Coevolution between eggs and sperm of insects.

Sexual selection is known to play a major role in the evolution of insect sperm size, whereas natural selection is thought to be a major driver of insect egg size. Despite these differing forms of selection operating, it is possible coevolution between male and female gametes can occur owing to their vital interactions during fertilization. We tested egg-sperm coevolution in insects and found that longer sperm correlated to longer and wider eggs. Moreover, the size of the entry point of sperm into insect eggs (micropyles), was positively related to the diameter of sperm, on average being approximately three times the diameter of the sperm. This suggests a function in reducing and channelling sperm entry, but potentially still leaving space for movement. Our work suggests that greater attention needs to be paid to egg-sperm interactions prior to the point of fertilization as they may influence the evolution of gametes.

Fine structure of micropylar regions of Cobitis hankugensis, Iksookimia longicorpa and their unisexual natural hybrids.

Iksookimia longicorpa and Cobitis hankugensis are two species of fish distributed only on the Korean Peninsula. They have a unique reproductive ecology that naturally hybridizes into three widely known unisexual types, maintaining populations of almost all females. In this study, the fine structure of the micropyles of I. longicorpa, C. hankugensis and their hybrids was analyzed to find out how egg-sperm interaction, a common interspecies isolation mechanism, is possible between heterogeneous species. Analysis of 30 eggs from five females of each species revealed that all had one funnel-shaped micropylar region and a manhole-shaped micropyle canal. With the exception of C. hankugensis, which had no spiral grooves or ridges, the rest had counterclockwise spiral grooves and ridges on the micropylar region. All five species, however, showed identical groove patterns for the micropyle canal. The egg size was the largest in HL (one from the C. hankugensis locus with one from the I. longicorpa locus) and the smallest in C. hankugensis. In the hybrids, the HL type had the largest egg and HHL (two from the C. hankugensis locus with one from the I. longicorpa locus) type the smallest. For the diameter of the micropylar region and micropyle canal, the diploid I. longicorpa, C. hankugensis and HL were smaller than those of the triploid. In addition, as the ratio of the canal diameter to the eggs was lower in I. longicorpa than in C. hankugensis, it was confirmed that I. longicorpa has a relatively small micropyle canal compared with C. hankugensis.

The Hippo pathway effector Taz is required for cell morphogenesis and fertilization in zebrafish.

Hippo signaling is a critical pathway that integrates extrinsic and intrinsic mechanical cues to regulate organ size. Despite its essential role in organogenesis, little is known about its role in cell fate specification and differentiation. Here, we unravel a novel and unexpected role of the Hippo pathway effector Taz (wwtr1) in controlling the size, shape and fate of a unique cell in the zebrafish ovary. We show that wwtr1 mutant females are infertile. In teleosts, fertilization occurs through the micropyle, a funnel-like opening in the chorion, formed by a unique enlarged follicle cell, the micropylar cell (MC). We describe here, for the first time, the mechanism that underlies the differentiation of the MC. Our genetic analyses show that Taz is essential for MC fate acquisition and subsequent micropyle formation in zebrafish. We identify Taz as the first bona fide MC marker and show that Taz is specifically and strongly enriched in the MC precursor. Altogether, we performed the first genetic and molecular characterization of the MC and propose that Taz is a key regulator of MC fate.This article has an associated 'The people behind the papers' interview.

Structural analysis and characterization of egg-envelope in the Indian freshwater murrel, Channa punctatus.

