ABSTRACT
This study focused on the reproduction and development of
three articulate brachiopods to enable rearing of young in the lab
which could later be used for transplant studies in the deep sea. The
three brachiopods studied are Terebratulina crossei, Laqueus
californianus var. vancouverensis and Laqueus californianus.
Specimens of each were collected from Monterey Canyon using a ROV
and maintained in chilled aquaria. Fertilization was accomplished by
stripping gonads from both sexes, macerating them through Nitex
and combining them in vitro. Successful fertilization of both T.
crossei and L. c. vancouverensis was achieved at 5°C and embryos
were raised to the larval stage.
INTRODUCTION
Most of the early work on deep sea species involved taxonomic
and distributional studies. Over the last few decades, scientists and
engineers have developed more sophisticated methods of studying
and sampling the organisms living in the deep ocean. Currently,
submersibles and remotely operated vehicles (ROVS) enable
scientists to observe deep sea organisms in their natural
environment and collect them unharmed for lab experiments such as
this one.
This study focuses on reproduction and development in three
articulate brachiopods: Laqueus californianus, from the continental
shelf, and Laqueus californianus var. vancouverensis and
Terebratulina crossei, from the walls of the Monterey Submarine
Canyon. Only one published study of deep sea brachiopods has ever
focused on reproduction and development (Rokop 1977);
reproduction and development in these three species has never been
the subject of a published paper.
It is possible that brachiopods will be used as model organisms
to raise to settlement to provide juveniles for experimental
transplant studies to various deep sea habitats. Brachiopods are
ideal for this purpose because of their abundance in the deep sea and
their ability to survive in aquaria. Furthermore, they are sessile
organisms, have a short developmental cycle and different species
are variable in abundance with habitat and depth.
The aim of this study was to develop methods for in vitro
fertilization of eggs and rearing them through to settlement. L. c.
vancouverensis and T. crossei were successfully fertilized in vitro
and reared to swimming larvae.
MATERIALS AND METHODS
Specimens were collected from Monterey canyon between
October, 1992 and May 1993 using a ROV (Remotely Operated
Vehicle) operated from the Monterey Bay Aquarium Research
Institute research vessel. L. californianus was found at a depth of
100-200 m, with total cover of its substrate, often attached to each
other in large chains. L. c. vancouverensis was found at a depth of
500-700 m and T. crossei at depths of 350-700m, in clusters and
small groups respectively. The boundaries of Laqueus and
Terebratulina overlap, and have been found together. This has been
seen in previous studies as well (Tunnicliffe and Wilson 1988)
Specimens were identified using a key by Bernard (1971) and
confirmed with a guide by Hertlein and Grant (1944). All specimens
were maintained in aquaria with chilled recirculating sea water at
approximately 5 °C until used.
Preliminary sexing of individuals was accomplished by
squeezing the brachiopod gently at the lateral sides to open the shell
a few millimeters and peeking through the gap to visually inspect
the gonads lining the mantle cavity for the presence of eggs. The
female’s gonads were full, fluffy and soft-looking compared to the
male’s. To confirm the gender, the specimen was squeezed again,
and fine forceps were used to remove a small piece of gonadal tissue
for viewing under the compound microscope. Males were identified
primarily by the absence of eggs, but it was possible to see sperm as
well under higher magnification. Upon positive identification of sex,
the individual was opened by inserting a scalpel through the gap and
severing the diductor muscle from the ventral valve. The two valves
were then easily pulled apart to reveal the gonads lining the mantle
cavity of both valves.
Once opened, a female’s gonads were macerated through 247
Um Nitex and allowed to soak in 300-450 mL of sea water. Once the
eggs settled to the bottom of the beaker most of the supernatant sea
water was decanted and replaced with fresh sea water. At this time,
fecundity measurements were taken by first stirring the egg water
to ensure an equal distribution of eggs. Several aliquots of known
volume were removed from the beaker (also of known volume) and
the eggs were counted under a compound microscope.
