133
RESEARCH NOTES
strombids. The slower velocities exhibited by crown
conchs moving through Spanina could be due to
several factors. Occasionally, Spartuw stems seemed
to impede a conch's progress, but that did not seem
to be a major factor There are undoubtedly many
sources of food odour m the Spamna zone, given the
abundance of prey living there and the accumulation
of dead animals deposited there during previous
tides; this multiplicity of odour sources may lead to
inconsistent orientation and slower movement Also,
neogastropods tend to orient into the current in
response to prey and food odours, and current flow
is certainly slower and more directionally variable
amid the Spamna, which could make oriented movements less precise and slower.
I thank D. Mernll for assistance, C. N D'Asaro
for editorial comments, and staff of the St Marks
National Wildlife Refuge for their cooperation.
REFERENCES
1 UNDERWOOD, AJ 1979 AUv Mar. Biol Ecol.,
16: 111-210
2. HESSE, K O 1979 Bull Mar Set, 29- 303-311
3. MAGALHAES. H 1948. Ecol Monogr, 18 377409
4. HATHAWAY, R.R.
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6.
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10
& WOODBURN, K.D
1961
BulL Mar Sa , 11: 45-65
BOWLING, C. 1994. J Exp Mar Biol Ecol, 197
181-195
HAMILTON, P.V 1978. Mar Biol, 46. 49-58.
BATSCHELET, E 1981 Circular statistics in biology. Academic Press N Y.
MILLER, S L. 1974 J Exp Mar Biol Ecol, 14
99-156.
BERG, CJ. 1974 Behaviour, 51.274-322.
VOLTZOW.J 1986 Can.} Zool, 94- 2288-2293
] MolL Stud. (1996), 62,133-135
Biochemical composition of prosobranch egg capsules
Patricia Miloslavich
Umversidad Simdn Bolivar, Instituto de TecnologCa y Ciencias Mannas (INTECMAR),
Postal 89 000, Caracas 1080, Venezuela
The egg capsules of gastropods are structurally and
chemically complex and energetically costly."
Among their attributed functions are the protection
of the embryos against bacterial attack, predators
and physical stress. ^ Egg capsules can also enclose
extraembryonic yolk,' nurse eggs and mtracapsular
liquid with nutritive value for the embryos. The egg
capsule walls have also been suggested to be a
source of extraembryonic nutrition for the developing embryos of some prosobranchs (Thais haemastoma canaliculate Duclos, 1832 and Adelomelon
brasiliana Lamarck, 1811).1J Studies on the egg capsule walls of prosobranchs have indicated that they
are microstructurally complex and composed of
three or four layers.*101"12 Histochemical and biochemical studies have indicated that these capsules
are composed by proteins and carbohydrates and
lack Upids."113-14-^"^
A previous biochemical study earned out in eggs
and hatchlings of several prosobranch species which
feed on nurse eggs (Miloslavich, unpublished) indicated that fasciolanid species do not have enough
biochemical material (protein, glycogen and lipid) in
eggs and nurse eggs to account for the total material
in the hatchlings, proposing that some of the material comes from another source within the egg capsule. The objective of this work is to determine the
protein and glycogen biochemical value of the egg
capsules and to verify the absence of upids.
The species used m this study were the buccinids
Buccinum undatum Linnaeus, 1758 from the Saint
Apartado
Lawrence estuary, Canada, Buccinum cyaneum
Bruguiere, 1792 from the Saguenay fjord, Canada,
Engoruophos unicmctus (Say, 1825) from Isla
Canbe-Chacopata, Venezuela, the fasciolanids Fasciolana tulipa hollisten Weisbord, 1962, Fusmus
closter (Phihppi, 1850) both from Isla Canbe-Chacopata, Venezuela, and the muncid Murex brevifrons
Lamarck from Isla Canbe-Chacopata, Venezuela.
The spawn masses were obtained both in the field
and under laboratory conditions. Recently deposited
egg capsules were carefully opened and their contents emptied. The egg capsule walls were nnsed
with distilled water and capsules were placed
individually in 1.5 ml eppendorfs for biochemical
determinations.
The protein determination followed the Bio-Rad
protein rrucroassay procedure based on protein dye
binding." Bovine serum albumin was used as a standard. Samples were left overnight in 1.5 ml of NaOH
0.5 N and thoroughly homogenized with the help of
a mortar. For spectrophotometer readings, a subsample of 10 ul was used. For the egg capsules of E.
unicmctus only 600 ul of NaOH were used- to homogenize the egg capsule and 80 ul sub-sampled for the
readings.
The glycogen determination was made by a modification of the iodine binding method." The procedure consists of the extraction of glycogen with
perchlonc acid (PCA) 10% and iodine binding to
produce colour Mussel VII glycogen (SIGMA) was
used as a standard. Samples were left overnight with
134
RESEARCH NOTES
Table 1. Weight and biochemical content of the egg capsule walls (in jig of protein, glycogen and lipid per mg of humid capsule). Numbers represent mean ± standard deviation, n
» sample size.
