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1. Introduction
The following study was undertaken to see if a hatching
substance similar to that reported by Crisp and Spencer (1958)
and Barnes (1959) for Balanus balanoides is also involved in
the hatching of nauplii larvae from the barnacle Chthamalus
dalli. Balanus balanoides has only one brood a year and the
whole population release their larvae synchronously. Barnes (1962)
has reported that this spawning is correlated with plankton
blooms. Crisp and Spencer proposed that a hatching substance,
possibly a metabolic product, formed by the adult animal
in response to feeding caused this synchronous release.
Barnes (1959) suggested that the substance might be a neurosec-
ratory substance.
This type of hatching substance would be beneficial to
barnacles such as Balanus balanoides which hatch synchronous-
ly at certain times of the year. It is not at all clear,
however, that the other barnacle genera exhibit this type of
hatching and have hatching substances. Indeed, the studies on
hatching and reproductive behavior of Chthamalus dalli reported
in this paper indicate there is no synchronous hatching or hatch-
ing substance in this barnacle.
The animals used in this research were collected from various
points on the Pacific coast in the area of Monterey, California.
2. Observations on ovigerous lamellae, egg case strength, and
hatching of nauplii in vitro
a. Ovigerous lamellae
The two ovigerous lamellae of Chthamalus dalli lie
free in the mantle cavity of the adult. In the early stages
of development (from the undivided eggs to the multicellular
stage) there is a transparent membrane enclosing the whole
lamella, but this membrane breaks down in the later stages,
with only scattered remnants of it remaining visible. There
is also a breakdown of the cementing substance holding the
eggs together. The lamellae containing fully-formed nauplii
can easily be broken apart into individual enbryos with a probe
or scalpel, but the early stage embryos remain together in clumps
after such treatment.
b. Egg case strength
An experiment was done to determine whether the strength
of the egg membranes changes during the course of development.
Enbryos in the early stages of development and nauplii ready to
hatch were broken free from a lamella and placed on a slide with
a drop of water and a coverslip over the. Small weights
were then pleced on the coverslip to determine the force
needed to rupture the menbrane. For both cases it was found
that the nenbranes first ruptured when the weight was between
twenty and twenty-five grams. The rupture occurred as a
small hole in one end and was always at the anterior end
of the nauplii enbryos.
c. Hatching of nauplii in vitre
For some barnacles it has been noted that hatching
in vivo is preceeded by the fragmentation of the egg mass,
and that both hatched and unhatched larvae are extruded from
the adult. I have not been able to observe the in vivo hatching
of Chthanalus dalli, but in vitro the hatching process is
not preceeded by such a fragmentation. When the larvae break
O
3.
out, the egg cases remain cemented together in a single
clump. The outer animals hatch first, but there are usually
a large number of larvae near the center of the lamella
which do not hatch, even if left in aerated sea water for
several days. Some of these inner larvae move in the egg
cases after the lamella is first placed in sea water but
then stop.
If completely developed lamellae are removed from the
barnacles and examined microscopically, no movement can usually
be detected. But if several drops of sea water are added,
movenent begins and the nauplii soon hatch. It is not
obvious why this hatching should occur, because according to
Crisp and Southward (1956) the mantle cavity is continually
being washed with sea water.
The hatching process is preceeded by vigorous movenents
of the nauplii. The larvae appear to use contractions of
the body and pressure of the appendages against the posterior
end of the egg case to rupture the anterior end of the case
and force thenselves through. Hatching always occurs through
this anterior end and appears to cause the same type of
splitting of the egg membrane each time. The split occurs as
a snooth line running both dorsally and ventrally from the
anterior end half-way down the egg case.
3. Factors affecting hatching in
tro
a. Physical factors
A number of physical factors might affect hatching. I
studied the effects of fragmentation of the lamellae, light,
and oxygen on the hatching process. Two lamellae from the
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4
same animal were placed in artificial sea water (Harvey, 1955)
and used for each trial. One lamella served as a control and
the other was subjected to the experimental conditions.
Preliminary experiments indicated that the percentage of
eggs that hatched in a given amount of tine varied considerably
between lamellae obtained from the same animal and subjected
to the same conditions. The time taken for the first release
of larvae and the total number of larvae released per unit
time were much more constant. The reasons for this are not
clear. One possibility is that the surface areas of the two
lamellae would be more nearly the same than their surface to
volume ratio. Since the outer larvae hatch first, hatching
seems to depend on surface area, and hence the percentage hatching
would depend on the surface to volume ratio. The effect of
a larger surface area is clearly seen when the lamellae
are broken into several pieces,as the rate of hatching greatly
increases with such fragnentation.
To analyse hatching rate in the following experiments,
I used both the time of release of the first nauplii and
the nunber of nauplii which would hatch in a unit of tine.
To determine if the hatching process depends on oxygen,
one lamella of each pair was placed in deoxygenated artificial
sea water (through which nitrogen had been bubbled) and the
other lamella was placed in artificial sea water through
which oxygen had been bubbled. The hatching was compared,
and in all cases there was no hatching in the lamellae in
deoxygenated water even though hatching occurred in the cor-
responding lamellae in oxygenated water.
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In another experiment animals were disected and one
lanella was quickly put in the dark, the other being left
in the light. No differences in hatching were found.
b. Chemical factors
In these experinents I attempted to discern whether an
extract made fron grinding up bodies, shells, or tissue of
Chthamalus dalli accelerated hatching, as found by Crisp and
Speneer (1958) for Balanus balanoides. Extracts of C.
dalli
were prepared by grinding the whole animal in a teflon
homogenizer in artificial sea water and then centrifuging
to remove any particulate matter. Two lamellae were taken
fron the same animal and washed several'tines in artificial
sea viter. They were then placed on microscope slides
and a drop of extract plus artificial sea water was placed
on one and a drop of artificial sea water on the other.
Hatching in both lamellae was observed under the compound
nicroscope and no consistant differences in the time of
release of the first nauplii were noted. The nunber of
nauplii released after 30 to 60 minutes was also not sig-
nificantly different.
4. Factors affecting hatching in vivo
I collected animals on small rocks and mussel shells
and placed groups of 40 in beakers of aerated artificial
sea water. The Chthamalus extract was then added to some
