Micropylar Apparatus Research Articles

Disruption of a BTB-ZF transcription factor causes female sterility and melanization in the larval body of the silkworm, Bombyx mori

The dilute black (bd) of the silkworm Bombyx mori is a recessive mutant that produces a grayish-black color in the larval integument, instead of the characteristic white color found in wild-type larvae. In addition, eggs produced by bd females are sterile due to a deficiency in the micropylar apparatus. We identified candidate genes responsible for the bd phenotype using publicly available RNA-seq data. One of these candidate genes was homologous to the maternal gene required for meiosis (mamo) of Drosophila melanogaster, which encodes a broad-complex, tramtrack, and bric-à-brac-zinc finger (BTB-ZF) transcription factor essential for female fertility. In three independent bd strains, the expression of the B. mori mamo (Bmmamo) was downregulated in the larval integument. Using a CRISPR/Cas9-mediated knockout strategy, we found that Bmmamo knockout mutants exhibit a grayish-black color in the larval integument and female infertility. Moreover, larvae obtained from the complementation cross between bd/+ mutants and heterozygous knockouts for the Bmmamo also exhibited a grayish-black color, indicating that Bmmamo is responsible for the bd phenotype. Gene expression analysis using Bmmamo knockout mutants suggested that the BmMamo protein suppresses the expression of melanin synthesis genes. Previous comparative genome analysis revealed that the Bmmamo was selected during silkworm domestication, and we found that Bmmamo expression in the larval integument is higher in B. mori than in the wild silkworm B. mandarina, suggesting that the Bmmamo is involved in domestication-associated pigmentation changes of the silkworm.

Fecundity and morphological description of the eggs of Psorophora cyanescens (Coquillett, 1902) (Diptera: Culicidae) by scanning electron microscopy

Taxonomic studies on mosquitoes are based on the morphological description of larvae and adults. However, few studies have focused on the morphological description of eggs despite their taxonomic value. The description of mosquito eggs by scanning electron microscopy (SEM) allows more detailed descriptions of the ornamentation of the egg, which can provide diagnostic characters of the species. The objective of the work was to describe the eggs of Psorophora cyanescens by SEM and provide fecundity data for the species. In general, Ps. cyanescens eggs are elliptical and very wide. The exochorion is characterized by polygon pattern, where each polygon consists of longitudinal ridges joined by cross-ridges defining regular "hexagonal" areas. Each longitudinal ridge consisting by a small tubercle. The small tubercles are irregular, rectangular, rounded, or tubular. The ornamentation of the exochorion also has long and conical tubercles throughout the egg region. The micropylar apparatus located in the anterior region of the egg has a prominent, continuous and thickness collar. Psorophora cyanescens laid mature eggs at 2.5 days post-feeding. The mean number of eggs per female was 82.20 (± 13.31). This is the first study that describes the ultrastructure of the morphology of the eggs of Ps. cyanescens by SEM. With the contribution of the present work, there are five species of the genus Psorophora whose eggs morphology is described by SEM: Ps. albigenu, Ps. albipes, Ps. columbiae, Ps. cyanescens, and Ps. ferox.

Ovarian development and the reproductive profile of Astyanax altiparanae (Teleostei, Characidae) over one year: Applications in fish farming

Astyanax altiparanae is a Brazilian species of substantial commercial, environmental and scientific importance; however, existing studies on its reproduction do not seem to provide enough details. In light of the increasing use of this species in fish farming and the need for basic studies for the development of new production technologies, we describe the structural and ultrastructural characteristics of the ovaries of A. altiparanae, and characterize the species' reproductive cycle. Females were collected monthly from March 2013 to February 2014, and reproductive management began in October 2013. The ovaries were removed, fixed in Karnovsky's fixative, and prepared for light microscopy, transmission electron microscopy and immunohistochemistry anti-PCNA. These techniques enabled us to characterize the ovaries, the germ cells, and the somatic cells in detail, as well as their changes over time. The reproductive cycle was characterized based on the monthly variation in the gonadosomatic ratio, the proportion of germ cells, and the rate of oogonium proliferation. The macroscopic analysis of the ovaries suggests that the vascularization pattern and color of the ovaries vary according to development. There are new types of analyses that can be applied even in the fish farming industry, such as a comparison between ovaries staining and weight or the frequency distribution of these colors throughout the year. This study also provides details on microscopic characteristics that have never before been reported for species of Astyanax, such as the presence of annulate lamellae in oogonia, the development of the zona pellucida from oocytes in the one-nucleolus step, and the development of the micropylar apparatus in oocytes in the cortical alveolar step. When the reproductive cycle was analyzed, this species was found to have a long period of spawning, with a reproductive peak from October to February and multiple spawning events, confirming the period already described in the literature. Variations in reproductive periods and the ability to reproduce in lentic environments suggest that A. altiparanae has the ability to respond quickly to environmental changes and exhibits high reproductive flexibility. All of these characteristics confirm the great potential of this species in the fish farming industry.

