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Ultrastructure of the fertilized egg envelope from Melanotaenia praecox, Melanotaeniidae, Teleostei

Ultrastructure of the fertilized egg envelope from Melanotaenia praecox, Melanotaeniidae, Teleostei We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using light and electron microscopes. The fertilized eggs were spherical with adhesive filament, transparent, demersal, and had a narrow perivitelline space and small oil droplets. The size of fertilized egg was 1.02 ± 0.18 mm (n = 30), and there were two kinds of adhesive filament on the fertilized eggs. The long and thick (diameter 12.22 ± 0.52 μm, n = 20) adhesive filaments were only at the area of animal pole, and short and thin (diameter 1.99 ± 0.23 μm, n = 20) adhesive filaments were around the long filaments. A micropyle was conical shaped with adhesive filament and located near the animal pole of egg. The outer surface of fertilized egg was rough side. Also, the total thickness of the fertilized egg envelope was about 7.46 ± 0.41 μm(n = 20), the fertilized egg envelope consisted of two layers, an inner lamellae layer and an outer layer with high electron-density. And the inner layer was 8 layers. Collectively, these morphological characteristics and adhesive property of fertilized egg with adhesive filaments, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity. Keywords: Egg envelope, Fertilized egg, Melanotaenia praecox, Melanotaeniidae, Ultrastructure Introduction and virus, and selective or passive transports (Laale Dwarf rainbowfish (Melanotaenia praecox Weber & 1980; Grierson and Neville 1981; Harvey et al. 1983; Beaufort, 1922) is a teleost belong to family Melanotae- Cameron and Hunter 1984). In teleost, there are two niidae, order Atheriniformes and class Actinopterygii, types of fertilized eggs, adhesive and non-adhesive types. and habits Mamberamo River in northern Irian Jaya, The adhesive type is one among these three types, with Indonesia (Fishbase contributors 2021). This species is adhesive filament or protuberance, or fertilized egg is an omnivorous species, the males tend to be brighter in adhesive in itself without adhesive structures (Kim et al. color and have deeper bodies than the females (Tappin 1996; Kwon et al. 2017, Choi et al. 2019). 2016). Hatching begins 119.50 h post-fertilization at The structure of fertilized egg has been showed family 28 °C and newly hatched larvae were excellent swimming specificity because the fertilized eggs had same morph- activity (Radael et al. 2014). ology in same family under the stereo microscopic ob- The eggs of teleost are surrounded by an egg envelope, servation. But ultrastuctures of outer surface, micropyle, it has some functions including protections of physical, special adhesive structures and section of fertilized egg chemicals and biological factors such as bacteria, fungus envelope were showed species, genus or family specific- ities (Kim et al. 1996, 1998; Kwon et al. 2015, 2017, Choi et al. 2019). This ultrastructural difference of fertilized * Correspondence: fish7963@yonsei.ac.kr egg envelope has been known to be related with physical Department of Environmental Medical Biology, Yonsei University Wonju and chemical properties of water environment, location College of Medicine, Wonju 26426, South Korea Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Sohn and Kim Applied Microscopy (2021) 51:3 Page 2 of 6 of habitat (Ivankov and Kurdyayeva 1973; Stehr and prefixation, the specimens were washed twice in the Hawkes 1979; Laale 1980) and species variation (Joo and same buffer solution for 20 min. and then postfixed in Kim 2013). 1% osmium tetroxide solution in same phosphate buffer M. praecox has been studied on screening of fresh- solution for 2 h at room temperature. Specimens were water fish species for their susceptibility to a betanoda- dehydrated in ethanol and the samples were replaced virus (Furusawa et al. 2007). Fertilized eggs of this with tert-butyl alcohol and dried with freeze dryer (ES- species had oil droplets and fixing filaments for adhesion 2030, Hitachi, Japan). The samples were coated with Pt and embryoic development was similar to that of other by ion sputter (E-1045, Hitachi, Japan). Subsequently, species in the genus Melanotaenia (Radael et al. 2014). the fertilized eggs were observed under the table top But there is no research on the ultrastructure of fertil- scanning electron microscope (TM-1000, Hitachi, ized egg envelope because it is hard to get fertilized eggs Japan). from this species in aquarium. Therefore, we studied the For transmission electron microscope (TEM) observa- morphology of fertilized egg with adhesive filaments, tion, prefixation, postfixation and dehydration were and the ultrastructures of micropyle, outer surface, inner conducted by following the same procedure as that for surface and section of fertilized egg envelopes under the SEM., cleared in propylene oxide, and embedded in an light and electron microscopes to find out species speci- Epon 812 mixture. Ultrathin sections of embedded fertil- ficity in dwarf rainbowfish, M. praecox belong to family ized egg envelope were taken