Egg-envelope, an acellular coat, surrounds the egg and is essential for vitellogenin incorporation. It also plays a pivotal role during fertilization and provides protection to the developing embryo. In the present study, scanning electron microscopy was used to elucidate the structural details of isolated egg-envelopes from the Indian freshwater murrel, Channa punctatus. Several pores and single micropyle were observed on outer surface, whereas inner layer indicated deposition of proteinaceous material. The constituent proteins of egg-envelope were further characterized by Fourier transform infrared (FT-IR) spectroscopy, and electrophoresis and mass-spectrometry (MALDI-TOF-MS/MS). The secondary structure of egg-envelope proteins showed the presence of antiparallel ß-pleated sheets and aromatic amino acids. These proteins resolved into two peptides (130 kDa and 68 kDa) under denaturing conditions, which exhibited glycoprotein nature. The peptide band with low molecular mass showed significant similarity with transmembrane protein, whereas peptide band with high molecular mass matched with choriogenin protein of other fishes. These results confirm that chorion is derived from precursor protein, Choriogenin, in murrel. Chemical composition of egg-envelope supports that chorion is responsible exchange material and chemical defence during embryogenesis.

The role of the integuments in pollen tube guidance in flowering plants.

In angiosperms, pollen tube entry into the ovule generally takes place through the micropyle, but the exact role of the micropyle in pollen tube guidance remains unclear. A limited number of studies have examined eudicots with bitegmic micropyles, but information is lacking in ovules of basal/early-divergent angiosperms with unitegmic micropyles. We have evaluated the role of the micropyle in pollen tube guidance in an early-divergent angiosperm (Annona cherimola) and the evolutionarily derived Arabidopsis thaliana by studying γ-aminobutyric acid (GABA) and arabinogalactan proteins (AGPs) in wild-type plants and integument-defective mutants. A conserved inhibitory role of GABA in pollen tube growth was shown in A. cherimola, in which AGPs surround the egg apparatus. In Arabidopsis, the micropyle formed only by the outer integument in wuschel-7 mutants caused a partial defect in pollen tube guidance. Moreover, pollen tubes were not observed in the micropyle of an inner no outer (ino) mutant in Arabidopsis, but were observed in homologous ino mutants in Annona. The similar distribution of GABA and AGPs observed in the micropyle of Arabidopsis and Annona, together with the anomalies from specific integument mutants, support the role of the inner integument in preventing multiple tube entrance (polytubey) in these two phylogenetically distant genera.

A function for the pleurogram in physically dormant seeds.

BACKGROUND AND AIMS: Different structures have been shown to act as a water gap in seeds with physical dormancy (PY), and in Fabaceae they are commonly located in the hilar region. However, the function of the pleurogram, a structure in the extra-hilar region that is common in legume seeds, remains unknown. Our aims were to review the literature for occurrence of the pleurogram in Fabaceae, determine if the pleurogram can open, and compare the functional morpho-anatomy of water gaps in seeds of 11 Senna species. METHODS: Imbibition tests showed that all 11 species had PY. Structural features of the hilar and extra-hilar regions of the seeds were investigated using light and scanning electron microscopy, and dye-tracking was performed to trace the pathways of water through the seed coat. KEY RESULTS: A pleurogram has been reported for 37 legume genera. Water gaps differed among Senna species, with lens, hilum, micropyle and pleurogram taking up water after PY was broken. In Senna alata seeds, only the pleurogram acted as a water gap, whereas in S. reniformis and S. silvestris water entered the seed through both the pleurogram and the hilar region. In the pleurogram of S. alata and S. reniformis, the palisade layer moved outward, exposing the hourglass cells, whereas in S. silvestris the palisade layer was broken. CONCLUSIONS: The pleurogram acts as a water gap in some of the 11 Senna species examined, but it is non-functional in others. Opening the pleurogram occurs due to the formation of a linear slit in the palisade layer. The pleurogram is of functional significance by creating a wide opening, whereby water can reach the embryo and start germination. This is the first report of the pleurogram functioning as a water gap. Because this structure is shared by at least 37 genera, it also may be a water gap in many other legume species.

Chemical and physical guidance of fish spermatozoa into the egg through the micropyle†,‡.

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.

Termite queens close the sperm gates of eggs to switch from sexual to asexual reproduction.

