3. Stages in the Development

Adults are easily maintained in an aquarium with little space. They are hardy fish, withstand water of lower oxygen content and tolerate a wide range of temperature and salinity.

Natural spawning in the temperate zones occurs daily from mid-April to mid-September. However, spawning can be induced all the year round by keeping them at the temperature of 25Ą-28ĄC.

Adults which have been maintained outdoors in the winter season begin to spawn within twelve days if they be kept indoors at 28ĄC.

The rate of development of the medaka is neither too rapid as some pelagic eggs nor too slow as heavily yolked salmonid eggs which have an incubation period of months. Since the demersal egg of the medaka is moderately yolked, its developmental rate is quite adequate for a study of embryology. Its incubation period is about a week when kept at 28ĄC.

A mature female usually deposits a cluster of usually 10 to 30 eggs at dawn in outdoor culture. Under the indoor controlled photoperiodicity the oviposition starts at an artificial light period (Robinson and Rugh, 1943; Egami, 1954). As a prominent characteristic of the medaka, a cluster of fertilized eggs reamains attached to the vent of the female for some hours (Fig. 3-1, Color photo Plate 1), during which time eggs can be picked off for study.

Matui (1934) reported an outline of development in the orange-red (=golden) race (bR) of the medaka with three Plates containing 54 illustrated figures. Later(1949) he published a revised paper illustrating 35 developmental stages, known as Matui's stages in the development. Usui (1962) in his book on animal development, presented re-drawn illustrations of the Matui's stages. These normal stages have been widely used in referring developmental stages of the medaka in Japan. Gamo and Terajima (1963) published photographs of normal developmental stages. They set up the stage 26-2.

An excellent reference written in English is Rugh's book (1962, Ch. 46). Kirchen and West (1969) presented photomicrographs of 26 developmental stages. These references furnish the reader and students with useful information. As to the culture of embryos New's book (1966, Ch. 6) may also be referred to.

Oppenheimer's (1937) normal stages of Fundulus heteroclitus (Cyprinodontidae) are instructive reference on early development of Oryzias (Oryziatidae), although there are differences between them in the fifth cleavage (32celled) and in embryonic circulatory system.

To facilitate the work of researchers and students who may find Oryzias egg as an excellent material for investigation, Matui's normal stages taken from Usui's book are reproduced in the 3-Plates. However, the writer is responsible for working of the text of this article. Each stage is specified by criteria of easily observable gross examination.

The chronological age of teleostean embryos, expressed in hours and days, is of little value unless the incubation temperature is controlled because developmental rate is profoundly varied with temperature. It varies also with oxygen centent of water. In a cluster of eggs, interior eggs develop slower than those of outer ones. So that it is advisable to keep eggs separated singly in a culture medium. Time sequence in the development at 25 ±0.1ĄC worked out by Gamo and Terajima (1963) is listed in Table 3-1.

The egg membrane or the envelope of teleostean ova has been given different names by different authors, e.g. Ôshell membraneŐ, ÔchorionŐ, Ôvitelline membraneŐ, Ôegg capsuleŐ and Ôzona radiataŐ. The presence of various names may partly be due to the fact that the origin of the membrane has not been fully clarified.

The primary egg membrane secreted by the superficial protoplasm of the oocyte is the vitelline membrane. The secondary egg membrane formed by the follicle cells is the chorion of the zona radiata. The egg membrane of the present fish seems to consist mainly of the chorion which is internally lined with the vitelline membrane as that of Fundulus (cf. Kellicott, 1913). For the sake of brevity, the term chorion is used in the present article.

There is a minute, funnel-shaped perforation in the chorion on the animal pole of the unfertilized ovum. This aperture is micropyle for the entrance of the sperm. The formation of the micropyle results from the fact that one of follicle cells, micropylar cell, usually acquires a very intimate relation with the ovum, through a fine pseudopodial process, so that at this point no membranes are laid down as first pointed out by Eigenmann (1890) in the ovarian eggs of the pike (Esox) and the stickleback (Pygosteus). The same mechanism of the formation of the micpropyl was observed by T.S. Yamamoto (1955) in the medaka.

