Sakai Harumi

To induce breeding of the freshwater pufferfish Colomesus asellus, artificial maturation by hormone injection (hCG, 10 IU per gram) and insemination were performed, and subsequent development observed. The rearing environment for the parental fish was freshwater, maintained at 26.0 ± 1.5 ℃ under L12：D12 lighting cycle. Two hormone treatments were applied, one month apart, to two females and four males, ovulation being confirmed 47 hours after the second treatment. Three hundred and sixty-three and 548 eggs with a mean diameter of 1.11 mm (n = 20) were collected from the two females, 334 (92.0 %) and 395 (72.1 %) being fertilized, respectively. The larvae and juveniles were fed with S-type Brachionus sp. fortified initially with freshwater chlorella, followed by Artemia larvae and Chironomid sp. larvae. The pufferfish were reared in a salinity of 7 ‰ for 24 days after hatching, the water then being gradually changed to freshwater over the following 10 days. Two individuals survived for 225 days after hatching, growing to average standard and total lengths of 29.24 mm and 38.72 mm, respectively. The Gonperz growth formulae were as follows：SL：Lt = 29.4075×exp (-exp (-0.02301(t-39.1213))) TL：Lt = 38.51177×exp (-exp (-0.023253(t-41.25841))) Further investigations of rearing conditions, such as salinity, are required to improve the breeding techniques for this species.

An unusual instance of biting behavior by the pufferfish Arothron meleagris, possibly related to spawning behavior was observed during low tide of new moon spring tide while scuba-diving off the Maldives Islands. Multiple males were following, biting and clinging persistently to a female. Subsequent internet searches revealed that similar biting behavior was common among several congeners, possibly being a characteristic spawning-related behavior of the genus.

To improve breeding techniques for aquarium freshwater pufferfishes, hormone injection (hCG, 10 IU per g)-induced maturity and insemination of Auriglobus modestus from Thailand were undertaken, and morphological development of eggs and early stage larvae observed. Artificial insemination was performed two to five days after hormone injection. Although no fertilized eggs resulted from artificial insemination utilizing a wet (freshwater) method, an isotonic (sodium lactate ringer solution) method produced 458 fertilized eggs out of 787 eggs (fertilization rate 61.7%) spawned by one female, although only 8 larvae (1.7%) eventually hatched. Eggs were oval [long axis 2.05 ± 0.06 mm, short axis 1.68 ± 0.06 mm (n = 5)], translucent, demersal, and adhesive, and contained a number of small yellow oil globules. Oval eggs are unusual among pufferfishes, although common in the genus Chonerinos, an indication of the genetic closeness of the latter and Auriglobus, but also suggesting ecological similarity. The eggs hatched after 2 days, larvae one day after hatching [4.54 ± 0.34 mm NL (n = 9)] having a large yolk sac. However, the mouth remained unopened, the optic vesicle uncolored, and the pectoral fin membrane undeveloped, except for small knob-like rudiments. Seven days after hatching, larvae [5.22 ± 0.10 mm NL (n = 3)] had lost the yolk sac and acquired rudimentary soft rays in the pectoral, dorsal and anal fins. All larvae died after eight days. DNA barcoding comparisons (COI gene, 652 bp) of several pufferfish genera indicated a close genetic relationship of Auriglobus and Chonerhinos, being nested in the same clade.