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THE VELIGER
© CMS. Inc., 2002
The Veliger45(2):171-174 (April 1. 2002)
NOTES, INFORMATION & NEWS
A Useful Marker for the Study of Neural Gray, 1849 (Octopoda). Dechorionated embryos at vari-
Development in Cephalopods ous stages of neurogenesis and hatchlings were fixed in
Shuichi Shigeno and Masamichi Yamamoto 4% paraformaldehyde (PFA) dissolved in phosphate buff-
ered saline (pH 7.6) (PBS) for 12-24 hr at 4°C. Samples
Ushimado Marine Laboratory, Okayama University, were washed with PBS, dehydrated in a methanol series,
Ushimado, Okayama, 701-4303 Japan and stored in 80% methanol at -20°C. Some samples
were also fixed in Bouin's solution/seawater, dehydrated
tInhrceeep-hdailmoepnosdiso,natlhenreeuriaslnpoatstueirtnasblienmtahrekperrestehartvevdiseumalbirzye-s with an ethanol series, and stored in 70% ethanol at room
temperature. The stored samples to be immunostained
os. Development of the nervous system has been studied
were placed as a whole, or after dissection into a few
using conventional histological techniques (Meister,
pieces, in ice-cold 50% dimethyl sulfoxide (DMSO)/
1972; Marquis, 1989). Silverimpregnation and methylene
methanol with 10% hydrogen peroxide for 5 min. They
blue staining have been used in anatomical studies ofthe were incubated for 30 min at 4°C in the DMSO solution
nervous system in cephalopods (Stephens, 1971) but the
with 1% Triton X-100, washed with Tris-buffered saline
former are applicable only to late embryonic stages (Mar- (TST; 20 mM Tris-HCl, pH 8.0, 150mM NaCl, 0.1% Tri-
btianc,kf1i9ll7i7n)ga(nBdudtehlemalanttner&, oYnoluyngt,o 1li9v8i7n)gannedurDoinls.injCeocbtailotn ton X-100) containing 5% DMSO, and blocked with 5%
non-fat dry milk (TSTM) overnight at 4°C. The speci-
(Robertson et al., 1993) only label local neuronalpatterns.
Wemebrtyroiesd awnhdolheatmcholuinntgsiwmimtuhnosctoammienricnigalolfycoebpthaailnoapbolde bmoednys (wSeirgemai)ncduiblautteedd w1i:t7h50a-nt1i0-0a0ceitnylTatSedTMa-tfuobrul2i-n4adntia-t
4°C (Gianni & Fuller, 1985). After being washed with
monoclonal antibodies and found acetylated a-tubulin a
suitable immunohistochemical marker to visualize the TSTM, some samples were incubated in pre-diluted goat
anti-mouse antibody conjugated to peroxidase (Envi-
overall pattern of developing neurons.
We used four sepiids, Idiosepius paradoxus Ortmann, sion-l-, DAKO) for 12—24hratroomtemperature, washed
1881; Euprymna morsei (Verrill, 1881); Sepia lycidas with TSTM, immersed in ice-cold 3,3'-diaminobenzidine
Gray, 1849; Sepiella japonica Sasaki, 1929; two teu- (DAB) (1 mg/ml TST) for 1 hr, and reacted by adding
thoids, Loliolusjaponica, Hoyle, 1885; Todarodes paci- hydrogen peroxide (0.01%) for 5-20 min in the dark. The
ficus Steenstrup, 1880; and an octopod. Octopus ocellatus other samples were stained with ABC high-HRP immu-
nostaining kit (TOYOBO) according to the standard pro-
tocol.
Anti-acetylated a-tubulin clearly stained peripheral
nerve fibers as well as neuropiles in the brain (Figure
lA). It also stained the epidermal cilia, lateral lines, ec-
todermal photosensitive vesicles, Kolliker's canals of the
statocysts, and olfactory organs. The antibody recognized
neurons even in Bouin-fixed specimens that had been
stored for 3 yr in ethanol, though not always consistently.
