Ultrastructure of the chorion and the micropyle of an endemic cyprinid fish, Cyprinion tenuiradius Heckel, 1849 (Teleostei: ) from southern Iran

Item Type article

Authors Esmaeili, H. R.; Gholamifard, A.

Download date 27/09/2021 12:43:02

Link to Item http://hdl.handle.net/1834/37309 Iranian Journal of Fisheries Sciences 11(3) 657-665 2012

Ultrastructure of the chorion and the micropyle of an endemic cyprinid fish, Cyprinion tenuiradius Heckel, 1849 (Teleostei: Cyprinidae) from southern Iran

Esmaeili H. R.*; Gholamifard A.

Received: January 2011 Accepted: September 2011

Abstract The scanning electron microscope was used to investigate the ultrastructures of the egg membrane surface (unfertilized egg) of an endemic cyprinid fish, Cyprinion tenuiradius Heckel, 1849. The eggs of this species were almost circular in shape, had a smooth surface and one type II micropyle consisting of the flat pit and a long canal in the polar region. The micropyle region was not flat, micropyle was circular or oval in shape and the micropyle canal was located in its center. Round or oval accessory pores were also observed in the pit around of the micropylar canal. The surface of zona radiata was smooth with a uniform distribution of almost round pores with lips.

Keywords: Morphology, Fertilization, Cyprinion tenuiradius, Micropyle, Accessory openings

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______Department of Biology, College of Sciences, Shiraz University, Shiraz, 71454, Iran. *Corresponding author’s email: [email protected]

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Introduction An important reason of teleost openings; the adhesive structures of egg evolutionary success is their reproductive and mode of them to the substrate; system, which has been functionally ornamentation and the thickness of the acclimated in all aquatic environmental membrane have been used for taxonomic conditions (Koc, 2010). Reproduction purposes (Hirai and Yamamoto, 1986; represents one of the most important Riehl, 1993; Giulianini et al., 1994; Chen aspects of the different species biology and et al., 1999; Li et al., 2000; Bless and the maintenance of viable populations Riehl, 2002; Esmaeili and Johal, 2005; depending on its success (Suzuki and Huysentruyt and Adriaens, 2005; Chen et Agostinho, 1997). al., 2007; Costa and Leal, 2009). Different fish species live in Some characters such as shape of the special ecological conditions, therefore external surface of the egg membrane, they have unique reproductive strategy, number and shape of micropyles, the with special anatomical, behavioral, branching pattern of the tubules in the physiological, and energetic adaptations primary membrane, the width of the (Moyle and Cech, 2004). The envelope of membrane and the degree of egg teleost fish’s egg has a key role in this adhesiveness are species typical, although reproductive success. Teleosts develop they represent a convergent similarity in mature eggs enclosed in a hard and some species (Vorobyeva and Markov, complex eggshell (chorion), which is 1999; Huysentruyt and Adriaens, 2005; formed by protein components organized Costa and Leal, 2009). into a complex structure. This structure The chorion surface has also been plays an essential role in control of the analyzed using scanning electron relation between the external and the microscopy (Ohta et al., 1983; Johnson internal egg environments. It allows for and Werner, 1986; Costa and Leal, 2009; Downloaded from jifro.ir at 13:47 +0330 on Wednesday February 14th 2018 respiratory gas diffusion, provides Koç, 2010). Johnson and Werner (1986) mechanical protection and thermal described the external morphology of the insulation, and permits sperm entry chorion of five freshwater fishes and (Hamodrakas, 1992). concluded that scanning electron Ultrastructural characteristics of the microscopy was a powerful tool for chorion and the micropyle of teleost eggs identifying fish eggs. On the other hand, differ in different species, and have the micropyle and its microstructures in recently been considered as a criterion for unfertilized eggs are important characters identification of eggs (Ohta et al., 1983; in gamete recognition and fish egg Chen et al., 2007). Shape, number and size identification, therefore its morphology of micropyle and also reinforcement type may be species specific (Ginsburg, 1968; of the micropyle canal; the number and Kobayashi and Yamamoto, 1981; Chen et length of the longest and shortest ridge in al., 2007). The microstructure of the micropyle region; the diameter, micropyles and surface ornamentations, number, and arrangement of the accessory however, may change during fertilization.

