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The ability to roll the body into a ball is a common defensive strategy in several taxa and has been reported in arthropods, such as pill millipedes (terrestrial species of the Sphaerotheriida and Glomerida), marine pill bugs (Isopoda), and the larval stages of mantis shrimp (Stomatopoda; Haug & Haug 2014). Among marine molluscs, some cephalopods, such as small paralarvae (e.g., young pelagic squids; Young & Harman 1988) that have been chemically preserved while alive typically remain in the ball posture (Young 1972, Goto 2005). In the paralarvae of ommastrephid squid, the posture was once considered a distinct developmental stage, the prerhynchoteuthis stage (Hayashi & Iizuka 1953a, 1953b). Rhynchoteuthion is a unique paralarval stage of ommastrephid squids, which is characterized by the tentacles fusing together to form a trunk-like proboscis, with a few suckers on the distal tip (Roper et al. 2010). Okiyama (1965) was the first to report that this morphological response could be observed because of preservatives. Since the 1960s, the ball posture behavior has been reported in many squid paralarvae (Table 1). Images of a few squid species exhibiting a similar behavior are shown in Figure 1. Nonetheless, these reports were all incidental observations (i.e., preserved specimens or chance observations), and the behaviors of these species were never part of designed experiments, except for that of Arkhipkin and Bizikov (1996) and Vijai et al. (2015b). The balling behavior involves retraction of the head, arms, and tentacles into the mantle cavity, which results in a deformation of the paralarval body into a ball-shaped squid, from which only the paralarvae's small, transparent fins protrude (squid, like any cephalopod, have a head-arm complex that moves together).

In cephalopods, head retraction has been proposed to serve two separate functions; defense and feeding. For example, partial head withdrawal by gonatid paralarvae is considered a defensive body posture that mimics the size and color of a small, stinging hydromedusan jellyfish (Aglantha digitalis; Arkhipkin & Bizikov 1996). The feeding process of the paralarvae of oceanic squids remains unknown and has been a major bottleneck for the development of their culture technology (Villanueva et al. 2014). According to O'Dor et al. (1985) and Vidal and Haimovici (1998), balling behavior facilitates feeding in ommastrephid paralarvae by improving their access to detritus that adheres to the mucous around the mantle edge.

Because of the largely incidental nature of previous observations, there has been the need for careful, methodical examination of this behavior. The present study aimed to determine which stimuli triggered balling behavior in the paralarvae of three oceanic ommastrephid squid species: Sthenoteuthis oualaniensis, Eucleoteuthis luminosa, and Todarodes pacificus.


Paralarvae of Sthenoteuthis oualaniensis and Eucleoteuthis luminosa were obtained via standard artificial fertilization techniques (Sakurai et al. 1995, Sakai et al. 2011, Villanueva et al. 2012) conducted on the research vessel Kaiyo Maru in December 2013 (Vijai et al. 2015a). Gametes for the fertilization were obtained from mature, copulated females that were captured using jigs on handlines in the northern region of Hawaii (Fig. 2). Meanwhile, the Todarodes pacificus paralarvae were collected from spawned egg masses from the Hakodate Research Center for Fisheries and Oceans, during September and October 2015 (Puneeta et al. 2016).

Investigations on whether the head withdrawal reflex, that is, the balling behavior, (Vijai et al. 2015b) could be triggered by different stimuli, including mechanical, chemical (alcohol and formalin), and light stimuli were conducted. For mechanical stimuli, needle tip touch (1-inch steel teasing needle) and water agitation (gentle shaking of a Petri dish; radius = 4.3 cm; height = 2 cm) were applied. All the experiments were performed using randomly selected individuals (n = 20 of each species, 1- to 3-day-old), and each individual was only subjected to a single stimulus. Afterward, the surviving paralarvae were reared separately. Details of the experiments are shown in Table 2.

The balling behavior of 1- to 3-day-old paralarvae (n = 40 for each species) in Petri dishes (radius = 4.3 cm; height = 2 cm) was observed under a stereomicroscope (Nikon SMZ 1500), and live videos were recorded at 30 frames per second using a Canon PowerShot A400 IS digital camera (Vijai et al. 2015b). Paralarvae sinking videos were recorded through the transparent sides of the Petri dishes. All the videos were annotated, reviewed, and analyzed, and to observe ball formation and record sinking velocity, selected sequences from the videos were captured using the VLC media player and exported as frames in ImageJ. Based on the frame rate, passive sinking velocity was measured as the number of mantle lengths traveled per second (Vijai et al. 2015b).


All stimuli triggered an immediate and rapid retraction of the head into the mantle, thereby, forming the ball posture (Fig. 3A-C). Differences between the shape and relative position of body parts in undisturbed and balled squid are presented in Figure 3D, E. During the ball posture, the width of the squid expanded to more than 50%. Ball formation was complemented by chromatophore expansion and the chromatophores were retracted when the paralarvae reclaimed the normal shape.

