Tokio YAMAMOTO

1. Intoductory Remarks on the Medaka

(Reproduced from)

"Medaka" in Methods in Developmental Biology

edited by F. H. Wilt, and N. K. Wessells, pp. 101-111. (1967).
T. Y. Crowell Co., New York.

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"Intoductory Remarks on the Medaka" in Medaka, Biology and Strains (T. Yamamoto, ed.), Yugakusya Publ. (1975), pp. 1-16.

The medaka is indeed useful to biologists of various fields, because it is small, hardy, and prolific. It has proved to be extremely useful in the study of fertilization (Yamamoto, 1939, 1944; cf. also 1961) and embryology because, unlike the guppy, the platyfish, and the swordtail, the medaka is an oviparous fish. The medaka is particularly suitable as biological material for laboratories with limited finances and space because it can withstand cold temperatures. The medaka is also useful in the study and practices of genetics because there are various mutants of this species. In fact, among fishes, it is the first in which the Mendelian laws have been proven to be valid (Toyama, 1916) and partially Y-linked inheritance has been established (Aida, 1921). It is also the first animal in which complete reversal of sex differentiation in both directions was successfully induced by administration of sex hormones during larval life (Yamamoto, 1953, 1955, 1958).

In view of these facts, the writer presents several hints on procurement, maintenance, care, and use of the fish for convenience of researchers. Brief surveys of culture methods, secondary sexual characters, and the method of artificial fertilization are made. In addition, some precautions necessary for breeding and some advanced technics for developmental biology are touched upon.

As to cleavage and later developmental stages, Rugh's book (1948, p.376) is an adequate reference written in the English language. In Japanese, two articles on normal stages of development have been published, one by Matui (1949) and another by Gamo and Terajima (1963). Briggs and Egami (1959) presented the most complete bibliography on the medaka.

Fig. 1.  Female and male of the medaka, Oryzias latipes. (Drawn by
Yamamoto, 1969)

Although the wild type (brown or olive) fish can be found and easily collected in Japan, some color varieties such as orange-red, (golden), white, and others have been kept in Japan. The orange-red type is sometimes referred to as "golden" medaka in some quarters. Thus, Innes (1935 and later editions) stated that "In the cultivated stock a pale gold replaces the olive... " and that there is said to be a deeper orange stock which the writer has never seen." This is because of the fact that orange color (carotenoid) tends to fade when the fish is cultured indoors.

The origin of orange-red and other color varieties of cultivated stocks is wrapped in a shroud of a mist. The orange-red fishes have been painted by Ukiyoe artists of the Yedo era, so the race must have arisen by mutation from the wild type more than a few hundred years ago. The orange-red stock and a few other varieties have since been kept by goldfish breeders.

Centers of farms of cutivated stocks are Yatomi near Nagoya, Koriyama in Nara Prefecture, and Urayasu in Chiba Prefecture. It must be borne in mind that the medaka breeders have cultivated so-called "Himedaka" collectively. Hence, it is natural that so-called "Himedaka" (orange-red) stocks include a few non- orange-red varieties. The Himedaka are available at goldfish shops and night stalls in any large city in Japan. This being so, the Himedaka are not a pure orange-red variety, but are comprised of heterozygous orange-red, white, and variegated races. To establish pure strains, medaka must be bred in the laboratory. The chromatophores responsible for body color of the fish are melanophores, xanthophores, and leucophores. The "brown" chromatophores described by Goodrich (1927) seem to be leucophores which are brown by transmitted light but opaque white by reflected light.

The inheritance of body colors of this fish has been studied by Toyama (1916), Ishiwara (1917), and Aida (1921). The excellent work by the last-mentioned author revealed that the various color effects are referred to the action of genes. When the genes are homozygous, the color varieties are assigned as follows : brown (wild type) BBRR; blue BBrr; orange-red bbRR; white bbrr; variegated orange-red B'B'RR; and variegated white B'B'rr. The triple alleles B, B', b control the formation of melanin in the melanophore and are autosomal. The gene B, which is dominant to both B' and b, permits full formation of melanin. The B' is dominant to b and causes variegation. There are pigmented and non- pigmented areas in the genotypes B'B' and B'b producing the mottled appearance. In the orange-red (bbRR or bbRr) and white (bbrr), the melanin formation is so scanty that melanophores are usually invisible in an "expanded" condition.

