Journal of Biotechnology and Sericology 86, 17-20 (2017)

Behavior of the larvae of wild mulberry silkworm mandarina, domesticated silkworm B. mori and their

Natuo Kômoto

Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan (Received September 8, 2016; Accepted September 27, 2016)

The behavior and horizontal dispersal of larvae of the wild mulberry silkworm Bombyx mandarina, the domes-

ticated silkworm B. mori, and their F1 hybrid were compared in the laboratory. B. mandarina larvae moved out of an experimental arena faster than B. mori and the hybrid. They tended to remain motionless for a period, and

then quickly move out of the arena, whereas F1 hybrids spent a longer time moving around before leaving the arena, and B. mori larvae showed frequent and short movements without changes in position. These results sug- gest that has led to behavioral changes that affect horizontal dispersal. Key words: larval behavior, dispersal, domestication, , silkmoth

INTRODUCTION MATERIALS AND METHODS The domesticated silkworm, (L.) (Lepi- doptera: ), is believed to have been bred from A laboratory strain of B. mandarina originating from a the wild mulberry silkworm, B. mandarina (Moore) (Lepi- female from Gunma Prefecture and a male from Yamanashi doptera: Bombycidae), in China 5,000 years ago (reviewed Prefecture was used in this study. The B. mori race J137 × by Kawarabata, 1998). Domestication has affected various C146 was also used. F1 hybrids were obtained by mating characteristics of the silkworm, for example, cocoon size. B. mori females and B. mandarina males. Diapause eggs of The weight of the cocoon shell is 10 times greater in B. each were treated with hydrochloric acid at room mori (0.5 g) than in B. mandarina (0.04-0.07 g) (Ômura, temperature for artificial hatching using the conventional 1950). It is also easier to rear the domesticated species: method for B. mori (National Institute of Sericultural and unlike B. mandarina, domesticated larvae can be kept in Entomological Sciences, 1995). Larvae were reared on an open containers because they are inactive and do not es- artificial diet (SilkMate PS, Nosan Corporation, Yokohama, cape (Kômoto et al., 2014). Furthermore, the of B. Japan) at 25°C under a light-dark cycle of 10L14D. mori cannot fly, although males actively flap their wings when they sense sex pheromone. Observation of horizontal movement To understand the effects of domestication, behaviors Larvae on the third day of the last instar were used for have been compared between the two species, and many observation of walking activity without distinguishing be- differences have been reported. For example, wild larvae tween sexes. A circle with a radius of 18.5 cm was drawn are solitary, whereas B. mori larvae aggregate, which on a sheet of cardboard as an experimental arena. Two such makes high-density rearing possible (Okui, 1963a, b, 1964, experimental arenas were placed under a video camera 1965). Newly hatched larvae of the domesticated silkworm (GZ-E565-T, JVC KENWOOD Corporation, Yokohama, disperse less and more slowly than those of the wild spe- Japan) and observed for 1 h. A was placed at the cies (Kakizaki and Nakada, 1981; Shimoda and Kanekatsu, center of each circle under light. After 1 h, the position of 2016). Low dispersal activity of late larvae is crucial for the larva was marked on the paper and its distance from the efficient management of sericulture. Thus, the walking the center was measured. The larva was also recorded on distance of B. mori larvae in rearing beds has been stud- time-lapse video at 5 s intervals to check for re-entry into ied from various aspects, including the effects of race, de- the circle. In addition, five experimental arenas were velopmental stage, density, temperature, light, and wind placed adjacent to each other and recorded with a video (Koizumi and Matsuda, 1960; Koizumi et al., 1962, 1963, camera for 7 h under light to obtain time lapse video at 1964; Shimoda and Kanekatsu, 2016). In this study, the 5 s intervals. walking activity of last-instar larvae was compared among B. mandarina, B. mori, and their F hybrid. 1 RESULTS  *To whom correspondence should be addressed. Walking activity Fax: +81-29-838-6263. Tel: +81-29-838-6285. Walking distances were compared among last-instar lar-

