Abotract
Different methods of inducing spawning in the gastropod Achea
incessa were investigated. No reliable method was found that could
be used as a generalized technique for mollusc spawning. To stain the
chromosomes of the eggs and developing zygotes, used a method that
required preliminary fixation in carnoys solution followed by DAPI
stain. The developing stages of the zygote chromosomes immediately
l17
after fertinzation up until the first cleavage are documented.
Introduction
Oogenesis, fertilization, and development of the embryo has
been studied extensively in echinoids (Strathmann, 1987 ). Part of
sse gametes have been used is the ease with which they
theresonth
can be studied There are standard methode for constanthy inducing
spawning and large amounts of gametes can be obtained. Also, the
eggs are transparent enough to get a clear view of the internal
ages of developr
organization of the egg during the first
ent

Molluscan egas have been studied little in the past because
working with the animaje and the eggs ie difficult. There are no

phabeme
hods for inducing spawning. Also, the egge are coaqde and


d
n
inquishable
theic

+
norder to learn more about spawn

zyocte development in molluscs,have used th
haveteted various epawning techniquee and aleo


egaed differenth

for
omosome aining proceud
i
obtained directions from Dr. Daniel Mazia and Dr. Linda Goff. With the
tools for chromosome staining,  have determined the nuclear
cytology of eggs and the early stages of developing zygotes.
Materials and Methods
Handling of gametes:
Acmea incessa were collected at the shorline in Pacific Grove
and Carmel California and kept on fronds of Egregia menziesi in an
outdoor running sea water tank. The tank was covered with a screen
mesh which diffused the bright afternoon sun but allowed enougn
illumination to keep the Egregia in good condition without allowing
the water in the tank to become too warm. In compliance with Sharon
Proctor 's thesis (1968), to induce spawning of the animals, six to
nine individuals were removed form the tank and placed in separate
glass dishes filled with fresh sea water that subsequently was
allowed to slowly reach room temperature (between 18eand 22 0.
As soon as a female animal spawned, the eggs were removed with a
Pasteur pipet and placed in filtered sea water (FSW) in a 16C cool
room. Spawned eperm was similarly removed but placed immediately
into a6Ccold refridgerator. To induce increased gamete release

fter an animal had apawned, some streams of water were auned at
the foot or the dish was jiggled.
Eggs were fertilized within one and a half to two hour
wring by adding a few drope of dilut sperm to the egg-row
mixture. Sperm stored for up to two days at 6 C could still be used
for fertilization.
In sgme cases, eggo were aloo acquired by disecting females in
ammonia sea water (sea water titrated to pH 9.5 with MOH). The
ammonia sea water causes gernimal vesicle (GV) breakdown. while
holding the animal in the ammonia sea water, an incision was made in
the gonad and oocytes flushed out of the opening. The released
oocytes were allowed to settle to the bottom of the dish and the
excess sea water was removed by aspiration. The eggs were then
placed in fresh ammonia FSW and allowed to sit for one to two hour
until GV breakdown which is necessary before normal fertilization
çan occur. After GV breakdown and before fertilizing, the eggs were
transferred to normal sea water.
Fixing and staining techniques:
Eggs suspended in FSW were centrifuged, the sea water
aoys fixative
remoyed by aspiration, and the eggs resuspended in Carii
(Jethapol; 1 acetic acid). After and hour of fixing in carnoys, the
eggs were washed in a 707 ethanol solution and recentrifuged. The
banol was removed by aspiration and the remaining
ces were
allowed to evaporate. The eggs were resuspended in FSW and
4,6-diamidino-2-phenylindole (DAPD) stain was added to make a final
concentration of Sugimi (Linda Goff, 1962)DAPI (Sigma Corporation /
wes in astock solution of 1ma/mi in dimethylsulfoxidet OMSO). After
fifteen minutes of staining, the eggs were viewed under a
fluoreceence mierescepe witha UV filter to determine the
cytological state of the chromosomes.
To examine fertilized eggs by the DAPI procedure thee

ronase (Sigma Corporanon) before fixation
we
ted wit
remoye excess sperm. Sixty ml of pronase stock (Img/mi) was added
to every Iml quantity of egg-FSW solution. Usually pronase stock was
added to egg-FSW mixture four minutes after insemination (in one
case at two minutes). The fertilized eggs were suspended in
propase-FSW mixture for one to two minutes and gently shaken. The
fertilized eggs were then washed twice with FSW to discard the
pronase.
Other fixing and staining techniques
Carpoys was also used without DAPI. Eggs suspended in F5w
were centrifuged and the FSW removed by aspiration. The eggs were
resuspended in carnoys solution for one hour before being washed
with 458 acetic acid. The eggs were viewed under a phase
microscope.
Adetergent-extacted-DAPl prdocedure was also Used to
visualize the chromosomes of the egg (Dr. Daniel Mazia). Eggs were
centrifuged in ESW and the pellet resuspended in KGE solution of ph
6.8 (29 potassium gluconate, 306M glycine, 2mM potassium ethylene
glycol bis-(B-aminoethylether) N,N,N,N-tetraacetic acid,VEGIA
MA MaSO Stock solution of EGTA is dissolved by titrating with For