with an ultramicrotome Melanotaeniidae. (Ultracut E, Reichert-Jung, Austria) at a thickness of about 60 nm. The ultrathin sections were mounted onto Materials and methods copper grids, double stained with uranyl acetate followed Animals by lead citrate, and observed with a transmission elec- Three pairs of dwarf rainbowfish, Melanotaenia praecox tron microscope (JEM-1400, JEOL, Japan). (total length: 4–6 cm) used in this study were purchased from TrofishNet Aquarium (Yongin, Korea). The tap Results and discussion water used for rearing was treated with carbon filter Structure of fertilized eggs (Pre-carbon filter, filter114 Co. Ltd., Korea) to remove The fertilized eggs of Melanotaenia praecox were spher- chlorine, and its temperature and pH were maintained ical with adhesive filament, transparent, demersal, and at 25 ± 0.5 °C and 6.5 ± 0.5, respectively. Biological filtra- had a small perivitelline space and small oil droplets tion was performed using a sponge filter (Tetra Twin- (Fig. 1). The size of fertilized egg was 1.02 ± 0.18 mm Brillant Super Filter™, Tetra Co. Ltd., Germany), and (n = 30). The fertilized eggs were hard enough to handle scraps and excrement settled on the bottom of the water tank were eliminated by exchanging one-third of the water every day. The artificial light was illuminated for 10 h per day to simulate a daytime environment using an electronic timer, and hatched Artemia nauplii (Great Salt Lake Artemia cysts, Saunders, U.S.A.) were provided as food three times a day at 9 a.m., 1 p.m., and 5 p.m. Collection of fertilized eggs It used the same rearing water as water of breeding tank (45X30X30 cm) and a bundle of acrylic knitting yarn was used as a spawning ground. The fertilized eggs were isolated using fingers being careful not to break the fertilized eggs from a bundle of acrylic knitting yarn. Fertilized eggs that confirmed the formation of perivitel- line space were measured for size (n = 30) under digital microscope (AD-7013MZT, Dino-Lite, Anmo, Taiwan) and used in this study as experimental samples. Electron microscopy For scanning electron microscope (SEM) observation, Fig. 1 Fertilized egg of dwarf rainbowfish (Melanotaenia praecox). White arrow; lipid droplets, E; egg envelope, Y; yolk. The perivitelline first fertilized egg envelopes were pierced a hole with 1 space (black arrow) was very small and a bundle of adhesive mL injection needle and fixed in 2.5% glutaraldehyde in filament (Af) was only at the area of animal pole 0.1 M phosphate buffer (pH 7.4) for 24 h at 4 °C. After Sohn and Kim Applied Microscopy (2021) 51:3 Page 3 of 6 it with fingers. Fertilized egg itself had no adhesive prop- 2002). But the external shape is different. Fertilized eggs erty, but the filaments continuously have been main- of A. frenatus and O. obscura are long ellipsoidal but tained the adhesive property after sample preparation that of M. praecox is spherical. Also, small oil droplets for scanning electron microscopic observation too. The were in vitelline membrane, these small oil droplets are fertilized eggs of most species belong to Belontiidae, thought to be used as a nutrient to embryonic develop- Characidae, Cyprinidae and Cichlidae have adhesive ment. In species belong to Belontiidae, the fertilized eggs property, but fertilized eggs of Hemigrammus caudovit- have a large oil droplet that provide buoyancy for their tatus (Characidae) and Danio rerio (Cyprinidae) are floating (Kim et al., 1999). non-adhesive type (Kim et al. 1996; Joo and Kim 2013). As mentioned above, morphological characteristics of The fertilized eggs have same morphology according to fertilized egg from M. praecox including external egg the genus or family (Deung et al. 1997; Kim et al. 1999; shape, a bundle of adhesive filament at the area of ani- Joo and Kim 2013; Kwon et al. 2017;Choi et al. 2019). mal pole, small perivitelline space and oil droplets, and The perivitelline space of fertilized egg of M. praecox was demersal types were showed species specificity. very small enough to vitelline membrane of egg stick to the fertilized egg envelope. The size of perivitelline space Micropyle and adhesive structures was related with their spawning habits. Most egg scatter Adhesive filaments were only near the micropyle on the species such as Cyprinidae and Characidae have a large animal pole of fertilized egg (Fig. 2a). Sometimes the perivitelline space for protection from the external phys- long adhesive filaments were coiled together (Fig. 2b). ical impacts (Kim et al. 1996; Chang et al. 2019). But that There were two kinds of adhesive filaments, one was of fishes belong to Belontiidae, Cichlidae, Callichthyidae, long whippy adhesive filaments (diameter in the middle Loricariidae and Nothobranchiidae have a habit that lay- 12.22 ± 0.52 μm, n = 20) and the other was short adhe- ing eggs on a spawning ground have a small perivitelline sive filaments (diameter in the middle 1.99 ± 0.23 μm, space (Deung et al. 1997, 2000; Kim et al. 2009; Kwon n =20). et al. 2015, 2017;Choiet al. 2019;Kim 2020). The adhe- But there were no adhesive filaments in other part siveness of fertilized egg from Ancistrus cirrhosis was excepting