Males and females are in conflict over genetic transmission in the evolution of parthenogenesis, because it enhances female reproductive output but deprives the males' genetic contribution. For males, any trait that coerces females into sexual reproduction should increase their fitness. However, in the termite Reticulitermes speratus, queens produce their replacements (neotenic queens) parthenogenetically while using normal sexual reproduction to produce other colony members. Here, we show that termite queens produce parthenogenetic offspring in the presence of kings by closing the micropyles (sperm gates; i.e., openings for sperm entry) of their eggs. Our field survey showed that termite eggs show large variation in numbers of micropyles, with some having none. Microsatellite analysis showed that embryos of micropyleless eggs develop parthenogenetically, whereas those of eggs with micropyles are fertilized and develop sexually. Surveys of eggs among queens of different age groups showed that queens begin to lay micropyleless eggs when they are older and thus, need to produce their replacements parthenogenetically. In addition, we found clear seasonality in new neotenic queen differentiation and micropyleless egg production. This micropyle-dependent parthenogenesis is the first identification, to our knowledge, of the mechanism through which females control egg fertilization over time in diploid animals, implying a novel route of the evolution of parthenogenesis in favor of female interests without interference from males.

Micropyle number is associated with elevated female promiscuity in Lepidoptera.

In the majority of insects, sperm fertilize the egg via a narrow canal through the outer chorion called the micropyle. Despite having this one primary function, there is considerable unexplained variation in the location, arrangement and number of micropyles within and between species. Here, we examined the relationship between micropyle number and female mating pattern through a comparative analysis across Lepidoptera. Three functional hypotheses could explain profound micropylar variation: (i) increasing micropyle number reduces the risk of infertility through sperm limitation in species that mate infrequently; (ii) decreasing micropyle number reduces the risk of pathological polyspermy in species that mate more frequently; and (iii) increasing micropyle number allows females to exert greater control over fertilization within the context of post-copulatory sexual selection, which will be more intense in promiscuous species. Micropyle number was positively related to the degree of female promiscuity as measured by spermatophore count, regardless of phylogenetic signal, supporting the hypothesis that micropyle number is shaped by post-copulatory sexual selection. We discuss this finding in the context of cryptic female choice, sperm limitation and physiological polyspermy.

New approaches for morphological diagnosis of bovine Eimeria species: a study on a subtropical organic dairy farm in Brazil.

Bovine eimeriosis or coccidiosis is an intestinal disease caused by Eimeria spp. which is related to gastrointestinal disorders and, in some cases, death. The current work aimed to identify and provide detailed morphological characteristic features of the different Eimeria spp. parasites of crossbred cows of a subtropical organic dairy farm in Brazil, offering tools for the diagnosis of bovine eimeriosis. Eimeria auburnensis, Eimeria bovis, Eimeria bukidnonensis, Eimeria canadensis, Eimeria cylindrica, Eimeria ildefonsoi, and Eimeria zuernii were identified. The application of line regressions and ANOVA provided a means for the identification of these species. Finally, the current work proposes a dichotomous key to assist in the morphologic identification of bovine Eimeria spp. oocysts.

Seed morphology and anatomy and its utility in recognizing subfamilies and tribes of Zingiberaceae.

