The sexes of both species of Fundulus are quite easily identified and obtained. The mature F. heteroclitus female is pale olive in color and usually has no definite bars or spots, although young females may have indistinct, dark, transverse bars on the sides; the dorsal fin is non-pigmented. The adult male of this species is a dull, dark green color, with narrow, ill-defined transverse bars composed of silvery spots; the dorsal fin is black-pigmented, in a mottled pattern.

The pale olive F. majalis female has a pattern of heavy, black longitudinal stripes on the sides, and a non-pigmented dorsal fin. The sides of the somewhat darker male bear approximately 12 broad, dark transverse bars, and there is a striking black patch on the dorsal fin.

Material is best and most abundant, as a rule, during the first three weeks of June, but small numbers of fertilizable eggs have been procured through July 15 at Woods Hole, Mass.

A. Care of Adults: Fish are usually delivered by the M. B. L. Supply Department in mixed lots, but it is advisable to segregate the sexes, to prevent spawning. Males and females should be placed in separate aquaria until needed, and after they have been stripped, they should be removed to a discard tank. An adequate supply of running sea water is, of course, essential.

B. Procuring Gametes: Both eggs and sperm are obtained by "stripping": the fish is held firmly in one hand while gentle pressure is applied to its abdomen with the thumb and forefinger of the other hand. As these fingers are drawn towards the anus of the fish, the pressure forces out the gametes. If the fish is held in front of a strong light source during the stripping process, the eggs may be seen passing through the oviduct which runs along the anal fin.

C.. Preparation of Cultures: Strip the eggs into a clean four-inch fingerbowl which has been moistened with filtered sea water. Strip the milt into a small amount of sea water, and mix the suspension with the eggs in 1/4 inch of sea water. The eggs should be inseminated as soon as possible after they are obtained from the body of the female. After 30-45 minutes, change the sea water and leave the eggs in about a 1/4 to 1/2-inch depth of sea water. Keep the fingerbowl covered with a glass plate to prevent evaporation; do not allow the eggs to clump or accumulate in one spot. The water should be changed at least twice daily.

D. Methods of Observation: To remove the sticky outer jelly layer, roll the eggs on a piece of filter paper or paper towel until the surface of the outer membrane is left smooth and clean. This same procedure should be followed daily for stock cultures, in order to prevent clumping of the eggs.

For experimental work, where it is essential to obtain development as nearly normal as possible, the eggs are usually examined uncovered in shallow depression slides; they may be manipulated with hair loops. For classroom study, when the eggs are to be observed over long periods of time and a specific orientation is desired, either of the following methods is suggested: (1) Place the eggs in sea water in special culture slides having a depression of 1.7 to 1.8 mm. (slightly less than the diameter of the eggs); it is then possible to roll the eggs to the desired position by moving the coverslip. (2) If these special slides are not available, the eggs may be placed in a drop of sea water on an ordinary glass slide and covered with a very thin, flexible sheet of mica; water is then withdrawn (using lens or filter paper) until capillary attraction causes a pressure on the egg, so that it can be rotated as in the previous method.

Recently, Trinkaus and Drake (1956) have described a method for the in vitro culture of Fundulus blastoderms isolated from the subjacent periblast and yolk mass.

E. Permanent Total Preparations: Fix the eggs in Stockard's solution (formalin, 5 parts; glacial acetic acid, 4 parts; glycerine, 6 parts; distilled water, 85 parts). This turns the protoplasm white but leaves the yolk transparent. The fixative may be used as a preservative, or the material can be transferred to 10% formalin after two days.

F. Preparation of Eggs for Sectioning: Eggs to be sectioned must be dechorionated before fixation, so that fluids can penetrate to the interior. (For details of this process, see the paper by Nicholas, 1927.) The following schedule for dehydration and embedding is useful.

1. Fix in Bouin's or Zenker's solution, 12-24 hours.

2. Dehydrate as usual through the alcohol series (up to and including 95% alcohol), leaving the eggs in each for one hour.

3. Absolute alcohol, two hours&emdash;use several changes.

4. Equal parts absolute alcohol and amyl acetate, two hours. 5. Amyl acetate, 24-48 hours.

6. Equal parts amyl acetate and paraffin, 12 hours (incubate at 30û).

7. Three changes of infiltrating paraffin (15 minutes in each); embed in 56-58û paraffin.

NORMAL DEVELOPMENT

A. The Unfertilized Ovum: Eggs stripped from a female fish into diluted sea water (70% fresh water, 30% sea water) retain the morphological characteristics of freshly-extruded eggs, including the yolk platelets, oil drops, membranes, etc. A micropyle is present, but it must be observed before removal of the chorionic jelly.

B. Fertilization and Cleavage: In order to follow all the pre-cleavage changes, it is important to (1) record the exact time of insemination, and (2) transfer the eggs immediately to a slide (see above) for observation. Polar bodies have not been described for Fundulus eggs, and it is not certain what stage the egg nucleus is in at the time of fertilization. No fertilization membrane is given off.

There is a gradual accumulation of the egg protoplasm at one pole of the egg, 25-35 minutes after fertilization, to form the blastodisc or germ-disc. A groove on the surface of this blastodisc is the first indication of cleavage; it usually occurs two to three hours after fertilization. The cleavages continue for a considerable period without much change in the over-all form from that of the original blastodisc; this is called the period of the high blastula. Details of the process of cleavage are given by Oppenheimer (1937).

C. Time Table of Development: The following schedule is based on observations made at room temperatures which approximated 22-25û C. Times are recorded from insemination.