the animal pole area. In teleost, there are known to disappeared after spawning excepting some adhesive fertilized eggs without adhesive filaments parts that fertilized egg contact with other egg or spawn- (Kwon et al. 2017; Choi et al. 2019) and with adhesive ing place (Kim 2020). long filaments. (Kim et al. 1998, 2002; Kwon et al. 2017) Although Amphiprion frenatus (Pomacentridae) and or adhesive reticular fibers (Deung et al. 2000, Kim et al. Odontobutis obscura (Eleotrididae) belong to different 2009). Nothobranchius foerschi and Nothobranchius family, the fertilized eggs have a bundle of adhesive fila- rachovii belong to Nothobranchiidae have a lot of adhe- ments similar to that of M. praecox (Kim et al. 1998, sive whip-like structures over the whole outer surface of Fig. 2 Long adhesive filaments (LF) from the fertilized eggs of M. praecox were only at the area of the animal pole including a micropyle (a) and the short adhesive filaments (arrows) were around long adhesive filaments. Sometimes the long adhesive filaments were coiled together (b) Sohn and Kim Applied Microscopy (2021) 51:3 Page 4 of 6 fertilized egg in both species (Kwon et al. 2017). The Outer surface of the fertilized egg envelopes fertilized eggs of tomato clown anemonefish and dark In this study, the outer surface was rough side without sleeper have a bundle of adhesive filament. But the adhesive filaments excepting micropylar region (Fig. 4). researchers were unable to find a micropyle using a The outer surface of fertilized egg was smooth and the stereo microscope and a scanning electron micro- egg envelope have a bundle of adhesive filaments (Kim scope (Kim et al. 1998, 2002). In our research, a et al. 1998, 2002). This difference of outer surface could micropyle was found in a bundle of adhesive fila- be species specificity. The ultrastuctures of outer surface ments (Fig. 3). The micropyle was conical shaped of fertilized egg envelope were vary according to the spe- with whippy adhesive filaments. The external diameter cies, genus and family. of micropyle was about 12.5 μm, and inner diameter In the fishes belong to Callichthyidae, Cichlidae and was about 4.73 μm. In most egg with a bundle of ad- Belontiidae, the ultrastructure of outer surface of egg hesive filaments, the micropyle was believed to be envelope was showed family specificity because the located in a bundle of adhesive filaments. In some structure of outer surface was same according to the species, these adhesive filaments plays a role of pro- family (Deung et al. 1997; Kim et al. 1999;Choi et al. tection of water loss when the water level is lowered 2019). That of Callichthyidae have adhesive protuber- by the tide (Dumont and Brummet, 1980). Even if it’s ances (Choi et al. 2019), that of Cichlidae have cov- the same species, the morphology was different from ered with adhesive reticular structures (Deung et al. distribution (Brummett and Dumont 1981). 1997; Kim et al. 2009), and that of Belontiidae have Micropyle location in the area of animal pole is many grooves (Kim et al. 1999). But the fine structure suitable for fertilization because the animal pole con- of outer surface of fertilized egg envelope were differ- tains cytoplasm with nucleus. In general, there are no ent according to the species in Cyprinidae. That of any special structures around the micropyle, but fer- Tanichthys alborubes have rod-like structures (Kim tilized eggs of some species have special structures in et al. 1998), that of pale chub have Indian club-like the vicinity of a micropyle. In Characidae, the micro- structures (Deung et al. 2000), that of Hemibarbus pyle have a spoke-like structure consisted of protu- longirostris have taste bud-like structures (Kim et al. berance lines of egg envelope in all species (Kim 2001), and that of Danio rerio have knob-like struc- et al. 1996, 2005; Chang et al. 2019). The micropyle tures (Joo and Kim 2013). Therefore, ultrastructures of pale chub belong to Cyprinidae is surrounded by of outer surface of fertilized egg envelope was showed five peaks of hill structures (Deung et al. 2000). The genus specificity in fishes belong to Cyprinidae. The micropyles were same funnel shape in Belontiidae ultrastructures of outer surface can be different in (Kim et al. 1999) and a plate coral mouth shape in same family or same in different family (Kim et al. genus Nothobranchius (Kwon et al. 2017). Therefore, 1996, 2005). Also the structure of outer surface of the structure of micropyle seems to be family, genus egg envelope could be changeable by species variation or species specificities. (Joo and Kim 2013). Fig. 3 Scanning electron micrograph of micropyle (arrow) on the Fig. 4 The outer surface of fertilized egg envelope. The outer fertilized egg envelopes from M. praecox. The micropyle was conical surface was rough and there were no adhesive filaments on other shaped with whippy adhesive filaments side of egg envelope excepting micropylar region Sohn and Kim Applied Microscopy (2021) 51:3 Page 5 of 6 Fine structure section of fertilized egg envelope head and tail light fish and glowlight tetra consisted of 3 Under the scanning electron microscope, the section