The unfertilized ovum

The ripe unfertilized ovum is oblate spheroid measuring about 1.1 mm in horizontal diameter and little smaller in vertical one. The egg proper is closely surrounded by a chorion. The micropyle is situated on the animal pole of the ovum. The chorion has a number of evenly distributed short villi consisting of three segments. Long sticky filaments are tufted on the vegetal pole of the ovum (Fig. 3-2), by which eggs can adhere to any water plants or solids. For the convenience of beginners of teleost embryology, Oppenheimer's (1936) representation of successive in the transformation of the blastoderm to the embryo in Fundulus heteroclitus is presented here (Fig. 3-3).

At fertilization, the cortical alveoli embedded in the cortical protoplasmic layer begin to break down. beginning near the animal pole and ending at the vegetal pole. As the consequence of the breakdown of the cortical alveoli, the chorion is separated from the plasma membrane forming the perivitelline space. As the result of the breakdown of the cortical alveoli, hitherto translucent ovum becomes transparent (Yamamoto, 1939, 1944).

2. One-celled ovum (Blastodisc)

Following fertilization the protoplasm, peripherally concentrated, flows towards the animal pole to form a raised blastodisc. However, a very thin protoplasmic layer still remains on the other parts of the surface. Numerous oil globules hitherto evenly distributed underneath the photoplasmic layer gradually fuse each other and migrate towards the vegetal pole, where a tuft of sticky filaments is localized on the chorion.

3. Two-celled ovum

The first cleavage plane, which is meridional, normally divides the blastodisc into two cells of equal size.

4. Four-celled ovum

The second cleavage plane, also meridional, is perpendicular to the first and divides the blastodisc into four cells of equal size.

5. Eight-celled ovum

The third cleavage plane, which is double, is parallel to the first, and results in an eight-celled blastoderm made up of two rows of four cells each. The blastoderm at this stage becomes bilaterally symmetrical, elongated in the axis of the second plane of cleavage (Fig. 3-4).

6. Sixteen-celled ovum

The fourth cleavage plane, double and parallel to the second, divides the blastoderm into four rows of four cells each (Fig. 3-4). It may be remarked that the cleavage furrows have not cut entirely through the blastoderm,but left a thin layer of protoplasm adjacent to the yolk joining the blastomeres at their bases. This protoplasmic layer is the central periblast.

In the sea-bass, Serranus atrarius (Wilson, 1891) and in the pointed- nosed sole, Paraphrys vetula (Budd, 1940), the four central blastomeres becomes detached from the underlying central periblast by the time of the completion of the fourth cleavage. The resulting space between the blastomeres and the central periblast is the segmentation cavity which can be observed in prepared material. In the medaka, the segmentation cavity seems to appear later than this stage (Kamito, 1928).

7. Thirty-two-celled ovum

The fifth cleavage plane is also meridional and parallel to the first, taking place on either side of the third. (Kamito, 1928). This results in a nearly round blastoderm usually made of 32 visible cells. This state of things is quite different from most teleosts. In the sea bass, Serranus atrarius (Wilson, 1891), Fundulus heteroclitus (Oppenheimer, 1937), the pointed-nosed sole, Porophrys vetula (Budd, 1940), and the paradise fish, Macropodus cupanus (Padmanabhan, 1955), the fifth cleavage plane divides the marginal twelve cells meridionally and the central four cells horizontally resulting in the formation of two layers, the outer and the inner in the central region. The blastoderm at this stage in these fishes as seen in living material, therefore, is made of 28 visible cells, the other four forming the inner layer of the central poriton. In the brown bullhead, Ictalurus nebulosus, however, the cleavage continues to be meridional at least through the 32-cell stage (Armstrong and Child, 1962), as in the medaka.

The mode of the sixth and later cleavages is difficult to follow exactly. It is certain, however, that besides the meridional division in the peripheral blastomeres, the horizontal division also takes places in the central ones.

At the seventh cleavage, marginal cells appear first. Their nuclear divisions result in the formation of periblast nuclei (Kamito, 1928).

9. Late blastula

The blastodermal cap at this stage consists of cells smaller than that of the previous stage and the number of the marginal cells increases.

10. Early high blastula

The blastodermal cap of this stage is still high as the late morula stage but is composed of smaller ones. Nuclei from its marginal cells wander out of the cells to enter the central periblast.