PREMISE OF THE STUDY: Recent phylogenetic analyses based on molecular data suggested that the monocot family Zingiberaceae be separated into four subfamilies and four tribes. Robust morphological characters to support these clades are lacking. Seeds were analyzed in a phylogenetic context to test independently the circumscription of clades and to better understand evolution of seed characters within Zingiberaceae. METHODS: Seventy-five species from three of the four subfamilies were analyzed using synchrotron based x-ray tomographic microscopy (SRXTM) and scored for 39 morphoanatomical characters. KEY RESULTS: Zingiberaceae seeds are some of the most structurally complex seeds in angiosperms. No single seed character was found to distinguish each subfamily, but combinations of characters were found to differentiate between the subfamilies. Recognition of the tribes based on seeds was possible for Globbeae, but not for Alpinieae, Riedelieae, or Zingibereae, due to considerable variation. CONCLUSIONS: SRXTM is an excellent, nondestructive tool to capture morphoanatomical variation of seeds and allows for the study of taxa with limited material available. Alpinioideae, Siphonochiloideae, Tamijioideae, and Zingiberoideae are well supported based on both molecular and morphological data, including multiple seed characters. Globbeae are well supported as a distinctive tribe within the Zingiberoideae, but no other tribe could be differentiated using seeds due to considerable homoplasy when compared with currently accepted relationships based on molecular data. Novel seed characters suggest tribal affinities for two currently unplaced Zingiberaceae taxa: Siliquamomum may be related to Riedelieae and Monolophus to Zingibereae, but further work is needed before formal revision of the family.

The chorion ultrastructure of ova of Lophius spp.

The chorion surface ultrastructure of unfertilized eggs of black anglerfish Lophius budegassa and white anglerfish Lophius piscatorius was examined by scanning electron microscopy. Species-specific differences were observed.

Envelope surface ultrastructure and specific gravity of artificially fertilized Pacific cod Gadus macrocephalus eggs.

The envelope surface ultrastructure and specific gravity of artificially fertilized eggs of the Pacific cod Gadus macrocephalus were examined. The unfertilized, demersal and slightly adhesive eggs of G. macrocephalus were almost spherical and had no oil globules. Wrinkled envelope surface with elaborated hexagonal reticulated patterns and type I micropyle were observed under a scanning electron microscope. The adhesiveness of the eggs was lost at the blastodermal-cap stage after fertilization. The micropylar canal was sealed by secretion of the perivitelline fluid, and the entire surface became rough. Numerous bacilli were deposited at the micropyle and the outer envelope surface at the late germ-ring stage and at the embryo five-eighths around the yolk stage. The micropyle was completely deformed at the embryo seven-eighths around the yolk stage. The specific gravity of the fertilized G. macrocephalus eggs ranged from c. 1·0316 to 1·0454. These values, however, sharply decreased towards the end stages of egg development to produce pelagic larvae. The ultrastructural changes in the micropyle and envelope surface of the G. macrocephalus eggs protected the embryo from microorganism infections and mechanical stress during the long incubation period. The adhesiveness and specific gravity of the eggs influenced their dispersion potential.

A Comparative Morphological Study on the Characteristics of Egg Envelopes of Three Cultrinae Fishes (Cyprinidae, Teleostei) in Korea.

Three species of subfamily Cultrinae currently live in Korea, but Erythroculter erythropterus has been introduced into the Nakdonggang River and has taken over the habitat, reducing the habitat of Culter brevicauda. Only the endangered species C. brevicauda still lives in the Yeongsangang River, and it is necessary to be careful not to introduce E. erythropterus in the future. Hemiculter eigenmanni is also found throughout the country. In order to effectively manage and conserve the species in its various habitats and against invasions, this study was initiated. The ultrastructure of the egg envelopes of three species of Cultrinae inhabiting the Geumgang and Yeongsangang Rivers-E. erythropterus, C. brevicauda, and H. eigenmanni-were observed. It was found that the zona radiata of the egg envelopes of all three species were divided into two layers, an outer and inner layer, with the outer surface having a non-structural form. This form is characteristic of fishes with muddy, stagnant habitats or spawning grounds. The number of pore canals on the surface of the egg envelopes was 83 for E. erythropterus, 75 for C. brevicauda, and 58 for H. eigenmanni per 10 µm2, and the thickness was 7.89 ± 0.34 µm, 12.27 ± 0.46 µm, and 7.42 ± 0.24 µm, respectively. The shape of the micropyle demonstrated a funnel shape narrowing toward the inner diameter in all three species, and the size of the inner diameter was 6.62 ± 0.29 µm in E. erythropterus, 4.19 ± 0.39 µm in C. brevicauda, and 3.98 ± 0.46 µm in H. eigenmanni. The differences between species were identified in the number of pore canals, thickness, and micropyle inner diameter of egg envelopes, which were species-specific. Our study reveals a morphological mechanism in the egg envelope that prevents the formation of interspecific hybrids, and these features can be taxonomic traits that clarify species names. It also provides useful data for the production (breeding) of the second generation in aquaculture.