The eggs were allowed to soak for 10-15 hours to facilitate the
loss of the follicle cells. Then the male's testes were removed and
placed in 15-25 mL of sea water brought to pH 9.0 with NHAOH. The
gonads were macerated and after about 15 minutes a sample of the
sperm solution was checked under 450X magnification for sperm
motility. Once motility was observed, the sperm solution was added
to the egg solution and stirred to facilitate fertilization. In most
cases, the water over the eggs was decanted and replaced an hour
after fertilization to prevent polyspermy.
Following fertilization, samples of eggs were viewed
periodically under a compound microscope to check for cleavage.
The water over the fertilized eggs was refreshed daily by decanting
it through a piece of 100 um Nitex to avoid pouring off embryos or
larva.
RESULTS
The fecundity and egg size of each of the three species
examined is compared in table 1.
Size distribution by ventral valve length of the males and
females of each species used in the study is given in figures 1 and 2.
With the small data set used to calculate the fecundity values,
there was not enough information to determine a correlation
between the ventral valve length and fecundity.
Table 2 compares the observed developmental times of T.
crossei and L. c. vancouverensis to other known species.
Terebratulina crossei
See table 3 for the environmental conditions of each of the
fertilizations of T. crossei.
The white to slightly yellowish gonads of T. crossei are often
visible through the shell. There are two branches on both the dorsal
and ventral valves, the dorsal branches being slightly smaller than
the ventral branches. The branches are lacy looking and somewhat
bushy (see figure 3). No unripe gonads were encountered and it
appears to be impossible to determine sex by external
characteristics.
Successful fertilization of T. crossei was achieved on three
occasions. In each case, the eggs were soaked prior to fertilization
for a period of 14 h - 14 h 15 min. and were fertilized and incubated
at a temperature of 5 °C. Figures 4-6 illustrates the various stages of
development observed.
Most unfertilized eggs were predominantly opaque with a
bumpy membrane surrounding them and a small polar body
protruding from one side. However, a few had a dark center
surrounded by a clear sac. Because it was impossible to remove all
sperm by decanting after one hour, it was also impossible to know
exactly when any specific fertilization took place or how long it took
the fertilized egg to reach any given stage of development. It was
determined that the first cleavage occurred sometime before 10
hours post fertilization, however, no observations were made
between 0 and 10 hours. The morula stage was first observed at
12.5 h and the embryo developed into a blastula by 36 h. The
embryos developed into tri-lobed, counterclockwise-rotating larva
around 164 hrs after fertilization. Mature larvae were seen 200
hours post fertilization.
It is interesting to note that the bumpy membrane first
observed surrounding the unfertilized egg was present until the free
swimming stage. However, as development progressed the
membrane seemed to diminish in thickness. Perhaps a related
observation is that when bacteria infestation occurred, the blastula
seemed much more susceptible to attack and subsequent degradation
than did the unfertilized eggs.
Laqueus californianus vancouverensis
See table 4 for the environmental conditions surrounding the
fertilizations of L. c. vancouverensis.
The gonads of L. c. vancouverensis line both valves with two
parallel branches on each side of the ventral valve and one branch
on each side of the dorsal valve (figure 3). In general, females
gonads are salmon or pinkish while males are paler and beigish, but
color is not a reliable indicator of sex. Ripe gonads are plump and
somewhat fluffy looking whereas immature, or spent gonads are
brownish and pencil-thin. In some cases it is possible to see the
gonads through the shell, but this is not always true.
Fertilization of L. c. vancouverensis was attempted over a
range of egg soaking times (12 h 5 min. - 15 h 45 min.) and
temperatures (2 °C - 11 °C). However, successful fertilization seemed
to occur only when the water temperature was maintained at 5 °C
from egg maceration through fertilization and development. In one
successful fertilization the eggs were soaked for 13 h 15 min. and in
the other for 15 h 45 min. In the first case, embryos at the 2- and 8-
cell stages were observed 23 h 45 min. and 26 h 15 min. after
fertilization respectively. In the second case, free swimming larvae
were observed and photographed approximately 100 h after
fertilization, yet, no earlier observations of fertilization were made.
Figures 7-9 illustrates the various stages of development observed.
Three larvae were observed at 100 h. One swam freely and
quickly throughout the water sample, a second moved little but
continually butted its apical lobe against the side of the dish and a
third possessed motile cilia but seemed to be in the pre-setae stage.
