Hard Chorion Research Articles

Visualization of Actin Cytoskeleton in Cellular Protrusions in Medaka Embryos.

Cellular protrusions are fundamental structures for a wide variety of cellular behaviors, such as cell migration, cell-cell interaction, and signal reception. Visualization of cellular protrusions in living cells can be achieved by labeling of cytoskeletal actin with genetically encoded fluorescent probes. Here, we describe a detailed experimental procedure to visualize cellular protrusions in medaka embryos, which consists of the following steps: preparation of Actin-Chromobody-GFP and α-bungarotoxin mRNAs for actin labeling and immobilization of the embryo, respectively; microinjection of the mRNAs into embryos in a mosaic fashion to sparsely label individual cells; removal of the hard chorion, which hampers observation; and visualization of cellular protrusions in the embryo with a confocal microscope. Overall, our protocol provides a simple method to reveal cellular protrusions in vivo by confocal microscopy.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis.

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are someobstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

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.

Taxonomic identification and biological traits ofPlatystethynium triclavatum(Donev & Huber, 2002), comb. n. (Hymenoptera, Mymaridae), a newly recorded egg parasitoid of the Italian endemic pestBarbitistes vicetinus(Orthoptera, Tettigoniidae)

The little known fairyfly (Hymenoptera, Mymaridae),Platystethynium(Platystethynium)triclavatum(Donev & Huber, 2002), comb. n. fromPseudocleruchusDonev & Huber, 2002, is newly recorded as an egg parasitoid ofBarbitistes vicetinusGalvagni & Fontana, 1993 (Orthoptera, Tettigoniidae). This bush-cricket is endemic to northeastern Italy (mainly Euganean Hills of Veneto Region), where it has recently become an economically significant agricultural and forest pest. Data on discovery, distribution, and some remarkable biological traits of this gregarious egg parasitoid are presented. Its identification and availability of many well-preserved fresh specimens have made possible to re-definePseudocleruchusDonev & Huber, 2002 syn. n., with type and the only described speciesPseudocleruchus triclavatusDonev & Huber, 2002, as a synonym ofPlatystethyniumOgloblin, 1946 and its nominate subgenus,P. (Platystethynium), and also to describe the brachypterous male ofP. (Platystethynium)triclavatum. It is the first known male for the entire genus. Enlarged mandibles of the megacephalous males are used to chew holes in the hard chorion of the host egg, allowing fully winged females, whose mandibles are strongly reduced and do not cross over, to emerge after mating with the males inside it. Up to 136 individual parasitoids (about 77 on average) can hatch from a single egg ofB. vicetinus, with their sex ratio being strongly female biased (80–97% females per egg).

Microinjection of Marine Fish Eggs.

Microinjection is a powerful tool for studying embryonic development and analyzing gene functions in fish. This technique was first applied to model species of fish such as zebrafish and medaka whose egg chorions could be removed or softened before microinjection. Recent progress in genome editing using TALEN and CRISPR has opened the opportunity to analyze gene functions in a much wider range of fish including those important to marine aquaculture. Therefore, application of the microinjection technique is also required in these species. However, the characteristics of fish eggs vary widely between species and several technical difficulties need to be overcome in order to use microinjection in a wider range of species. To obtain consistent results with microinjection, an optimal method has to be developed for each target species. In this chapter, we describe the physical characteristics of the eggs of fish species that have been used in microinjection experiments in our laboratory and detail the microinjection system we developed for fish eggs with a hard chorion, such as those of marine species.

Multiparasitism with Trichogramma dendrolimi on egg of Chinese oak silkworm, Antheraea pernyi, enhances emergence of Trichogramma ostriniae

Trichogramma parasitoids are important biological control agents for insect pest management. However, efficient means for mass production of Trichogramma parasitoids are needed, particularly for T. ostriniae, which often fail to form emergence holes on host eggs of Chinese oak silkworm, Antheraea pernyi, because of the hard chorion. In the present study, we evaluated the possibilities to enhance adult emergence of T. ostriniae by utilizing emergence holes made by T. dendrolimi during multiparasitism on host eggs of A. pernyi and other biological parameters such as pre-emergence time, parasitism rate, and emergence rate of the two parasitoids, which were also evaluated between the two parasitism regimes of multi- and monoparasitism. Our data showed that multiparasitism on host eggs of A. pernyi significantly facilitated the emergence of adult T. ostriniae. Generally, both Trichogramma species favored parasitizing A. pernyi eggs, with multiple parasitoid adults emerged per host egg. The overall performance of T. dendrolimi was similar between the two parasitism regimes, although a significantly higher number of unemerged adults were found in the multiparasitized eggs than in the monoparasitized. For T. ostriniae, significantly more adults emerged, and a higher emergence rate was recorded on host eggs multiparasitized with T. dendrolimi than that on monoparasitized. Furthermore, significantly fewer unemerged adults were found in multi- than monoparasitized host eggs. The results of this study provided useful information on the feasibility of mass production of T. ostriniae via multiparasitism with T. dendrolimi on large eggs of A. pernyi.

Electro-microinjection of fish eggs with an immobile capillary electrode.

Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a μm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.

Visualization of primordial germ cells in the fertilized pelagic eggs of the barfin flounder Verasper moseri.

