 cDNA predicts a low molecular weight protein of 11.8 kDa. Real-time reverse transcription-quantitative PCR analysis performed with different silk-producing glands revealed ECP3 mRNA is predominantly expressed within tubuliform glands of spiders. Taken together, these findings reveal a novel protein that is secreted into black widow spider tubuliform silk.

Metal ion fluxes controlling amphibian fertilization.

Mammalian oocytes undergo major changes in zinc content and localization to be fertilized, the most striking being the rapid exocytosis of over 10 billion zinc ions in what are known as zinc sparks. Here, we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese. Synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through electron-nuclear double-resonance studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs, but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.

Micropylar and Perimicropylar Regions of the Egg Exochorion in Six Genera of Dismorphiinae (Lepidoptera: Pieridae).

Using scanning electron microscopy (SEM), we examine and compare the micropylar and perimicropylar regions of the egg exochorion in six genera of the subfamily Dismorphiinae (Pieridae): Leptidea Billberg, Enantia Hübner, Pseudopieris Godman Salvin, Lieinix Gray, Dismorphia Hübner, and Moschoneura Butler. We provide the first descriptions of the morphological structures of the chorion in these genera. Leptidea is the most primitive genus, and Dismorphia represents the most diverse. We examined the jethys complex of Enantia to study diversity of characters within the genus. We conclude that the exochorion of Dismorphiinae is the most plesiomorphic in relation to Coliadinae and Pierinae, owing to its simple morphology without a transition zone surrounding the wreath (perimicropylar region). Leptidea (Leptideini) shows the least derived characters of the subfamily, followed by Enantia (Dismorphiini). The latter genus exhibits several typical characters of the tribe that are distinctive from other more specialized genera. Exochorionic similarities among the four species of the jethys complex support their close relationship; E. mazai exhibits the most derived features among the species of the complex. Pseudopieris and Moschoneura exhibit several plesiomorphies, such as a large number of openings (micro-grid), but they also show some derived features, such as three semi-rings in the wreath. Dismorphia and Lieinix exhibit characters in their most derived states, such as a striated micro-grid (most conspicuous in Lieinix) and basal aeropyles (most conspicuous in Dismorphia), a character shared with Moschoneura. The purpose of this work is exploratory and descriptive and would be improved by a larger sample size. This contribution is the third in a series of studies on the chorion of Dismorphiinae eggs.

Formins control dynamics of F-actin in the central cell of Arabidopsis thaliana.

In the female gamete of flowering plants, sperm nuclear migration is controlled by a constant inward movement of actin filaments (F-actin) for successful fertilization. This dynamic F-actin movement is ARP2/3-independent, raising the question of how actin nucleation and polymerization is controlled in the female gamete. Using confocal microscopy live-cell imaging in combination with a pharmacological approach, we assessed the involvement of another group of actin nucleators, formins, in F-actin inward movement in the central cell of Arabidopsis thaliana. We identify that the inhibition of the formin function, by formin inhibitor SMIFH2, significantly reduced the dynamic inward movement of F-actin in the central cell, indicating that formins play a major role in actin nucleation required for F-actin inward movement in the central cell.

Mesozoic cupules and the origin of the angiosperm second integument.

The second integument of the angiosperm ovule is unique among seed plants, with developmental genetics that are distinct from those of the inner integument1. Understanding how the second integument should be compared to structures in other seed plants is therefore crucial to resolving the long-standing question of the origin of angiosperms2-6. Attention has focused on several extinct plants with recurved cupules that are reminiscent of the anatropous organization of the basic bitegmic ovules of angiosperms1-6, but interpretations have been hampered by inadequate information on the relevant fossils. Here we describe abundant exceptionally well-preserved recurved cupules from a newly discovered silicified peat dating to the Early Cretaceous epoch (around 125.6 million years ago) in Inner Mongolia, China. The new material, combined with re-examination of potentially related fossils, indicates that the recurved cupules of several groups of Mesozoic plants are all fundamentally comparable, and that their structure is consistent with the recurved form and development of the second integument in the bitegmic anatropous ovules of angiosperms. Recognition of these angiosperm relatives (angiophytes) provides a partial answer to the question of angiosperm origins, will help to focus future work on seed plant phylogenetics and has important implications for ideas on the origin of the angiosperm carpel.