In several of the fertilization attempts, structures resembling a
loosely held together blastula were observed. Although these
structures appear to be slightly irregular and ill-formed, there is a
slight possibility that they are in fact blastulae, however, as of yet,
none of them has been followed and observed to develop to the
larval stage.
Laqueus californianus
Table 5 presents the environmental conditions surrounding the
fertilization attempts of L. californianus.
The gonads of L. californianus were quite similar in
appearance to those of L. c. vancouverensis but tended to be more
deeply or intensely colored. Furthermore, the gonads of several of
the specimens were blotchy in appearance: thick in some areas and
pencil thin in others (figure 3). Finally, the eggs seemed quite small
and immature compared to those of the other species, and the
literature values for Laqueus. All of the fertilizations attempted with
these specimens were unsuccessful.
DISCUSSION
Most articulate brachiopods, including those studied here, are
dioecious. In previous studies, males and females have been shown
to exist in a 1:1 ratio (James et al., 1992). Some articulates are free
spawning, while others brood their larvae. L. californianus has been
shown to be free spawning (Reed 1987), as is Terebratulina retusa
(James et al. 1992) while Terebratulina unguicula (Long 1964) and
Terebratulina septentrionalis (Conklin 1902) have been shown to
brood their larvae inside the lophophore. In either breeding
strategy, articulate brachiopods produce demersal lecithotrophic
larvae, which have a brief free swimming phase before
metamorphosis.
Spawning has rarely been observed in brachiopods, and the
natural cues for spawning remain unclear. However, in some species
spawning can be induced by agitation, slight elevation of water
temperature, or the addition of sperm suspensions (James et al.,
1992, Long 1964). Gametes are released through the metanephridia
into the mantle cavity and expelled by an exhalant current of the
lophophore. This fertilization strategy is effective because most
articulates occur in dense patches, sometimes even attaching to one
another’s shells. Females of brooding species release their eggs into
the mantle cavity. Sperm are believed to be taken in through the
inhalant current of the lophophore, and the fertilized eggs are
maintained in a brood pouch or among the tentacles of the
lophophore (Long and Stricker 1991).
Once fertilization occurs, cleavage is holoblastic, nearly equal,
radial and somewhat asynchronous (Long and Stricker 1991).
Cleavages beyond the 8-cell stage in articulates are often irregular
and asynchronous (Conklin 1902). Despite irregular cleavage, these
tend to develop normally, becoming blastula and eventually, free
swimming larvae. The mature larvae of articulate brachiopods are
tripartite, possessing an apical lobe, a mantle lobe, and a pedicle lobe.
When a suitable substrate is found, a mature larvae begins the
process of metamorphosis (Reed 1987). Suitable substrates include
the shell of an adult brachiopod, the shell of bivalve mollusks, or the
tube of the polychaete Sabellaria cementarium. Metamorphosis
involves the reversal of the mantle lobe and the attachment of the
pedicle lobe to a suitable substrate (Long and Stricker 1991).
Metamorphosis may also be induced by placing mature larvae in
high K+ sea water (Freeman 1993). Within 24 hours of
metamorphosis, the mantle has secreted a bivalved protegulum, and
within 4 days post-metamorphosis, the beginnings of a new shell
have begun to form around the edges of the protegulum. (Stricker
and Reed 1985).
Terebratulina crossei
T. crossei was relatively easy to fertilize in vitro and
developed at a reasonable pace to the blastula stage when it
suddenly seemed to cease development completely. However, after
approximately 164 hours several rotating larva were finally
discovered. Although the reason for this apparent slowing in the
developmental process is unknown, it may be related to the fact that
some species of Terebratulina are known to brood their young (Long
1964, Conklin 1902). If T. crossei is one of these species, the female
may hold the developing embryos in her brood pouch for several
days, protecting them from the harsh deep sea environment and
releasing them only when they reach the larva stage. Whereas, non-
brooded embryos may need to mature more quickly in order to
survive in the open ocean, brooded embryos would experience less
environmental pressure to develop quickly and would thus be
expected to develop more slowly. This phenomenon is seen in
another brooding species Hemithyris psittacea, which takes 187 hour
before setae develop on the mantle lobe, and a full 200 hours elapse
before metamorphosis is initiated (Long 1964)
The fact that T. crossei was observed at nearly all major stages
of development from fertilization to larva is quite significant for
several reasons. First, it is quite reasonable to believe that T. crossei
could be successfully raised in significant numbers in the lab and
used as a model organism for the study of environmental processes.