The genes R and r govern the deposition of an orange-red carotenoid pigment in xanthophores and are partially sex-linked. Both the X and Y chromosomes have a locus for either R or r. The Y chromosome usually carries R. We will touch upon the occurrence of the Yr chromosome later. When only the orange-red and white varieties are referred to, the symbol of the former can be denoted simply as R and the latter r because both of them are carrying the common bb genes. The partially sex- linked R and r genes can be used as excellent marker genes.

In the cultivated stock available from dealers, a white (bbrr) fish is rare. In addition, rare white fish are usually females with the constitution bbXrXr (see Aida, 1921). This might be because of the fact that the gene r must have arisen by mutation of XR but not from the YR. The Yr chromosome is produced only by crossing over between the Xr and YR. Both in Aida's and our breeds, white males (bbXrYr) have appeared by allosomal crossing over.

In consequence of this fact, the white medaka is rare and white males are even rarer than white females. As an example, we may mention our scores made in 1961 (Tomita and Yamamoto, unpublished). Of 4,000 cultivated stock obtained from a dealer at Yatomi, we found only 19 white fish, all of which were females. It follows that the white medaka in cultivated stock is usually present at the level of only 0.5 percent. By use of the Hardy-Weinberg law (Hardy, 1908; Weinberg, 1908; see also Stern, 1943), it is estimated that about 13 percent of the cultivated stock is heterozygous (bbRr).

When we can obtain even a single white male, it is easy to establish a pure white strain (bbXrXr and bbXrYr). To establish homozygous stock of orange-red fish, one should mate individually an orange-red fish with a white fish in order to test whether the former has the genotype RR or Rr. If their offspring are all orange-red, it is certain that the orange-red parent is homozygous. When, however, no white fish is available, breeding of a single bR female and bR male and their offspring must be continued for several generations. If no white fish appear, it is almost certain that the stock breeds true. Our d-RR strain has been established by the latter alternative.

Genealogy of our d-rR stock in which the female is white (bbXrXr) and the male is orange-red (bbXrYR), showing father-to-son inheritance, is described in one of my papers (Yamamoto, 1958). The original progenitors of the stock were purchased in 1946 from a dealer at Yatomi near Nagoya. First, a white female (bbXrXr) was mated with a homozygous orange-red male (bbXRYR). Then, an F1 heterozygous orange-red male (bbXrYR) was backcrossed to a white female (bbXrXr) to get equal numbers of white females and orange-red males. Thereafter, interbreeding of the offspring has been repeated generation after generation by mass matings.

The d-rR strain is very useful for the study of sex differentiation because the presumable sex genotype can be distinguished by body color. It is important to make a stock census before the breeding season because there appear some exceptions (about 0.5 percent) which are either crossover white males (XrYr) or XrYR females arising from genic outbalance of sex genes. These exceptions must be removed from the population. The crossover XrXR females cannot be distinguished except by test crosses.

For experimental embryology, the gene B of the wild type can be used as an excellent marker. This gene manifests itself within 48 hours after fertilization when eggs are kept at 25 o-28 oC. On the yolk sac of embryos from the age of 2 days onward, there appear embryonic melanophores. Goodrich (1927) used this gene to score F2 segregation ratios of the hybrids of bb�~BB and bb�~Bb. One of my students (Uwa, 1965) has performed an experiment of gynogenesis using b-eggs inseminated by B- sperm weakened by treatment with toluidine blue to ascertain that karyogamy did not take place. Thus, he has succeeded in producing haploid embryos. Use of the wild-type males (BB) is promising for other experimental approaches.

Anesthesia

For observation, operation, and sexing of immature fish or adults, anesthesia is necessary. Either phenylurethane (not ethylurethane) or chloretone (chlorobutanol) can be used as an anesthetic. Recently, MS-222 (Tricaine methansulfonate, Sandoz Laboratories) has been widely used as a fish anesthetic. MS-222 of 0.1 percent may be used. For this fish, however, phenylurethane is the best. Phenylurethane of 0.015 percent may be used satisfactorily to immobilize the fish. Since the melting point of phenylurethane is about 50 oC and its solubility is low, it is wise to make 0.1 percent solution dissoloved at a temperature of 60 oC as a stock solution. Take 15 parts of this solution and dilute it with 85 parts of water. Chloretone (0.035 percent) is also useful.

To preserve the life of anesthetized fish, they should be returned to water before the extent of breathing movements reaches minimum as judged by overall feebleness and irregularity of breathing. It is harmful to anesthetize fish more than once a day.