The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo.

Canonical Wnt/ß-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with ß-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

New Insights into the Mammalian Egg Zona Pellucida.

Mammalian oocytes are surrounded by an extracellular coat called the zona pellucida (ZP), which, from an evolutionary point of view, is the most ancient of the coats that envelope vertebrate oocytes and conceptuses. This matrix separates the oocyte from cumulus cells and is responsible for species-specific recognition between gametes, preventing polyspermy and protecting the preimplantation embryo. The ZP is a dynamic structure that shows different properties before and after fertilization. Until very recently, mammalian ZP was believed to be composed of only three glycoproteins, ZP1, ZP2 and ZP3, as first described in mouse. However, studies have revealed that this composition is not necessarily applicable to other mammals. Such differences can be explained by an analysis of the molecular evolution of the ZP gene family, during which ZP genes have suffered pseudogenization and duplication events that have resulted in differing models of ZP protein composition. The many discoveries made in recent years related to ZP composition and evolution suggest that a compilation would be useful. Moreover, this review analyses ZP biosynthesis, the role of each ZP protein in different mammalian species and how these proteins may interact among themselves and with other proteins present in the oviductal lumen.

New data on eggshell structure of capillariid species: a SEM perspective.

Capillariidae is a group of nematode parasites of vertebrates with a complex taxonomy. The structure of the eggshell, which was indicated as the most important characteristic for identification of genus or species through eggs, is very diverse among genera. The visualization and characterization of eggshell by light microscopy (LM) are a challenging task since different planes of the egg surface are needed. Nevertheless, categories of eggshell ornamentation were proposed by LM: smooth, punctuated, reticulated type I, and reticulated type II. The present study aimed to characterize the eggshell structure of Capillariidae species, parasites of mammals and avians, deposited in a helminthological collection using scanning electron microscopy (SEM). Institutional Biological Collections are taxonomic repositories of specimens described and strictly identified at the species level by systematics specialists. SEM eggshell images were obtained from 12 species belonging to 5 genera (Aonchotheca, Baruscapillaria, Capillaria, Echinocoleus, Eucoleus) and compared to their respective LM images. Eggshell patterns observed using SEM were associated categories of eggshell ornamentation previously proposed by LM images. The SEM data indicate that eggshell categories are not in agreement with capillariid genera or sites of infection. However, the study provides previously unknown SEM eggshell information from curated species, which contributes with a specific and supplementary taxonomic feature at the species level of Capillariidae.

Egg structure of five antarctoperlarian stoneflies (Insecta: Plecoptera, Antarctoperlaria).

The egg structures of five antarctoperlarian species - Stenoperla prasina of Eustheniidae; Austroperla cyrene of Austroperlidae; and Zelandobius truncus, Megaleptoperla grandis, and Acroperla trivacuata of Gripopterygidae, were examined in detail, and the groundplan of the egg structure was considered within the representative lineages of Antarctoperlaria and Plecoptera. The flattened egg shape and the circular arrangement of micropyles along the equator are regarded as potential autapomorphies for not only Eustheniidae but also for Eusthenioidea. Austroperlidae has eggs with thin, less-sclerotized chorion, a gelatinous layer on the surface, and micropyles roughly and randomly arranged along the equator. A significant ultrastructural difference between the attachment disc in Gripopterygidae and the anchor plate of arctoperlarian Systellognatha suggests that these structures were independently derived. The thin less-sclerotized chorion represents a groundplan feature in Plecoptera, along with micropyles arranged in a circle, including those circularly arranged along the equator of the egg. On the other hand, in contrast to previous understanding, the sclerotized hard chorion is regarded as a derived feature, having been independently acquired in each of Eustheniidae and Gripopterygidae of Antarctoperlaria and Systellognatha of Arctoperlaria.

The External Morphology of the Eggs of Culex (Culex) saltanensis (Diptera: Culicidae) Under Scanning Electron Microscopy.