Further research would need to focus on inducing metamorphosis
and on increasing the fertilization rate. Secondly, if T. crossei is a
brooding species, these results show that brooding species do not
necessarily need to be brooded in order to develop; they can be
raised successfully in vitro.
Laqueus californianus vancouverensis
One difficulty of studying the development L. c.
vancouverensis in the lab was its extremely low rate of fertilization,
perhaps 1% at best. Thus it was difficult to determine whether a
sample containing no cleaved eggs indicated that the fertilization was
unsuccessful or that the few eggs which did cleave were still in the
beaker rather than the sample. Nonetheless, examination of the
experimental conditions surrounding the two fertilizations known to
produce embryos gives some indication of what is necessary for a
successful fertilization.
Both of the successful fertilizations were carried out in water
that remained at 5 °C from egg soaking through early development.
Furthermore, all of the fertilizations believed to be unsuccessful were
carried out at temperatures either slightly higher or lower than 5 °C
(e.g. 7 °C, 10 °C, 2 °C). Therefore it seems that the fertilization of L. c.
vancouverensis is very temperature sensitive.
One result of carrying out fertilization in such cold water is that
development begins much more slowly for L. c. vancouverensis than
for other warmer water species (table 2). However, observations
indicate that L. c. vancouverensis "catches up" with its warmer water
counterparts and reaches the larval stage around the same time as
Terebratalia transversa and Terebratulina unguicula (Long 1964).
Egg soaking time is believed to be a second factor in
vancouverensis fertilization. Although the range of acceptable
soaking times is likely broader than these results indicate, eggs
soaked for approximately 13-16 h prior to fertilization are known to
be viable. Eggs which are stripped from the specimens (as opposed
to being spawned) seem to require this amount of time to shed their
follicle cells.
Further studies with L. c. vancouverensis should incorporate a
better system for checking for fertilization (e.g. petri dish under a
high power dissecting microscope). If fertilized eggs could be
separated early on in the developmental process, they would be
much easier to follow.
Laqueus californianus
The spawning season of L. californianus is unknown however,
all the L. californianus eggs observed were quite small and
irregularly shaped, thus it is believed that the specimens in captivity
had spawned prior to collection. Thus, most oocytes remaining in the
gonads were immature and not yet capable of participating in
successful fertilization. Another problem could have been that all
fertilization attempts were attempted at 5 °C, while L. californianus
lives at temperatures of 9 or 10 °C.
Of the three species studied, only L. californianus seems to be
a poor candidate for deep sea studies. This is partly due to the fact
that it is generally found in shallower water than the other two
species and partly because fertilization attempts were unsuccessful.
Perhaps, if ripe oocytes were found in L. californianus, it too could
have also been successful. Both T. crossei and L. c. vancouverensis
show potential as model organisms for the study of larval settlement
and distribution. T. crossei were easy to fertilize and achieved a fair
fertilization rate and a good yield of larvae. L. c. vancouverensis is
more common in the Monterey Submarine Canyon, more easily
sexed, and reaches the larval stage more rapidly. Keeping the
characteristics of each species in mind and finding ways to increase
the rate of fertilization and decipher the potential bacteria problem,
both of these species could prove valuable in a variety of studies
exploring the processes and interactions which occur in the deep
ocean.
LITERATURE CITED
Bernard, F.R. (1972). The living Brachiopoda of British Columbia.
Syesis, 5, 73-82.
Conklin, E.G. (1902). The embryology of a brachiopod, Terebratulina
septentrionalis Couthouy. Proceedings of the American
Philosophical Society, 41, 41-76.
Freeman, Gary. (1993). Metamorphosis in the brachiopod
Terebratalia: Evidence for a role of Calcium channel function and
the dissociation of shell formation from settlement. Biological
Bulletin, 184, 15-24.