of layers, that of black tetra consisted of 4 layers, that of fertilized egg envelope from M. praecox was a multi- Buenos aires tetra consisted of 5 layers, and that of layer and the thickness was about 74.46 ± 0.41 μm(n = serape tetra consisted of 5–6 layers (Kim et al. 1996, 20) (Fig. 5a). Also, the fertilized egg envelopes consisted 2005; Chang et al. 2019). As mentioned above, the of 2 layers, an inner lamellae layer and an outer layer number of layers on fertilized egg envelope or section with high electron-density on TEM image. And the structure are showed species specificity, genus specificity inner layer was 8 layers (Fig. 5b). The inner lamellar or family specificity. But more structural research on the layer tended to be wide on the outside and narrow on other species belong to Melanotaeniidae is needed to de- the inside in particular. But this inner lamellar layer termine whether it is family specificity or not. had equal spacing in all species belong to Cichlidae The structure of egg envelope was related with (Kim et al. 1999). external environment including intensity of radiation, In general, the number of layer of fertilized egg enve- hydraulic pressure, water wave and current, and spawn- lope was different according to the family or species but ing behavior. The thickness of egg envelope was known sometimes it is same in a family such as Belontiidae, to be thick in floating type than demersal (Stehr and Cichlidae, Callichthyidae, and Nothobranchiidae (Kim Hawkes 1979), in oviparity than ovoviparity (Riehl and et al. 1999, 2009; Kwon et al. 2015, 2017; Choi et al. Greven 1993) and in the fast stream (Ivankov and 2019). The fertilized egg envelope consisted of 2 or 3 Kurdyayeva 1973). layers in most teleost and the inner layer was showed la- mellar structure alternated high and low layers due to Conclusions the difference of the electron-density. Unusually, the The fertilized eggs of dwarf rainbowfish (Melanotaenia fertilized egg envelope from Ancistrus cirrhosis (Loricar- praecox) belong to Melanotaeniidae were spherical with iidae) have counter structure from other species (Kim adhesive filament, transparent, demersal, and had a small 2020). In species belong to Characidae and Cyprinidae, perivitelline space and oil droplets. And there were two the fertilized egg envelopes have different structure ac- kinds of adhesive filament, those of long and thick, and cording to the species. In Characidae, those of head and short and thin on the fertilized eggs. A micropyle was tail light fish, serape tetra, Buenos aires tetra consisted conical shaped with adhesive filament and located at the of 3 layers, and those of black tetra and glowlight tetra area of the animal pole. The outer surface of fertilized consisted of 2 layers. But the number of inner layer is egg was rough side. Also, the fertilized egg envelope different according to the species. The inner layers of consisted of two layers, an inner lamellae layer and an Fig. 5 The electron micrographs of the fertilized egg envelopes section. The fertilized egg envelope was a multi-layer lamellar structure in SEM image (a). The egg envelope consisted of two layers, an inner lamella layer (IL) and an outer layer with high electron-density (arrow). And the inner layer was 8 layers on TEM image (b) Sohn and Kim Applied Microscopy (2021) 51:3 Page 6 of 6 outer layer with high electron-density. And the inner K.B. Joo, D.H. Kim, Comparative ultrastructures of the fertilized egg envelopes in Danio rerio and Danio rerio var. frankei, Cyprinidae, teleostei. Appl. Microsc 43, layer was 8 layers. Collectively, these morphology of 14–20 (2013). https://doi.org/10.9729/AM.2013.43.1.14 fertilized egg including egg size, adhesive property, oil D.H. Kim, Ultrastructure of the fertilized egg envelopes in Ancistrus cirrhosus, droplets, small perivitelline space, a bundle of adhesive Loricariidae, Teleostei. Appl. Microsc 50, 13 (2020). https://doi.org/10.1186/s42 649-020-00034-7 filaments on the animal pole, and ultrastructural charac- D.H. Kim, B.S. Chang, Y.K. Deung, S. Kim, K.B. Joo, K.J. Lee, Ultrastructure of the teristics of egg envelope including micropyle, outer sur- fertilized egg envelope in Cichlasoma managuensis, Cichlidae, teleost. 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Deung, A comparative study on the ultrastructures of the Funding egg envelope in fertilized eggs of fishes, Characidae, three species. Kor. J. No funding was received for this work. Electron Microsc. 26, 277–291 (1996) D.H. Kim, D.S. Reu, Y.K. Deung, Comparative ultrastructures of the fertilized egg Availability of data and materials envelopes in three species, Cyprinidae, teleost. Kor. J. Electron Microsc. 28, No applicable. 237–253 (1998) D.H. Kim, D.S. Reu, Y.K. Deung, Ultrastructure of the fertilized egg envelope from Declarations dark sleeper, Eleotrididae, teleost. Kor. J. Electron Microsc. 32,39–44 (2002) J.K. Kwon, H.S. Jung, D.H. Kim, Comparative ultrastructures of the fertilized egg Competing interests envelopes in Nothobranchius guentheri and Nothobranchius patrizii, The authors declare that they have no competing interests. Nothobranchiidae, teleostei. Appl. Microsc. 