Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 53

Establishment of large mutant lines on rotifer
using heavy-ion-beam irradiation
K. Tsuneizumi,∗1 M. Yamada,∗1 K. Ichinose,∗1 H. Ichida,∗1 and T. Abe∗1
In larviculture, rotifers (Brachionus plicatilis sensestrict) are generally used as the initial feed for hatchedlarvae,1) while larger preys (e.g., Artemia) are neededwith larval growth. The improper management of feed
size and density results in mass mortality, a high incidence of morphologically abnormal development, and asubstantial depletion of ﬁsh larvae.2) To improve the survival and growth of ﬁsh larvae, prey of appropriate size
between those of rotifer and Artemia is needed. The aimof this study was to apply heavy-ion-beam-based mutation breeding techniques3,4) for the selection of rotifer
mutants with larger body sizes.
The size distributions of the lorica length of rotifers
are 170–320 µm in individuals carrying amictic eggs.
The next food item Artemia nauplii has a body length of
400–1,000 µm, and thus, there is a large size gap (320–400 µm) between rotifer and Artemia nauplii. The rotifer strain is widely divided into two types, obligate andcyclically parthenogenesis, based on their life cycle.5) In
aquaculture facilities, the intensive mass culture of live
food rotifers is performed via parthenogenetic reproduction to induce their rapid proliferation. The Notojima
strain is known as the largest rotifer strain used in Japanand has obligate parthenogenesis. Based on these characteristics, this study used the Notojima strain as theraw material for ion-beam irradiation.
In our previous study, we measured the biological effect of heavy-ion-beam irradiation on rotifers under different conditions.6) In this study, a large-scale screening
method was developed, and mutant lines with increased
body sizes were selected using the length of the lorica asindicators. Heavy-ion-beam irradiation was performed
using carbon (C) (1.62 GeV, LET = 23 keV/µm) at six
irradiation doses of 100, 150, 200, 300, 400, and 600 Gyand argon (Ar) (3.8 GeV, LET = 312 keV/µm) at six
irradiation doses of 25, 50, 75, 100, 150, and 200 Gy. After irradiation, morphometric characteristics were compared with control groups without irradiation.
The results of categorising lorica lengths of the control and the 56 selected larger mutant strains are shownin Fig. 1. Strains were classiﬁed according to their average lorica length: Class I for 340–350 µm, Class II for
350–360 µm, and Class III for 360–370 µm. Class I corresponds to over 11% elongation, Class II to 15%, andClass III to 18% compared to the lorica length of thecontrols. The large mutant lines from 1968 rotifers irradiated with C-ion beams were categorized as follows:
30 lines to Class I, 19 lines to Class II, and 3 lines toClass III. In the large mutant lines from 1080 rotifers irradiated with Ar-ion beams, there were 3 lines in Class I∗1Fig. 1. Comparison of lorica lengths between large mutantstrains and the control.

and 1 line in Class II (Fig. 1). Steel’s multiple comparison tests showed a diﬀerence at 0.001 signiﬁcancelevel in lorica lengths of all larger mutant strains fromClass I to Class III compared with the control group(305.6 ± 3.0 µm).
The size range of lorica length of the larger mutant
strains was 320–400 µm, which is not covered by thesize ranges of the wild-type rotifer and Artemia nauplii in seedling production (Fig. 1, red bars). Various
larger mutant strains have been established by mutagenesis with heavy-ion-beam irradiation, and these strains
may help reduce the mass mortality of domestic ﬁsh if
used during the intermediate feeding stage between rotifers and Artemia nauplii.
The lorica lengths of all large mutant strains showedsigniﬁcant diﬀerences compared to the control group(Steel’s multiple comparison tests, p < 0.001, n = 20).
Boxplots indicate the lorica lengths of rotifers (n = 20).
Strains were separated into classes according to the average lorica length.
References

1) S. Mills et al., Hydrobiologia 796, 39 (2017).
2) T. Kotani et al., J World Aquac. Soc. 40, 383 (2009).
3) T. Abe et al., in Plant Mutation Breeding and Biotechnology, edited by Q. Y. Shu et al. (CABI, Oxfordshire,2012), p. 99.
4) A. Tanaka et al., J. Radiat. Res. 51, 223 (2010).
5) T. Yoshinaga et al., Hydrobiologia 412, 103 (1999).
6) K. Tsuneizumi et al., RIKEN Accel. Prog. Rep. 52, 221(2019).

RIKEN Nishina Center- 213 -