Sexing of fish

The sex of fully grown fish can be determined by putting the fish into a beaker of water and observing it from the side. The male and female are easily distinguished by the outline of the anal and dorsal fins (Fig. 1-1). It is wise and certainly safe, however, to observe an anesthetized fish under a low-power microscope. This method is especially useful for sexing an immature fish.

For detailed points of secondary sexual characters, the reader is referred to Oka (1931) and Yamamoto (1953, 1958). The female is characterized by having less developed anal and dorsal fins and well developed urinogenital papilla. In the full-grown female, all anal fin rays except the first two-to-four, are usually bifurcated. The male has enlarged anal and dorsal fins and poorly developed urinogenital papilla. The anal fin rays of the male are single except the last one, which is usually bifurcated. The margin of these fins in the male has a saw- toothed appearance, and the dorsal fin has a deep cleft between the last ray and the one preceding it.

The most prominent feature of the male sexual character is the presence of numerous small papillar processes on the posterior region of the anal fin. At the posterior margin of the caudal and at the distal margin of the anal fin, there appear a series of leucophores, which become conspicuous in the breeding season.

Spawning

Spawning usually takes place at dawn. Prior to spawning, the male displays a characteristic sexual maneuvering. The male approaches the female and performs swift circular movements around its mate. Then the male assumes a position slightly below and behind the female. When the female is receptive both sexes juxtapose themselves and the male embraces its mate by bending the anal and dorsal fins at the position of homologous female fins. Both dorsal and anal fins of the male are broader than those of the female, and the anal fin of the male has numerous papillar processes on the posterior half of the fin. These secondary sexual characters are undoubtedly adapted to clasping its mate firmly. During the embrace, their bodies twist in a characteristic S-curve, in which the head of the male is bent somewhat toward the female with its tail projecting outward. In this juxtaposition, accompanied by vibrating movements of the posterior parts of the pair, ova and sperms are released.

After the ova are expelled en masse at the time of mating, a cluster of eggs remains attached to the belly of the female for some hours, suspended from the oviduct pore by fine threads attached to the chorion. Finally, the egg mass is detached by the action of the female in swimming and contacting roots of water plants, or if there is no vegetation by contacting the bottom of the container. The water hyacinth,Pontederia (Eichhornia) crassipes, by virtue of its brushlike root system serves as an admirable receiver of fish spawn. The foxtail, Myriophyllum verticilatum, can also be used as a suitable water plant.

Breeding technic

In outdoor mass breeding, a number of males and females are placed in a concrete pond with a base or in a larger earthenware bowl. A frame covered by a wire netting is laid over the container. This is necessary to protect fish against such predatory birds as the bull-headed shrike and Japanese wagtail which pick up the fish, and the dragonfly which deposits its eggs in the water. Nymphs of dragonflies are plagues for young fish. Aside from protecting the fish, the frame is useful to prevent them from leaping out and from running over the sides during heavy rains.

The detritus or mulm accumulating on the bottom should be siphoned off by a hose. It is recommended to have a water hyacinth float on the water surface. When eggs deposited on the roots are "eyed", the plant should be removed to another container. This must be done because newly hatched fry are preyed upon by their parents. Because of this parental cannibalism, fry will usually not survive when they are allowed to live together with adults.

In indoor breeding, every care must be taken as to container, water, temperature, and lighting because indoor conditions cannot duplicate natural ones. In later pages, necessary conditions are described. Because medaka are inhabitants of stagnant waters or slowly streaming waters rather than rapidly flowing rivers, they do not need much oxygen. Hence, if fish are not unduly crowded, aeration and filtration are unnecessary. However, mulm should be siphoned off and the coral or ramshorn snail (Planorbis corneus) is a useful scavenger. The more light the better. Propagation of unicellular algae (producing so-called green water) is an indication that lighting conditions are sufficient.

For the purpose of genetic studies, one to five females of known genotype may be mated with one male. For carrying this out, the male and the females are put simultaneously into a container. When it is necessary to place the two sexes in the container at different time, it is better to introduce females to a male that has become accustomed to the container rather than the reverse, because a larger female accustomed to the container sometimes attacks the newly arrived male.

Care must be taken to avoid possible mixing of different breeds. In breeding season, the same net used to take fish out of one container should not be employed to take fish from other containers until the net is completely dried. This is to guard against allowing eggs adhering to the net to be transferred.