45, 144–149 (2015). https://doi. org/10.9729/AM.2015.45.3.144 Author details O. Kwon, J.H. Shon, D.Y. Chung, E.J. Kim, D.H. Kim, Comparative ultrastructures of Institute of Lifestyle Medicine, Yonsei University Wonju College of Medicine, the fertilized egg envelopes in Nothobranchius foerschi and Nothobranchius Wonju 26426, South Korea. Department of Environmental Medical Biology, rachovii, Nothobranchiidae, teleostei. Appl. Microsc. 47,70–74 (2017). https:// Yonsei University Wonju College of Medicine, Wonju 26426, South Korea. doi.org/10.9729/AM.2017.47.2.70 H.W. Laale, The perivitelline space and egg envelopes of bony fishes: A review. Received: 3 February 2021 Accepted: 23 March 2021 Copeia 1980, 210–226 (1980) M.C. Radael, L.D.C. LD, D.R. Andrade, D. Mattos, J.H. Motta, J.V. Manhães, M.V. Vidal, Morphophysiological characterization of the embryonic development References of Melanotaenia praecox (weber & de Beaufort, 1922). Zygote 22, 533–539 A.R. Brummett, J.N. 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Gamete Res. 3,25–44 (1980) Fishbase contributors, Melanotaenia praecox (Weber & de Beaufort, 1922), Dwarf rainbowfish, Fishbase, https://www.fishbase.se/summary/10516. Accessed 12 Jan 2021 R. Furusawa, Y. Okinaka, K. Uematsu, T. Nakai, Screening of freshwater fish species for their susceptibility to a betanodavirus. Dis. Aquat. Org. 77, 119–125 (2007). https://doi.org/10.3354/dao01841 J.P. Grierson, A.C. Neville, Hellicoidal architecture of fish eggshell. Tissue Cell 13, 819–830 (1981) B. Harvey, R.N. Kelley, M.J. Ashwood-Smith, Permeability of intact and dechorionated zebrafish embryos to glycerol and dimethyl sulfoxide. Cryobio. 20, 432–439 (1983) V.N. Ivankov, V.P. Kurdyayeva, Systematic differences and the ecological importance of the membranes in fish eggs. J. Ichthyol. 13, 864–873 (1973) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Microscopy Springer Journals

Ultrastructure of the fertilized egg envelope from Melanotaenia praecox, Melanotaeniidae, Teleostei

Applied Microscopy , Volume 51 (1) – Apr 1, 2021

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Abstract

We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using light and electron microscopes. The fertilized eggs were spherical with adhesive filament, transparent, demersal, and had a narrow perivitelline space and small oil droplets. The size of fertilized egg was 1.02 ± 0.18 mm (n = 30), and there were two kinds of adhesive filament on the fertilized eggs. The long and thick (diameter 12.22 ± 0.52 μm, n = 20) adhesive filaments were only at the area of animal pole, and short and thin (diameter 1.99 ± 0.23 μm, n = 20) adhesive filaments were around the long filaments. A micropyle was conical shaped with adhesive filament and located near the animal pole of egg. The outer surface of fertilized egg was rough side. Also, the total thickness of the fertilized egg envelope was about 7.46 ± 0.41 μm(n = 20), the fertilized egg envelope consisted of two layers, an inner lamellae layer and an outer layer with high electron-density. And the inner layer was 8 layers. Collectively, these morphological characteristics and adhesive property of fertilized egg with adhesive filaments, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity. Keywords: Egg envelope, Fertilized egg, Melanotaenia praecox, Melanotaeniidae, Ultrastructure Introduction and virus, and selective or passive transports (Laale Dwarf rainbowfish (Melanotaenia praecox Weber & 1980; Grierson and Neville 1981; Harvey et al. 1983; Beaufort, 1922) is a teleost belong to family Melanotae- Cameron and Hunter 1984). In teleost, there are two niidae, order Atheriniformes and class Actinopterygii, types of fertilized eggs, adhesive and non-adhesive types. and habits Mamberamo River in northern Irian Jaya, The adhesive type is one among these three types, with Indonesia (Fishbase contributors 2021). This species is adhesive filament or protuberance, or fertilized egg is an omnivorous species, the males tend to be brighter in adhesive in itself without adhesive structures (Kim et al. color and have deeper bodies than the females (Tappin 1996; Kwon et al. 2017, Choi et al. 2019). 2016). Hatching begins 119.50 h post-fertilization at The structure of fertilized egg has been showed family 28 °C and newly hatched larvae were excellent swimming specificity because the fertilized eggs had same morph- activity (Radael et al. 2014). ology in same family under the stereo microscopic ob- The eggs of teleost are surrounded by an egg envelope, servation. But ultrastuctures of outer surface, micropyle, it has some functions including protections of physical, special adhesive structures and section of fertilized egg chemicals and biological factors such as bacteria, fungus envelope were showed species, genus or family specific- ities (Kim et al. 1996, 1998; Kwon et al. 2015, 2017, Choi et al. 2019). This ultrastructural difference of fertilized * Correspondence: fish7963@yonsei.ac.kr egg envelope has been known to be related with physical Department of Environmental Medical Biology, Yonsei University Wonju and chemical properties of water environment, location College of Medicine, Wonju 26426, South Korea Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Sohn and Kim Applied Microscopy (2021) 51:3 Page 2 of 6 of habitat (Ivankov and Kurdyayeva 1973; Stehr and prefixation, the specimens were washed twice in the Hawkes 1979; Laale 1980) and species variation (Joo and same buffer solution for 20 min. and then postfixed in Kim 2013). 