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Therefore, the microstructural features of River and drains to Persian Gulf. The the micropylar canal are not reliable tools gravid females were distinguished from for the identification of fertilized eggs but males by application of slight pressure on can be used for unfertilized eggs. The the abdominal region of the alive fish, thickness of the egg membrane in different leading to the release of ripe oocytes. kinds of fishes might be also affected by Following a modified Nikolsky’s scheme ecological environments and (Nikolsky 1963) we assigned a maturity developmental stages (Ivankov and stage by recording color, and texture of the Kurdyayeva, 1973; Riehl, 1978; Chen et gonads in dissected specimens. For SEM al., 2007). study, we removed the gravid eggs from As mentioned above, SEM studies are ovaries, fixed and preserved them in 10% an important prerequisite to obtain further formalin. After fixation, we cleaned the insight into the fine structures of teleost gravid eggs with a fine, short-haired brush eggs. The aim of this study is description and fine forceps, washed them with of morphology and ultrastructures of egg distilled water and then dehydrated them envelope of poorly known endemic in a graded series of ethanol (30, 50, 70, species, Cyprinion tenuiradius Heckel, 90, and 100%). We placed egg samples in 1843 for the first time using scanning a freeze drier system (30 min) and then electron microscope. Qarah Aqhaj botak, mounted dried eggs on aluminum stubs. Cyprinion tenuiradius is the only endemic These mounts were then sputter-coated species of the genus Cyprinion (locally with gold, and were observed and named ‘botak’) in Iran, which has been photographed with a Cambridge 180 reported from the Persian Gulf in southern scanning electron microscope (SEM) at Iran (Esmaeili et al., 2010; Coad, 2011). voltage of 20 KV (Esmaeili et al., 2007). Description of morphological The measurements were performed using characteristics of this fish may be useful to Image Tool software and the data were Downloaded from jifro.ir at 13:47 +0330 on Wednesday February 14th 2018 have clear picture of taxonomic position of analyzed in SPSS 15 software. Cyprinion taxa in Iran when the comparative data are completed on C. Results macrostomum, C. kais and C. watsoni The unfertilized eggs of Qarah Aghaj which are closely related species to C. botak at stage V were spherical in shape tenuiradius. (Fig. 1a), and ranging between 0.07-1.3 mm in diameter. The ripe stage (stage V) Material and methods of gonad was recognized macroscopically To investigate the morphology and surface according to Nikolsky (1963) method. At ultrastructures of ripe eggs (stage V), we this stage, ovaries were dark yellowish and caught female specimens of C. tenuiradius occupied most of the body cavity. They from Rudbal River (28º41'36.18" N, had achieved their maximum weight and 52º37'11.52" E, at an altitude of 1168 m), size. Large, dark yellowish and spherical Firooz Abad, Fars Province, southern Iran. oocytes, full of yolk were distinguishable. Rudbal River is a branch of Qarah Aghaj

660 Esmaeili & Gholamifard, Ultrastructure of the chorion and the micropyle of cyprinid fish…

Using SEM, the micropyle region was regularly in the pit, around the micropyle not flat (Figs. 1b, c). The micropyle was canal (Fig. 1e). The outer terminus of the almost circular or oval in shape and the micropyle, measuring about 18.55 μm in micropyle canal was located in its center diameter. The micropyle was of type 2. In (Figs. 1c-e). The outer opening diameters this type, micropyle has a flat pit and a of the micropylar canal ranged from 2.56 long canal. The surface of the zona radiata μm to 18.28 μm with a mean of 10.42 ± (chorion) was smooth with the large 11.11 in circular and oval shape number of regularly distributed lipped oval micropyles, and the canal tapering toward pores at a density of 213 per 400 μm2 and the inner membrane surface (Figs. 1d, e). pore openings were 0.155- 0.235 μm in The micropyle region was curved, porous diameter (0.20± 0.02) (Fig. 1f). Some without any microvilli – like structures other morphometric measurements for (Figs. 1b, d, e). Round or oval accessory ultrastructures of ripe eggs are given in pores of various sizes were distributed Table 1.