SPECIES
Egg
capsule
weight
(mg)
PROTEIN
ngP
/mg of
capsule
GLYCOGEN
HQG
/mg of
capsule
UPID
ugL
/mg of
capsule
TOTAL
I P+G+L
ug/mg
(%)
TOTAL
I P+G+L
per
capsule
Buccinum
undatum
24.8 ±
10.4
n-9
12.9 *
2.9
n =. 20
2.6 ±
1.1
n - 18
97.4 ±
19.9
n o 11
21.1 ±
10.9
n• 4
29.1 ±
8.3
n=9
35.5 *
15.2
n = 10
6.12 *
2.5
n-3
14.3 *
2.2
n=6
11.0*
6.7
n- 2
60.9 ±
30.4
n-3
108.6 ±
27.6
n=5
27.8 *
17.0
n- 6
21.5 ±
2.0
n=3
63.6 ±
14.0
n-6
26.6 ±
4.5
n-2
5.5 *
4.1
n»2
19.0 ±
11.1
n- 4
87.6
(8.8%)
2174.1
156.6
(15.7%)
2016.0
153.8 ±
65.4
n=9
2.1 ±
C.7
n-3
15.0*
10.3
n -4
6.5*
1.0
n-2
217.0
(21.7%)
570.8
29.8
(3.0%)
2898.1
92.9
(9.3%)
2545.5
44.1
(4.4%)
2357.5
Buccinum
cyaneum
Engoniophos
unicinctus
Fasciolana
t. hollisten
Fusinus
closter
Murex
brevifrons
27.4 ±
85
n-23
53.5 ±
7.1
n-7
1.5 ml of 10% PCA, then homogenized with the help
of a mortar. Samples were then centnfuged for 2 mm
at 30,000 rpm, the supernatant was saved for glycogen determination The pellets were twice reextracted with 1 ml of PCA 10% (1J ml in the case
of F. L hollisten). The three glycogen extractions of
the egg capsules of E unicinctus were made with 600
ul of PCA 10%. For spectrophotometer readings,
the procedure described in Miloslavich and
Dufresne20 was followed.
The lipid assay was made by modifying the
sulphophosphovamlhn methods.21^2 Olive oil (10
mg/ml in chloroform) was used as a standard. Samples were homogenized using a mortar with 1.5 ml of
a mixture chlorofornrmethanol (2.1 v/v) and centnfuged for 10-15 s at 30,000 rpm. The supernatant
was saved in a dry pyrex test tube. The pellets were
twice re-cxtracted with 1 ml of the mixture. The rest
of the procedure is described in detail by Miloslavich
and Dufresne.20
The protein, glycogen and lipid content of the egg
capsules expressed in u.g of protein, glycogen and
lipid per mg of humid weight of the egg capsule of
the six studied species is presented in Table 1. The
tropical buccinid £. unicinctus presents the richest
capsules in biochemical terms. This is the only
species in this study that does not feed on nurse eggs
and in this case, the egg capsule could have a more
important nutritive role for the embryos than in
nurse egg feeding species. It is very interesting to
observe that the total protein, glycogen and lipid per
capsule is very similar between the rest of the species
even with different egg capsule weights, varying
between 2000 and 2900 ug. The results indicate that
glycogen is the most important component in terms
of quantity followed by proteins and lipids. The
exception to this trend is E. unicinctus for which
lipids are the most important component. Previous
results on the biochemical content of the spawn of
the prosobranchs Littonna obtusata Linnaeus, 1758
and Lacuna pallidula da Costa, indicated that even
when the egg masses of these species are not egg
capsules but a jelly matrix, lipids make little contribution to either egg mass."
The most outstanding result is the presence of
lipids in the egg capsules of all the species. Previous
studies on the egg capsules of the prosobranchs
Ocentbra erinacea Linnaeus," Ilyanassa obsoleta
(Say, 1822)'6 and Micella laptllus (Linnaeus, 1758)13
have indicated the absence of lipids in all capsule
layers even if the raethanol:chloroform dissolvent
was also used." This discrepancy with our results
could probably be due to an insufficient extraction
of the lipids in the chloroformnnethanol solvent Egg
capsules need the help of mechanical action in order
to completely dissolve in the solvent mixture. The
resistance of the egg capsule to go into solution in
several organic and inorganic solvents has been previously tested in B undarum."
The quantification of these biochemical substances
in the egg capsule is relevant as it estimates the
amount of protein, glycogen and lipid potentially
available for the embryos to feed on. The demonstrated presence of lipids in the egg capsule of prosobranch species is very important, as these provide a
potential energy reserve for the embryos during
RESEARCH NOTES
development and could also be participating in the
embryo-egg capsule dynamic metabolism.