At this point, presence of peculiar canals in the yolk in the egg of the present fish may be pointed out (Fig. 3-5). These canals open underneath the central periblast in blastodermal stages and underneath the embryonic body in later stages. Although these canals cannot be seen in living materials, they can be easily observed in formalin-fixed eggs as whitish canals.

The blastodermal cap has not changed its shape but is composed of still smaller cells than before.

12. Late flat blastula

The blastoderm has flattened down capping the yolk sphere. Well- defined blastocoel is visible in sections.

13. Expanding blastula

Epiboly is the process by which the blastoderm expands over the surface of the yolk sphere. Epiboly begins at this stage. The rim of the blastoderm becomes thick by piling up cells to form early germ ring while the other central area becomes thin forming the extra-embryonic membrane. As to the mechanism of epiboly the reader is referred to Trinkaus (1966).

At the stage when the blastoderm covers about one fourth of the yolk sphere, gastrulation begins at one portion of the germ ring where the thickning is greater than other parts, forming the embryonic shield. A longitudinal section of Oryzias early gastrula is shown in Figure 3-6. Diagram of a median section of blastoderm at the stage of early gastrula of Fundulus is shown in Figure 3-7.

At the stage of 1/3 epiboly which is between the stages 14 and 15, weak undulating rhythmical movements begin to appear on the blastoderm but not on the uncovered yolk sphere. The characteristic movements are due to the photoplasmic contractions of the periblast underlying the blastoderm. The surface of the blastoderm is changed passively by the underlying syncytial periblast contractions. These rhythmical movements are very conspicuous in the egg of the present fish and will be considered later in a separate chapter.

The blastoderm at this stage covers about half of the yolk sphere. The germ ring is well-defined and the embryonic shield increases in size. A refractive streak visible in the middle of the shield is the neural keel which is the anlage of the central nervous system.

Kupffer (1868) was the first to notice that in the development of the central nervous system, the teleosts show a marked difference from the rest of the Chordates in that this structure is primarily formed as a solid cord of cells. The structure is, therefore, called as neural keel. The neural tube or neurocoele appears in the solid mass in a later stage.

16. Late gastrula (2/3 epiboly)

When the germ ring reaches two thirds of the yolk sphere, the embryonic shield becomes narrow and the neural keel is more clearly visible.

17. Blastopore nearing closure

By the time when the yolk sphere is nearly covered by the blastoderm excepting a small vegetal area, the embryonic shield develops to form the embryo. The entire embryonal area at this stage raises slightly over the yolk sac and appears to consist of the neural keel which is slightly broader at the cephalic end. The underside of the neural keel at the anterior part projects into the yolk.

18. Just after closure of blastopore

Three parts of the caphalic area become distinct, of which the fore brain (prosencephalon) is the widest, the mid-brain (mesencephalon) and the hind brain (rhombenceophalon) taper posterioly. Small vesicles (two or three) appear as small vacuoles underneath the posterior end of the embryo. These vesicles later become a single Kupffer's vesicle.

In teleostean eggs, Kupffer (1868) first found this peculiar vesicular structure beneath the caudal end of the embryo after the closure of the blastopore. According to Ziegler (1887) and Wilson (1891) Kupffer's vesicle represents the vestige of the archenteron (gastrocoele) since it appears as a transitory space between the endoderm and the periblast. In teleostean eggs. the typical archenteron is obliterated by the presence of the periblast and heavily loaded yolk. According to Agassiz and Whitman (1884), Cunningham (1885) and Wilson (1891), the roof of Kupffer's vesicle is formed by the median endodermal cells and its floor is lined by the periblast.

Eigenmann (1892) stated that a mass of vesicles appear at the posterior end of the embryo of the tide-pool cottid (Clinocottus analis), larger vesicles of which represents Kupffer's vesicle. Budd (1940) observed also several vesicles at the caudul end of the embryos in the same fish and others. According to him larger vesicles of Eigenmann seem to be small oil globules.

19. Optic buds

A pair of solid optic buds appear at the cephalic end.

20. Optic vesicles

A slit appears in each of the optic lobes, which represents the cavity. It is the most characteristic feature in teleostean development that the central nervous system and its associated sense organs at first form as solid structures and their cavities are formed later by vacuolization. Simultaneously with this process mesodermal segmentation begins. At this stage the embryo has usually 3 to 4 somites.