Hertlein, L.G. and Grant, U.S. (1944). The Cenezoic Brachiopoda of
Western North America. Publications in Mathematical and
Physical Sciences, 3, 1-236, 21 pl.
James, M.A., Ansell, A.D., Curry, G.B. (1991). Reproductive cycle of
the brachiopod Terebratulina retusa on the west coast of
Scotland. Marine Biology, 109, 441-451.
James, M.A., Ansell, A.D., Collins, M.J., Curry, G.B., Peck, L.S., and
Rhodes, M.C. (1992). Biology of Living Brachiopods. Advances in
Marine Biology, 28, 175-387.
Law, R.H. and Thayer, C.W. (1991)
Articulate fecundity in the
Panerozoic: Steady state of what? In "Brachiopods Through
Time" (D.I. McKinnon, D.E. Lee and J.D. Campbell, eds.), pp 183-
190. Balkema, Rotterdam
Long, J.A. (1964) The embryology of three species representing
three superfamilies of articulate brachiopoda. Ph.D. Dissertation,
University of Washington.
Long, J.A., and Stricker, S.A. (1991). Brachiopoda. In "Reproduction
in Marine Invertebrates, Vol. 6 Echinoderms and
Lophophorates. (A.C. Giese, J.S. Pearse, and V.B. Pearse, eds), pp.
47-84. Boxwood Press, Palo Alto, CA.
Reed, C.G. (1987). Phylum Brachiopoda. In "Reproduction and
Development of Marine Invertebrates of the Northern Pacific
Coast. (M. Strathmann, ed.), pp. 486-493. University of
Washington Press, Seattle.
Rokop, F.J. (1977). Seasonal Reproduction of the Brachiopod Frieleia
halli and the Scaphopod Cadulus californicus at Bathyal Depths in
the Deep Sea. Marine Biology, 43, 237-246.
Stricker, S.A. and Reed, C.G. (1985) The ontogeny of shell secretion in
Terebratalia transversa (Brachiopoda: Ariculata) II. Formation of
the protegulum and juvenile shell. Journal of Morphology, 183,
251-271.
4
Tunnicliffe, V. and Wilson, K. (1988). Brachiopod populations:
distribution in fjords of British Columbia (Canada) and tolerance
of low oxygen concentrations. Marine Ecology -Progress Series,
47, 117-128.
Figure Legend
Figures
Figure 1 - Male Size Distribution by Ventral Length
Figure 2 - Female Size Distribution by Ventral Length
Figure 3 - Shell and Gonad Morphology of Terebratulina crossei,
Laqueus californianus, and Laqueus californianus
vancouverensis
Figure 4 - Developmental Stages of Terebratulina crossei
Figure 5 - Terebratulina crossei blastula and early free swimming
larva
Figure 6 - Terebratulina crossei late and mature free swimming larva
Figure 7 - Developmental Stages of Laqueus californianus
vancouverensis
Figure 8 - Laqueus californianus vancouverensis unfertilized egg,
and early free swimming larva
Figure 9 - Laqueus californianus vancouverensis mature free
swimming larva and Laqueus californianus abnormal cleavage.
Tables
Table 1 - Average Fecundities and Egg Sizes
Table 2 - Developmental Timetable
Table 3 - Terebratulina crossei Fertilization Conditions
Table 4 - Laqueus californianus vancouverensis Fertilization
Conditions
Table 5 - Laqueus californianus Fertilization Conditions
Figure 1
MALE SIZE DISTRIBUTION BY VENTRAL LENGTH
T. crossei
L. californianus
L. californianus vancouverensis

—E
c
ktatatataa-
atataaaa-
Ventral Length (cm)
Figure 2
FEMALE SIZE DISTRIBUTION BY VENTRAL LENGTH
T. crossei
L. californianus
L. californianus vancouverensi

L
0-
ktatatataa-
ataataavaa-
Ventral Length (cm)
Figure 3: Shell and Gonad Morphology of Terebratulina crossei,
Laqueus californianus vancouverensis, and Laqueus
californianus
1-3: Ventral views of each species respectively
4-6: Side views of each species respectively
7-9: View of gonads in ventral valve for each species
respectively
Figure 3
Shell and Gonad Morphology of
Terebratulina crossei, Laqueus californianus
and Laqueus californianus vancouverenis
1 crossei
L. cali fornianus
L. c. vancouverensis
Ventral vieu
ventral view
ventml vie
side vied
side vieu
side view

ventral gonads
Ventral gonads
ventml gonads
Figure 4: Developmental stages of Terebratulina crossei
1. Unfertilized egg
2. Unfertilized egg with polar body
3. 2-cell stage
4. 3-cell stage
5. 4-cell stage
6. 6-cell stage
7. early blastula
8. late blastula
9. immature larva
Key: bm - bumpy membrane
pb - polar body
al - apical lobe
ml - mantle lobe
pl - pedicle lobe
7.
