JOURNAL OF CRUSTACEAN BIOLOGY, 20(3): 453–459, 2000
RECIPROCAL TRANSPLANTATION OF LEG TISSUE BETWEEN ALBINO
AND WILD CRAYFISH PROCAMBARUS CLARKII
(DECAPODA: CAMBARIDAE)
Isamu Nakatani
Department of Biology, Faculty of Science, Yamagata University, Yamagata 990–8560 Japan
(e-mail: [email protected])
A B S T R A C T
To examine albinism, cheliped tissue was reciprocally transplanted into the autotomized stump of
the walking leg (and vice versa) between albino and wild crayfish. Two of the 24 walking-leg stumps
of albino crayfish formed a claw, and three of the 49 leg stumps of wild crayfish formed claws. Dactyl
and pollex tissues of wild (and albino) crayfish were transplanted into an eyestalk stump and rostrum stump of 20 albino (and wild) crayfish, respectively. In albino crayfish, one normal claw and
six abnormal chelipeds developed from the eyestalk stump, and seven abnormal chelipeds developed from the rostrum stump. In wild crayfish, one normal and seven abnormal chelipeds developed
from the eyestalk stump, and four abnormal chelipeds developed from the rostrum stump. Dactyl
tissue of wild (and albino) crayfish was inserted into the carapace near the eyestalk of 11 albino
(and wild) crayfish. In six of each of the albino and wild crayfish, a dactyl-like structure developed
from the graft. All of the regenerated structures were the same color as the host. These results suggest that the albinism in this crayfish is caused by a deficiency of some hormonal factor(s).
Albino specimens of the crabs Chionoecetes japonicus Rathbun (see Muraoka and
Honma, 1993) and Portunus (Portunus) trituberculatus Miers (see Ariyama, 1997) and of
the spiny lobster Panulirus japonicus Von
Siebold (Okamoto and Misyuku, 1998) have
been reported. A male albino crayfish Procambarus clarkii Girard that lacked melanin
and red pigments except in its compound eyes
was captured, and the albino phenotype was
segregated (Nakatani, 1999). Based on the
phenotypes of the first and second filial generations, the albino trait is recessive to normal pigmentation in crayfish and its inheritance is controlled by Mendelian laws
(Nakatani, 1999). However, the cause of albinism in crayfish and crabs is unknown. In
the migratory locust, Locusta migratoria, albinism is caused by a deficiency of some hormonal factor(s), because implantation of the
corpora allata from a normal locust causes
it to turn as dark as that in the normal strain
(Tanaka, 1993, 1996).
The claw tissues of the crabs Cancer anthonyi Rathbun, C. gracilis Dana, and C. productus Randall formed a complete claw when
they were autotransplanted into the autotomized stump of the walking leg (Kao and
Chang, 1996). In C. gracilis, the tissue from
the claw or walking leg, when autotrans-
planted into the eye socket, formed a complete claw or walking leg, respectively (Kao
and Chang, 1997). If albinism is caused by a
deficiency of some hormonal factor(s), the
leg, formed by reciprocal transplantation between albino and wild crayfish, should be the
same color as the host. Crayfish have five
pairs of thoracic legs: the ends of the anterior first to third legs are pincer-like structures
formed by the dactyl and pollex (Fig. 1). The
ends of the fourth and fifth legs lack a pollex.
Thus, the origin of a regenerated leg, after
transplantation of cheliped tissue into the
walking leg stump (or vice versa), is clear.
In this study, the possibility that albinism
in crayfish is due to some hormonal defect
was examined by reciprocal transplantation.
Leg tissue was transplanted into the stump
of an autotomized leg, removed eyestalk or
removed rostrum, or the carapace.
MATERIALS AND METHODS
Crayfish.—Albino crayfish Procambarus clarkii were obtained by pairing albino crayfish of the second filial generation. Wild crayfish were collected from a pond in the
suburbs of Yamagata City, Japan. Albino and wild crayfish with carapace lengths of 19.7–44.0 mm were used.
Transplantation of Cheliped Tissue into a Leg Stump.—
Autotomy of the leg was achieved by crushing the merus
segment with forceps. A piece of the dactyl, pollex, propodus, carpus, merus, or ischium segment of the first, sec-
453
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 20, NO. 3, 2000
Fig. 1. Structure of the second cheliped of Procambarus
clarkii. The parts enclosed in solid lines were transplanted
into the autotomized stump of a leg.
