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９愛媛大学農学部紀要（Mem. Fac. Agr., Ehime Univ.）
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The Relation of Fragmentation Frequency to Fragment Number
in Enchytraeus japonensis NAKAMURA, 1993 (Oligochaeta, Enchytraeidae)
cultured Several Years under Laboratory Conditions
Yoshio NAKAMURA＊
Abstract
Over a period of six and a half years, Enchytraeus japonensis fragmented repeatedly with a wide range in thenumber of survival days under laboratory conditions. In the case of head fragments, in which only the head fragmentwith an original head was transferred to new media after each fragmentation, the maximum fragmentation frequency
until death was 122 times while cultured with ground oats at 20℃. The fragmentation frequency, fragment number per
fragmentation, and the interval between successive bouts of fragmentation averaged 35.3 times, 6.3 fragments and 20.4
days, respectively. In the case of tail fragments, in which only the tail fragment with an original tail was transferred to
new media after each fragmentation, the highest fragmentation frequency was 85. The fragmentation frequency,
fragment number, and the interval averaged 11.4, 6.1 and 24.0, respectively. In both cases, there was no tendency forthe fragment number or interval days to increase or decrease with the increase of fragmentation frequency.
Key words: fragmentation frequency, fragment number, interval, enchytraeids, Enchytraeus japonensis, Japan

Introduction
In 1959, the enchytraeid species, Enchytraeus fragmentosus, which reproduces asexually by fragmentation was
firstly recorded (Bell, 1959). Since then, seven species
have been reported to use this reproductive mode (Christensen, 1959; Nielsen and Christensen, 1963; Bouguenec
and Giani, 1987; Nakamura, 1993; Dózsa-Farkas, 1995a).
Christensen (1961) states that the normal method ofreproduction in enchytraeids is by mutual transference of
spermatozoa between hermaphrodites. But various reproductive modes are reported in addition to fragmentation(parthenogenesis by Christensen, 1961; self-fertilization by
Dózsa-Farkas, 1995b).
Enchytraeids are small terrestrial and aquatic worms of
the phylum Annelida, class Oligochaeta. They belong tomicrodrilids and are often called ‘potworms’ or ‘enchy’.
Most species are 3-15 mm long with a body diameter of
0.03-0.5 mm, and are usually white in color. Up to thepresent, about six hundred species have been described(Nielsen and Christensen, 1959; Nakamura, 2000). In
terrestrial ecosystems, enchytraeids are actively involved
in decomposition of organic matter, in nutrient cycling,
and in soil formation (Kasprzak, 1982).
The first evidence in Japan of the fragmenting species,
Received 8 March 2004.
Accepted 31 May 2004.
＊
Laboratory of Crop Sciences, Faculty of Agriculture, Ehime
University, 3-5-7 Tarumi, Matsuyama city, Ehime, 790-8566,
Japan

Enchytraeus japonensis, was collected from an ecofarmed crop field under conditions of no-tillage agriculture, an organic mulch of chopped crop residues andmowed weed, and no chemicals (Nakamura, 1993). The
specimen was obtained in the second year after the change
from conventional farming to eco-farming. Finding thisspecies was serendipitous. During a routine survey of soil
animals at the above mentioned crop field, several
enchytraeid larvae were extracted and cultured, because
identification of the enchytraeid is based on live and fullgrown specimens. One of the cultured specimens isbroken into several fragments, and continued to fragment.
Thereafter Enchytraeus larvae collected from routine sampling over nine years were also cultured, but no additional
specimens which carried out fragmentation were found(Nakamura et al ., 2003). It is unknown why it disappeared.
This species resembles another fragmenting species
obtained from culture, Enchytraeus bigeminus NIELSEN et
CHRISTENSEN, 1963, in morphological characteristics, but
the analysis of general protein and isozyme patterns showsthat the two species are different (Schmelz et al ., 2000).
E. japonensis is a widely used new experimental material
for regeneration studies (Myohara et al ., 1999). Its fragmentation can be induced by decapitation, and its egg
production by re-feeding starved worms in wet leaf moldor in aged agar medium (Inomata et al ., 2000).
In Oct. 1991, one specimen of this species, E. japonensis, was collected, and now, in Dec. 2003, it is cultured atvarious laboratories. One question about these cultured
worms is whether they will continue to fragment eternallyto support their population. When a worm is cut into

２０

Yoshio NAKAMURA

several fragments, the most anterior fragment with thehead or the most posterior one with the tail regenerates
new segments except the head or tail, respectively.
Another question is whether the original head or tailfragment is endowed with a fragmentation limit. The
present study investigates the fragmentation potential in
the original head and original tail fragments under laboratory conditions.