21. Otic (auditory) vesicles

A pair of otic vesicles appear. The pericardial sac (pericardium) makes its appearance. From above, it is seen as an elliptic structure underneath the embryonic body. Kupffer's vesicle attains its maximum size. About 6 somites.

22. Lens formation

The optic vesicles are converted into the optic cups and the lens is formed. Divisions of the brain into the fore-brain, mid-brain and hind-brain become well-defined. The neurocoele is formed throughout in the nerve cord except the relatively undifferentiated tail-bud region. It is the widest in the head region. The brain develops a dorso-median slit which widens in the mid-brain to a ventricle. Tubular heart is formed underneath the mid- brain. The amplitude of the rhythmical contractile movements becomes largest at this stage.

23. Expansion of mid-brain

The mid-brain expands and forms a pair of the optic lobes. The third brain vesicle appears between the fore- and the mid-brains, and the ventricular aequeduct (Aqueductsylvii) widens at the end of the mid-brain. (cf. Fig. 3-8). Heart becomes a long tube and its anterior part reaches beneath the cephalic end. The vesicular otocyst (otic capsule) becomes well-defined.

24. Heart begins to pulsate

Heart begins to pulsate beneath the head region. The pulsations at this stage can be seen by a closer observation. Left and right ducts of Cuvier are formed as semi-circular tubules on the yolk sac between the head and the middle part of the embryo. Somites are 13 or 14 in number.

Brick-red colored leucophores (larval form) appear beneath the mid- brain region. Kupffer's vesicle disappears. Somites are 15 in number. In the wild type embryo, melanophores appear diffusely on the yolk sac.

25. Vitello-caudal vein

The vitello-caudal vein is formed, which drains the caudal vein and circulates around the median part of the yolk sac and reaches to the heart.

At this stage all the three vitelline veins, i.e. left and right ducts of Cuvier and the vitello-caudal vein are formed. A pair of the nasal sacs are formed between the fore brain and eyes. Two otoliths are discernible in the otic capsule. The third brain ventricle widens.

26. Circulation in three vitelline veins

Circulation in the three vitelline veins is established (Fig. 3-9). Melanin begins to deposit in the eye. Somites are 22-23 in number.

The embryonic circulatory system of the medaka has certain charactesistic features. First, the vitelline veins consists of left and right ducts of Cuvier and a vitello-caudal vein. They have no ramifications in contrast to vitelline veins of Fundulus (cf. Oppenheimer, 1937), the platyfish Xiphophorus maculatus (cf, Tavolga, 1949) and other fishes which have prominent ramifications. Second, at the stage when circulation is established, the duct of Cuvier drains only the anterior cardinal vein in contrast to most teleostean embryos in which the dust drains both anterior and posterior cardinal veins. Judging from the figure of embryos of Horaichthys setnai (Fam. Horaichthyidae) illustrated by Kulkarni (1940), the situation seems to be the same in the fish and the medaka (Fam. Oryziatidae). This fact seems to support the new classification of killifishes as established by Rosen (1964) in which both families are belonging to the superfamily Adrianichthyidea.

The tail tip becomes free of the yolk sac. The third brain ventricle enlarges. A pair of olfactory lobes appear in front of the eyes.

26-2. Embryo encircles 2/3 of yolk sac

Pectoral fin rudiments are formed behind the otic capsules. The trunk of embryo begin to move. Ducts of Cuvier begin to meander. In adults of the orange-red (golden) race, melanophores are almost colorless. In their embryos, however, light colored epineural melanophores as well as dorsal ones appear. In adults, leucophores are white in the reflected light. In their embryos, however, they are brick-red colored. Colored leucophores become prominent beneath the mid-brain at this stage. In embryos of the brown (wild) type hitherto diffusely distributed melanophores on the yolk sac begin to gather around the three vitelline veins.

One thirds of tail becomes free of the yolk sac. The pectoral fins become well-defined. Pigmentation of the eyes increases.

28. Embryo encircles 7/8 of yolk sac

Posterior half of embryo becomes free of yolk sac. The median fin fold is formed on the ventral side of tail and around the tail tip. The brain broadens greatly. Rhythmical contractile movements cease.