1% osmium tetroxide solution in same phosphate buffer M. praecox has been studied on screening of fresh- solution for 2 h at room temperature. Specimens were water fish species for their susceptibility to a betanoda- dehydrated in ethanol and the samples were replaced virus (Furusawa et al. 2007). Fertilized eggs of this with tert-butyl alcohol and dried with freeze dryer (ES- species had oil droplets and fixing filaments for adhesion 2030, Hitachi, Japan). The samples were coated with Pt and embryoic development was similar to that of other by ion sputter (E-1045, Hitachi, Japan). Subsequently, species in the genus Melanotaenia (Radael et al. 2014). the fertilized eggs were observed under the table top But there is no research on the ultrastructure of fertil- scanning electron microscope (TM-1000, Hitachi, ized egg envelope because it is hard to get fertilized eggs Japan). from this species in aquarium. Therefore, we studied the For transmission electron microscope (TEM) observa- morphology of fertilized egg with adhesive filaments, tion, prefixation, postfixation and dehydration were and the ultrastructures of micropyle, outer surface, inner conducted by following the same procedure as that for surface and section of fertilized egg envelopes under the SEM., cleared in propylene oxide, and embedded in an light and electron microscopes to find out species speci- Epon 812 mixture. Ultrathin sections of embedded fertil- ficity in dwarf rainbowfish, M. praecox belong to family ized egg envelope were taken with an ultramicrotome Melanotaeniidae. (Ultracut E, Reichert-Jung, Austria) at a thickness of about 60 nm. The ultrathin sections were mounted onto Materials and methods copper grids, double stained with uranyl acetate followed Animals by lead citrate, and observed with a transmission elec- Three pairs of dwarf rainbowfish, Melanotaenia praecox tron microscope (JEM-1400, JEOL, Japan). (total length: 4–6 cm) used in this study were purchased from TrofishNet Aquarium (Yongin, Korea). The tap Results and discussion water used for rearing was treated with carbon filter Structure of fertilized eggs (Pre-carbon filter, filter114 Co. Ltd., Korea) to remove The fertilized eggs of Melanotaenia praecox were spher- chlorine, and its temperature and pH were maintained ical with adhesive filament, transparent, demersal, and at 25 ± 0.5 °C and 6.5 ± 0.5, respectively. Biological filtra- had a small perivitelline space and small oil droplets tion was performed using a sponge filter (Tetra Twin- (Fig. 1). The size of fertilized egg was 1.02 ± 0.18 mm Brillant Super Filter™, Tetra Co. Ltd., Germany), and (n = 30). The fertilized eggs were hard enough to handle scraps and excrement settled on the bottom of the water tank were eliminated by exchanging one-third of the water every day. The artificial light was illuminated for 10 h per day to simulate a daytime environment using an electronic timer, and hatched Artemia nauplii (Great Salt Lake Artemia cysts, Saunders, U.S.A.) were provided as food three times a day at 9 a.m., 1 p.m., and 5 p.m. Collection of fertilized eggs It used the same rearing water as water of breeding tank (45X30X30 cm) and a bundle of acrylic knitting yarn was used as a spawning ground. The fertilized eggs were isolated using fingers being careful not to break the fertilized eggs from a bundle of acrylic knitting yarn. Fertilized eggs that confirmed the formation of perivitel- line space were measured for size (n = 30) under digital microscope (AD-7013MZT, Dino-Lite, Anmo, Taiwan) and used in this study as experimental samples. Electron microscopy For scanning electron microscope (SEM) observation, Fig. 1 Fertilized egg of dwarf rainbowfish (Melanotaenia praecox). White arrow; lipid droplets, E; egg envelope, Y; yolk. The perivitelline first fertilized egg envelopes were pierced a hole with 1 space (black arrow) was very small and a bundle of adhesive mL injection needle and fixed in 2.5% glutaraldehyde in filament (Af) was only at the area of animal pole 0.1 M phosphate buffer (pH 7.4) for 24 h at 4 °C. After Sohn and Kim Applied Microscopy (2021) 51:3 Page 3 of 6 it with fingers. Fertilized egg itself had no adhesive prop- 2002). But the external shape is different. Fertilized eggs erty, but the filaments continuously have been main- of A. frenatus and O. obscura are long ellipsoidal but tained the adhesive property after sample preparation that of M. praecox is spherical. Also, small oil droplets for scanning electron microscopic observation too. The were in vitelline membrane, these small oil droplets are fertilized eggs of most species belong to Belontiidae, thought to be used as a nutrient to embryonic develop- Characidae, Cyprinidae and Cichlidae have adhesive ment. In species belong to Belontiidae, the fertilized eggs property, but fertilized