Materials and Methods
Animals and cultures
The enchytraeid worm, E. japonensis, which had been
cultured for several years in the laboratory of Tohoku
National Agricultural Experiment Station, was used (Fig.
1A). One fully grown worm from a stock culture wasplaced in a small glass dish (30 mm diam., 15 mm high)with a 1.5% agar solution. The dish was covered with

H

A

1mm

B

2mm

T

D

C

T

H

E

1mm

F

1mm

Fig. 1 (A-F). A: Stock culture of Enchytraeus japonensis; B: Head fragment (arrow shows nerve cord); C: Body
circular muscle constriction before fragmentation; D: Tail fragment with an expanded epidermis which connectswith the other fragment; E: Fragmentation finished (tail fragment is transported to a new dish); F: Worms
staying in jellied ground oats (white bubbles). H: head, T: tail

Relation of fragmentation frequency to fragment number in Enchytraeus japonensis

２
１Fig. 2. Fragmentation frequency, fragment number, interval, and number of survival days in Enchytraeus japonensis
under laboratory conditions. Standard errors of the mean appear as vertical bars.
Upper: head fragment group; lower: tail fragment group２
２

Yoshio NAKAMURA

Fig. 3. Relationship among fragmentation frequency, fragment number, interval, and number of survival days in
Enchytraeus japonensis under laboratory conditions.

Relation of fragmentation frequency to fragment number in Enchytraeus japonensis

２
３Fig. 4. Fragment number and interval in Enchytraeus japonensis with a large number of survival days under laboratory
conditions. H: head fragment; T: tail fragment; The Arabic numerals show the culture number in Fig. 1

２
４

Yoshio NAKAMURA

another of the same size and sealed with vinyl tape toprevent desiccation. Ground oats, in a quantity in excessof the worm’s needs, were placed on the agar substrate toprovide food, and the dishes were stored in the dark at 20℃. One hundred dishes were prepared, and half of themwere used for a head group, the rest for a tail group.
When the experiment commenced, a bamboo stick wasused to gently place the head (Fig. 1B) or tail fragment on
the agar substrate in a new small dish. Thereafter, thehead fragment with an original head in the head group or
the tail one with an original tail in the tail group was
repeatedly transported into a new glass dish with an agar
substrate until death. At each daily inspection under alight microscope it was ascertained whether fragmentation
had taken place. When fragmentation had taken place, thenumber of fragments and the number of days between
successive bouts of fragmentation (interval) were recorded.
Statistical evaluation
Data were subjected to an analysis of variance, andFisher’s least significant difference for means and coefficient of correlation were calculated.
Results
In the initial phase of fragmentation under laboratory
conditions, body circular muscle constriction occurred inseveral parts of the body (Fig. 1C). Thereafter, the fragmented body moved backward and forward (Fig. 1D), or
again constricted in the middle of the body and twisted inorder to promote the succeeding separation. Each fragment moved here and there, and one could not distinguishwhere the fragment was located (Fig. 1E). In a new dish,the transported head or tail fragment also moved here andthere, and sometimes went under a part of the sealed vinyl
tape. After regeneration of the tail or head had finished,worms gathered to the food. The ground oats foodchanged gradually into jelly, which resulted from preoral
digestion (Reichert et al ., 1966). Several worms did not
depart from the jelly oats until the next fragmentation(Fig. 1F). Such behaviour was often observed in bothgroups throughout the experimental period.
Head fragment (Head group)
Of the 50 specimens examined, three broke intofragments more than 110 times, and survived beyond
2200 days (Fig. 2). The fragmentation frequency increased
with an increase in survival days, and the maximum
fragmentation frequency was 122, associated with a survival period of 2459 days. The fragmentation frequency of
the remaining worms was less than 59 times, and thesurvival period was less than 1433 days. The interval
between successive bouts of fragmentation ranged from165 to 7 days, with a mean of 20.4 days at 20℃. The
fragment number in one fragmentation ranged from 14 to2, with a mean of 6.3. There was no tendency for the
fragment number or the interval to increase or decrease

