Mature animals are obtained from the fish traps at Barnstable and Buzzards Bay, and at Menemsha Bight, Martha's Vineyard, Mass. The sexes are separate. Among mature animals the males are usually longer and more slender, with the milk-white testis visible through the mantle near the posterior end; this region in the female is filled with a uniformly transparent mass of eggs. The accessory nidamental glands of the female are red during the breeding season and can usually be seen through the mantle.

Egg-strings can be collected at low tide along the sandy beaches of Nonamessett Island, where they are found floating free or attached to submerged objects in the shallow water.

Females with mature eggs are available at Woods Hole, Mass., from May through July, although the majority of the animals are spent by mid-July.

PROCURING AND HANDLING MATERIAL

A. Care of Adults: In order to keep adults even a day or two, they must be transported to the laboratory without undue disturbance in an adequate amount of sea water. Then they should be transferred to large aquaria with a constant supply of running sea water. On several occasions, the sexual activities of the adults and deposition of the egg-masses have been observed in the laboratory' (Drew, 1911, 1919).

B. Procuring Gametes: Open both male and female by making a longitudinal section through the mantle from the siphon to the tip, cutting along the posterior (funnel) wall; remove the ink sac. In the female, tear the thin wall of the oviduct with forceps and shake the eggs into a large fingerbowl of sea water. If the eggs are fully mature, they separate readily from the ovary and appear as beautifully transparent as glass. Immature eggs are not transparent and will not develop. In the male, collect the bundles of spermatophores from the opening, of the sperm duct, and transfer them to a watch glass of sea water. The spermatophores "explode" on contact with sea water, and a concentrated sperm suspension is thus obtained. If males are not available, sperm may be obtained from the sperm receptacle of the female, which is located in the collar between the head and the free edge of the mantle.

C. Preparation of Cultures: Add several drops of sperm suspension to the fingerbowl of eggs. After 20 to 30 minutes, transfer to a large dish filled with clean sea water. Keep the culture on a sea water table, and leave undisturbed for 2-1/2 to 3 hours; thereafter change the sea water at least twice a day.

Embryos of all stages are readily obtained from naturally-laid egg-strings, which can be kept in aquaria of running sea water. The egg-strings containing the older stages are usually darker and more weathered in appearance than those containing the young ones. To release the embryos proceed as follows: Place an egg-string in a Syracuse dish. Use two beading needles in the manner of knives cutting against one another, and cut the string in half. Place the left needle so that the pressure forces several embryos clear of the jelly at the open end of one of the halves. Keeping this needle in hand, puncture the chorion of one of the eggs with the tip of the right needle, and tear the chorion with a sharp jerk. The pressure of the enclosed fluid will pop the embryo from the membrane. When the exposed row of embryos has been removed, cut off the empty jelly and repeat the procedure. If the eggs are not first forced clear of the jelly, the embryos are difficult to remove without injury; the older stages are procured more easily than the very young ones.

D. Methods of Observation:

To study intact spermatophores: Transfer intact spermatophores quickly into concentrated (40%) formaldehyde and fix for ten minutes. (They will "explode" in a weaker solution.) Rinse with distilled water for several minutes, transferring the spermatophores gradually from the formaldehyde. Stain with Ehrlich's triacid for 5 to 10 minutes (6 drops of the stock solution to 8 cc. of distilled water; this amount will half-fill a Syracuse dish). Rinse off the stain with distilled water and place the spermatophores on a slide under a coverslip.

To observe early development: For short periods, the developing eggs can be examined in depression slides. To obtain a polar view of the cytoplasmic cap (which alone undergoes cleavage), it is necessary to mount the eggs in an upright position. Place a small amount of vaseline (enough to cover the surface with a film) in a depression slide, fill the depression with sea water, add a few eggs and manipulate them with hair loops or fine needles so that they stand up.

Preparation of Slides: Because of the large amounts of yolk which they contain, squid embryos have a tendency to be friable and difficult to section, especially in the younger stages. The amyl acetate technique may be used (see p. 225 of this manual) or the dioxan technique as outlined below:

1. Fix embryos in Bouin's solution, first anaesthetizing in sea water containing chloretone, if the embryos are highly motile.

2. Transfer the embryos from the fixative into pure dioxan. Make two changes to fresh dioxan, at hourly intervals (total of three hours in dioxan).