Some precautions with this fish are of paramount importance for an accurate genetic study. Although fertilization is usually external, internal fertilization and development of eggs may occur in extremely rare cases, as first pointed out by Amemiya and Murayama (1931). Eggs fertilized within the female are usually shed together with ripe ova at the next spawning (usually on the next day). In order to avoid the possible mixture of zygotes resulting from promiscuous mating, it is recommended that the first batch of eggs or the first hatched fry should be excluded from the container. This procedure is necessary when previously mated females are used for genetic work. It is, therefore, advisable to use virgin females when possible. In this case, however, virgin females prior to maturity, or before breeding season, should be used. A mature female does not usually lay eggs when she is kept isolated from the male. Only on rare occasions does she lay some infertile eggs in swimming in contact with the wall of the container. When a mature female is kept isolated from the male for long, her abdomen becomes greatly distended because of an accumulation of daily ovulated and decaying ova within the ovary. A female with an abnormally plump abdomen becomes barren, and even if she is given a mate she does not spawn and her death is hastened as a result of rapture of the abdominal skin.

Artificial fertilization

The female, once it has become mature, spawns at dawn of succeeding days during the breeding season. In the day time, the mature female living together with the male contains no ripe ova. In performing artificial fertilization, therefore, females which have spawned one or more times should be isolated from males during the day before the experiment is to be performed. Females are sacrificed to obtain ripe ova because the ordinary stripping method is not only harmful to fish but frequently injures ova and causes them to manifest mechanical activation.

The brain of the isolated female is pithed with a needle to immobilize it. Upon dissecting such a female, one will find in the ovary ripe eggs which would have been laid at the dawn of the day of the experiment if the female had been living with the male. The testis of the male is isolated in the same manner.

Isolated ovary and testis are separately immersed in an isotonic balanced salt solution for the medaka, composed of 0.133 M*1 NaCl (100 parts) + 0.133 M KCl (2.0 parts) + 0.099 M*2 CaCl2 (2.1 parts) (pH adjusted to 7.3 by about 0.002 percent of NaHCO3). This solution, later known as Yamamoto's solution, is equal to the following weight mixture: NaCl, 0.75 percent; KCl, 0.02 percent; CaCl2, 0.02 percent; and NaHCO3, 0.002 percent. The isolated ovary is torn with a pair of glass needles in the solution in order to separate ripe ova from the rest. The ripe ova can be distinguished from unripe ones by their large size and translucent nature. Sperms are liberated by tearing the testis in the solution, and they are used to inseminate the ova.

The above solution was formulated by the writer on the basis of osmotic pressure of Oryzias eggs (Yamamoto, 1941) and is found to be most appropriate for keeping the unfertilized egg in fertilizable condition and as a medium for artificial insemination (Yamamoto, 1939a, 1944a, cf. also 1963). Moreover, this solution is better than fresh water for development of Oryzias eggs.

*1  M/7.5       *2  M/11

The egg of the medaka is surrounded by the chorion (egg membrane). It consists of a thinner outer and a thicker inner layer. For experimental embryology it is desirable to remove the chorion. However, within a few minutes after fertilization the chorion becomes so tough and the egg proper is so delicate that mechanical removal without injuring the egg is almost always unsuccessful.

The use of hatching enzyme to dissolve the chorion proved to be satisfactory. Ishida (1944a, 1944b) found the hatching glands in the medaka while he was staying in our laboratory. If we observe advanced embryos under a low-power microscope, the upper roof of the buccal cavity looks opaque. This is due to the fact that there are numerous hatching glands in that region. If, however, we observe embryos ready to hatch or newly hatched fry the buccal cavity is clear. This indicates that the hatching enzyme has been already discharged.

A simple method for chorion removal is to put three advanced embryos with opaque buccal cavities into a deep hollow slide containing the balanced salt solution for the medaka. Crush embryos to obtain a crude enzyme solution. Then, put one intact egg into the solution. Within 3 hours the outer layer may be perforated here and there, and the inner layer is dissolved. Then, the thinner outer layer may be torn easily by a pair of forceps or by gently blowing, using a pipette. The enzyme acts mainly on the inner layer.

Sakai (1961) reported another method to remove the chorion. Hardening of the chorion after fertilization is due to toughening of the thicker inner layer. Hence, if slits are made in the chorion, access of the enzyme is quickened with the result that the dissolution of the chorion becomes faster. Using this procedure and putting eggs in the balanced salt solution containing hatching enzyme and pancreatin, she has succeeded in removing the chrion not only from fertilized eggs but from unfertilized ones. Fertilization of denuded eggs of the medaka was accomplished.

Smithberg (1966) reported an enzymatic procedure for dechorionating this fish egg, using "pronase," a proteolytic enzyme of bacterial origin.