with an increase in the fragmentation frequency or survival days (Fig. 3).
Three heads which survived beyond 2200 days showeda wider variation in fragment number in the first half of
their lives than in the latter half, and converged on anaverage fragment number as time went by (Fig. 4). Eachof the other specimens showed no tendency for the
fragment number and interval to increase or decrease as
time went on.
Tail fragment (Tail group)
Of the 50 specimens examined, only one fragmented
more than 80 times and survived beyond 2400 days (Fig.
2). The fragmentation frequency increased with the expansion of survival days, and the maximum fragmentation
frequency was 85, while the maximum number of days
survived was 2463. The fragmentation frequency of theremaining worms was less than 32 times, and the numberof days survived less than 1003. The range was from 126
to 10 days, and the mean was 24.0 days. The fragment
number ranged from 14 to 2, and the mean was 6.1. There
was no tendency for the fragment number or interval toincrease or decrease with the addition of fragmentation
frequency or survival days (Fig. 3).
The tail with the longest survival period showed awider variation of fragment number in the first half of its
life than in the latter one, and converged on an average
fragment number with the lapse of time (Fig. 4). Each of
the remaining specimens showed no tendency for the
fragment number or interval to increase or decrease as
time went on.
Discussion
Since the first description of fragmenting enchytraeid
species (Bell, 1959), seven fragmenting species have beenreported. Studies on them have established that they areable to reproduce sexually in addition to fragmenting(Christensen, 1959 and 1973; Vena et al ., 1969; Hamiltonand Hess, 1971; Dózsa-Farkas, 1996; Myohara et al .,1999). In the case of the present species, E. japonensis,
fragmentation was also induced by re-feeding starved
worms in wet leaf mold or in aged agar medium; also, thestarvation was not essential for, but did promote theinduction of sexual maturation (Myohara et al ., 1999;
Inomata et al ., 2000). Sexual maturation was density dependent, as in E. bigemius (Christensen, 1973) and E.
dudichi DÓZSA-FARKAS, 1995 (Dózsa-Farkas, 1996). The
fragmentation of another species, Cognettia sphagnetorum(VEJDOVSKY, 1877), the predominating species in a pine
forest soil in Sweden, would be advantageous in recovering after the dry periods that occur with fairly highregularity at the site (Lundkvist, 1982). During the present
experimental period of seven and a half years, however,specimens of both groups, head and tail, reproduced
asexually. And they certainly regenerated tails from headfragments or heads from tail fragments. This is thought toindicate that the conditions did not change abruptly or２５

Relation of fragmentation frequency to fragment number in Enchytraeus japonensis

Table 1. Fragmentation frequency, fragment number, number of survival days and interval between fragmentation bouts in
Enchytraeus japonensis under laboratory conditions. (Each group consists 50 specimens)
Head group
Fragmentation frequency until death
Fragment number (per one fragmentation)
Survival days
Interval (days between fragmentations)

Tail group

Max

Min

Mean

(sd)

Max

Min

Mean

(sd)

122
14
2459
165

11
2
265
7

35.3
6.2
726
20.4

( 22.6)( 0.5)(476 )( 2.7)

85
14
2463
126

2
2
44
10

11.4
6.0
275
24.0

( 12.3)( 0.9)(358 )( 4.7)