Table 1: Microstructure characters of Cyprinion tenuiradius eggs (means± SD) Egg diameter (μm) 1153.09± 65.55 Micropyle region diameter (μm) 47.29± 4.38 Diameter of micropyle pit (μm) 18.41± 0.27 Pore openings diameter of micropyle pit (μm) 0.49± 0.14 Distance between pores inside pit of micropyle (μm) 0.55± 0.20 Diameter of superficial pore openings (μm) 0.20± 0.05

Downloaded from jifro.ir at 13:47 +0330 on Wednesday February 14th 2018 Distance between superficial pores (μm) 0.65± 0.12 Number of pores in pit of micropyle (25 μm2) 25 Number of pores around of micropyle pit (25μm2) 53 Number of pores in edge of micropyle pit (25μm2) 23

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Figure 1: Scanning electron microscopy of egg surface structures of Cyprinion tenuiradius: (a) whole view of egg; (b) micropyle region; (c) micropyle region and oval micropylar canal; (d) higher magnification of micropyle region showing oval micropylar canal and accessory pores in pit around of it; (e) higher magnification of micropyle showing round or oval accessory pores in pit around micropyl canal; (f) Regular distribution of lipped oval pores on the egg surface. Ap, accessory pore; Mc, micropyle canal; Mp, micropyle pit; Mr, micropyle region; P, pore

662 Esmaeili & Gholamifard, Ultrastructure of the chorion and the micropyle of cyprinid fish…

Discussion Number, shape and size of micropyle and coiooides (9.06 lm) (Li et al., 2000). As a ultrastructere of egg membrane have been fertilizing spermatozoon gains access to used as taxonomic tool in ichthyology the egg surface only by passing through (Esmaeili and Johal, 2005). In the present the micropyle, therefore, we concluded work it was found that the unfertilized that the diameter of the micropyle canal eggs of the C. tenuiradius possess only should be slightly larger than that of the one micropyle at the animal polar region. sperm head. The size of the micropyle is The same is reported in many other fish species-dependent but is usually only wide species. According to Ginsburg (1968) and enough to allow one sperm through at a Lahnsteiner (2003) most fish species have time. This adaption is crucial in the only one micropyle, but a few species may prevention of polyspermy (Coward et al., have up to 52 micropyles. Among the 2002). The type of micropyle is another cyprinid species, presence of only one useful character for identification of fish micropyle has also been reported in eggs eggs. Riehl and Schulte (1978) described of Aulopyge huegeli (Bless and Riehl, three types of micropyles: type I, 2002) and Hypophthalmichthys molitrix micropyles with a deep micropylar pit and (Esmaeili and Johal, 2005). The existence short micropylar canal; type II, micropyles of only one micropyle in eggs of this carp with a flat pit and a correspondingly longer may be one of the mechanisms which canal; type III, micropyles without a pit, prevent polyspermy. During fertilization a only with a canal. Our observations single sperm enters the micropyle, then the showed that the micropyle of C. inner part of the micropylar canal becomes tenuiradius should be classified as type II, narrower and a plug-like blockage quickly because micropyles were found with a flat forms on the micropyle to prevent pit and a longer canal than type I. In two polyspermy (Chen et al., 2007). other cyprinid species, A. huegeli and H. Downloaded from jifro.ir at 13:47 +0330 on Wednesday February 14th 2018 Diameter of micropyle is another aspect molitrix, the type of micropyles have been which is been considered for egg recorded as type I and III, respectively identification. Base on the obtained results, (Bless and Riehl, 2002; Esmaeili and the mean of outer opening diameters of the Johal, 2005). According to Chen et al. micropyles canal of Qarah Aghaj botak (1999), the micropyles of the four species was about 10.5 μm. These amounts have of Sparidae including: Acanthopagrus been recorded 7.5 μm and 9.5 μm for A. latus, A. schlegeli, Pagrus major and huegeli and H. molitrix, respectively Sparus sarba were classified as type III. (Bless and Riehl, 2002; Esmaeili and Teleostean eggs are known to possess a Johal, 2005). The diameter of the number of pores or knobs uniformly micropyle canal was larger than in some distributed on the surface of the egg fishes such as Epinephalus malabaricus membrane. As stated earlier, many oval (6.36 μm), Mugil cephalus (3.55 μm) and pores having lips were scattered uniformly near to the same as that of Epinephalus on the egg envelopes but, any special