Microinjection of chemicals into fertilized egg

Shortly after fertilization the chorion is soft. Hardening of the membrane is completed within some minutes after fertilization. After the completion of the toughening process, the chorion becomes so tough and elastic that injection is difficult. Injection of chemicals immediately after fertilization is relatively easy. At this stage it can be done without using a micromanipulator.

Artificial insemination should be accomplished by the method described in the section on artificial fertilization, and injections should be made immediately after the breakdown of cortical alveoli. A suitable tool must be devised to hold the egg tightly. A loop made of glass is recommended. Injection can be made under a binocular microscope using a micropipette with a thick gum bulb, the tip of which is 20� in diameter. After the injection, eggs may be kept in our 0.133 M balanced salt solution containing 100 I.U. of penicillin per milliliter.

Recently, microinjection of oily substances has successfully been accomplished in our laboratory. Hishida (1964) has performed the injection of a droplet of olive oil containing estrogens stained with Sudan III. About 10 percent of injected eggs hatched out and grew into larvae of 15 mm in length. Takeuchi (1965) has injected carotenoids into the egg.

Container

For indoor culture, all-glass vessels are the best. We use squat round glass bowls with a diameter of 24 cm. A container of this size can hold about 15 adults at a temperature of 25 oC - 28 oC. The usual rectangular aquarium with steel frames can be used only after every trace of potentially poisonous substance is completely diffused out. So that used aquaria are preferable.

Feeding

The medaka is omnivorous; it takes both animal and vegetable foods. As live foods, certain aquatic worms (Tubifex, Limnodrilus) and the white worm (Enchytraeus) are adequate. The ideal live foods for both adults and young are certain water fleas (Entomostraca) and nauplii of the brine shrimp (Artemia salina). Among water fleas, Moina macrocopa has proved to be the best. It is smaller than Daphnia, so that it is readily taken by the fish. Although adult Moina cannot be eaten by larvae, it is recommended that they be put in the containers, because tiny larvae of Moina, which are parthenogenetically produced, are easily eaten by juvenile fish.

For newly hatched fry, large protozoans, especially Paramecium caudatum, are adequate as live food. In ordinary culture of fish, live foods have beneficial effects. The medaka, however, can thrive safely on dried foods provided that water contains unicellular green algae. For instance, in our experiments on the effect of sex hormones, it was necessary to force the fry to eat a standard dry diet containing hormones. The writer has formulated a standard diet which consists of the following ingredients: 60 gm shrimp powder, 30 gm parched barley flour, 6 gm dried yeast preparation, 4 gm powdered green tea (Yamamoto, 1955). The mixture should be sieved by a sieve with 100-150 mesh. Fry are fed on this diet with or without hormone (control) until they reach the 12-mm stage or older. While unicellular algae in the aquarium may be taken by fry to some extent even under this condition, it is almost certain that the main nutriments are the dried foods.

Thereafter, the fry are reared outdoors on a mixture of shrimp powder and parched barley flour with the occasional addition of some live foods. Some natural live foods, such as mosquito larvae, are taken by fish under outdoor conditions. Chironomid larvae are not taken by the fish.

Water

Well or pond water is perfectly satisfactory. Tap water cannot be used immediately after it is drawn, because it is usually treated with chlorine and other chemical agents. By drawing a quantity of tap water and exposing it to sunlight for 2 or 3 days or allowing it to stand longer, it then becomes fit for use because volatile chlorine will have evaporated within that period. Bubbling of air quickens elimination of chlorine. For a rapid remedy for harmful chlorine, addition of 0.1- 0.3 gm of sodium hyposulfite, Na2S2O3, per 10 liters is generally recommended.

Although habitats of the medaka are mainly waters of low lands and brackish water, it is also known to inhabit tide pools of coasts of certain regions in Japan and Korea. The medaka withstands a wide range of salinity, and a diluted sea water is rather better than the fresh water. In indoor culture, therefore, the use of a balanced salt solution is recommended, if the tap water proves to be unsuitable.