drastically during the experimental period. Because in thepresent study only one worm was cultured in each dish,density dependence of the type in sexual reproduction is
unknown, and further investigation is needed.
Inomata et al . (2000) cultured E. japonensis, which
was acquired from the same original stock as the present
study, and reported that the fragmentation interval wasfrom 13 to 15 days at 24℃. This is a fair amount shorter
than what was found in the present study: 20 days in thehead group, and 24 days in the tail group at 20℃. This
may due to the difference in temperature, as demonstrated
in E. dudichi by Dózsa-Farkas (1996), or the fragments
without head and tail segments may regenerate more
quickly. Comparing the data concerning the fragment
number of other fragmenting Enchytraeus species and thepresent species, E. japonensis, the latter had a high capability for fragmentation, since it was 4-7 in E. bigeminus(Christernsen, 1964 and 1973), 3-11 in E. fragmentosus(Bell, 1959), 3-6 in E. variatus (Bouguence and Giani,
1989) and 7-18 in E. dudichi (Dózsa-Farkas, 1996).
The fragmentation frequency increased with the increase in survival days. Such a trend was clear in the tailgroup. The fragmentation frequency, survival days, andfragment number were more numerous or longer in thehead group than in the tail group. On the other hand, theinterval between successive bouts of fragmentation was
longer in the head group than in the tail group. No
significant difference was observed between the twogroups in any of the four factors mentioned above. But
there was a slight tendency for the fragment number todecrease in the head group and increase in the tail groupwith the increasing number of days. The specimens of thehead and the tail groups were each divided into two
categories, long and short life spans. In the case of aworm with an exceptionally large number of survivaldays, as in three of the head and one of the tail group, thefragment numbers showed a wider variation in the firsthalf of their lives than in the latter half, and converged on
an average fragment number with the increasing numberof survival days. Such a tendency was clearly observed in
the worm with the largest number of survival days in the
tail group. These four worms lived for between 2200 and2500 days, although there was a large difference in the
fragmentation frequency between the two groups, head
and tail.
It was believed that the maximum number of generations would be 167 to 196 from the first fragmentation ina stock culture under laboratory conditions; this was basedon the interval calculated in the present study, although an
accurate interval was not known except for the head andtail fragments. The cause of death could not prove in thepresent study. But the maximum fragmentation frequency,
122 in the head group and 85 in the tail group, indicatesthat the fragmentation capacity is higher in the headfragment than in the tail one. And its capacity may becharged with a limit, like the fission limit of Paramecium
aurelia, Protozoa (Sonneborn, 1954) and the phenomenonof Hayflick (1965). Since fragmentation of the present
species can be induced by decapitation (Inomata et al .,2000), the question is whether the present fragmentation
capacity is higher or lower than that in fragmentation
stimulated by decapitation. The fragmentation frequency
and interval were many and short in the head group, andfew and long in the tail group, respectively. This suggests
the existence in both groups of some substance like
telomerase, and further studies, especially on biogenic
activity, are needed.
Acknowledgements: My thanks are due to Dr. T.
FUJIKAWA of Shigenobu-cho, Ehime pref. and Mr. K.
NAKAMURA of Kyusyu Univ. for the stock management
and observation of specimens, and Dr. Y-N. NAKAMURAof Kinki Univ. and Dr. S. TOCHINAI of Hokkaido Univ.
for their kind advice.
References
Bell, A.W. (1959) Enchytraeus fragmentosus, a new species ofnaturally fragmenting Oligochaete worm. Science, 129:
1278.
Bouguenec, V. and N. Giani (1987) Deux nouvelles espècesd’Enchytraeus (Oligochaeta, Enchytraeidae) et redescriptiond’E. bigeminus Niel. & Chr. Remarques sur le genre
Enchytraeus. Annls Limnol., 23: 9−22.
Bouguenec, V. and N. Giani (1989) Biological studies upon
Enchytraeus variatus Bouguenec & Giani 1987 in breeding
cultures. Hydrobiol., 180: 151−165.
Christensen, B. (1959) Asexual reproduction in the Enchytraeidae (Oligo.). Nature, 184: 1159−1160.
Christensen, B. (1961) Studies on cyto-taxonomy and reproduction in the Enchytraeidae with notes on parthenogenesis
and polyploidy in the animal kingdom. Hereditas, 47: 387−
450.
Christensen, B. (1964) Regeneration of a new anterior end in
Enchytraeus bigeminus (Enchytraeidae, Oligochaeta). Vidensk. Medd. Dansk aturh. Foren., 127: 259−273.
Christensen, B. (1973) Density dependence of sexual reproduction in Enchytraeus bigeminus (Enchytraeidae). Oikos,