Identification and characterization of ten new water gaps in seeds and fruits with physical dormancy and classification of water-gap complexes.

BACKGROUND AND AIMS: Physical dormancy (PY) occurs in seeds or fruits of 18 angiosperm families and is caused by a water-impermeable palisade cell layer(s) in seed or fruit coats. Prior to germination, the seed or fruit coat of species with PY must become permeable in order to imbibe water. Breaking of PY involves formation of a small opening(s) (water gap) in a morpho-anatomically specialized area in seeds or fruits known as the water-gap complex. Twelve different water-gap regions in seven families have previously been characterized. However, the water-gap regions had not been characterized in Cucurbitaceae; clade Cladrastis of Fabaceae; subfamilies Bombacoideae, Brownlowioideae and Bythnerioideae of Malvaceae; Nelumbonaceae; subfamily Sapindoideae of Sapindaceae; Rhamnaceae; or Surianaceae. The primary aims of this study were to identify and describe the water gaps of these taxa and to classify all the known water-gap regions based on their morpho-anatomical features. METHODS: Physical dormancy in 15 species was broken by exposing seeds or fruits to wet or dry heat under laboratory conditions. Water-gap regions of fruits and seeds were identified and characterized by use of microtome sectioning, light microscopy, scanning electron microscopy, dye tracking and blocking experiments. KEY RESULTS: Ten new water-gap regions were identified in seven different families, and two previously hypothesized regions were confirmed. Water-gap complexes consist of (1) an opening that forms after PY is broken; (2) a specialized structure that occludes the gap; and (3) associated specialized tissues. In some species, more than one opening is involved in the initial imbibition of water. CONCLUSIONS: Based on morpho-anatomical features, three basic water-gap complexes (Types-I, -II and -III) were identified in species with PY in 16 families. Depending on the number of openings involved in initial imbibition, the water-gap complexes were sub-divided into simple and compound. The proposed classification system enables understanding of the relationships between the water-gap complexes of taxonomically unrelated species with PY.

The orchid seed coat: a developmental and functional perspective.

Orchid seeds are 'dust-like.' The seed coat is usually thin, with only one to a few cell layers. It originates from the integuments formed during ovule development. In orchids, the outer integument is primarily responsible for forming a mature seed coat. The inner integument usually fails to develop after fertilization, becomes compressed, and collapses over the expanding embryo. Hence, the seed coat is formed from the funiculus, chalaza, and outer integumentary cells. The outermost layer of the seed coat, the testa, is lignified, usually at the radial and inner tangential walls. The subepidermal thin-walled layer(s), the tegmen, subsequently cold, resulting in seeds having only a single layer of seed coat cells. In some species, cells of the inner integument remain alive with the ability to synthesize and accumulate lipidic and or phenolic compounds in their walls covering the embryo. This cover is called the 'carapace,' a protective shield contributing to the embryo's added protection. A developmental and functional perspective of the integuments and seed coat during seed development and germination is presented in this review.

Identification of the initial water-site and movement in Gleditsia sinensis seeds and its relation to seed coat structure.