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attachment structures such as microvilli- completed on other related taxa such as C. like structures were not found in the pore macrostomum, C. kais and C. watsoni. opening region or other part of chorion; Hence, study of fine structures of egg hence the egg surface is completely envelop in other members of Cyprinion is smooth. These pores may increase the gas highly recommended. exchange between egg and water. The presence of pores has been reported on the Acknowledgments egg envelopes of Epinephelus malabericus The authors would like to thank the and E. coioides while only pore-traces Engineering College for providing the have been observed on the egg envelope of SEM facilities and Shiraz University for Sciaenops ocellatus and no pores have its financial support. been found on the envelopes of Mugil cephalus (Li et al., 2000). According to References Chen et al. (1999) and Li et al. (2000), Bless, R. and Riehl, R., 2002. Biology pore diameter did not differ significantly and egg morphology of the Dalmatian for fishes in the same genus, but was barbelgudgeon Aulopyge huegeli, an significantly different for different genera, endangered endemic species in Croatia. even when the genera were in the same Environmental Biology of Fishes, 63, family. 451-456. Some cyprinid fishes, such as Rutilus Chen, K. C., Shao, K. T. and Yang, J. S., rutilus, and Leuciscus frisii meidingeri 1999. Using micropylar ultrastructure (Patzner et al., 1996), Alburnoides for species identification and bipunctatus (Glechner et al., 1993), Vimba phylogenetic inference among four vimba (Riehl et al., 1993c), have strong species of Sparidae. Journal of Fish attachment extensions on their eggs while Biology, 55, 288-300. others, such as Alburnus chalcoides mento Chen, C. H., Wu, C. C., Shao, K. T. and Downloaded from jifro.ir at 13:47 +0330 on Wednesday February 14th 2018 (Riehl et al., 1993a), Squalius cephalus Yang, J. S., 2007. Chorion (Riehl et al., 1993b), Cyprinus carpio microstructure for identifying five fish (Riehl and Patzner, 1994) and Leucaspius eggs of Apogonidae. Journal of Fish delineatus (Riehl et al., 1995), do not have Biology, 71, 913-919. any special attachment structures and Coad, B. W., 2011. Freshwater Fishes of whose eggs appear completely smooth. Iran. www.briancoad.com, downloaded Cyprinion teniradius is also included in 15 January 2011. this group. Costa, W. J. E. M. and Leal, F., 2009. It seems that C. tenuiradius egg shows Egg surface morphology in the special characteristics in terms of shape Neotropical seasonal killifish genus and size of micropyle and absence of Leptolebias (Teleostei: Aplocheiloidei: special attachment structure. These Rivulidae). Vertebrate Zoology, 59, 25- characteristics may be useful to have clear 29. picture of taxonomic position of Cyprinion Coward, K., Bromage, N. R., Hibbitt, O. taxa in Iran when the comparative data are and Parrington, J., 2002. Gamete

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