Figure 4
Developmental Stages of
Terebratulina crossei


bm
L

Om
bm
pm


S
J
int
bi
bm
bm
1al

ml-

S
/
Figure 5: Terebratulina crossei blastula and early free swimming
larva
A: blastula seen - 36hrs (20x
B: unfertilized egg and early free swimming larva :
164hrs (20x)
Figure 5
Figure 6: Terebratulina crossei late and mature free swimming larva
A: late free swimming larva - 190hrs (20x
B: mature free swimming larva - 200hrs (20x)
Figure 6
Figure 7: Developmental Stages of Laqueus californianus
vancouverensis
1. 2-cell stage
2. 8-cell stage
3. irregular cleavage (possibly blastula)
4. mature larva
Key: cm - cell membrane
al - apical lobe
ml - mantle lobe
pl
pedicle lobe
setae
Figure 7
Developmental Stages of
Laqueus californianus vancouverenis
N

.
41



ml
5
em
Figure 8: Laqueus californianus vancouverensis unfertilized egg,
and early free swimming larva
A: unfertilized egg - Ohrs (40x)
B: early free swimming larva - 96hrs (20x)
Figure 8
Figure 9: Laqueus californianus vancouverensis mature free
swimming larva and Laqueus californianus abnormal cleavage.
A: mature free swimming larva - 100hrs (20x)
B: abnormal cleavage - 40hrs (40x)
Figure 9
Table 1
Average Fecundity and Egg Size
Standard
Avg. egg
Standard
Avg.
Abrachs
Feggs
Species
Reggs
size
Deviation
Deviation
examined
examined
Laqueus
2
14500
20-30
18,000
=100um
NA
californianus
Laqueus
40-50
10,000
+1500
-140um
410um
californianus
vancouverensis
Terebratulina
8,500
+150
20-30
NA
2210um
crossei
Laqueus
140um
35,000f
californianus
170um
(Lit. values)
Terebratulina
NA
170um
unguicula
Terebratulina
NA
160um
septentrionalis
Terebratulina
8000-
130umf
retusa
150001
Hemithyris
190um
NA
psittacea

f Law and Thayer (1991)
+ Reed (1987
Long (1964)
** Conklin (1902)
+ James (1991)
This table lists the fecundity and average egg size for each of the
species studied, and some relevant literature values. It should be
noted that the Laqueus californianus data was taken form
individuals with immature eggs while the eggs of the other two
species were mature. Standard deviations for egg size for L. c.
californianus, and T. crossei could not be calculated, for although
multiple eggs were observed, only an average size was recorded
rather than individual sizes. NA indicates information is not
available.
Table 2
Developmental Timetable
First
Species
Second
Larva
Metamor.
Blastula
phosis
Cleavage
Cleavage
Terebratulina crossei
10.75
11.25
164
NA
36
Laqueus californianus
26.25
100
NA
NA
NA
vancouverensis
Terebratalia
2-3
8-14
100
92
transversat
Terebratulina
118
NA
20
102
unguicula
Hemithyris Psittaceat
NA
NA
1200
123
1187—
ILong (1964)
This table presents the number of hours post fertilization when
various developmental events occur. The Terebratalia transversa,
Terebratulina unguicula and Hemithyris Psittacea data is taken from
Long (1964). The Terebratulina crossei and Laqueus californianus
vancouverensis values, which are original, give the time at which
each stage was first observed. It is possible, however, that the
developing embryo reached a given stage prior to the actual time of
observation. NA indicates data that was not available.