BACKGROUND: Water uptake is essential for seed germination. However, Gleditsia sinensis seeds have a water-impermeable seed coat, which is beneficial for its adaption to the environment, but prohibits its germination without treatment. This feature may be associated with the structure of the seed coat. Thus, the aim of this research was to identify and describe the initial water uptake site and water movement and to determine the relationship between seed coat structure and water absorption. RESULTS: A water temperature of 80 °C was optimal to break the hardseededness of G. sinensis seeds. Scanning electron microscopy (SEM) images revealed that the seed coat consisted of a palisade layer and light line that can hinder water entry into the seed. Also, a structure of vascular bundles existed in the hilar region. Hot water treatment caused the tightly closed micropyle to open and the cavity beneath it expanded; the layer of palisade cells in the lens was raised. The embryo dye-tracking tests showed that the radicle tip was the initial region to be stained red. After staining for 24 h, the red-stained area on the vascular bundle side of cotyledon was more extensive than that on the other side. Further studies by MRI maps indicated that the micropyle was the initial site for water imbibition. Some water then migrated along the space between the seed coat and the endosperm to the chalazal; simultaneously, the rest of the water reached the embryonic axis and spread into cotyledons. The maps of 20-24 h showed that water was unevenly distributed within the cotyledons in a way that the edge parts were more hydrated than the center. Blocking tests showed that the hilar region was the initial and an important region during seed imbibition. The medial region and chalazal portion were capable of imbibing water when the hilar region was blocked, but water absorption was later and slower than that through the hilar region. CONCLUSION: MRI technology provides a promising and non-invasive technique to identify the water gap and the path of water movement in the seed. Combined with the results of SEM, the relation between seed coat and its imbibition can be inferred.

Labeling the Micropylar Cell in Zebrafish Whole-Mount and Cryo-sectioned Follicles.

In some animal species, fertilization occurs through a funnel-like canal called the "micropyle." In teleost fishes, the micropyle is formed by a very specialized follicle cell, called the micropylar cell (MC). Very little is known about the mechanisms underlying the specification and differentiation of the MC, a unique cell among hundreds that compose the follicle cell layer. The Hippo pathway effector Taz is essential for this process and is the first reported MC marker. Here, we describe a method to identify and mark the micropylar cell following the immunostaining procedure on cryosections or combining it with the RNA in situ hybridization on whole-mount follicles.

Chorion exploration in the tribe Anthocharidini (Lepidoptera: Pieridae) and their possible importance in its systematics.

We describe and compare the morphology of the chorion in nine species belonging to five genera of the tribe Anthocharidini (Pieridae: Pierinae), from a sample of 12 females with mature eggs, the bibliographic record of oviposited eggs, and photographs of scanning electron microscopy (SEM). The eggs examined come from Mexico, Spain, Brazil and Costa Rica. Its characterization was made considering the main structural features of the chorion in this tribe; it includes a distinction of structures in the apical zone and differentiation between the chorionic regions. We attached to this work sheets, diagrams, and terminology to understand and clarify the descriptions. Our results agree with the proposal of Anthocharidini as the least derived tribe of the Pierinae, considering that Hebomoia, a specialized genus, is not part of it. Tribes such as Leptosiaini, Elodinini or Nepheroniini also have more chorionic characteristics related to more derived tribes of the Pierinae.

The Drosophila micropyle as a system to study how epithelia build complex extracellular structures.

Dynamic rearrangements of epithelial cells play central roles in shaping tissues and organs during development. There are also scenarios, however, in which epithelial cell movements synergize with the secretion of extracellular matrix to build rigid, acellular structures that persist long after the cells are gone. The formation of the Drosophila micropyle provides an elegant example of this epithelial craftsmanship. The micropyle is a cone-shaped projection of the eggshell through which the sperm will enter to fertilize the oocyte. Though simple on the surface, both the inner structure and construction of the micropyle are remarkably complex. In this review, I first provide an overview of egg development, focusing on the key events required to understand micropyle formation. I then describe the structure of the micropyle, the cellular contributions to its morphogenesis and some interesting open questions about this process. There is a brief discussion of micropyle formation in other insects and fish to highlight the potential for comparative studies. Finally, I discuss how new studies of micropyle formation could reveal general mechanisms that epithelia use to build complex extracellular structures. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.