eggs of Hemigrammus caudovit- have a large oil droplet that provide buoyancy for their tatus (Characidae) and Danio rerio (Cyprinidae) are floating (Kim et al., 1999). non-adhesive type (Kim et al. 1996; Joo and Kim 2013). As mentioned above, morphological characteristics of The fertilized eggs have same morphology according to fertilized egg from M. praecox including external egg the genus or family (Deung et al. 1997; Kim et al. 1999; shape, a bundle of adhesive filament at the area of ani- Joo and Kim 2013; Kwon et al. 2017;Choi et al. 2019). mal pole, small perivitelline space and oil droplets, and The perivitelline space of fertilized egg of M. praecox was demersal types were showed species specificity. very small enough to vitelline membrane of egg stick to the fertilized egg envelope. The size of perivitelline space Micropyle and adhesive structures was related with their spawning habits. Most egg scatter Adhesive filaments were only near the micropyle on the species such as Cyprinidae and Characidae have a large animal pole of fertilized egg (Fig. 2a). Sometimes the perivitelline space for protection from the external phys- long adhesive filaments were coiled together (Fig. 2b). ical impacts (Kim et al. 1996; Chang et al. 2019). But that There were two kinds of adhesive filaments, one was of fishes belong to Belontiidae, Cichlidae, Callichthyidae, long whippy adhesive filaments (diameter in the middle Loricariidae and Nothobranchiidae have a habit that lay- 12.22 ± 0.52 μm, n = 20) and the other was short adhe- ing eggs on a spawning ground have a small perivitelline sive filaments (diameter in the middle 1.99 ± 0.23 μm, space (Deung et al. 1997, 2000; Kim et al. 2009; Kwon n =20). et al. 2015, 2017;Choiet al. 2019;Kim 2020). The adhe- But there were no adhesive filaments in other part siveness of fertilized egg from Ancistrus cirrhosis was excepting the animal pole area. In teleost, there are known to disappeared after spawning excepting some adhesive fertilized eggs without adhesive filaments parts that fertilized egg contact with other egg or spawn- (Kwon et al. 2017; Choi et al. 2019) and with adhesive ing place (Kim 2020). long filaments. (Kim et al. 1998, 2002; Kwon et al. 2017) Although Amphiprion frenatus (Pomacentridae) and or adhesive reticular fibers (Deung et al. 2000, Kim et al. Odontobutis obscura (Eleotrididae) belong to different 2009). Nothobranchius foerschi and Nothobranchius family, the fertilized eggs have a bundle of adhesive fila- rachovii belong to Nothobranchiidae have a lot of adhe- ments similar to that of M. praecox (Kim et al. 1998, sive whip-like structures over the whole outer surface of Fig. 2 Long adhesive filaments (LF) from the fertilized eggs of M. praecox were only at the area of the animal pole including a micropyle (a) and the short adhesive filaments (arrows) were around long adhesive filaments. Sometimes the long adhesive filaments were coiled together (b) Sohn and Kim Applied Microscopy (2021) 51:3 Page 4 of 6 fertilized egg in both species (Kwon et al. 2017). The Outer surface of the fertilized egg envelopes fertilized eggs of tomato clown anemonefish and dark In this study, the outer surface was rough side without sleeper have a bundle of adhesive filament. But the adhesive filaments excepting micropylar region (Fig. 4). researchers were unable to find a micropyle using a The outer surface of fertilized egg was smooth and the stereo microscope and a scanning electron micro- egg envelope have a bundle of adhesive filaments (Kim scope (Kim et al. 1998, 2002). In our research, a et al. 1998, 2002). This difference of outer surface could micropyle was found in a bundle of adhesive fila- be species specificity. The ultrastuctures of outer surface ments (Fig. 3). The micropyle was conical shaped of fertilized egg envelope were vary according to the spe- with whippy adhesive filaments. The external diameter cies, genus and family. of micropyle was about 12.5 μm, and inner diameter In the fishes belong to Callichthyidae, Cichlidae and was about 4.73 μm. In most egg with a bundle of ad- Belontiidae, the ultrastructure of outer surface of egg hesive filaments, the micropyle was believed to be envelope was showed family specificity because the located in a bundle of adhesive filaments. In some structure of outer surface was same according to the species, these adhesive filaments plays a role of pro- family (Deung et al. 1997; Kim et al. 1999;Choi et al. tection of water loss when the water level is lowered 2019). That of Callichthyidae have adhesive protuber- by the tide (Dumont and Brummet, 1980). Even if it’s ances (Choi et al. 2019), that of Cichlidae have cov- the same species, the morphology was different from ered with adhesive reticular structures (Deung et al. distribution (Brummett and Dumont 1981). 