I wish to thank Marie Pascal Monn, University of
Quebec at Rimouski for her help with the biochemical assays. I am also grateful to Louise Dufresne for
her assistance with the procedures and Pablo Penchaszadeh, Universidad Simon Bolivar, for commenting on the manuscript.
REFERENCES
135
9 TAMARIN. A. & CARRIKER, M.R. 1967. J. Ultrast
Res., 21:26-40
10. FLOWER, N.E. et al. 1969 / . Ultrast Res., 26 262-
273
D'ASARO. C.N. 1988. Nautilus, 102(4)-134-148.
RAWUNGS. T.A. 1990. BioL Bull., 179: 312-325.
HUNT, S. 1966 Nature, 210(5034) 436-437.
HUNT, S 1971 Comp Btochenu Phystol, 40B
37-46
15 BAYNE, CJ. 1968 Proc Malac. Soc Lond., 38
199-212.
11.
12.
13.
14.
16 SULLIVAN, C.H. & MAUGEL, T.K. 1984 BioL
1 PECHENIK, J.A. 1986. Am. Mai BulL, 4 165-172.
Bull, 167- 378-389
2. PERRON, F.E. 1981. Am. Sat., 118:110-118.
17 HAWKINS. L.E & HUTCHINSON, S. 1988. J. Exp
3. DESLOUS-PAOU,
Mar BioL Ecol, 119 269-283
18. BRADFORD. M 1976 Anal Btochem , 71(1/2)
248-254
19 DREIUNG, C.E et al. 1987. Meat Sci.. 20-167-177
J.M
&
HERAL,
M. 1986
OccanoL Ada, 9: 305-311.
4 PECHENIK, J.A. 1979 Am. Nat, 114:859-870
5 PECHENIK, J A. 1982. / Exp. Mar BioL Ecol, 63
195-208.
6 PECHENIK, J A. 1983 J Exp Mar BioL Ecol., 71
165-179
7 ROLLER, R.A. & STICKLE, WB
1988. Am.
Make. Bull, 6.189-197
20. MILOSLAVICH, P. & DUFRESNE, L. (m press)
Can. J. Fish Aq SCL
21. BARNES, H & BLACXSTOCK, J 1973. J Exp Mar
Biol Ecol, 12:103-118.
22. BLIGH, E.G. & DYER, W J
8 DE MAHIEU, G. et al. 1974. Can. Biol. Mar, XV
215-227
1959. Can. J
Biochem. Physiol.,31 911-917
23 GOODWIN, BJ 1979.7 Moll Stud,4S 1-11.
J. Moll Stud. (1996), 62,135-137
Sexual differentiation of Crassostrea tulipa in two contrasting brackishwater
environments
Kobina Yankson
Department of Zoology, University of Cape Coast, Ghana, West Africa
The West African mangrove oyster, Crassostrea
tulipa (Lamarck, 1819) provides a cheap source of
protein for many coastal communities in the
region IJU Other benefits derived from the species
include the use of the shell in poultry and building
industries and in traditional medicine' Breeding of
the species has been found to be continuous in open
lagoons but seasonal in estuaries.4"" The Lagos Harbour population of the species (referred to as
Cryphaea gasar Adanson) has been reported to differentiate sexually through 'male', 'intermediate'
and 'female' phases.9 Preliminary observations on
samples from Ghanaian waters, however, showed a
deviation from this pattern Since the only available
report (to my knowledge) on the sexual differentiation of the species is that of Sandison,5 tt was deemed
necessary to undertake a histological analysis of the
gonads of juvenile oysters as part of an extended
study on the biology of the species in Ghanaian
waters.
Specimens were collected in May 1992 from two
brackishwater bodies (Benya Lagoon and Pra Estuary) located between latitudes 5° and 5° 30T^ and
longitudes 1° and 2°W. The lagoon is of the 'open'
type and hence subjected to the daily tidal influence
of the adjacent sea. while the estuary is a 'positive'
one formed by a moderately large nver (Pra). The
period of sample collection coincided with the beginning of the wet season (May-July) which also
marked the end of oyster spat settlement in the estuary.4 Oysters measuring between 5 and 40 mm in
shell height (from the umbo to the ventral shell margin) and not more than 4 months old were fixed in
Bouin'j fluid within 24 hours of collection. They
were subsequently processed by standard histological techniques before sectioning (6-8 urn) and staining with Ehrlich's haematoxylin and eosin Four
'serial' sections were made from each oyster and
examined microscopically at magnifications ranging
from 50 to 400 to determine their sex and level of
gonadal development. Slides from 386 and 154 juvenile oysters from the lagoon and estuary, respectively, were analysed.
Oysters with no traces of gonadal material in the
visceral mass were classified as 'undifferentiated'
The smallest identifiable male oysters measured 8
mm and 14 mm in the lagoon and estuary respectively, while their female counterparts were 10 mm
and 19 mm. again respectively (Fig. 1). The proportion of females increased with size in both popula-