Rugh (1948) suggested use of the following salt solution: NaCl, 1.0 gm; KCl, 0.03 gm; CaCl2, 0.03 gm; MgCl2, 0.08 gm; and distilled water to 1 liter. This solution is osmotically equivalent to about 0.02 M NaCl solution (1/25 of sea water) and is quite satisfactory. However, the use of a "bicarbonated" distilled water may be recommended (0.02 gm of NaHCO3 per liter, pH 7.3) in making the above mentioned solution. Practically, it is convenient to make the following stock solution, which is osmotically equivalent to a 1 M NaCl solution: NaCl, 500 gm; KCl, 15 gm; CaCl2, 15 gm; MgSo4, 40 gm; and distilled water to 10 liters. This solution is diluted to 1/50 with "bicarbonated" distilled water of pH 7.3. For rearing of eggs, the balanced salt solution equivalent to 0.133 M NaCl solution (pH adjusted to 7.3) is better.

Keeping one or more extra containers filled with reservoir water is recommended. These may be placed close to fish-containing aquaria, and used for changing water. This procedure is desirable for two reasons: first, because the harmful effect of chlorine is avoided, and second, because the same temperature as that of fish-containing aquaria is obtained. For the latter reason even when using a balanced salt solution, keeping a reservoir is preferable. Any sudden change in water temperature may prove fatal to fish.

Temperature

Since the medaka is native to the Far East, it can withstand the winter in temperate zones without artificial heat. This fact gives it a higher scientific value than other tropical fishes. In temperate climates, the stock can be kept in out-of- door conditions throughout the whole year. They can survive at a wide range of temperatures, tolerating temperatures as low as 1 oC. The maximal thermal point is 37 oC - 38 oC. Temperatures of 25 oC - 28 oC have been found to be suitable for breeding.

In Tokyo and Nagoya, the breeding season extends from mid-April to mid- September. If a temperature of 25 oC -28 oC is controlled by artificial means, maturation is quickened and the seasonal reproductive habit is destroyed; spawning can then be induced in any season even winter. The fish can stand a wide range of temperature fluctuations providing the change is gradual; sudden changes of temperature are harmful to fish.

Light

Because the medaka is probably tropical or subtropical in origin and is a typical surface swimmer, it likes plenty of sunlight. In culturing of this fish, light has a beneficial effect. In rearing the medaka indoors, containers should be placed under windows in the area where pollution of air is not serious.

In culturing the fish in the interior of a room, "day-light" fluorescent tubes or "warm white" tubes must be used. The latter, which resembles sunlight more closely, is better. Continuous lighting, however, upsets the rhythm of reproduction of the fish. The tubes must be switched on and off; 12 hours of light and 12 hours of dark are recommended.

Sufficient light induces growth of unicellular algae and turns the water greenish. Light green water is favorable for fish because uni-cellular algae serve not only as oxygenators but their food.

Diseases

It is not my intention to give a detailed description of fish diseases and fish therapy, since they are common to many fresh-water fishes and may be found in any book on aquarium keeping or fish culture. Therefore, only a few hints on remedies for the most common diseases are given.

Among many diseases, the most common ones are caused by a fungus (Saprolegnia) and a parasitic ciliate (Ichthyophthirius), or "Ich." Saprolegnia infects the chorion of eggs, young fish, and adults. Two methods are recommended for combating the fungus. Eggs or fish should be put temporarily in a remedial bath. One method is to bathe them in 0.02 percent (1 : 5,000) solution of Mercurochrome (dibromohy droxymercurifluorescein, sodium) for 30-40 minutes. The other is to bathe them in 0.0005 percent (1 : 200,000) solution of Malachite green for 30-40 minutes.

The "white spots," or "Ich," is a highly contagious disease and is difficult to cure. Formerly, treatment with a 0.001 percent solution of quinine sulfate for a week was recommended. Nowadays, however, effective medications for treatment may be obtained from any tropical fish shop.

References

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sex-linked inheritance.  Genetics 6 : 554-573.  

Amemiya, I., and S. Murayama, 1931 Some remarks on the existence of
developing embryos in the body of an oviparous cyptinodont, Oryzias
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Briggs, J.C. and N. Egami, 1959 The medaka (Oryzias latipes).  
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Gamo, H. and I. Terajima, 1963 The normal stages of embryonic development
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Hardy, G.H., 1908 Mendelian proportions in a mixed population.  Science 28 :
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Innes, W.T., 1935 Exotic Aquarium Fishes.  Aquar. Publ, Co., Norristown, Pa. 

Ishida, J., 1944a Hatching enzyme in the fresh-water fish, Oryzias
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Rugh, R., 1948 Experimental Embryology.  Burgess, Minneapolis, Minn.


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Stern, C., 1943 The Hardy-Weinberg law.  Science 97 : 137-138. 

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Weinberg, W., 1908 Ueber den Nachweis des Vererbung beim Menschen.
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