The aim of the present study was to describe the morphology of the eggs of Culex (Culex) saltanensis Dyar that occurs in the Neotropical region. Eggs of the Cx. (Cux.) saltanensis were collected at the Mata Atlântica FIOCRUZ campus, fixed in 1% osmium tetroxide, prepared for mounting on metal supports, observed under a scanning electron microscope, and described morphologically. The eggs had a coniform shape with a length of approximately 0.5 mm (505-510 µm) and a width in the median portion of 117 µm (113-123 µm). Upper portion is lined with tubers of irregular shape and varying sizes (0.64-1.31 µm), located on a cross-linked matrix forming bands observed under optical microscopy. The micropyle is encased in a necklace of approximately 6.6-µm plates arranged in a flower-like shape. Comparing Cx. (Cux.) saltanensis eggs with several species of different genera, important divergent characteristics can be observed. However, this study points to the need for new descriptions of eggs of species belonging to the same subgenus in order to analyze if there will be differences between them. Culex (Cux.) saltanensis eggs have particular characteristics not observed in eggs of other Culicidae genera.

Success rates of inoculation of the various compartments of embryonated chicken eggs at different incubation days.

Inoculation of embryonated chicken eggs has been widely used during the past decades; however, inoculation success rates have not been investigated systematically. In this study named success rates were assessed in brown eggs incubated between 5 and 19 days, which were inoculated with 0.2 ml methylene blue per egg. Inoculations were performed in a simple and fully standardized way. Five embryonic compartments were targeted blindly (amniotic cavity, embryo, allantoic cavity, albumen and yolk) with needles of four different lengths; albumen and yolk were targeted with eggs in upside down position. Three compartments were inoculated within sight (air chamber, chorioallantoic membrane and blood vessel). Twenty embryos were used per incubation day, intended deposition site and needle length. Success rates were assessed by visual inspection after breaking the eggs. The inoculations targeting albumen, yolk, amniotic cavity and embryo yielded low scores. Magnetic resonance imaging was performed to elucidate the reason(s) for these low success rates: needles used were of appropriate length, but embryo and amniotic cavity had variable positions in the eggs, while albumen and yolk rapidly changed position after turning the eggs upside down. The latter led to adjustment of the inoculation method for albumen and yolk. Failures to inoculate compartments within sight were immediately visible; therefore, these eggs could be discarded. Except for the amniotic cavity, full scores (20/20) were obtained for all compartments although not always on every day of incubation. In conclusion, the present study may serve as a guide to more accurately inoculate the various chicken embryo compartments. RESEARCH HIGHLIGHTS Blind inoculation of embryonated egg compartments was successful, except for the amniotic cavity. MRI showed rapid position change of albumen and yolk after turning eggs upside down. In ovo vaccination against Marek's disease might be improved by using 38 mm needles.

In Vitro Culture of Mouse Blastocysts to the Egg Cylinder Stage via Mural Trophectoderm Excision.

The developmental transition from the blastocyst to the egg cylinder stage is associated with stark changes in the overall shape of the embryo, as well as with reorganization of the transcriptional network and epigenetic landscape in the pluripotent and the supportive extraembryonic lineages. To directly analyze this pre- to postimplantation switch, culture conditions are needed that can support mouse embryogenesis beyond the blastocyst stage without maternal input. Here we provide a step-by-step protocol describing an experimental pipeline for isolating late blastocysts, excising (manually or via laser assistance) the mural trophectoderm, and, finally, culturing the embryo to the egg cylinder stage.

Morphology of the Eggs of Ornithocoris pallidus (Hemiptera, Cimicidae, Haematosiphoninae).

Morphology of cimicid eggs are scarce, and this is the first record for the genus Ornithocoris Pinto, 1927 (Hemiptera: Cimicidae), using scanning electron microscopy (SEM). This genus comprises two species: Ornithocoris toledoi Pinto, 1927 (Hemiptera: Cimicidae) and Ornithocoris pallidus (Usinger, 1959). The eggs of O. pallidus are ellipsoid, and an evident 'lateral flattening', which may give clear asymmetry from the longitudinal axis. The exochorion of the body of the egg and operculum of O. pallidus present spherical or polygonal structures in relief, and pseudomicropyles on the border of the operculum, both differing from Cimex lectularius. The internal face of the operculum is smooth and the border also presented three layers. It was not possible to observe micropyles in the egg of O. pallidus. Bed bugs have a great psychological impact on people, and some people can develop a more or less severe allergic reaction against the bite. Successful control of bed bugs needs serious organization. Studies on egg morphology will add more information to assist in taxonomy studies of O. pallidus and can serve as a basis for control studies because eggs are resistant to insecticides.