Ultrastructure of egg chorion of castor butterfly Ariadne merione (Crammer) (Lepidoptera: Nymphalidae)

The egg chorion of the castor butterfly Ariadne merione exhibits well organized regional and radial complexity. The anterior pole or egg pole contains a micropylar apparatus bearing three circular micropylar openings and a micropylar rosette consisting of three to four rows of polygonal cells or rosette petals. The posterior pole or egg base is membranous and bears numerous pores and is firmly glued to the substrate. The egg surface bears longitudinal ridges, grooves, protuberances and several respiratory filaments bearing aeropyles. The chorion consists of the four distinct layers such as internal layer, trabecular layer having trabecular air spaces, principal layer and external layer.

Scanning Electron Microscopic Studies on Egg Surface Morphology and Morphometrics of Culex Pipiens Pipiens ( Lin. ) ( Northern House Mosquito ) and Culex Quinquefasciatus ( Say ) ( Southern House Mosquito ) ( Diptera : Culicidae )

During this study the surface morphology and morphometric of Culex pipiens pipiens (Lin.) and Culex quinquefasciatus (Say) eggs were described and compared using scanning electron microscopy for the first time. The results indicate that eggs of both species appear to be similar to great extent in surface morphology. However, morphometric provide 71.621% demarking attributes out of 44 attributes at various significant levels (P < 0.05-0.001), i.e., egg length, width and ratio of length/width, attributes of micropylar apparatus including corolla, disc, mound, tubercles size, and length of tubercular rows in micropylar region, size and density of tubercles, exochorionic pore in conical-shaped regions of eggs, and size of tubercular wheel units. Structurally, the additional presence of large tubercles strengthens the micropylar region to bear various collapsing forces in these species.

Differentiation of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) with egg surface morphology and morphometrics using scanning electron microscopy

Aedes aegypti and Aedes albopictus are potential arboviral vectors leading to high human fatality worldwide. Efforts in the present study were made to differentiate the eggs of A. aegypti and A. albopictus morphologically and morphometrically using scanning electron microscopy (SEM). Morphometrically, these species’ eggs were 48.48% significantly different of the 33 attributes including egg dimensions, micropylar apparatus, dimensions and density of outer chorionic cells (OCCs), tubercles and width of exochorionic network. In comparison to A. aegypti eggs, A. albopictus eggs were significantly smaller and more tapered at the posterior end; however, the micropylar disc of A. aegypti was wider and had incomplete circular sectors whereas it was a narrower polygon without sectors in A. albopictus. These species were also significantly different with regards to OCC which enclose both large central and small peripheral tubercles. Specifically, the exochorionic networks in A. aegypti were interwoven, reticulated and extensively wide whereas they were narrow, prominent and solid-wall-like in A. albopictus. This feature may strengthen A. albopictus eggs against desiccation, when they are laid in containers. The morphometrical and morphological analysis of the egg’s attributes of A. aegypti and A. albopictus may be helpful in understanding egg biology as well as in species confirmation.

