The spawning behavior of ommastrephid squids has never been observed under natural conditions. Previous laboratory observations of Japanese flying squid (Todarodes pacificus) suggest that prespawning females might rest on the continental shelf or slope before they ascend above the pycnocline to spawn, and that the egg masses might settle in the pycnocline. Here, two mesocosm experiments were conducted in a 300 m 3 tank that was 6 m deep to investigate this hypothesis. In the first experiment, a thermocline (2.5-3.5 m) was established in the tank by creating a thermally stratified (17-22°C) water column. In the second experiment, the temperature was uniform (22°C) at all depths. Prior to spawning, females did not rest on the tank floor. In the stratified water column, egg masses remained suspended in the thermocline, but in an unstratified water column, they settled on the tank bottom, collapsed and were infested by microbes, resulting in abnormal or nonviable embryos. Eleven females spawned a total of 18 egg masses (17-80 cm in diameter), indicating that females can spawn more than once when under stress. Paralarvae hatched at stage 30/31 and survived for up to 10 days, allowing us to observe the most advanced stage of paralarvae in captivity. Paralarvae survived after consumption of the inner yolk, suggesting they might have fed in the tank.

14Batch spawning, intermittent spawning and multiple spawning represent common 15 reproductive strategies among cephalopods. These flexible spawning strategies are also common 16 in fishes, and are thought to be a female trait that is programmed depending on environmental 17 parameters. The ommastrephid squid Todarodes pacificus, being a terminal spawner, is 18 considered to have a single spawning event, extruding one large egg mass and dying soon 19 thereafter. Females that are interrupted by males exhibiting mating behavior, while extruding the 20 egg mass, instead spawn multiple egg masses over the course of 2-3 days instead of dying soon 21 after spawning the first egg mass. We demonstrate that male mating behavior causes "forced" 22 intermittent spawning by females (i.e., more than one spawning event). We hypothesize that in T. 23 pacificus, some males use this strategy to mate with females unable to repel advances while 24 spawning, and thus providing the male with the opportunity to contribute sperm and enhance 25 gene flow. 26Keywords: batch spawning, spawning interruption, multiple paternity, egg mass, oceanic squid 27 INTRODUCTION 28Coleoid cephalopods are considered semelparous (i.e., no gonadal resting phase) 29 (Mangold 1987), with the exception of the recently reported iteroparous vampire squid (Hoving 30 et al. 2015). Although the spawning pattern of cephalopods is monocyclic (single spawning 31 season), semelparity occurs in species that spawn eggs in single or multiple events, with their 32 reproductive strategies being considered very flexible (Pecl 2001). The terms "batch spawning," 33 "intermittent spawning," and "multiple spawning" are common in studies of cephalopod 34 reproductive biology, particularly among species of the family Ommastrephidae, with these 35 3 terms being used interchangeably (Rocha et al. 2001). This family contains a few species that 36 spawn (lay egg masses) intermittently, with no somatic growth between spawning events 37 (Nigmatullin & Laptikhovsky 1994, Nigmatullin 2011. For instance, Todarodes pacificus, an 38 ommastrephid squid, is a semelparous cephalopod mollusk that lays eggs by embedding oocytes 39 inside a large egg mass (Sakurai et al. 2013). Being an intermittent terminal spawner, the 40 female's feeding ceases prior to spawning (Bower & Sakurai 1996) in order to allocate all 41 available energy to spawning, during which the female undergoes strenuous muscular 42 contractions, involving the mantle, arms, and tentacles, while extruding the egg mass (Hamabe 43 1962). 44The multiple spawning events of semelparous species are assumed to be a programmed 45 reproductive strategy (Rocha et al. 2001); however, the reason why all spawning events by these 46 individuals occur in the final days or weeks of their life cycle remains unknown (lifespan ≈ 1 y) 47 (Rocha et al. 2001). For T. pacificus, the reported residual fecundity and potential fecundity are 48 >150,000 and 320,000-470,000 (Soeda 1956) respectively. Thus, it is possible that the multiple ...

The Japanese flying squid, Todarodes pacificus, is thought to spawn neutrally buoyant egg masses that retain a specific location in the water column by floating at the interface between water layers of slightly different densities. It is important to understand the physical process that determines the vertical distribution of the egg masses to predict their horizontal drift in relation to embryo survival and subsequent recruitment. Here, mesocosm experiments were conducted in a 300 m3 tank by creating a thermally stratified (17–22°C) water column to obtain egg masses. A cage net methodology was developed to sustain egg masses for detailed observation. We measured the density of the egg masses of T. pacificus, and used this information to infer the vertical distribution patterns of the egg masses at the spawning grounds (Tsushima Strait, Japan). When measured separately, the density of the outer jelly of each egg mass was 2.7 σ units higher than that of the surrounding water. The outer jelly and the specific gravity of embedded individual eggs (~1.10) cause the egg masses to have very slight negative buoyancy relative to the water in which they are formed. Analysis of the vertical profile of the spawning ground showed that water density (σθ) increased sharply at ~30 m depth; thus, egg masses might settle above the pycnocline layer. In conclusion, we suggest that T. pacificus egg masses might retain their location in the water column by floating at the interface between water layers of slightly different densities, which happen to be above the pycnocline layer (actual depth varies seasonally/annually) in the Tsushima Strait between Korea and Japan.

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The spawning behavior of a Japanese flying squid (Todarodes pacificus) is described based on up-close observation of a captive female. The squid was first transferred from a 10-ton tank to a polystyrene plastic box containing 45 liters of seawater. About one hour later, the mantle-contraction rate increased rapidly, followed by a brief convulsion of the mantle and arms and a whitening of the body. The mantle contractions become shallow and rapid, and several seconds later, semitransparent jelly presumably from the nidamental glands emerged from the funnel and passed between the ventral pair of arms. Approximately 90 seconds after the egg mass first emerged, the female began ejecting oocytes through the funnel into the egg mass using rapid, powerful mantle contractions. Soon after the oocytes were ejected, translucent strands (presumably sperm) emanated from the buccal membrane. The female continued to eject oocytes for approximately two minutes, after which the mantle convulsed, and the mantle-contraction rate decreased slowly for about one minute until the contractions stopped. The squid died soon afterwards.