3. Transfer to pure paraffin for one hour; change to a fresh paraffin bath for one hour, and then to a mixture of paraffin containing 8-10% bayberry wax for one hour.

4. Embed in paraffin-bayberry wax mixture.

5. Section at 5 or 6 microns and stain with Heidenhain's haematoxylin or with Prenant's triple stain.

A. The Unfertilized Ovum: The mature egg, from the oviduct of the female, is surrounded by a thick, closely applied, transparent chorion. At the pointed end of the egg, there is a depression in the chorion and a minute canal, the micropyle, extending through it. The opposite end of the egg is blunt, and the region between shows a bilaterality. The more convex side is the future "anterior" or mouth side of the embryo. The egg is large and somewhat elongated in shape; it measures 1500 to 1600 microns in length and 1000 to 1200 microns in diameter (Williams, 1910). A thin cytoplasmic cap covers the yolk at the pointed pole; this cap will give rise to the embryonic structures.

The inseminated eggs are embedded in a gelatinous matrix, which is produced by glands of the oviduct, and covered by a jelly-membrane produced by the nidamental glands. The eggs in their jelly-coats are wound spirally around a central core.

B. The Spermatophore: This unique structure consists essentially of an outer envelope and a central core, with a fluid-filled space between. The envelope has a double wall, the outer and middle tunics, and at its small tip-end, an opening sealed by a cap bearing a long thin cap thread. The central core is attached at this "oral" end. It is made up of the elongated, opaque sperm mass and an ejaculatory apparatus, with flask-shaped cement body and spiral filament. The ejaculatory apparatus is enclosed within three membranes, the most conspicuous being the middle membrane. It is relatively thick and extends from the cement body to the cap end where it is permanently fused to the outer tunic. The outer membrane also begins at the cement body, but it is closely applied to the inner tunic, a structure which also encloses the sperm mass. The "oral" end of the inner tunic and outer membrane can be easily identified as a thickened ring around the middle membrane at a short distance from the cap.

When the spermatophore "explodes," the entire contents of the capsule evaginate at the "oral" end. The evaginated inner tunic and outer membrane form the sperm reservoir after the "explosion." During evagination the cement gland reaches the surface and bursts, releasing cement which forms a seal at one end of the reservoir. The middle membrane opens and is lost, along with the outer envelope of the spermatophore. The sperm, mixed with a gelatinous mass, ooze out slowly from the opposite, open end of the sperm reservoir; this process may extend over a period of hours or even days. Further details and illustrations of the spermatophore are to be found in papers by Drew (1911, 1919).

C. Fertilization and Cleavage: The entrance of the sperm through the micropyle of an inseminated egg is soon followed by a withdrawal of the cytoplasmic cap from the chorion, leaving a clear perivitelline space. Within an hour, two polar bodies are formed (Hoadley, 1930).

Cleavage is meroblastic, and, in contrast to other molluscan eggs, not spiral. The first cleavage plane coincides with the median plane of the future embryo (Watase, 1891). At the end of the first day, there is a gradual extension of the blastoderm about the yolk. The "blastocones," which are supposed to give rise to the yolk epithelium, are not very distinct in Loligo. The thickening of the margin of the blastoderm denotes the formation of the entomesoderm, and is thus the beginning of gastrulation.

D. Time Table of Development: There is considerable variation due to temperature differences. The following table gives only a rough approximation of the times after insemination at which given stages are reached.

Stage First polar body Second polar body

Time 20 minutes 1 hour

Stage First cleavage Blastoderm over top of egg "Gastrula :" thickened peripheral ring Blastoderm halfway over egg Blastoderm nearly covers egg Shell glands and eye-stalks appear Siphonal folds and arms appear, eyes project Siphonal folds fuse into a tube, eye-stalks prominent Hatching

Time 3 hours 12 hours 24 hours 2 days 3 days 3-1/2 days 5-1/2 days 6-1/2 days 11-12 days

E. Later Stages of Development: The blastoderm grows down to- completely enclose the yolk at the ventral pole. Meanwhile at the opposite, or dorsal, pole the developing shell gland can be seen, with the mantle primordium underneath. The primitive mouth, seen as a slight ectodermal invagination, and a pair of large, rounded projections, the eye primordia, lie toward the anterior side. On the posterior side are the gill primordia, otocysts and the anterior and posterior siphonal folds, and near the equator the primitive arms can be seen. Rhythmical contractions of the yolk epithelium serve to circulate liquefied yolk material in the yolk sac vessels, which are continuous with the embryonic vessels (Portmann, 1926). As the embryo develops, the yolk sac gradually constricts. It continues into the embryo which is thus formed around a yolk core. The mantle and fins are easily recognized at this stage, but the shell gland has invaginated and is not visible. On the posterior side, the siphon is forming by concrescence of the anterior siphonal folds. The posterior siphonal folds, which will form the retractor muscles, continue as ridges to the anterior side. The anus is located between the gill primordia.