Primordial germ cells (PGCs) appear during early embryogenesis and differentiate into gametes through oogenesis or spermatogenesis. Teleost PGCs can be visualized by injecting RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3). Although this method has been widely applied to teleost PGCs, the visualization of PGCs in pelagic species that have eggs with a hard chorion is more problematic due to the technical difficulty of microinjection into their eggs. In this study, we developed a reliable method for microinjection of fertilized eggs in a pelagic species, the barfin flounder. Using a microneedle with a constriction "brake", we were able to introduce gfp-nos3 3'UTR mRNA into embryos and to determine the origin and migration route of PGCs. We also isolated the barfin flounder nos3 (bf-nos3) gene to compare its 3'UTR sequence with that of zebrafish. The 3'UTR of the bf-nos3 sequence was longer than that of zf-nos3. However, PGCs were also visualized after injection of gfp-bf-nos3 3'UTR mRNA both in zebrafish and barfin flounder. These results suggest that the function of nos3 is conserved between these species regardless of the sequence differences. The method developed here for labeling PGCs with gfp-nos3 mRNA will provide a means to study PGC development in the embryos of a wide range of marine fish species.

Caracterización electroforética de las proteínas del corion normal y del corion duro de Salmo salar

SUMMARY The chorion is an egg envelope that allows the early development of teleostean fishes. After reaching the alevin stage, hatching enzymes are secreted in order to digest the chorion and release the alevin. Massive mortality events have occurred in a number of salmon farms as a consequence of hatching problems, with alevins being unable to degrade the chorion. A previous study on the chorion structure of Salmo salar has shown that protein fibers forming the inner layer of non-degraded chorion are thicker than those observed in normal chorion, suggesting that polypeptides forming the choriogenin could exhibit an altered structure. It would be interesting to make a biochemical comparison between polypeptides forming protein fibers of the inner layer of normal and hard chorion in order to establish differences in the electrophoretic pattern of Salmo salar ova. According to our results, fibers forming the inner layer of normal chorion are thinner than those building the hard chorion. The electrophoretic pattern for normal chorion is described as four bands of molecular weight 107 kDa, 92 kDa, 38 kDa, and 31kDa in contrast to the 179 Kda, 157 Kda, 55 Kda and 54 Kda for non-hatched chorion. Consequently, protein fibers with higher molecular weight turned into thicker fibers with a different three dimensional structure, but also with different glycosylic radicals compared to normal fibers. These features make the inner layer of hard chorion different to normal chorion since it is very difficult for the hatching enzyme to dissolve it, leading the alevin to fail in the hatching process.

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Dechorionation of medaka embryos and cell transplantation for the generation of chimeras.

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out (1). Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species (2), thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps (3) so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Salmo salar: morfología ultraestructural de la pared del corion en ovas normales y con problemas de eclosión

SuMMaRy Either eyed alevins unable to digest the chorion or those digesting it partially, are both suffering the “hard chorion” phenomenon; in both cases alevins can not survive producing high mortality rates. During recent years, the salmon farming industry in Southern Chile has been facing massive mortality events caused by “hard chorion”. Since “hard chorion” is a recently observed phenomenon there are no literature reporting its origin. The aim of this study was to find differences in the ultrastructure of chorion fibers from Salmo salar ova at different stages of development by using Scanning Electron Microscopy and image analysis technique. Early results here reported indicated no differences in thickness of chorion fibers for Salmo salar ova at different stages of development, however differences in spatial arrangement of fibers forming the inner layer of chorion seem to explain the “hard chorion” occurrence. No pores were observed in the inner layer of chorion of ova exhibiting “hard chorion” as opposed to normally developed ova which have pores and allow hatching of alevins. Since choriogenin is a protein formed by a number of sub-units, it is possible that failure in synthesis of one of these sub-units might be responsable for an anomalous arrangement of fibers of chorion causing the “hard chorion” phenomenon.

Reprodução induzida da piabanha, Brycon insignis (Steindachner, 1876), mantida em cativeiro

Estudos de reprodução induzida da piabanha, Brycon insignis, foram realizados em janeiro-fevereiro/96 e fevereiro/97, utilizando-se exemplares de 3 a 5 anos de idade, mantidos em cativeiro na Estação Experimental da Seção de Hidrobiologia e Aqüicultura, pertencente à CESP (Companhia Energética de São Paulo), localizada na cidade de Paraibuna, SP. As fêmeas selecionadas para indução da reprodução apresentaram ventre abaulado, papila genital saliente e avermelhada e 60% dos ovócitos com núcleo em posição excêntrica. Trinta e três fêmeas foram induzidas à reprodução com dose única de hCG de 5 UI/g (gonadotropina coriônica humana); 17 receberam duas aplicações de extrato bruto de hipófise de carpa, de 0,5 e 5,0 mg/kg, com intervalo de 10 horas entre as aplicações; 18 receberam 0,5 mg/kg de extrato bruto de hipófise de carpa e, após 10 horas, 5 UI/g de hCG; 5 fêmeas receberam dose única de solução salina a 0,9%. Cerca de 10 horas após a última aplicação hormonal, 43 fêmeas responderam positivamente aos tratamentos hormonais liberando óvulos por extrusão. A distribuição de freqüência porcentual dos diâmetros dos ovócitos da amostra inicial apresentou tendência unimodal, com moda em 1250 µm. Entre os hormônios testados, o mais eficiente foi o hCG, em dose única, e o menos eficiente, o de hipófise de carpa, com aplicação de duas doses. Os machos receberam dose única de 3 UI de hCG/g. Cada fêmea liberou, em média, 24.690 óvulos, que, após a fertilização e hidratação, se apresentaram esféricos, demersais e não adesivos, com diâmetro médio de 3785 a 3900 µm, grande espaço perivitelínico e córion resistente. A taxa de fertilização oscilou entre 20 e 96% e a de eclosão, entre 0 e 85%. A temperatura da água foi de 26ºC ± 1ºC.