to about pH 6.5). The eggs were centrifuged again and restien
suspended in
detergent-DAPI soluticn (made with ten volumes of FGE-pH58 and
one volume 103 Triton X-100). Then DAPI was added to make a final
concentration of lugml The staineg ec
reviewed

fuorescent microscope.
Reoulto
Spawning:
Spawning induced by placing individuals in warm sea water was
incopsistent. Often times no spawning would occur at all while at
other times, every individual in the dishes would spawn. My
impressions of the phenomina were that Acmea incessa released
gametes more often if the animals were used on the same day of
collection than if they had been stored in the outdoor tank for a few
days before being used.
Disection:
The limpets that did not spawn were saved for disection. 10
distinquish between male and female gonads, the animal was cut out
of it'sshell. On the dorsal surface, aside the visceral mass, the gonad
was visible. The male gonad appeared orange with smalk creamy
white dots. The femal gonad appeared brown.
The disected female gonads provided eggs with intact GVs. he
GV broke down after an egg sat in ammonia FSW pH9.S for one to twe
hours. Out of four separate attempts at fertilizing disected eggs,

only one attempt was successful.
The disected male gonads provided mostly nonmotile eperm. A
few procedures were tried in order to induce motility. Asmall drop
of EDEA PH 8.0, a crystal of cysteine, and a small drop of spawned
sperm supernatant were all separately added to large drops of
disected sperm on microslides and viewed under a dark field
microscope. All of these procedures produced negative resulte.
One more method of spawning induction was tried. Ten mM
gamma amino butyric acid (GABA) was injected into the foot. The
response was foot flexion and inability to grip the glass dish. No
spawning occured.
Light microcope observations:
Spawned unfertilized eggs appeared opaque and orange-brown
under the phase microscope. No GV was present and internal
structures were not easily visible. A jelly coat surrounded the eggs
but was not apparent unt il sperm was added. The coat was made
apparent by the clear separation between the surrounding sperm and
the egg surface.
Eggs obtained by disection had a jelly coat but also had a GV
that was seen with dark-field. With this illumination, the GV was
seen as a white-orange circle inside the darkere colored egg. Äfter
GV breakdown, the eggs obtained by disection appeared identical to
spawned eggs.
Fixation and staining techniques:
The carnoys-DAPI staining technique provided the best
ar behavior. The chrome
visualization of chromosomes and nucle
and sperm DNA were seen in great detail as fluorescent blue
t

uctures under the fluorescent microscope. Chromosome?
waseesiettoeithe sarpeofeg ore had acover p
and the egas were flattened by pressing againet a paper towe
Carnoys alone (no DAPI or other dyes) did not make the
chromosomes as visible as the carnoys -DAPI technique. Carnoye
fixed the egg and the chromosomes appeared black under phase
microscopic illuminations. However, the yolk granules and other
cellular structures also appeared black, resulting in difficulty in
finding the chromosomes.
The detergent-DAPI procedure stained the chromosome
similarily to the carnoys-DAPI procedure. However, the fine
structure of the chromosomes was not as apparent, appearing as
plurred fluorescent dots.
Nuclear events.
The carnoys-DAPl technique was used to visualize the egg and
sperm chromosome structure before and af ter fertilization. Portions
of pewly fertilized eggs were fixed with carnoys every five minutes
up to the first cleavage. Unfertilized eggs were found to be in
rested metaphase IFigure 1). They had not yet gone through the
mejotic divisions that produce poiar bodies. Within five minutes
after
perm addition, cells had bare ly begun to enter anaphase
(Figure 2). Between five and fifteen minutes after fertilization, the
zygote had formed one polar body (PB) which stayed intact at the edge
of the zygote (Figures Jand 4). At twenty five minutes, the meiotic
apararus of anaphase Il occured next to the site where the firet PE
asformed (Figure 5). Between twenty-five and thirty-five
minutes after fertilization, the second PB had formed and remained at
the outer edge of the zygote next to the first PE (Eigure eE
minutes after fertilization, the zygote was already dividing (Figure
7) The mitotic events must have been rapid, as they were missed.
sed DNA was visible as tear-shaped, lobes on each side 04
Deconder
the dividing zygote (Figure 7). The two PB's were still attached to
the egg and were almost always located at the site of cleavage
(Figures 7 and 8).
Discussion
Acmea incessaindividuals proved to be a difficult species with
which to work because all attempts to induce spawning wer
upreliable, Disection provided large quantities of gametes, but they
were usually not usable. The sperm was always nonmotile and the
eggs did not ferilize successfully. There was really no good method
of obtaining gametes. With the gametes that were obtained, however,
the chromosomes were successfully fixed and stained with the
carnoys-DAPI preparation. Using this staining method,was able to
account for the cytological changes in the zygote chromosomes up to
the first cleavage.
As already mentioned, there was a correlation between
spawning frequency and the day of collection. It is my impression
that the animals kept in the tank spawned there and resultant ly did
not have any more ripe gametes ready for release when later
subjected to spawning induction methods. This hypothesis is fürther
tthe disected gametes from individuals t
upported by the fa
whpaturally were not functional (except one ce
would not sp
—heresome dicetedegeeid ferilze).
at some hormone other than GAEA may activate
tispiet
t