The pre-hatching embryo has very prominent eye-stalks, which contain primordia of the optic and cerebral ganglia, the so-called "white bodies," and a separate mass of yolk. The inner sector of the lens, which is formed by the outer part of the optic vesicle, is clearly visible as a club-shaped rod extending into the eye vesicle. The contractile mantle has overgrown the anus and gills, and later develops chromatophores which are equipped with muscles and innervated. The otocysts lie close together. Feather-like gills can be observed through the mantle. At their bases lie the branchial hearts and, between them, the systemic heart; all three pulsate.

When the yolk sac is nearly absorbed, the embryos adhere to the chorion by Hoyle's organ. This is a T-shaped structure, which is located on the posterior dorsal side of the mantle. The secretion from Hoyle's organ dissolves the chorion and the jelly, leaving a hole through which the embryo emerges, aided by pulsations of its mantle. Adherence to the chorion can be stimulated in late embryos by stroking the egg-string.

Illustrations of these stages may be found in a paper by Brooks (1880).

BROORS, W. K., 1880. The development of the squid Loligo pealii (Lesueur). Anniv. Mem. Boston Soc. Nat. Hist., 1880, pp. 1-22.

DREW, G. A., 1911. Sexual activities of the squid, Loligo pealii (Les.). I. Copulation, egglaying and fertilization. J. Morph., 22: 327-359.

DREW, G. A., 1919. Sexual activities of the squid Loligo pealii (Les.). II. The spermatophore; its structure, ejaculation and formation. J. Morph., 32: 379-435.

HOADLEY, L., 1930. Polocyte formation and the cleavage of the polar body in Loligo and Chaetopterus. Biol. Bull., 58: 256-264.

KORSCHELT, E., 1892. Beitrage zur Entwicklungsgeschichte der Cephalopoden. I. Die Entstehung des Darmkanals und Nervensystems in Beziehung zur Keimblatterfrage. Festschrift R. Leuckart, S. 347-373.

KORSCHELT, E., 1936. Cephalopoden. In: Vergleichende Entwicklungsgeschichte der Tiere, Gustav Fischer, Jenz, Bd. 2, S. 968-1009.

MACBRIDE, E. W., 1914. Text-Book of Embryology. Vol. I. Invertebrata. Macmillan and Co., Ltd., London.

NAEF, A., 1928. Die Cephalopoden, Bd. 2, Embryologie. Fauna und Flora des Golfes von Neapel, 35: 1-347.

PORTMANN, A., 1926. Der embryonale Blutkreislauf und die Dotterresorption bei Loligo vulgaris. Zeitschr. f. Morph. u. Okol. d. Tiere, 5: 406-423.

PORTMANN, A., AND A. M. BIDDER, 1928. Yolk-absorption in Loligo and the function of the embryonic liver and pancreas. Quart. J. Micr. Sci., 72: 301-324.

RANZI, S., 1931a. Duplicitas cruciata in embrioni di Cefalopodi. Pubbl. Staz. Zool., Napoli, 11: 86-103.

RANZI, S., 1931b. Sviluppo di parti isolate di embrioni di Cefalopodi. (Analisi sperimentale dell'embriogenesi.) Pubbl. Staz. Zool., Napoli, 11: 104-146.

RANZI, S., 1937. Ricerche sulfa fisiologia dell'embrione dei Ce&lopodi. Pont. Acad. Scient., Acta, 1: 43-49.

SPEK, J., 1934. Die bipolare Differenzierung des Cephalopoden- und des Prosobranchiereies. Arch. f. Entw., 131: 362-372.

WATASE, S., 1891. Studies on Cephalopods. I. Cleavage of the ovum. J. Morph., 4: 247-302.

WILLIAMS, L. W., 1910. The anatomy of the common squid, Loligo pealii, Lesueur. E. J. Brill, Leiden, Holland, pp. 1-92.