Ultrastructural analysis of Rhynchospora ovules: The first record of Cyperaceae megagametophyte on transmission electron microscope.

Microsporogenesis and microgametogenesis are unusual in sedges (Cyperaceae), the third largest monocotyledonous family, as three microspores are aborted in favor of a single functional microspore. However, studies using light microscopy show that megasporogenesis and megagametogenesis occur normally. Nevertheless, the lack of ultrastructural details limits our knowledge of female gametophyte development in this family. Given the importance of morphological studies of reproductive structures, ovules and megagametophytes of Rhynchospora pubera were analyzed under transmission electron microscopy for the first time. Overall, ovules presented features similar to those described for the family, but ultrastructural details revealed an absence of a clear boundary between the egg cell and the central cell cytoplasm. Most interestingly, antipodal and nucellar cells showed several signs of vacuolar cell death, which suggest that programmed autolysis in sporogenous and gametophytic tissue is common in gametophyte development in the Cyperaceae. This may be related to the reproductive success of this family.

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae), an important vector of Leishmaniosis in South America, described by scanning electron microscopy.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes-trichoidea, basiconica, coelonica and campanoformia-were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species.

Automontage microscopy and SEM: A combined approach for documenting ancient lice.

Human ectoparasites, including lice, have been recovered from a wide range of archaeological materials. The human head louse, Pediculus humanus capitis, has been identified from mummies and sediments for decades. Louse eggs are the body part most commonly encountered and therefore the most frequently quantified. Typically, several types of microscopy are applied for egg documentation. For studies in which quantification of infestation is a goal, counting is done with the naked eye or with the aid of handheld lenses. For determination and stage classification, stereomicroscopy is commonly used. For more detailed examination of microstructure, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) can be employed. In most reports, researchers use two or more techniques to accomplish interrelated goals. Automontage microscopy is used to document prehistoric arthropods with good success. Herein, we report the results of a combination of SEM and automontage microscopy to document lice and eggs recovered from South American mummies. This combined approach allows for simultaneous examination of internal and external characteristics. Thirty automontage composite images of 2 adult lice and 16 eggs showed that egg internal morphologies were easily examined showing the within-egg anatomy of emergent nymphs. SEM imaging of 9 lice and 129 eggs was completed. In the case of two adults and several eggs, SEM imaging was accomplish after automontage image capture of the same specimens. This one-to-one image comparison of SEM and automontage shows that transmitted light of automontage reveals egg internal structures and details of the adult lice. SEM allows for high magnification examination of egg, nymph and adult microstructures. We conclude that automontage imaging followed by SEM results in efficient graphic documentation of rare louse specimens.

Eye to eye: classification of conjunctival sac polystomes (Monogenea: Polystomatidae) revisited with the description of three new genera Apaloneotrema n. g., Aussietrema n. g. and Fornixtrema n. g.

Polystomes (Monogenea: Polystomatidae) of freshwater turtles are currently represented by five genera, namely Neopolystoma, Polystomoides, Polystomoidella, Uropolystomoides and Uteropolystomoides. These parasites can infect the urinary, oral and/or the conjunctival sac systems of their hosts, showing strict site specificity. A recent phylogenetic study showed that the two most diverse genera within chelonian polystomes, i.e. Neopolystoma and Polystomoides, are not monophyletic. Furthermore, polystomes infecting the conjunctival sacs of their host, except for one species, formed a robust lineage. A fusiform egg shape has been reported for conjunctival sac polystomes and it was assumed that this characteristic could be a good character for the systematics of polystomes. Our objective in the present work was, therefore, to study more in depth the morphology of polystomes collected from the conjunctival sacs of chelonians to find characters defining a putative new genus. To achieve this objective, more specimens were collected in 2018 and 2019 from turtles sampled in North Carolina and Florida (USA) to extend taxon sampling for the phylogenetic analysis. Morphological characters of relevant polystome specimens were re-examined from several collections from Asia, Australia, Europe, South Africa, South America and North America. Based on a Bayesian tree inferred from the analysis of four concatenated genes, namely 12S, 18S, 28S and COI, polystomes found in the conjunctival sacs were grouped in three distinct lineages, the first one including a single species infecting an Australian pleurodire turtle; the second one including eleven species infecting cryptodire turtles of South America, North America and Asia; and the last one including a single species infecting a softshell cryptodire turtle of North America. Based on observations of live specimens by Dr. Sylvie Pichelin and our morphological analysis, the conjunctival sac polystomes from Australian turtles are small, cannot extend their body significantly, have a spherical ovary and egg, have a large genital bulb and possess latero-ventral vaginae at the level of the testis. Based on observations of live specimens and morphological analysis of whole mounted specimens, polystomes of the second lineage share the following morphological characteristics: the ability to stretch out and double their length, a long oval ovary, a separate egg-cell-maturation-chamber, fusiform to diamond-shaped eggs with acute tips, small genital bulb and vaginae peripheral on the side of the body at the level of the testis. The polystome species of the third lineage occupies a basal position, has the ability to stretch out and possess an elongated ovary, a large fusiform egg with rounded tips, a small genital bulb and small latero-ventral vaginae at the level of the ovary. These three distinct conjunctival sac polystome lineages are herein described as separate new genera, Aussietrema, Fornixtrema and Apaloneotrema, respectively.