Scanning Electron Microscopy of the Eggs of Aedes scapularis from Southern South America

The eggs of Aedes scapularis analyzed by scanning electron microscopy are black and elliptical in outline, measuring approximately 620.4 +/- 16.74 microm long and 163.7 +/- 16.90 microm (n = 10) wide, with an egg index (length/width ratio) of 3.79. The anterior extremity tapered abruptly from a width of 51.6 microm, while such tapering was more gradual at the posterior extremity, from a width of 61.4 microm. The ventral surface of the chorionic coating presented cells with a tubular aspect containing tubercles in rows at a density of 5 to 9 per cell with 2 different sizes, the largest measuring 7.23 +/- 0.98 microm in a longitudinal diameter and the smallest 4.15 +/- 0.53 microm (n = 30). In the dorsal region, the external chorionic reticulum had a porous appearance, and its thickness ranged from 2.5 to 4.1 microm. Isolated tubercles presented wide variation per cell. In the central region of some chorionic cells were tubercles of greater diameter, characterized as central tubercles of 8.45 +/- 0.67 microm, and around them 3 to 5 smaller tubercles measuring 2.57 +/- 0.26 microm. The micropylar apparatus presented a collar with a very evident molding and edges with defined margins for the transition area and a thickness of around 11.1 microm. The micropyle disc margins were raised, measuring around 17.8 microm in diameter and 229 microm in circumference. The micropyle orifice was very evident, with a diameter of 1.41 microm.

Morphological Variation of Micropylar Apparatus in Bombyx mori eggs

Morphological Variation of Micropylar Apparatus in Bombyx mori eggs

Morphology of the egg shell and the developing embryo of the Red Palm Weevil, Rhynchophorus ferrugineus (Oliver)

The harvested eggs of Rhynchophorus ferrugineus are ovo-cylindrical shaped, averaged 1.09 mm in length and 0.43 mm in width, with ratio of L \\ W 4.42. The chorionic layer of electron dense material is seen covering the exochorion structure of the eggs. The egg main body chorion exhibits a polygonal pattern and architecture surface of the egg is supported by a system of irregular interconnecting grooves. The micropylar apparatus of the eggs of the Red Palm Weevil, R. ferrugineus is described in the present study for the first time. Two micropylar openings are found closed to the center of the posterior wide pole of the egg. Each micropylar opening presents a single small orifice and its surrounding chorion is porous and densely set with tiny projections allowing the spermatozoa to penetrate the egg. Respiratory aeropyles are distributed on the borders of reticulations in the area chorionic surface of egg capsule. The hatching region is detected on the anterior part at the opposite side of the egg. Changes in the appearance and shape of R. ferrugineus eggs as well as the incidence of embryonic development are observed.

Variation in morphology and morphometrics of eggs of Culex quinquefasciatus mosquitoes from different ecological regions of India

Variation in egg surface morphology and morphometrics of Culex quinquefasciatus mosquitoes of the Jodhpur, Bikaner, Jamnagar, and Bathinda strains were correlated with geographical distribution in different ecological regions of India. We report the geographic variation in Cx. quinquefasciatus based on 44 attributes of micropylar and conical-shaped regions of eggs, including micropylar apparatus (corolla, disc, and mound), micropylar tubercles, and the exochorionic tubercle, pores, and network in anterior, middle, and posterior regions. No remarkable differences were observed in the surface morphology of eggs of these strains except the absence of small tubercles in the anterior and middle region of the JMN strain. However, a statistical analysis indicated significant morphometric variations in 66% of the attributes of the eggs. The cluster analysis of all egg attributes showed that the JD, BKN, and BTH strains are closer to each other than the JMN strain. The positive correlation (r = 0.95) also indicated an effect of geographical distribution on morphometry of various egg attributes of these strains. The present study suggests that ecological variation may have affected the morphometric attributes of the egg of four strains of Cx. quinquefasciatus from different geographical areas.

Scanning Electron Microscopy of the Egg of &lt;I&gt;Ochlerotatus albifasciatus&lt;/I&gt; (Diptera: Culicidae)

Ochlerotatus albifasciatus (Macquart) (Diptera: Culicidae) is a mosquito species of medical and veterinary importance, usually associated with temporary groundwater habitats. Eggs obtained from females collected in Córdoba city, in the center of Argentina, were studied using scanning electron microscopy. The eggs are elliptical in outline, measuring approximately 615.7 +/- 37.24 microm in length (1) and 180.7 +/- 22.87 microm in width (w), with an egg index (l/w ratio) of 2.9-3.7. The outer chorionic sculpture has racquet-shaped and wristwatch-shaped cells consisting of a small central tubercle surrounded by larger tubercles and followed by a row of smaller tubercles on one or both sides, respectively, although variations between regions of the egg were observed. The micropylar apparatus has a collar with evident molding and edges of determined length, albeit irregular, with defined margins for the transition area and a thickness of approximately 10.7 microm. The margins of the micropylar disc are raised and the disc measures approximately 21.1 microm in diameter. The micropyle is distinct.