of the beakers, with the remaining beakers left as controls.
The beakers were checked hourly for release of larvae, but at
no time was there any noticible difference in the number
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6.
of larvae in each beaker. After a day the animals were disected
and the percentage of animals without lamellae computed.
This method proved to be invalid, however, because the controls
differed among thenselves as much as did the experimental
populations from the controls.
A feeding experiment similar to the above extract
experiment was run at the same time to deternine whether
feeding triggered larval release as in Balanus balanoide
(Crisp and Spencer, 1958). Barnacles on rocks and shells
were placed in filtered sea water and some were fed phyto¬
plankton. These experiments were inconclusive for the same
reason as the experiment above, because an adequate control
could not be found.
5. Field work
To explain this apparent lack of factors affecting
hatching, I carried out a field study to get information
about the in vivo brooding of embryos by Chthanalus dalli
My main study was done on a vertical rock face along Cannery
Row in Monterey, California. This rock face was covered by
hthamalus
illi from 0.5 foot to 5.5 feet above the mean low
tide level. I divided this height up into eight eight-inch
zones, with zone one being the lowest. Animals were collected
from each of the zones and the percent of animals with
ovigerous lanellae was calculated. (see table 1). The stages
of the embryos were deternined under a microscope. Four
stages were considered: 1.one- and two-celled e¬
gs 2.nulti-
cellular to limb buds of nauplii 3.early nauplii 4. fully-
formed nauplii. The results of this study are in table 2,
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7.
expressed as percent of the total number of animals with
lamellae in each zone.
Examination of table 2 indicates that there is a decrease
in the number of lamellae found in the higher aninals.
Further observations on the C. dalli populations at Hopkins
Marine Station and at a point on the Pacific coast about three
niles south of Carnel, California, yielded similar results.
At Hopkins Marine Station two horizontal rock faces in the
same area and subjected to the same wave action were examined.
The lower rock was at 1.5 feet above the mean low tide and
the higher rock was at the 5 foot level. The higher C.
dalli
population had no animals with lamellae, while 93.43 of the
lower population had lamellae. At the other location I sampled
various populations, some on mussel shells at a 1 foot level,
some on rocks at 4 feet, and others on rocks at 8 feet.
The upper population had no animals with lamellae, the niddle
population had 36.73 with lamellae, and the lower one had
86.73 with lanellae.
In addition to the above work, I sought to correlate the
observed hatching behavior with the data obtained by Galen
Hilgard of Hopkins Marine Station on the yearly pattern of
dalli populations
brooding enbryos. Her data on Chthanalu
at Hopkins Marine Station is shown in figure 1. Figures 2
and 3how the plankton readings and mean shore tenperatures
for the period shown in figure 1, as regrded at Hopkins
Marine Station for the California Cooperative Fisheries
Investigation.
101
8.
6.
Discussion
Findings on possible physical factors affecting hatching
in Chthanalus dalli were similar to those obtained by Crisp
and Spencer (1958) in their work on Balanus balanoides.
The liberation of the larvae from the egg nembranes is
apparently an oxygen-requiring process carried on by the larvae.
This process is not affected by light. The nost important
physical factor affecting hatching appears to be the frag-
mentation of the egg mass, since breaking up the lamellae causes
a narked increase in the rate of hatching and the total
number of larvae which hatch.
I found no chemical substance from barnacle extracts
which affected hatching. I cannot conclusively rule out the
possibility that some chemical does affect the whole animal:
I simply could not find any way to set up an experimental
population of animals with consistent controls. It seens
as if these sane problems must have been present for Barnes
(1959) and Crisp and Spencer (1958), but no mention of any
such problem is made in their papers.
Perhaps one factor which could explain the difference
in hatching behavior between Balanus balanoides and Chthanalus
lalli is the difference in the reproductive behavior of the
two animals. The animals that both Barnes and Crisp worked
with were animals living in an environment where they only
liberated larvae in response to plankton bloons. Data on
the reproductive behavior of Chthamalus dalli, however, shows
that this barnacle posesses lamellae throughout the year,
with the exception of the late fall and early winter. There
is apparently no major larval release correlated with the
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9.
peak plankton bloon. There also appears to be no correla¬
tion between temperature and larval release during the year.
The above conparisons are tenuous without knowing the period
of larval development in vivo, which might be difficult to
obtain in a non-synchronously reproducing population.
The study done on the Chthamalus population at
Cannery Row also indicates that there seems to be no factor
synchronizing hatching. Lamellae were equally abundant at
all stages of development, indicating that developnent was
not arrested at the nauplius stage in preparation for some
environnental trigger as in B. balanoides.
One factor which I cannot explain is that the percentage
of lamellae decreases greatly with the height of the animals
above the water. Barnes (1962) found the same situation to
exist for Balanu
balanoides and postulated that it night
be due to earlier hatching triggered by a concentration of
hatching substance because of longer periods out of water or
by longer periods of time in oxygen. Neither of these
reasons seens likely for C. dalli.
In order to understand the hatching behavior of nauplii
tha
malus dalli, more data is required on the repro-
fron C
ductive and hatching behavior in the field. Laboratory
experiments vith adequate controls must also be devised.
7. Sunnary
Experiments were done with Chthamalus dalli to determine
what factors affect the rate of both in vivo and in vitro
hatching of nauplii. Aside from mechanical fragnentation of
the lamellae,which increases the rate of hatching, no
106
10.
physical or chemical factors were found to affect the rate.
From the field work done, there is no evidence to suggest
that a factor regulating the hatching is necessary.
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Barnes, H.2W. 1956. The fornation of the egg-mass in Balanus
palanoides (L). Arch
30c. 7001. Bot. fenn. Vanamo 1(1):
11-10.
Barnes, H.åW. 1958. In vitro development of cirripede eggs.
Vidensk. Medd. fra Dansk naturh. Foren. Bd. 1.
: 94-100.
Barnes, H.2M. 1959. Note on stinulation of cirripede
nauplii. Oikos, 10 Fasc. 1: 19.
Barnes, H. 1962. Note on variations in the release of nauplii
of
balanoides with special reference to the
Bilanu.
spring diaton outburst. Crustaceana, 3
: 118- 122.
Crisp, D.J. 1956. A substance promoting hatching and liber-
ation of young in cirripedes. Nature, Lond.
18: 263.
Crisp, D.J. £ Jouthward, A.J. 1956. Demonstration of small-scale
water currents by means of milk. Nature, 178: 1076.
Crisp, D.J. £ Spencer, C.P. 1958. The control of the hatching
process in barnacles. P.
roc. roy. soc. Lond. 1498:278-299.