Do all geckos hatch in the same way? Histological and 3D studies of egg tooth morphogenesis in the geckos Eublepharis macularius Blyth 1854 and Lepidodactylus lugubris Duméril & Bibron 1836.

The egg tooth of squamates evolved to facilitate hatching from mineralized eggshells. Squamate reptiles can assist their hatching with a single unpaired egg tooth (unidentates) or double egg teeth (geckos and dibamids). Egg tooth ontogeny in two gekkotan species, the leopard gecko Eublepharis macularius and the mourning gecko Lepidodactylus lugubris, was compared using microtomography, scanning electron microscopy, and light microscopy. Investigated species are characterized by different hardnesses of their eggshells. Leopard geckos eggs have a relatively soft and flexible parchment (leathery) shell, while eggshells of mourning geckos are hard and rigid. Embryos of both species, like other Gekkota, have double egg teeth, but the morphology of these structures differs between the investigated species. These differences in shape, localization, and spatial orientation were present from the earliest stages of embryonic development. In mourning gecko, anlagen of differentiating egg teeth change their position on the palate during embryonic development. Initially they are separated by condensed mesenchyme, but later in development, their enamel organs are connected. In leopard geckos, the localization of egg tooth germs does not change, but their spatial orientation does. Egg teeth of this species shift from inward to outward orientation. This is likely related to differences in structure and mechanical properties of eggshells in the studied species. In investigated species, two hatching mechanisms are possible during emergence of young individuals. We speculate that mourning geckos break the eggshell through puncturing action with egg teeth, similar to the pipping phase of chick and turtles embryos. Egg teeth of leopard geckos cut egg membranes similarly to most squamates. Our results also revealed differences in egg tooth implantation between Gekkota and Unidentata: gekkotan egg teeth are subthecodont (in shallow sockets), while those in unidentates are acrodont (attached to the top of the alveolar ridge). © 2020 Wiley Periodicals LLC.

Ultrastructure of the Immature Stages of Musca domestica (Diptera: Muscidae: Muscinae).

Musca domestica (Linnaeus, 1758) is a muscoid species that is widespread throughout the world and acts as a mechanical vector of different enteropathogens primarily in underdeveloped countries. The adult and its immature forms are associated with decaying organic matter and can be seen visiting human corpses and animal carcass, and the larvae can also cause an infestation on human and animal wounds, feeding on the tissues. These characteristics make them have a forensic significance and, mainly, a great sanitary importance. This study aimed to analyze and describe morphological aspects of their immature stages of M. domestica, including the eggs, the first-, second-, and third-instar larvae, and the puparium using scanning electron microscopy (SEM). The eggs have standard format that all muscoid flies shared. The exochorion has some modified cells that were used for embryonic respiration. The first-instar larvae present two openings on the posterior spiracle as in the second-instar larvae, but with the last one, we can observe the anterior spiracle structure. The third-instar larvae, as in some other Muscidae species, have a posterior spiracular opening with a sinuous form that is located near the edge of the spiracular plate. The puparium morphology is equal to the third instar with a respiratory structure that helps the breathing during the metamorphosis process. This article supports the need for knowledge over the morphological characteristics of the immature forms of the muscoid Diptera (Linnaeus, 1758) at the same time helping with the correct identification of this insect phase.