Scanning electron microscopic studies on egg surface morphology and morphometrics of Culex tritaeniorhynchus and Culex quinquefasciatus (Diptera: Culicidae)

Culex tritaeniorhynchus Giles, 1901 and Culex quinquefasciatus Say, 1823 is an important vector of Japanese encephalitis and Bancroftian filariasis, respectively in India and South East Asian countries. In this paper, we are describing the surface morphology and morphometrics of the egg of C. tritaeniorhynchus in comparison with C. quinquefasciatus for the first time. The results indicated that eggs of both the species appears to be similar to great extent in surface morphology, however, morphometrics provide 56.81% demarking attributes out of 44 attributes at various significant levels (p < 0.05-0.001), i.e., egg length, width and ratio of length/width, attributes of micropylar apparatus including corolla, disc, mound, tubercles size, and length of tubercular rows in micropylar region, size and density of tubercles, exochorionic pore in conical-shaped regions of eggs, and size of tubercular wheel units. Structurally, the additional presence of large tubercles strengthens the micropylar region to bear various collapsing forces in these species.

Scanning Electron Microscopy of the Egg of Haemagogus Tropicalis

Haemagogus tropicalis is strictly a forest-dwelling species from the fertile valley area of the Amazônia forest. It is a diurnal mosquito, and the oviposition sites for the species include tree holes. The eggs of Hg. tropicalis used in this study were from females captured on Combú Island, situated across from the city of Belém, Guajará Bay, state of Pará, at 1 degrees 25'S latitude and 48 degrees 25'W longitude. The eggs are elliptical and approximately 575 microm long with a width of approximately 144 microm. The ventral surface of the chorionic reticulum has regular chorionic cells with hexagonal and sometimes pentagonal ornamentation. Each chorionic cell has a thick external chorionic reticulum with regular borders. The interior of the chorionic cells have small, evenly distributed tubercles, and the dorsal external chorionic reticulum appears porous. The micropylar apparatus, located on the anterior area of the egg, was formed by a collar with a well-developed frame. Centrally, the micropylar disc had a diameter of approximately 20 microm and the micropylar orifice is 2.1 microm in diameter. These data may enable construction of taxonomic keys for identifying eggs of Haemagogus species.

Scanning electron microscope study on the egg chorion of silkmoth, Antheraea assamensis Helf. (Lepidoptera : Saturniidae)

AbstractThe eggshell of Antheraea assamensis Helf. has a highly decorated chorion and its micropylar apparatus is situated at the cephalic end. Scanning electron microscopic investigation on the eggshells of A. assamensis reveals distinct patterns of structural elements viz., central cavity, micropylar rosette encircled the central cavity, and the aeropyles. The micropylar structures are at the anterior pole of the oval shaped eggs opposite to its side of distal pole. Centrally located central cavity leads into eight micropylar openings which further radiate down to the eight micropylar canals. The central cavity (5.28±0.21 μm in diameter) is encircled by a primary rosette of 16-18 cell imprints and each primary cell imprint measured 21.08±3.13 μm in length. The primary petal shaped cell imprints are followed by secondary and tertiary rows of cell imprints. Except the central cavity and micropylar rosette, the entire surface of the chorion is marked by a circular shell imprints which are boarded by aeropyles. Each cell imprint has 4-6 aeropyles and each aeropyle measured 1.06±0.01 μm in diameter. The aeropyles of A. assamensis are covered with a cup shaped like structures and the terminals of these structure has many finger like projections which may help to check the entry of pathogens or dust particles while taking the oxygen from the outside environment to the oocyte.