Table 1. Results of the transplantation of tissue from wild
crayfish Procambarus clarkii into albino crayfish.
PL1→L5: Pollex tissue from the first leg was transplanted
into the autotomized stump of the fifth leg. DL2: Dactyl
tissue from the second leg. DL3: Dactyl tissue from the
third leg. PL1, PL2, and PL3 are pollex tissues from the
first, second, and third legs, respectively. CarL2: Carpus
tissue from the second leg. CarL5: Carpus tissue from the
fifth leg. ProL3: Propodus tissue from the third leg. ProL5:
Propodus tissue from the fifth leg. IscL3: Ischium tissue
from the third leg. L1, L4, and L5 are the first, fourth, and
fifth legs, respectively.
Type of leg regenerated
ond, or third cheliped of wild (and albino) crayfish was
dissected out and inserted completely into the stump of
the autotomized fourth or fifth leg of albino (and wild)
crayfish (Fig. 1). A piece of the propodus, carpus, or ischium of the fifth leg was transplanted into the autotomized stump of the first cheliped. In crayfish with a
carapace length of greater than 31 mm, both eyestalks
were removed after transplantation to promote molting.
Transplantation of Dactyl and Pollex Tissue into the Eyestalk and Rostrum Stumps, Respectively.—The right eyestalks of wild (and albino) crayfish were removed near
the base with small scissors. Before eyestalk removal, the
rostrum was removed, which revealed the base of the eyestalks. Dactyl and pollex tissues from the right second
cheliped of the albino (and wild) crayfish were inserted
into the eyestalk stump and into the wound that was made
by removal of the rostrum (rostrum stump), respectively.
The transplanted dactyl was in the base of the removed
eyestalk and not in the carapace.
Transplantation of Dactyl Tissue into the Carapace.—A
pore was made on the carapace of wild (and albino) crayfish near the right eyestalk with the end of fine forceps.
Dactyl tissue from the first right cheliped of the albino
(and wild) crayfish was inserted into the pore. The eyestalk was not removed from the crayfish.
Rearing Experimental Crayfish.—The crayfish in which
cheliped tissue had been transplanted into the stump of
an autotomized leg were kept separately in individual containers (200 × 130 × 130 mm) under a 12 h light/12 h dark
cycle at 24 ± 2°C. Other operated crayfish were kept separately in individual containers (435 × 296 × 140 mm)
and reared in the open air from June to September 1999.
Prawn pellets (Royal B; Nihon Nosan Industry, Japan)
and dry persimmon leaves were placed in the rearing containers so that the crayfish could feed ad libitum.
RESULTS
Transplantation into the Leg Stump
Of 24 leg stumps of albino crayfish that
were transplanted with tissue from wild crayfish, two regenerated a claw. In seven cases,
the host crayfish died before regenerating a
leg. The remaining 15 stumps regenerated a
normal leg (Table 1). In one case, tissue from
the dactyl of the left second cheliped of wild
crayfish was transplanted into the left fifth leg
stump of an albino crayfish. A claw developed
Operation
n
Death
Normal
Claw
PL1→L5
DL2→L4
DL2→L5
PL2→L5
CarL2→L5
DL3→L5
IscL3→L5
PL3→L5
ProL2→L5
CarL5→L1
ProL5→L1
1
3
6
5
1
1
1
1
1
2
2
1
1
2
2
0
0
0
0
1
0
0
0
2
3
2
1
1
1
1
0
2
2
0
0
1
1
0
0
0
0
0
0
0
on the regenerated fifth leg at the first postoperative molt (Fig. 2). In another case, tissue from the pollex of the right second cheliped was transplanted into the left fifth leg
stump. A claw developed on the regenerated
leg at the first postoperative molt. The coxa,
basis, and ischium of the regenerated leg were
of normal size. However, the apical part from
the merus was smaller than normal and was
bent and positioned between the base of the
regenerated leg and the fourth leg.
In wild crayfish, three of 49 leg stumps that
were transplanted with tissue from albino
crayfish formed claws. In four cases, the host
crayfish died before regenerating a leg. The
remaining 42 stumps regenerated a normal
leg (Table 2). The three transformed legs belonged to the same crayfish. The stumps of
the right fourth, right fifth, and left fifth legs
were transplanted with tissue from the base
of the propodus, tissue from the dactyl, and
tissue from the base of the carpus of the left
second leg of albino crayfish, respectively.