As for the secondary antibodies. Envision-I- (DAKO) was
slightly more effective than ABC high HRP (TOYOBO)
kit. The intensity andthe extent ofvisualizationdepended
on the limit of penetration of the antibodies. In small
specimens, such as the embryos and hatchlings of/. par-
adoxus and the embryos of E. morsei. all neuronal ele-
ments, i.e., peripheral nerves and neuropiles in the brain,
were observable in the samples mounted as a whole (Fig-
Figure 1. Neuropiles in whole-mount of specimens immuno- ure IB). In larger specimens, such as O. ocellatus, S.
stained with acetylated a-tubulin antibody. A. The vertical lobe lycidas, S. japonica, and L. japonica, dissection was nec-
(vn) and the superior buccal lobe (sb) in a late embryo ofSepia essary before immunostaining permitted visualization of
Bly.ciTdhase. sTthelelactreangiaanlglciarotnil(asgteg)isarnedmotvheedp.osStcearlieorbasrub=eso3p0h0ag[examl. the neuropiles in the deep portion ofthe brain and all the
mass (pms) in an Idiosepius paradoxus hatchling. Scale bar = peripheral nerve fibers in the body. We tested two other
30 |j.m. monoclonal antibodies, anti-neurofilament 200 (Sigma)
Page 172 The Veliger, Vol. 45, No. 2
and anti-HRP (Sigma) (Jan & Jan 1982), but they did not cific. The distribution ranges from the type locality,
recognize any neuronal elements. 4rs, to 45°S off the Chilean coast. According to Gal-
Acknowledgments. We thank Mr. W. Godo and Dr T. Akiyama lardo (1979). C. dilatata Lamarck, 1822, can only be
for their help in collecting cephalopods, and Ms. M. Yoshioka, differentiated by its direct development and thepresence
Dr. S. Segawa, Dr K. Fujita, and Mr K. Kidokoro forsupplying ofembryos consuming nurse eggs. Adult morphological
some cephalopod samples. Thanks are also due to Dr Sv. Bol- features are identical. Therefore, earlier records refer-
etzky for his kind help in preparing the manuscript. ring to the presence of C. dilatata in the Atlantic coast
of South America (Parodiz, 1939) need validation. The
Literature Cited type locality of C. dilatata remains unknown. Mermod
BuDELMANN, B. U. & J. Z. YouNG. 1987. Brain pathways ofthe (1950) mentioned in a commented list ofthe types from
brachial nerves of Sepia and Loligo. Philosophical Trans- Lamarck's collection, the Western coast of South Amer-
actions ofRoyal Society ofLondon Series B, 315:345-352. ica as a probable type locality. Gallardo (1979) recorded
Gianni, P. & M. T. Fuller. 1985. Monoclonal antibodies specific C. dilatata Lamarck, from 2ril'S to 43°47'S. He also
for an acetylated form ofa-tubulin recognize the antigen in stated (in Spanish in the original); "It is probable that
cilia and flagella from a variety of organisms. Journal of
Cell Biology 101:2085-2094. future studies including developmental stages, will ex-
Jan, L. Y. & Y. H. Jan. 1982. Antibodies to horseradish perox- pand this distribution particularly towards the Argentine
idase as specific neuronal markers in Drosophila and in Atlantic coast."
grasshopperembryos. Proceedings ofthe National Academy This note confirms the presence of C. dilatata (Figures
of Science ofthe United States ofAmerica 79:2700-2704. 1-9) in Argentine waters and restricts C. fecunda to
Marquis, F. 1989. Die Embryonalentwicklung des Nervensys- Chile.
tems von Octopus vulgaris Lam. (Cephalopoda, Octopoda).
