NOTE Munehara: Reproductive biology and early life stages of Podothecus sachi 
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Figure 3 
Photomicrograph of the eggs internally asso- 
ciated with spermatozoa. (A) Irregular par- 
ticles (arrows) on and near the vestibule, and 
micropylar canal (MC). EE = egg envelope, O 
= ooplasm. (B) High magnification of micro- 
pylar cone in A. Spermatozoa (SP) have en- 
tered into micropylar canal. (C) Chromosomes 
and metaphase spindle of second meiotic di- 
vision. Bars in A and B indicate 50 pm and 
bar in C indicates 10 pm. 
thick egg envelope (Fig. 3 A). The external opening 
of the canal was centrally located at the bottom of 
the vestibule. The canal was slightly tapered, and 
its inner opening was situated at the center of the 
micropylar cone. The external opening of the canal 
was about 5 pm in diameter. Many irregular par- 
ticles stained with hematoxylin were deposited on 
and near the vestibule (Fig. 3A). 
In eggs fixed before exposure to seawater, a num- 
ber of spermatozoa were found to have entered the 
micropylar cone (Fig. 3B), but membrane fusion had 
not yet occurred. Furthermore, in the region of the 
ooplasm near the micropylar cone, chromosomes and 
a metaphase spindle of the second meiotic division 
were detected (Fig. 3C). 
Embryonic development 
After developing to the 2-cell stage after 20 hours of 
immersion in seawater, eggs reached the 32-cell, 
morula, and blastula stages on the 1st, 5th, and 8th 
days, respectively (Fig. 4A). The embryo became vis- 
ible on the 13th day. The early embryo was smaller 
than the egg size, its length approximately 1/5 of the 
yolk’s circumference. A pair of optic vesicles and op- 
tic lenses appeared on the 16th and the 21st day, 
respectively. Myomeres began forming on the 22nd 
day (Fig. 4B). On the 29th day, the tail of the embryo 
began to grow free from the yolk. The heart was pul- 
sating and the embryo was moving occasionally on 
the 31st day. On the 35th day, a pair of otoliths was 
observed and the eyes began blackening. The em- 
bryo elongated to encircle the yolk completely by the 
42nd day (Fig. 4C). A pair of pectoral fins began to 
extend at this time. Melanophores first appeared on 
the abdominal membrane on the 57th day. They be- 
gan forming on the side of the trunk on the 62nd 
day. On the 76th day, the intestine and the liver were 
differentiated, and blood vessels appeared along the 
yolk below the thoracic region of the embryo. Just 
before hatching, the embryo measured 1.5 times the 
yolk circumference, and some projections of supra- 
lateral and infralateral bony plates were observed 
(Fig. 4D). Hatching began on the 92nd day and ended 
by the 104th day. 
Larvae and juveniles 
Newly hatched larvae were 6.9-7. 1 mm in notochord 
length (NL) (Fig. 4E). Their bodies were slender, 
white, and pigmented on the head, trunk, and finfold. 
Yolks had not been completely absorbed yet, and an 
oil droplet remained in each yolk’s anterior part. The 
larvae were weak swimmers and usually lay on the 
bottom of the tank. Most hatched 101 days after fer- 
tilization. On the 3rd day of hatching, not only 
supralateral and infralateral bony plates, but dorsal 
and ventral bony plates began to form. The larvae 
occasionally fed on Artemia salina. The urinary blad- 
ders of larvae were always swollen with transparent 
liquid. On the 14th day, the yolk was completely ab- 
sorbed; a 7.4-mm-NL specimen had melanophores on 
the lateral sides of the abdomen and two pairs of fronto- 