The duration of the ball posture was strongly dependent on the duration of the applied stimulus, and the time required to recover from the ball posture triggered by the mechanical and light stimuli ranged from almost instantaneous to several minutes, whereas the chemical stimulus led to death or permanent deformity. While in the ball posture, the squid's funnel was concealed to prevent jet propulsion, and the paralarvae sank passively, with a maximum measured velocity of 0.5 cm [sec.sup.-1].


Because the ball posture increased the width of the squid by greater than 50%, predators would need a sufficiently large mouth (relatively) to engulf the whole squid. In addition, the appearance of the balled squid was completely different from that of the undisturbed squid. Ball formation was always synced with chromatophore expansion, which might result in Batesian mimicry, which involves the imitation of aposematic coloration (Wiister et al. 2004), as observed in Ommastrephes bartramii (Vijai et al. 2015b).

Because ommastrephid paralarvae lack morphological defenses, the orange color and ball posture they adopt when threatened by predators may mimic the appearance of unpalatable organisms that share the same habitat (Vijai et al. 2015b), as has been observed in other species. For example, the balloon response of deep-sea cirrate octopods, in which the organism expands its web in response to tactile stimuli, is also assumed to be a defense mechanism (Boletzky et al. 1992, Villanueva 2000).

Despite the slow speed, the passive sinking might enable the paralarvae to move away from dangerous situations (Vijai et al. 2015b) while remaining disguised. The ball formation helps oceanic squids to increase their chances of survival during the vulnerable paralarval phase (Arkhipkin & Bizikov 1996). Additional research is needed to understand the contractive capacities of head retractor muscles that enable the paralarvae to adopt the defensive body posture. The similarity of dorsal light reflex reported in Loliginid squid (Preuss & Budelmann 1995) also worth further investigation. Preuss and Budelmann (1995) stated that the directional effects of light and gravity as well as input from the neck receptor organs are integrated centrally to maintain normal orientation.


We would like to thank Richard E. Young for his very useful comments and suggestions, which helped us improve the quality of our paper, and also J. R. Bower and Erica A. G. Vidal for stimulating discussions. We are grateful to the captain and crew of the RV Kaiyo Maru as well as all cruise participants. We sincerely appreciate the generosity shown by R. E. Young, D. Shale, and H.-K. Yoo for sharing their images.


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(1) Department of Marine Bioresources and Ecology, School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041-8611, Japan; (2) Hakodate Cephalopod Research Center, Fisheries and Oceans Hakodate, Benten-cho 20-5, Hakodate, Hokkaido 040-0051, Japan

(*) Corresponding author. E-mail:

DOI: 10.2983/035.037.0513
TABLE 1. Oceanic squid species whose paralarvae have been reported to
perform incidental head withdrawal behavior (ball posture).

Species              Family            Source

Cranchia scabra      Cranchiidae       Young (1972), Young
                                       and Mangold (2016)
Taonius megalops     Cranchiidae       Dilly (1972)
Teuthowenia          Cranchiidae       lmber(1978)
Gonatus onyx         Gonatidae         Kubodera and Okutani (1981)
Gonatus onyx and     Gonatidae         Arkhipkin and Bizikov (1996)
seven other species
Dosidicus gigas      Ommastrephidae    Yatsu et al. (1999)
Itlex argentinus     Ommastrephidae    Sakai and Brunetti (1997),
                                       Vidal and Haimovici
Illex illecebrosus   Ommastrephidae    O'Dor et al. (1985)
Ommastrephcs         Ommastrephidae    Vijai et al. (2015b)
Omithoteuthis sp.    Ommastrephidae    Nesis (1987)
Todarodes pacificus  Ommastrephidae    Hamabe (1962), Okiyama
                                       (1965), Shigeno et al.
                                       (2001), Puneeta et al.
Onychoteuthis        Onychoteuthidae   Nesis (1987)
Thysanoteuthis       Thysanoteuthidae  Miyahara et al. (2006)

TABLE 2. Effects of various stimuli applied to trigger the head
withdrawal reflex in paralarvae of three oceanic squid species
(Sthenoteuthis oualaniensis, Eucleoteuthis luminosa, and Todarodes

            Stimulus          Duration/concentration

Mechanical  Needle tip touch  Touch and withdraw
            Needle tip touch  2-3 sec
            Needle tip touch  Greater than 5 sec
            Water agitation   3 sec
            Water agitation   Greater than 5 sec
Chemical    Ethanol (95%)     2-3 drops in Petri dish (~150 mL)
            Ethanol (95%)     Greater than 5 drops
            Formalin          2-3 drops in Petri dish (~150 mL)
            Formalin          Greater than 5 drops
Light       Flash             1-2 sec
            Flash             Greater than 5 sec

            Ball posture duration/fate
            of paralarvae

Mechanical  ~2 sec
            4-6 sec
            ~10 sec (stimulation ignored after)
            ~5 sec
            ~5 sec (stimulation ignored after)
Chemical    5-10 sec, affects health
            All died
            ~5 sec
            Permanent deformity/death
Light       ~5 sec
            ~5 sec (stimulation ignored after)
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Author:Vijai, Dharmamony; Puneeta, Pandey; Sakurai, Yasunori
Publication:Journal of Shellfish Research
Article Type:Report
Date:Dec 1, 2018

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