Table 3
Terebratulina crossei Fertilization Conditions
Egg soaking
Calendar
Time of
Water
Fert? Notes
Fert.
time (hrs)
temp
date
0
k
5/13/93
14:00
YES
2:00 AM
blastula stage. Cells appeared
lysed.
Counted 17 fertilized eggs out of
5/18/93
12:40 AM
YES
14:15
entire batch, pre-setae larvae
found after 164 hours. Mature
larvae seen at 200 hours
5/18/93
2:30 PM
14:10
YES
This table shows the conditions under which the Terebratulina
crossei fertilizations were performed.
Table 4
Laqueus californianus vancouverensis
Fertilization Conditions
Water
Calendar
Time of
Egg soaking
Notes
Fert?
date
Fert.
time (Hrs)
Temp.
O
Carried out in water
4/15/93
N
daytime
NA
NA
contaminated by sulfur and
decaying clams
Carried out in water
4/19/93
NA
5:15 PM
NA
N
contaminated by sulfur and
decaying clams
Carried out in water
4/22193
NO
10:45 AN
18:45
contaminated by sulfur and
decaying clams
New, uncontaminated water
4/26193
12:00 PM
5 and
YES.
13:15
source Two- and eight-cell stage
NO
observed
Sperm motility low
4/28193
N
NA
9:20 AM,
10,13,16,18
12:15 PM
3:30 PM,
5:30 PM
Perhaps some abnormal cleavage
13:25
5/3/93
11:10 AN
5-10
N
Perhaps some abnormal cleavage
5/3/93
5-10
NO
11:50 PN
12:05
Free swimming larvae observed
YES
5/6193
2:00 PM
15:45
5/11/93
10:20 AM
NO
12:20
5/23193
NO
12:15 AN
10,15
5-6
N
5/24193
10:43 AN
13:08
5/24193
NO
0:00
1:05 PM
This table gives the conditions under which each fertilization of
Laqueus californianus vancouverensis was attempted. NA indicates
information which was Not Available.
Table 5
Laqueus californianus Fertilization Conditions
Calendar
Time of
Water IFert? INotes
Egg soaking
Fert.
temp
date
time (hrs)
(0C
Sperm only slightly motile
5/4/93
9.55 AM 18:25
N
Small, immature looking eggs
Small, immature looking eggs
N
577193
11:25 AM 11:25
4-5
This table lists the conditions under which the fertilization of
Laqueus californianus was attempted. Note that in both cases the
eggs used were quite immature.
ACKNOWLEDGMENTS
We would like to thank our advisor Jim Barry for creating this
project and keeping us well stocked with brachiopods; Chuck Baxter
for his suggestions and the fact that he was always truly excited to
see a cleaved brachiopod egg; Dr. Dan Mazia for his help in getting
our sperm to move; Kurt Buck for his assistance on microscopic
photography and his undying wit; Dr. Richard Strathmann at Friday
Harbor Laboratories for his advice and reassurance; Dr. David Epel
for letting us borrow his pH meter and ammonium hydroxide; Judy
Connor for patiently searching the ROV footage for us; Molly
Cummings and the rest of the 1993 Bio 175H students for being such
a cool and crazy group of people.
Dana-Lynn would like to especially thank her house mates, Linda,
Christine, and Maria for making 122 18th St. so much more than just
a place to sleep. From now on, whenever I drink Citrus Punch or eat
Stove Top, l’Il think of you guys. I would also like to thank Jim for
patiently enduring my attempts to get him to lab before noon and for
his real enthusiasm for and knowledge of all the critters in the deep
sea lab-it was catching. Finally, I thank God for using this quarter
to teach me to: "Rejoice evermore. Pray without ceasing. In
everything give thanks: for this is the will of God in Christ Jesus.'
I Thessalonians 5:16-18
Jim would like to thank Chuck Baxter again, who has helped me
figure out what I want in life; my parents, Jim and Sylvia, who have
supported me in whatever ventures I have embarked upon; Dana
Lynn, for putting up with my idiosyncrasies; and all of the
brachiopods, great and small, who sacrificed their lives to make this
project possible. Thank you all!