Email: [email protected] vae of B. mandarina, B. mori, and their F1 hybrid for the 18 Kômoto

Fig. 2. Time to leave the experimental arena. Percentage of Walking distance in 1 h. Percentage of larvae at each Fig. 1.  larvae at each time point of 7-h observations are plotted as distance from the center of the experimental arena at the end histograms. Larvae that did not leave the experimental arena of the 1-h observation are plotted as histograms. Larvae that were recorded as having stayed “within the arena.” Sample did not move in the time-lapse video were recorded as “no numbers are written in parentheses. movement.” Those that exited the arena were recorded as having moved “out of the arena.” Sample numbers are written in parentheses. of the body from side to side (waving). The time-lapse video recordings were analyzed minute by minute (Fig. 3b). 1-h observation (Fig. 1). Larvae that left the experimental Turning and waving were grouped together because turning arenas were recorded as having moved “out of the arena” was accompanied by waving in most cases: larvae changed even if they re-entered the arenas later. More B. mandari- direction after bending the body to one side and placing na larvae left their arena than hybrid larvae (p < 0.001, χ2 their thoracic legs on the substrate. test). B. mandarina larvae were clearly separated into two B. mandarina larvae left the experimental arenas about groups: those that did not move at all or stayed within 1 h after the beginning of movement, before which they 2 cm of their original position, and those that left their remained motionless. They first started turning and- wav arena. No B. mori larvae left their arena within the 1-h ing without changing their position and then increased the observation. frequency of walking to leave the experimental arena. F1 Distances were also compared for the 7-h observation hybrid larvae showed a similar pattern, in which turning

(Fig. 2). More than 90% of B. mandarina and F1 hybrid and waving preceded walking, and they left the arena about larvae left their arena, with the former leaving earlier 3 h after the beginning of movement. The behavior of B. (p = 0.019, Mann-Whitney U test). Half of each strain left mori larvae differed from the other two. The domesticated their arena within 2.5 h and 3.5 h, respectively. Almost all B. silkworms showed frequent and short actions without con- mori larvae stayed at the center of the experimental arena. tinuous rest but remained in a limited area.

Behavioral patterns DISCUSSION Larval behavior was classified into three types of move- ment (Fig. 3a): moving in a specific direction (walking), It has been reported that newly hatched larvae of B. changing direction (turning), and moving the anterior half mandarina walk faster and farther than those of B. mori Movement patterns of Bombyx larvae 19

Fig. 3. Larval behavioral patterns. (a) Examples of three types of movement. (b) Examples of behavioral patterns in 7-h observations. Black vertical bars indicate movement in that minute. Bars above and below the horizontal lines indi- cate walking and turning/waving, respectively. White triangles show the time to exit the experimental arena.