The left fourth leg was not autotomized.
These three regenerated legs formed a claw
at the first postoperative molt. The right
fourth and fifth legs formed three and one
claws, respectively. The left fifth leg formed
one claw, one dactyl, and one propodus with
a dactyl. The extra propodus developed from
NAKATANI: TRANSPLANTATION OF LEG TISSUE IN CRAYFISH
455
Fig. 2. An albino crayfish Procambarus clarkii with a transformed leg after the third postoperative molt. (A) The
left fifth leg with a claw regenerated from grafted tissue, i.e., the dactyl of the second cheliped of a wild crayfish,
which had been transplanted into an autotomized stump. (B) The left fifth leg with a claw.
the base of the primary propodus. However,
this claw, dactyl, and extra propodus were
torn off after the second postoperative molt
before a photograph could be taken (Fig. 3).
Table 2. Results of the transplantation of tissue from
albino crayfish Procambarus clarkii into wild crayfish.
DL1→L5: Dactyl tissue from the first leg was transplanted
into the autotomized stump of the fifth leg. DL1, DL2, and
DL3 are dactyl tissues from the first, second, and third legs,
respectively. PL1, PL2, and PL3 are pollex tissues from the
first, second, and third legs, respectively. ProL2, ProL3, and
ProL5 are propodus tissues from the second, third, and fifth
legs, respectively. CarL5: Carpus tissue from the fifth leg.
MerL2: Mersus tissue from the second leg. IscL5: Ischium
tissue from the fifth leg. L1, L4, and L5 are the first, fourth,
and fifth legs, respectively.
Type of leg regenerated
Operation
DL1→L5
PL1→L5
DL2→L4
PL2→L4
PL2→L5
DL2→L5
ProL2→L1
ProL2→L4
ProL2→L5
MerL2→L1
DL3→L5
PL3→L5
ProL3→L1
ProL3→L5
CarL5→L1
IscL5→L1
ProL5→L1
n
Death
Normal
Claw
3
5
2
1
2
8
1
1
4
2
1
2
2
1
6
3
5
0
1
0
0
1
2
0
0
0
0
0
0
0
0
0
0
0
3
4
2
1
1
5
1
0
3
2
1
2
2
1
6
3
5
0
0
0
0
0
1
0
1
1
0
0
0
0
0
0
0
0
Transplantation into the Eyestalk and
Rostrum Stump
One of 20 eyestalk stumps of albino crayfish formed a claw with almost normal form,
but only up to the propodus of the cheliped,
after the fourth postoperative molt (Fig. 4A).
In four crayfish, both eyestalk and rostrum
stumps formed an abnormal claw, and a total
of six eyestalk stumps and seven rostrum
stumps formed an abnormal claw. One of
these abnormal claws consisted of a dactyl
and pollex, but these could not contact each
other (Fig. 4C). Other claws had a dactyl but
lacked a pollex (Fig. 5).
In wild crayfish, one of 20 eyestalk stumps
formed a cheliped, although with a bump on
the merus (Fig. 4B). Six eyestalk stumps and
seven rostrum stumps formed an abnormal
claw without the pollex or proximal portion
beyond the propodus. One of seven rostrum
stumps formed a pair of double dactyls and
double pollexes, although other portions of
the cheliped did not develop (Fig. 4D).
Each of three eyestalk stumps of the albino
and wild crayfish formed an antennule. These
results are summarized in Table 3.
Transplantation into the Carapace
In albino crayfish, a regenerate developed
from each of six grafts after the second or third
postoperative molt. The regenerate was similar to the dactyl of the first cheliped (Fig. 6A).
One wild crayfish died. Furthermore, six
wild crayfish developed a dactyl-like struc-
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 20, NO. 3, 2000
Fig. 3. A wild crayfish Procambarus clarkii with transformed legs after the second postoperative molt. (A) The
right fourth, right fifth and left fifth legs regenerated from graft tissue from the second cheliped of an albino crayfish. The right fourth and fifth legs have three claws and one claw, respectively. The left fifth leg had one claw, one
dactyl and one propodus with a dactyl, but these structures were torn off before a photograph could be taken. (B)
The fourth right leg.