Stage Blastodisc formation First cleavage Four-cell stage Eight-cell stage Sixteen-cell stage Early high blastula (Oppenheimer Stage 8) Late blastula (Oppenheimer Stage 9) Expanding blastula (Oppenheimer Stage 11) Early gastrula; embryonic shield (Oppenheimer Stage 12) Middle gastrula; keel (Oppenheimer Stage 13) Late gastrula, closure of blastopore (Oppenheimer Stages 14-15) Formation of brain and auditory capsules; 4-14 somites (Oppenheimer Stage 18) Heart-beat, embryonic circulation (Oppenheimer Stage 20)

Time 25-35 minutes 2-3 hours 2-1/2-3-1/2 hours 4-5 hours 4-1/2 - 5-1/2 hours 10 hours 12 hours 17 hours 1 day 2 days 2-1/2 - 3 days 3-1/2 days 4 days

D. Later Stages of Development: The periblast appears 16-24 hours after fertilization. The uncleaved protoplasm around the margin of the group of blastomeres is called the marginal periblast, while that beneath the blastodisc (visible only in sections) is the central periblast. At about this same time, the large, pinkish periblast nuclei may be visible. The nuclei of the marginal row of cells gradually become free of cell outlines, continue their divisions and migrate into the marginal periblast, converting it into a nucleated but non-cellular structure. Subsequent to the nucleation of the periblast, the blastoderm changes in form and size, and the embryo is now referred to as a blastula. Soon the margin of the blastodisc thickens (due both to a peripheral increase in cells and to a thinning of the central part of the disc), to form the germ-ring; this structure is best observed in eggs of F. majalis

During the next few hours, the germ-ring grows completely over the surface of the yolk mass, so that the uncovered portion of the egg (the blastopore) is finally covered. This process of blastopore closure occurs after the first stages of formation of the embryonic axis. Under favorable conditions, it is sometimes possible to observe the beginning of gastrulation; a slight indentation appears at the edge of the germ-ring, usually when the yolk is about one-fourth covered. Staining with neutral red (one or two drops of a 0.5% solution in a Syracuse dish of sea water) may make easier the identification of the germ-ring and periblast.

While the germ-ring is extending around the yolk, the embryonic axis is being established. The first indication of this process is a cellular thickening, the embryonic shield, resulting from a more active movement of cells in one region of the germ-ring It is usually initiated when the blastoderm has covered from onequarter to one-third the surface of the yolk. When the blastoderm has spread to cover approximately one-half the yolk, the embryonic shield has become a bluntly triangular area, extending from the margin of one portion of the germ-ring almost to the center of the blastoderm. The shield can best be identified in profile view. As the blastoderm spreads over the surface of the yolk, the embryo grows rapidly in length, and becomes segmented; this segmentation is confined to the mesoderm. It is suggested that embryos be removed from the chorion for observation of the later developmental stages. Although this de-chorionation is rather difficult at early stages, it can readily be accomplished later, with the use of sharpened forceps or beading needles. Injury to the yolk sac should be avoided.

After hatching, the young fish may be studied in detail if they are anaesthetized with chloretone. The paper by Oppenheimer (1937) contains further details of developmental stages.

AGASSIZ, A., AND C. O. WHITMAN 1885. The development of osseous fishes. I. The pelagic stages of young fishes. Mem. Mus. Comp. Zool., Harvard, 14: no. 1, part 1, pp. 1-56.

AGASSIZ, A., AND C. O. WHITMAN, 1889. The development of osseous fishes. II. The preembryonic stages of development. Part First. The history of the egg from fertilization to cleavage. Mem. Mus. Comp. Zool., Harvard, 14: no. 2, part 2, pp. 1-40.

BREDER, C. M., JR., 1948. Field Book of Marine Fishes of the Atlantic Coast from Labrador to Texas. G. P. Putnam's Sons, New York. (Rev. ea.)

CLAPP, C. M., 1891. Some points in the development of the toad-fish (Batrachus tau). J. Morph., 5: 494-501.

CLAPP, C. M., 1898. Relation of the axis of the embryo to the first cleavage plane. Biol. Lectures M. B. L., Wood's Holl, Mass., pp. 139-151.

NEWMAN, H. H., 1907. Spawning behavior and sexual dimorphism in Fundulus heteroclitus and allied fish. Biol. Bull., 12: 314-348.

NEWMAN, H. H., 1915. Development and heredity in heterogenic teleost hybrids. J. Exp. Zool., 18: 511-576.

NEWMAN, H. H., 1918. Hybrids between Fundulus and mackerel. A study of paternal heredity in heterogenic hybrids. J. Exp. Zool., 26: 391-421.

NICHOLAS, J. S., 1927. The application of experimental methods to the study of developing Fundulus embryos. Proc. Nat. acad. Sci., 13: 695-698.

NICHOLAS, J. S., AND J. M. OPPENHEIMER, 1942. Regulation and reconstitution in Fundulus. J. Exp. Zool., 90: 127-157.

OPPENHEIMER, J. M., 1937. The normal stages of Fundulus heteroclitus. Anat. Rec., 68: 1-15.

RUSSELL, A., 1939. Pigment inheritance in the Fundulus-Scomber hybrid. Biol. Bull., 77: 423-431

SOLBERG, A. N., 1938. The development of a bony fish. Prog. Fish. Cult., no. 40, pp. 1-19.

SUMNER, F. B., 1903. A study of early fish development. Experimental! and morphological. Arch. f. Entw., 17: 92-149.

TRINKAUS, J. P., AND J. W. DRAKE, 1956. Exogenous control of morphogenesis in isolated Fundulus blastoderms by nutrient chemical factors. J. Exp. Zool., 132: 311-347.

WILSON, H. V., 1889. The embryology of the sea bass (Serranus atrarius). Bull. U. S. Fish Comm., 9: 209-278.