29. Embryo encircles yolk sac entirely

The pectoral fins begin to move. The optic capsules enlarge.

30. Encircling tail tip reaches eyes

The fin fold is formed on dorsal of tail also. Circulation in pectoral fins. Grayish hatching glands are seen in the buccal region. Reflective tissue is formed on the eye-ball. Embryonic melanophores extends to distribute to the tail.

31. Tail tip reaches beyond eyes

The lower jaw is discernible both from top and side. Eye movements begin. Air bladder is visible beneath the embryo. Greenish gall bladder is discernible in the left side of embryonic body. Opaque hatching glands are seen in the buccal cavity.

32. Reflective tissue on air bladder

One day before hatching. The yolk is significantly decreased in amount. Guanine-laden reflective tissue (iridocytes) on the air bladder. Spleen is visible as a red spot.

33. The last embryonic stage

A few hours before hatching. The yolk is decreased more than the preceding stage. About an hour before hatching, hitherto opaque buccal region becomes clear because of discharge of hatching enzyme from the hatching gland.

34. Hatching

In the larva just hatched, the yolk is greatly decreased in amount while the oil globule is as jet not significantly decreased in size. The peritoneum on the dorsal part of the swim bladder is pigmented. A detailed description of the newly hatched fry is given in the ensuing pages.

35. Yolk absorbed nearly completely

Five days after hatching reared at 22ĄC. The swim bladder becomes extensively pigmented. From this stage onwards, many more changes must ensue before a miniature adult is formed.

Newly hatched fry

The larva of the medaka hatches in an advanced state. This feature seems to be related to a moderate incubation period as well as a moderate size of the ovum. The average total length (TL) of newly hatched fry is about 4.6 mm. The well-developed fry swim about actively immediately after hatching. Their movements are perfectly directed and they meet their new environment much more completely.

The body of the larva tapers gently from the nape to the caudal tip. The reduced yolk sac extends from beneath the pectoral fins to the anus (rectum) which is situated slightly anterior to the mid-point of the body.

The transparent median (unpaired) fin fold is still continuous. The dorsal fin fold arises at slightly posterior to the mid-point of the body and extends along whole dorsal and ventral rim of the tail. It is broadest at the prospective dorsal fin region, just posterior to the level of the anus. The ventral fin fold extends from the anus to the caudal end. It is broadest at the prospective anal fin region. The prospective caudal fin area is broader than the peduncular area and mesenchymal rudiments of the caudal fin-rays can be seen at the ventro-posterior part.

The pectoral fins are transparent fan-like structures just posterior to the auditory capsules and usually escape notice unless the larva is top viewed. The ventral (pelvic) fins are not yet been formed and differentiate during larval stage.

As to chromatophores of just hatched fry, the reader is referred to Goodrich (1927). His Ôbrown chromatophore' is nothing else but the leucophore which appears brown in transmitted light and white in falling light. The primitive organ systems are all represented at the newly hatched fry although they are not yet miniature adults.

The chronological age of a teleortean embryo as expressed in hours and days is of little value unless the incubation temperature is controlled and described because the developmental rate is an exponential function of temperature. It varies also with oxygen content of water.

The relationship between incubation temperature and time required to reach each developmental stage in the present fish is presented in Fig. 3-10. The graphs are made by the present writer, basing on the data (Table 3-1) by Gamo and Terajima (1963) who recorded time required to reach each stage at 25 ± 0.1 ĄC and temperature characteristics ( ”) for developmental rate as worked out by Shirai (1937).

Arrhenius (1915) - Crozier (1924) equation is

Fig. 3-eq. where K is rate of development (reciprocial of developmental time), T is absolute temperature, C is a constant and ” is temperature characteristic. The equation is expressed in a convenient form

Fig. 3-eq2. where K2 > K1 and T2 > T1.

Shirai found that there are two critical temperatures at 19ĄC and 27.5ĄC in the temperature range 14-36ĄC. The ”s were found to be 29,000 between 14-19ĄC, 20,000 between 19-27.5ĄC and 10,000 between 27.5-34ĄC. These values are coincide with ”s for developmental rates in various animals as compliled by Needham (1931).

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