1997; Kim et al. 2009), and that of Belontiidae have Micropyle location in the area of animal pole is many grooves (Kim et al. 1999). But the fine structure suitable for fertilization because the animal pole con- of outer surface of fertilized egg envelope were differ- tains cytoplasm with nucleus. In general, there are no ent according to the species in Cyprinidae. That of any special structures around the micropyle, but fer- Tanichthys alborubes have rod-like structures (Kim tilized eggs of some species have special structures in et al. 1998), that of pale chub have Indian club-like the vicinity of a micropyle. In Characidae, the micro- structures (Deung et al. 2000), that of Hemibarbus pyle have a spoke-like structure consisted of protu- longirostris have taste bud-like structures (Kim et al. berance lines of egg envelope in all species (Kim 2001), and that of Danio rerio have knob-like struc- et al. 1996, 2005; Chang et al. 2019). The micropyle tures (Joo and Kim 2013). Therefore, ultrastructures of pale chub belong to Cyprinidae is surrounded by of outer surface of fertilized egg envelope was showed five peaks of hill structures (Deung et al. 2000). The genus specificity in fishes belong to Cyprinidae. The micropyles were same funnel shape in Belontiidae ultrastructures of outer surface can be different in (Kim et al. 1999) and a plate coral mouth shape in same family or same in different family (Kim et al. genus Nothobranchius (Kwon et al. 2017). Therefore, 1996, 2005). Also the structure of outer surface of the structure of micropyle seems to be family, genus egg envelope could be changeable by species variation or species specificities. (Joo and Kim 2013). Fig. 3 Scanning electron micrograph of micropyle (arrow) on the Fig. 4 The outer surface of fertilized egg envelope. The outer fertilized egg envelopes from M. praecox. The micropyle was conical surface was rough and there were no adhesive filaments on other shaped with whippy adhesive filaments side of egg envelope excepting micropylar region Sohn and Kim Applied Microscopy (2021) 51:3 Page 5 of 6 Fine structure section of fertilized egg envelope head and tail light fish and glowlight tetra consisted of 3 Under the scanning electron microscope, the section of layers, that of black tetra consisted of 4 layers, that of fertilized egg envelope from M. praecox was a multi- Buenos aires tetra consisted of 5 layers, and that of layer and the thickness was about 74.46 ± 0.41 μm(n = serape tetra consisted of 5–6 layers (Kim et al. 1996, 20) (Fig. 5a). Also, the fertilized egg envelopes consisted 2005; Chang et al. 2019). As mentioned above, the of 2 layers, an inner lamellae layer and an outer layer number of layers on fertilized egg envelope or section with high electron-density on TEM image. And the structure are showed species specificity, genus specificity inner layer was 8 layers (Fig. 5b). The inner lamellar or family specificity. But more structural research on the layer tended to be wide on the outside and narrow on other species belong to Melanotaeniidae is needed to de- the inside in particular. But this inner lamellar layer termine whether it is family specificity or not. had equal spacing in all species belong to Cichlidae The structure of egg envelope was related with (Kim et al. 1999). external environment including intensity of radiation, In general, the number of layer of fertilized egg enve- hydraulic pressure, water wave and current, and spawn- lope was different according to the family or species but ing behavior. The thickness of egg envelope was known sometimes it is same in a family such as Belontiidae, to be thick in floating type than demersal (Stehr and Cichlidae, Callichthyidae, and Nothobranchiidae (Kim Hawkes 1979), in oviparity than ovoviparity (Riehl and et al. 1999, 2009; Kwon et al. 2015, 2017; Choi et al. Greven 1993) and in the fast stream (Ivankov and 2019). The fertilized egg envelope consisted of 2 or 3 Kurdyayeva 1973). layers in most teleost and the inner layer was showed la- mellar structure alternated high and low layers due to Conclusions the difference of the electron-density. Unusually, the The fertilized eggs of dwarf rainbowfish (Melanotaenia fertilized egg envelope from Ancistrus cirrhosis (Loricar- praecox) belong to Melanotaeniidae were spherical with iidae) have counter structure from other species (Kim adhesive filament, transparent, demersal, and had a small 2020). In species belong to Characidae and Cyprinidae, perivitelline space and oil droplets. And there were two the fertilized egg envelopes have different structure ac- kinds of adhesive filament, those of long and thick, and cording to the species. In Characidae, those of head and short and thin on the fertilized eggs. A micropyle was tail light fish, serape tetra, Buenos aires tetra consisted conical shaped with adhesive filament and located at the of 3 layers, and those of black tetra and glowlight tetra area of the animal pole. The outer surface of fertilized consisted of 2 layers. But the number of inner layer is egg was rough side. Also, the fertilized egg envelope different according to the species. The inner layers of consisted of two layers, an inner lamellae layer and an Fig. 5 The electron micrographs of the fertilized egg envelopes section. The fertilized egg envelope was a multi-layer lamellar structure in SEM image (a). The egg envelope consisted of two layers, an inner lamella layer (IL) and an outer layer with high electron-density (arrow). 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