Harvey, H.W. 1955. The Chemist
and Fertility of Sea Waters,

Cambridge University Press. p. 29.
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with lamellae in each
han
Perent of
Table 1
lus
zone of annery Rowtudy
nimals with lamellae

Zone
87
76.53
76.53
76.0
60.58
55.1
31.07
0.06
als in zone 1 heavily parasitized by Hemioniscus
ilan
with various stage
Table 2 Percent of Chthanalus da
enbryos in eaen zone of Cannery Row study
Stage 2
tage 3
e
tage
Zone
12.
14.3
21.4%
21.48
1.73
30.13
. 570
30.43
21
30.48
9.1/
21.88
31.87
11.0%
13.63
3.6%.
30.02
10.02
5.09
12.5
5.0%
33.13
2.27
22.2%
22.27
100
10


MsoNM
POUTHS
FIGURE ! -
75 oF CATHANALOS DALLI ST REPRIDS MARINE STATON OITH OVIGEROOS
LANE — FRO TD. 164 ro na 1967
100
mRMaso
HOOTHS
FiCURE X - PNYTOPLAMKTON PROFILE FOR MODTEREY BRY MEAR HoPROS
NAUNE STATIOD - NS. 1966 r0 Faxe 1757
na3
HONTHS
PIGORE 3 - HEAN SHDRE TEHPEROTTORE PROEILE EOR HOPRISS HARINE
STATOD - JO. 1966 70 MARCR 1167
2

+
0
0
tkatatatatao-
HONTRS
7 eF CATHANALOS DALLL AT ROPKISS HARINE STATIOD OTTH OUIGEROOS
PGORE !
LANEE — FRON JAN. 164 T HARE 1967
00
80

ktatatakatkaavavakaa-
HOOTHS
s  - rоrоо нооуив оов ооуеову вну ооав оооS
NOSNE STATION - TAN. 1966 ro MAReN 1767
snaaso
HONTHS
EICURE 3 PaR SHORE TENPERATORE PROFILE FeR HOPKIDS HARIDE
5TAT.OO - JAN. 1166 To MNRCR 1167
14