Fig. 4. Examples of regenerates from the stump of an eyestalk or rostrum of the albino and wild crayfish Procambarus clarkii with the reciprocal transplantation of dactyl or pollex tissue. (A) The eyestalk stump of albino crayfish
generated a claw. (B) The eyestalk stump of wild crayfish generated a cheliped. (C) The eyestalk stump of albino
crayfish generated an abnormal cheliped. d and p are the generated dactyl and pollex, respectively. (D) The rostrum
stump of wild crayfish generated an abnormal cheliped. d and p are double dactyls and double pollexes, respectively.
NAKATANI: TRANSPLANTATION OF LEG TISSUE IN CRAYFISH
Fig. 5. Example of an abnormal cheliped from an eyestalk stump of the albino crayfish Procambarus clarkii.
The abnormal cheliped has a dactyl, but not a pollex.
ture on the first cheliped (Fig. 6B). Each of
the remaining five grafts of albino crayfish
and four grafts of wild crayfish formed nothing (Table 4).
All of the regenerated structures from
grafts were the same color as the host crayfish rather than that of the donor.
DISCUSSION
In the present study, regenerated structures
from reciprocally transplanted grafts between
albino crayfish, segregated by Nakatani
(1999), and wild crayfish were the same color
as the host. In previous studies, the external
morphology of a regenerated leg was affected
by the grafted tissue, if the leg tissue was
transplanted inter- or intraspecifically in
crabs, Uca pugilator Bosc, U. pugnax Smith
(see Trinkaus-Randall, 1982), C. anthonyi, C.
gracilis, and C. productus (see Kao and
457
Chang, 1996). In the crayfish P. clarkii, a regenerated leg may have the morphology of
the donor, the host, or both when the tissue
from the first cheliped is transplanted into the
autotomized stump of the second cheliped or
vice versa (Mittenthal, 1980). Furthermore,
in the crayfish, jointed and jointless regenerates formed when a dactyl segment was inserted into the proximal part of the propodus
segment in the second leg (Mittenthal, 1981).
The results shown in Figs. 4–6 show that the
morphology of regenerated structures was determined by the donor because regenerates
developed from different regeneration fields
of the cheliped. In the present study, however,
the antennule that developed from the eyestalk stump did not develop from a transplanted graft. The graft may have died, and
the eyestalk stump may have formed the antennule. It has been shown that a heteromorphic antennule develops from an eyestalk
stump in spiny lobster Panulirus argus Latreille (see Maynard, 1965; Maynard and Cohen, 1965), in freshwater shrimp Caridina
weberi sumatrenisis De Man (see Ravindranath, 1978), in crayfish P. clarkii (see Mellon et al., 1989), and in freshwater prawn
Macrobrachium rosenbergii De Man (see
Nevin and Malecha, 1991).
The present results suggest that albinism in
crayfish is caused by a deficiency of some
hormonal factor(s) related to the synthesis of
melanin and red pigments in chromatophores.
If this albinism is caused by any other factor, a cheliped that has regenerated from
grafted tissue should be the same color as the
donor. The albino swimming crab Portunus
trituberculatus reported by Ariyama (1997)
had dark brown and dark red pigments on the
dorsal surface of the chela and on each dactyl,
respectively. Therefore, it is possible that the
Table 3. Results of the transplantation of dactyl and pollex tissue from wild (or albino) crayfish Procambarus clarkii into
the base of the eyestalk and the carapace in 20 albino (or wild) crayfish, respectively.
Type of regeneration
Recipient crayfish and
region of transplantation
Albino
Eyestalk stump
Rostrum stump
Wild
Eyestalk stump
Rostrum stump
Normal
cheliped
Normal
claw
Abnormal
claw
Antenna
Nothing
0
0
1
0
6*
7*
3
0
10
13
1
0
0
0
7**
4**
3
0
9
16
* In four crayfish, both the eyestalk and rostrum stumps generated an abnormal claw.
** In three crayfish, both the eyestalk and rostrum stumps generated an abnormal claw.
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 20, NO. 3, 2000
Fig. 6. Examples of regenerates from the carapace of albino and wild crayfish Procambarus clarkii with the reciprocal transplantation of dactyl tissue. A dactyl-like structure generated from the graft (arrow heads). (A) and (B)
are the albino and wild crayfish, respectively.
cause of albinism is different between the present albino crayfish and the albino crab reported by Ariyama (1997). Furthermore, both
the present albino crayfish and the crabs reported by Muraoka and Honma (1993) and
Ariyama (1997) have melanin pigments in
their compound eyes. These facts show that
albinism occurs with slight differences in hormonal factor(s) in hemolymph.