Cortical reaction and solicitation mechanism in Hemibarbus labeo ovum

There are 1 to 4 rows and five types of cortical alveoli in the cortex of the pallas (Hemibarbus labeo) egg. From the outer to the inner of the cortex, the diameter of the cortical alveoli decreases gradually. We found a new structure named solicitation speckle at the low latitude of the animal pole and near the micropylar apparatus between the ovum envelope and cell membrane in the zygote of fish. The solicitation speckle similar to type I cortical alveoli was very purple in H.E. chromosome, and it had no obvious boundary with the ovum envelope and membrane. Moreover, no membrane around the solicitation speckle in TEM was found. In SEM, the solicitation speckle looked flocculent. During cortical reaction, the solicitation speckle played a very important function in arousing cortical reaction. Thirty-five seconds after fertilization, cortical alveoli began to break down near the low latitude of the animal pole. At the same time, the same thing happened near the micropylar apparatus before cortical reaction. Both starting points encountered and healed up at the vestibule of the micropylar apparatus. The cortical reaction that happened near the low latitude of the animal pole was another new pattern. The cortical reaction was divided into four parts that included latent period, developmental period, climactic period and declining period. In the latent period, no cortical alveoli were released. In the developmental period, a few cortical alveoli were released outside the cortex. In the climactic period, several cortical alveoli were inosculated into a big vesicle and released intensely. In the declining period, the type V cortical alveoli and the other remnant cortical alveoli were released. Five minutes after fertilization, cortical alveoli were released entirely in the animal pole. Five minutes after fertilization, all of the remnant cortical alveoli were released. This leads us to conclude that cortical reaction is induced by type I cortical alveoli, and the solicitation speckle is a volcanic chain reaction under water or the other lower osmotic pressure of fluids. The outer cortical reaction can accelerate the inner cortical reaction. While cortical alveoli releases in batches, the cell plasma membrane is reorganized over and over. No cortical alveoli were found below the micropylar tube where sperm enters the ovum, which suggests that the cortical reaction prevents polyspermy.

SCANNING ELECTRON MICROSCOPY OF EGGS OF OCHLEROTATUS (PROTOMACLEAYA) TERRENS WALKER

Observations on the morphological ultrastructure of eggs of Ochlerotatus (Protomacleaya) terrens (Diptera: Culicidae) were conducted by using scanning electron microscopy. Morphometry of the principal structures was obtained with the aid of Semafore analysis software. Eggs of Oc. terrens from females caught in the Biological Reserve of Tinguá, State of Rio de Janeiro, Brazil, were utilized. The eggs presented elliptical outlines, with a length of approximately 649.0 microm and width of 168.7 microm. The egg index (length to width ratio) was 3.85 microm. The exochorion had hexagonal and sometimes pentagonal ornamentation. Inside the chorionic cells were small, well-distributed tubercles with a large range of sizes. The micropylar apparatus, located in the anterior region of the egg, presented a collar with a poorly visible frame, with borders of indeterminate extent and margins without a defined transition area, and a thickness of approximately 1.8 microm. The micropyle was plugged.

Micropyle in neuropterid insects. Structure and late stages of morphogenesis

The eggshells of five representatives of neuropterid insects belonging to three orders and five families (Neuroptera: Ithonidae and Myrmeleontidae; Raphidioptera: Raphidiidae and Megaloptera: Corydalidae and Chauliodidae) have been analyzed with various microscopic techniques. These studies have revealed that the eggshells of all investigated species are morphologically similar and always equipped with an anteriorly located micropylar apparatus. The eggshell surface is usually uniform, smooth or densely set with small, raised projections. Only in some species ( Euroleon nostras, Oliarces clara) these projections are more regularly arranged and form a characteristic polygonal pattern on the eggshell surface. The shape and size of the micropyle vary from species to species but it always consists of two parts: the core and cortex. In the investigated representatives of Neuropterida the micropyle is pierced by several (about 20) micropylar canals. These canals converge radially in the central part of the core, where they bend and run towards the anterior pole of the egg cell. In the majority of studied species the orifices of the canals are located circumferentially at the base of the micropyle, whereas in the corydalid, Corydalus peruvianus, they have been found on a swollen apical part of the micropyle. Analysis of late choriogenic oocytes have revealed that the micropyle is always formed by two distinct subpopulations of the follicular cells. The micropyle forming cells (MFCs) synthesize and secrete the macromolecules that are subsequently incorporated into the micropyle, while micropylar canal forming cells (MCFCs) send long cytoplasmic projections that mould the micropylar canals. Formation and/or maintenance of these extensions depend on microtubules and/or microfilaments.