jon procees.
y
gsor speed up the matt

theeg
for Acmea incessa spert
Since there is no known method of inducing spawning reliably,
suggest that the best way of obtatning functional gametes is to
induce spawning by letting the animals sit in warm sea water on the
same day that the animals are collected. Also, the water that they
are transported in from the intertidal zone should not be allowed to
become warmer than 16'C.
Spawned eggs were opaque orange and contained no GV contrary
t
to findings by Sharon Proctor (1968). The eggs had a jelly coat diat
was shed after fertilization.
Eggs obtained by disection clearly had a GV before spending one
to two hours in ammonia sea water at pH 9.5. The eggs also had a
jelly coat and not a thick chorion as Sharon Proctor reported (1968).
Nuclear evente
Carpoys-DAPI was the most lucrative method of fixing and
staining Acmea incessa eggs and zygotes. Using this method, the
events of egg maturation after sperm entry were visualized cleary.
Unfertilized eggs were in arrested metaphase as reported by wilson (
1919). After insemination, I found that two polar bodies were formed
efore the nuclear fusion of the sperm and egg. However, did not fix
any eggs at the correct time to capture the mitot
t

ore detailedaccount ofthenucleare
btan a
T00
e zygotes could be fixed at narrower time inter
fertilization th
thanfive minuteshecarnoys-DADtechnige eeprege
reliable method for future cytological studies on damea incessa.

ed staining
my could be a lucrative study using th
Poly
jethod. However, without a more reljable methe
d for obtaining
gametes, do not recommend using Acmea incessa as a research
subject.
References
Dube.F., Dufresne-Dube,L., and Guerrier, P.C1982). Sperm Nuclear
Decondenstion in Barnia candida (Mollusca, Pelecypoda) Oocytes
does not require germinal vesicle breakdown. JExp. Zoology
9
:383-387.
2
Goff.L., (1988). Carnoys-DAPl staining. Personal reference.
Hipkley, R.E., wright, B.D., and Lynn, J.W.(1986). Rapid visual
detection of sperm-egg fusion using the DNA-specific
77
fluorochrome Hoechst 55942. Devel. Bio. 118,148-154.
Kanatani,H. (1985), Oocyte growth. Oocyte maturation. Biology or
0
Fertil
tion vol.1,225-240.
Mazia.D. (1988). Detergent-extraction-DAPI staining methods.
Personal reference
Proctor.S. (1968). Studies on the stenotopic marine limpet Aced
incessa (Mollusca, Gastropoda, Prosobranchia) and it's host Egregla
menziesil (Phaeophyta) Thesis.
per
oment of
ann (1987). Beproduction and Develof
a
Strathm
rthern Pacific Coast.
ofthe
invertebra

ent
Wilson,E. (1919). Ihe Cell in Developinenand in
Figure Legendo
Figures:
1. Lwo upfertilized eggs with the chromosomes visibly in metaphase.
2. Five minutes post-insemination, the sperm nucleus is seen as the
smaller dot. The egg nucleus is just beginning to enter anaphase !.
3. Ten minutes post-insemination, the egg nucleus has already funly
entered anaphase 1 of mejosis to form the first polar body.
4. Fifteen minutes post-insemination, the eggs nucleus has a formed
polar body.
S. Twenty-five minutes post-insemination, the egg nucleus is seen
in apaphase Il of meiosis. The chromosomes divide next to the
site of the first meiotic cleavage. The smaller spot is the sperm
nucleus.
6. Thirty-five minutes after insemination, two polar bodies are seen.
Sixty minutes after fertilization, mitosis is already complete as
can be seen here where two lobes of decondensed DNA have
recently separated The third spot is the combination of two polar
bodies out of focus.
9. Seventy minutes post-insemination, cleavage is finished and the
two polar bodies are seen at the site of the cleavage.
Figure 1
Figure 2.
12
Figure 3
Figure
13
Figare 5
Figure .
14
Figare
Figure 8.
15