Egg capsules and adult males and females were col-
eine histologische Analyse. Verhandlungen der Naturfor-
Martsicnh,enRd.en19G7e7s.elTlhsechgaifatntinnBearsveelf9ib9e:r23s-y7s5t.em ofcephalopods, BleActeddivfirnogmiBnah3i-a4Emnsednepatdha,, aUtsthaucaheida t(o~5t5h°eS)roobtyoSfCtUh-e
recent structural findings. Symposia of the Zoological So- common kelp Macrocystis pyrifera (Linnaeus); Punta
ciety ofLondon 38:261-275. Peiias, Puerto San JuUan (49°15'S-67°39'W) in 2 m
Meister, G. 1972. Organogenese von Loligo vulgarisLam(Mol- depth; and Punta Dos Hermanas, Puerto Deseado
lusca. Cephalopoda, Te—uthoidea, Myopsida, Loliginidae). (47°10'S; 2-3 m depth) in Santa Cruz province; and sev-
ZtoogoelnoigeisdcehreTiJearherb8li9c:h2e4r7-3A0b0t.eilung fiir Anatomic unt On- eral localities around Golfo Nuevo (~42°30'S) in Chubut
Robertson, J. D., G. M. Schwartz & P. Lee. 1993. Carbocy- province (subtidal). All collections were made during
anine dye labeling reveals a new motor nucleus in Octopus February 2000.
brain. Journal ofComparative Neurology 328:485-500. We studied more than 100 brooding females (vouch-
Stephens, R R. 1971. Histological methods. Pp. 646-649 in J. er material was deposited in Museo Argentino de Cien-
Z. Young (ed.). The Anatomy of the Nervous System of cias Naturales, number MACN 33901). Most females
Octopus vulgaris. Clarendon Press:
were brooding egg masses at advanced stages of em-
bryonic development, containing embryos and un-
cleaved nurse eggs orcrawlingjuveniles. This factcon-
Crepidula dilatata Lamarck, 1822, Truly Living in firms the presence of C. dilatata in the southern Atlan-
the Southwestern Atlantic tic, and as far as we observed, restricts C. fecunda to
Pablo E. Penchaszadeh,' Guido Pastorino.- and the Pacific.
Maximiliano Cledon' The observed material was completely homogeneous,
FCEyN—UBA—CONICET, with only one developmental mode characterized by the
' presence of nurse eggs. Each egg capsule (n = 150) con-
Museo Argentine de Ciencias Naturales, tained 203-375 eggs (mean = 303, SD = 54) with only
Av. Angel Gallardo 470 3° piso lab 57,
C1405DJR Buenos Aires. Argentina; two to 12 developing embryos, representing as an average
2.4% of the initial egg number. The average uncleaved
^CONICET,e-Mmuasile:[email protected] Naturales, egg diameter was 214 (xm (SD = 13 (Jtm, n = 72). The
egg diameterdistribution adjustedto anormal distribution
AvC.14A0ng5eDlJRGaBlulaerndoos4A7i0res3," pAirsgoenltaibna5;7, with a single mode was 212 |jLm. Hatching occurred at a
crawling juvenile stage. The egg capsule size averaged
e-mail; FrvCpaEsytoNr—@cUrBibAa,.edu.ar 3873 jjum in length and 3954 iJim in width (SD = 648 and
Museo Argen'tino de Ciencias Naturales, 53227m|mjLm,(mreeaspnec=tiv2e2lym).m)Brionodsihenlgl fleenmgatlhe,sbmuteaisnutrheids p1r1o--
Av. Angel Gallardo 470 3° piso lab 57,
tandric species the loss ofpenis can be already observed
C1405DJR Buenos Aires, Argentina mm
at 11 of shell length. Males (with a penis) measured
mm
Crepidula fecunda Gallardo, 1979, was described from 7-19 of shell length.
Bahia Chinquihue (4]°3rS-73°03'W) in the Chilean Pa- The Argentine material agrees with Gallardo's (1976,