In the cockroach Blatella germanica Linnaeus, a segment structure regenerated from
graft tissue was also the same color as the
donor. In this case, tissue from a leg was
transplanted reciprocally into the tibia between dark- and light-cuticle mutants
(French, 1976). Furthermore, in crabs, the regenerated leg from reciprocally transplanted
grafts between C. productus and C. gracilis,
faint brown spots were characteristic of the
donor (Kao and Chang, 1999). These facts
may show that the color of the donor is not
necessarily affected by the host, if the composition of hemolymph in the host and donor
is normal.
Table 4. Results of the transplantation of dactyl tissue
from wild (or albino) crayfish Procambarus clarkii into the
carapace in 11 albino (or wild) crayfish.
Regeneration
Recipient
crayfish
Death
Dactyl
Nothing
Albino
Wild
0
1
6
6
5
4
To further examine the cause of albinism
in crayfish, it may be necessary to implant endocrine organs from wild crayfish into albino
crayfish.
LITERATURE CITED
Ariyama, H. 1997. Albino of the swimming crab Portunus (Portunus) trituberculatus caught in Osaka
bay.—Cancer 6: 9–10.
French, V. 1976. Leg regeneration in the cockroach,
Blatella germanica II. Regeneration from a non-congruent tibial graft/host junction.—Journal of Embryology and Experimental Morphology 35: 267–301.
Kao, H.-W., and E. S. Chang. 1996. Homeotic transformation of crab walking leg into claw by autotransplantation of claw tissue.—Biological Bulletin 190:
313–321.
———, and ———. 1997. Limb regeneration in the eye
sockets of crabs.—Biological Bulletin 193: 393–400.
———, and ———. 1999. Limb regeneration following auto- or interspecies transplantation of crab limb
tissues.—Invertebrate Reproduction and Development
35: 155–165.
Maynard, D. M. 1965. The occurrence and functional
characteristics of heteromorph antennules in an experimental population of spiny lobster, Panulirus argus.—
Journal of Experimental Biology 43: 79–106.
———, and M. J. Cohen. 1965. The function of a heteromorph antennule in a spiny lobster, Panulirus argus.—Journal of Experimental Biology 43: 55–78.
Mellon, De F., Jr., H. R. Tuten, and J. Redick. 1989. Distribution of radioactive leucine following uptake by olfactory sensory neurons in normal and heteromorphic
crayfish antennules.—The Journal of Comparative Neurology 280: 645–662.
Mittenthal, J. E. 1980. On the form and size of crayfish
legs regenerated after grafting.—Biological Bulletin
159: 700–713.
———. 1981. Intercalary regeneration in legs of crayfish:
distal segments.—Developmental Biology 88: 1–14.
NAKATANI: TRANSPLANTATION OF LEG TISSUE IN CRAYFISH
Muraoka, K., and H. Honma. 1993. Albino of the tanner crab, Chionoecetes japonicus Rathbun collected
from Hokkaido.—Cancer 3: 23–25.
Nakatani, I. 1999. An albino of the crayfish Procambarus clarkii (Decapoda: Cambaridae) and its offspring.—Journal of Crustacean Biology 19: 380–383.
Nevin, P. A., and S. R. Malecha. 1991. The occurrence
of a heteromorph antennule in a cultured freshwater
prawn, Macrobrachium rosenbergii (De Man) (Decapoda, Caridea).—Crustaceana 60: 105–107.
Okamoto, K., and A. Misyuku. 1998. Molting and
growth of the albino spiny lobster, Panulirus japonicus collected from Southern coast of Izu Peninsula,
Shizuoka prefecture.—Cancer 7: 17, 18.
Ravindranath, K. 1978. An unusual abnormality in a
freshwater shrimp (Decapoda, Atyidae).—Crustaceana
35: 92, 93.
459
Tanaka, S. 1993. Hormonal deficiency causing albinism in
Locusta migratoria.—Zoological Science 10: 467–471.
———. 1996. A cricket Gryllus bimaculatus neuropeptide induces dark colour in the locust, Locusta migratoria.—Journal of Insect Physiology 42: 287–294.
Trinkaus-Randall, V. 1982. Regeneration of transplanted
chelae in two species of fiddler crabs Uca pugilator and
Uca pugnax.—The Journal of Experimental Zoology
224: 13–24.
RECEIVED: 11 May 1999.
ACCEPTED: 11 February 2000.