Microscopia eletrônica de varredura de ovos de Haemagogus leucocelaenus (Diptera: Culicidae)

To observe morphological details of the eggs of Haemagogus (Conopostegus) leucocelaenus, seen for the first time via scanning electron microscopy (SEM), with morphometric analysis of the main structures. Eggs of Hg. leucocelaenus were obtained from females captured in the Biological Reserve of Tinguá, State of Rio de Janeiro. Some of the eggs were kept for hatching and others underwent processing for scanning electron microscopy studies. Three eggs were submitted to morphometric analysis. The material was fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide, both in 0.1M, pH 7.2 sodium cacodylate buffer, then dehydrated in ethanol and dried using the critical point method. This was then set up on metallic supports, covered with gold and observed using the Jeol 5310 scanning electron microscope. Measurements were made with the aid of the Semafore analysis software coupled to the electron microscope. The eggs presented elliptical outlines of approximately 574 mm in length and 169 mm in width, with an egg index (l/w ratio) of 3.39 mm. The exochorion was extremely regular and had ornamentation that was usually hexagonal but sometimes pentagonal. Tubercles were observed on the chorionic cells, symmetrically arranged in relation to the longitudinal axis. Inside the cells, there were smaller, individualized tubercles, some arranged peripherally and others grouped to a greater or lesser extent in the center. The surface of the chorionic reticulum did not present rugosity. The micropylar apparatus was formed by a prominent continuous collar of 8.32 mm in thickness, with a slightly irregular surface. The micropylar disk was very evident, and was continuous with the collar. The micropyle was seen at the center of this disk, measuring 1.6 mm and with a micropylar apparatus of 27.3 mm in diameter. The ornamentation of the exochorion presents differences in relation to the tubercles of chorionic cells and the external chorionic reticulum between the eggs of Hg. Leucocelaenus, in comparison with the eggs of Hg. janthinomys and Hg. equinus, and also in relation to those of Aedes aegypti, Ae. albopictus and Ae. bahamensis. In various aspects, the eggs of Hg. leucocelaenus have more resemblance to those of Hg. Equinus than those of Hg. janthinomys, with greater differences presented in relation to the eggs of Hg. spegazzinii and Hg. lucifer.

Short-term storage of oocytes from the neotropical teleost fish Prochilodus marggravii

The loss of oocyte viability after ovulation is one of the limiting factors in controlled reproduction of several fish species. Experiments were performed with 15 feral Prochilodus marggravii female fish induced to spawn with crude carp pituitary extract to evaluate the viability of oocytes retained within the ovarian cavity (in situ storage) and outside of the ovarian cavity (ex situ storage). Because fertility rates rapidly declined after ovulation, simultaneously with an increase in the number of deformed larvae, P. marggravii oocytes could only be successfully stored for 1 h ex situ at room temperature (∼26 °C). There was a highly negative correlation ( r=−0.82) between fertilization and deformed larvae during in situ storage at ∼26 °C. Ex situ cooling (18 °C) caused a drastic reduction in fertilization rates as compared with storage at ∼26 °C. Oocyte structure was preserved during 2 h storage and the cortical reaction was induced before spawning. Since the micropylar apparatus remained open, it was not the primary cause for the loss of oocyte fertility. The cytoskeleton of the oocyte appeared to be affected since ooplasmic segregation was altered after 2 h storage.

Structure of micropyle surface on oocytes and caviar grains in sturgeons

The structure of the surface and the micropylar apparatus of intra follicular and ovulated oocytes from six sturgeon and one paddlefish species were studied. Likewise changes in these stuctures were examined after eggs were processed into caviar. The objective was to establish some criteria of caviar quality and to identify interspecific differences. The presence (Acipenser) or the absence (Huso) of a funnel on the micopyle allows the discrimination of these two genera. The oocyte diameter, the size of the micropylar fields (surface perforated by micropyles) and the ultrastructure and size of the external micropylar opening allows the discrimination of several species. The oocyte surface of all species investigated looks very similar. After caviar processing, changes in the oocyte envelopes occurred which makes it difficult to recognise structural differences between species.