and the F1 hybrid (Shimoda and Kanekatsu, 2016). The within small areas, less than 2 cm from the center (Fig. 1). present study found a similar pattern for last-instar larvae: F1 hybrids showed intermediate behavioral patterns, spend- B. mandarina left an experimental arena faster than B. ing a longer time turning and waving than B. mandarina mori and the F1 hybrid (Figs. 1 and 2). However, activity before actively leaving the experimental arena. It has been did not correlate simply with dispersal rate. Although B. reported that F1 larvae do not escape from rearing pads al- mori moved frequently without continuous rest, they though they actively walk (Yagi, 1922; Ishihara, 1941). stayed in narrow areas because they spent most of the time These results suggest that the difference in behavior be- turning around and waving the anterior body (Fig. 3b). In tween B. mandarina and F1 hybrid larvae is a conse- contrast, B. mandarina larvae walked actively once they quence of the genetic influence of B. mori. started moving after a motionless period (Fig. 3b). This The short and frequent motions of B. mori larvae sup- characteristics explains the clear separation of B. manda- port previous observations that they wave their heads rina larvae into two groups in 1-h observations, in which without walking if mulberry leaves are absent in rearing 40% of the larvae left their arenas, while the others stayed beds (Okui, 1964). This behavior aids in the effective con- 20 Kômoto sumption of mulberry leaves because the larvae are able Kanda, K. (1988) Location of the armyworm, Pseudaletia sep- to find leaves soon after feeding by sericulture farmers. arata Walker, on pasture grasses. Jpn. J. Appl. Entomol. However, in nature, the busy waving could be disadvanta- Zool., 32, 267-271. (in Japanese with an English abstract) Kawarabata, T. (1998) Biological studies on the ancestral in- geous as it could attract predators. Wild silkworms must sect species of the silkworm, Bombyx mori. Report of a find their own food while also hiding from enemies. The Grant-in-Aid for Scientific Research (A) No. 07406004 (in behavioral pattern of B. mandarina observed in the pres- Japanese). ent study reflects their natural habits. In mulberry trees, B. Koizumi, J. and Matsuda, Y. (1960) Horizontal movement of mandarina larvae remain motionless for long periods and silkworm larvae in rearing beds. Sansi-Kenkyû (Acta Serico- logica), 36, 3-12. (in Japanese) mimic twigs by stretching the anterior half of the body Koizumi, J., Shiomi, A. and Kobari, Y. (1962) Horizontal from the second abdominal segment during the daytime, movement of silkworm larvae in rearing beds (II) Relation- particularly in late instars (Nawa, 1936; Ishihara, 1943). ship of movement with temperature, silkworm races, and When they start feeding, they will first search for leaves density. Sansi-Kenkyû (Acta Sericologica), 40, 23-34. (in around them and then move to another place if no leaves Japanese) Koizumi, J., Hashizume, T. and Kobari, Y. (1963) Horizontal are available. Further genetic and genomic analyses of F2 movement of silkworm larvae in rearing beds (III) Move- or backcross generations will help further clarify effects ment on sloped rearing beds and so on. Sansi-Kenkyû (Acta of domestication on larval behavior. Various lepidopteran Sericologica), 47, 44-50. (in Japanese) larvae have been observed be active in the night to feed Koizumi, J., Takano, K. and Hashizume, T. (1964) Horizontal and get motionless to hide in the daytime in order to es- movement of silkworm larvae in rearing beds (IV) Wind and movement. Sansi-Kenkyû (Acta Sericologica), 50, 6-11. cape from predators. Larvae of the armyworm, Pseudale- (in Japanese) tia separata, were observed to move up to leaves to feed Kômoto, N., Tsuda, M., Okada, E., Iizuka, T., Kuwabara, N., soon after sunset and go down closer to the ground at Sezutsu, H. and Tabei, Y. (2014) Development of methods sunrise (Kanda, 1988). Unlike mulberry trees, there is no for risk assessment of transgenic silkworms rearing on bio- place to hide in the experimental arena of the present study. diversity. Sanshi-Konchu Biotec, 83, 171-179 (in Japanese with an English abstract). It is possible that the absence of refuge induced quick es- National Institute of Sericultural and Entomological Science cape of some larvae of B. mandarina (Fig. 1). Field inves- (1995) Handbook of silkworm rearing (in Japanese). tigations to compare B. mandarina and the F1 hybrid will Nawa, U. (1936) On the wild mulberry silkworm, an insect complement the laboratory studies. pest of mulberry trees. Konchu Sekai (Insect World), 40, 80- 83. (in Japanese) Okui, K. (1963a) Studies on aggregative behaviour of the silk- ACKNOWLEDGMENTS worm, Bombyx mori L. I. On behaviour patterns in a certain period. Appl. Entomol. Zool., 7, 102-108. (in Japanese with I thank Ms. Naomi Yasuda for her help in the observa- an English abstract) tion of larval behavior. I am also grateful to Ms. Mayumi Okui, K. (1963b) Studies on aggregative behaviour of the silk- Hiraide and Ms. Satomi Okadome for rearing the insects. worm, Bombyx mori L. II. On the sense organs related to the aggregative behaviour. Appl. Entomol. Zool., 7, 321-326. 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