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Yoshio NAKAMURA

24: 287−294.
Dózsa-Farkas, K. (1995a) Enchytraeus dudichi sp. n., a new
fragmenting Enchytraeus species from Iran (Oligochaeta,
Enchytraeidae). Opusc. Zool. Budapest, 27/28: 41−44.
Dózsa-Farkas, K. (1995b) Self-fertilization: an adaptive strategy in widespread enchytraeids. Eur. J. Soil Biol., 31:
207−215.
Dózsa-Farkas, K. (1996) An interesting reproduction type inenchytraeids (Oligochaeta). Acta Zool. Acad. Sci. Humgaricae, 42: 3−10.
Hamilton, M. & R. T. Hess (1971) Axenic production of ova
by Enchytraeus fragmentosus (BELL). Canadian J. Zool., 49:
1199−1120.
Hayflick, L. (1965) The limited in vitro lifetime of human
diploid cell strains. Experim. Cell Res., 37: 614−636.
Inomata, K., F. Kobari, C. Yoshida-Noro, M. Myohara and S.
Tochinai (2000) Possible neutral control of asexually reproductive fragmentation in Enchytraeus japonensis (Oligochaeta, Enchytraeidae). Invertebrate Repro. Develop., 37:
35−42.
Kasprzak, K. (1982) Review of enchytraeids (Oligochaeta,
Enchytraeidae) community structure and function in agricultural ecosystems. Pedobiologia, 23: 217−232.
Lundkvist, H. (1982) Population dynamics of Cognettia
sphagnetorum (Enchytraeidae) in a Scots pine forest inCentral Sweden. Pedobiologia, 23: 21−41.
Myohara, M., C. Yoshida-Noro, F. Kobari & S. Tochinai(1999) Fragmenting oligochaete Enchytraeus japonensis: a
new material for regeneration study. Develop. GrowthDiffer., 41: 549−556.
Nakamura, Y. (1993) A new fragmenting enchytraeid species,
Enchytraeus japonensis from a cropped kuroboku soil in
Fukushima, northern Japan (Enchytraeids in Japan 5).
Edaphologia, 50: 37−39.
Nakamura, Y. (2000) Checklist of enchytraeids (Oligochaeta:
Enchytraeidae) of the world. Misc. Publ. Tohoku Natl.
Agrc. Exp. Stn., 24: 29−104. (In Japanese with English
abstract.)
Nakamura, Y., H. Shiraishi & M. Nakai (2003) Earthworm
and enchytraeid numbers in soybean-barley fields under tilland no-till cropping systems in Japan during nine years.
Mem. Fac. Agr., Ehime Univ., 48: 19−29.
Nielsen, C. O. and B. Christensen (1959) The Enchytraeidae,

critical revision and taxonomy of European species. Natura
Jutlandica, 8/9: 1−160.
Nielsen, C. O. and B. Christensen (1963) The Enchytraeidae,
critical revision and taxonomy of European species. Natura
Jutlandica, 10 (suppl. 2): 1−19.
Reichert, A., U. Mothes-Wagner & K. -A. Seitz (1996) Ecohistological investigation of the feeding behaviour of theenchytraeid Enchytraeus coronatus (Annelida, Oligochaeta).
Pedobiologia, 40: 118−133.
Schmelz, R. M., R. Collado & M. Myohara (2000) A taxonomic study of Enchytraeus japonensis (Enchytraeidae,
Oligochaeta): morphological and biochemical comparisonswith E. bigeminus. Zool. Sci., 17: 505−516.
Sonneborn, T. M. (1954) The relation of autogamy to senescence and rejuvenescence in Paramecium aurelia. J.
Protozool., 1: 38−53.
Vena, J. A., R. T. Hess and M. L. Gotthold (1969) Attainmentof sexuality in Enchytraeus fragmentosus Bell underlaboratory conditions. Experimentia (Basel), 25: 761.
摘

要中村好男：長期飼育条件下におけるヤマトヒメミミズの死亡までの砕片分離回数，砕片数および間隔
わが国ではじめて記録されたヒメミミズ科の砕片分離
種，ヤマトヒメミミズ Enchytraeus japonensis NAKAMURA，
１
９
９
３は，１
９
９
１年に畑土壌から採集された．砕片分離で増殖する個体群から成長した１００個体を選び出し，個体ごとに寒天培地上に添加した粉砕オートミールを餌に飼育した．砕片分離直後の５０個体の砕片から頭部を含む頭部砕片を，新しい容器で飼育し，その後毎回の砕片分離時に頭部砕片のみを，新しい容器に移し継代飼育した．また同じように５０個体の砕片から尾部を含む尾部砕片のみを継代飼育した．その結果，死亡までの砕片分離回数，１回の砕片数，砕片間隔日数および生存日数の平均は，頭
部砕片が３
５．
３回，６．
２片，２
０．
４日および７２６日；尾部砕片
が１
１．
４回，６．
０片，２
４．
０日および２
７５日であった．最大の砕片分離回数と生存日数は，頭部砕片が１
２
２回と２
４
５
９日，
尾部砕片が８５回と２
４
６３日であった．頭部砕片と尾部砕片
とも，１回の砕片数と砕片間隔日数が，砕片回数の増加，
すなわち生存日数の増加にともなって，それぞれ増加あるいは減少するという傾向は認められなかった．