Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Studies on the Embryological Development of the Squid
Loligo opalescens and Symbiotic Sheath Bacteria
Abstrac
Encased within a bacteria associated sheath, the embryos of the
squid Loligo opalescens are able to withstand a three to five week
incubation period on the ocean floor. It appears that the unidentified
bacteria are symbionts of the developing embryos; the longer the
association with the sheath, the further the development of the
embryos. When the sheath remained, but the bacteria was removed
with antibiotics, the stripped fingers showed a greater susceptibilty
when exposed to fungal-rich solutions. The experimental evidence
suggests that the sheath bacteria are symbionts of the developing
Loligo opalescens embryos.
Introduction
The market squid Loligo opalescens inhabits the waters along the
west coast of North America (Hixon, 1983). After mating, females
deposit twenty or more finger-like gelatinous masses containing
between 115 to 300 embryos (Strathmann, 1987). Inexplicably, the
embryos of the squid Loligo opalescens withstand a three to five
week incubation period on subtidal sandy substrates resisting fungal,
microbial, and animal predation.
Discovered and described several years ago, the egg capsule
sheath of Loligo opalescens contains one to five layers of bacteria
(Biggs and Epel, 1991). With the structural properties of a secretory
organ (Bloodgood, 1977), the female squids’ accessory nidamental
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
(AN) gland was pointed to as the probable source of the sheath
bacteria. It has been observed that the AN lumen becomes filled
with several types of symbiotic bacteria with sexual maturation
(Richard et al, 1979; Buchner, 1965). However, the function of the
AN gland and the sheath bacteria is unknown (Lum-Kong, 1992a).
Symbiotic marine bacteria provides protection for other systems.
The embryos of the shrimp Palaemon macrodactylus (Gil-Turnes et
al, 1989) and lobster Homarus americanus (Gil-Turnes and Fenical,
1992) demonstrate a remarkable ability to resist infection by the
fungus, Lagenidium callinectes. In the Hawaiian squid, Euprymna
scolopes, luminous symbiotic bacteria are needed for the maturation
and development of the light organ (McFall-Ngai and Ruby, 1991).
The following experiments attempted to determine the
relationship between the sheath bacteria and the squid embryos. By
separating the bacteria-associated sheath from the developing
embryos, and later eliminating all bacteria with antibiotics, the
development of the squid embryos was observed. When separated
from the sheath, the embryos with the least sheath association
suffered the earliest mortality. The longer the development with
the encasing the sheath, the greater the development of the embryos.
In another experiment, the sheath remained while the sheath
bacteria were removed with the use of antibiotics. The fingers that
were stripped of bacteria showed a greater susceptibility when
exposed to two fungal-rich solutions. The following experiments
support the symbiotic relationship of the bacteria with the
developing embryos.
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Materials and Methods
General: Loligo opalescens were captured in Monterey Bay during
May 1994, placed in circular, 2000 L holding tanks with continual
flow, and induced to mate by the presence of egg fingers gathered by
staff scuba divers. Additional egg fingers were collected from scuba
divers offshore from Hopkins Marine Station in Monterey Bay.
Freshly dead squid were collected from fishing boats for in vitro
fertilization.
The standard arrangement for following development and effects
of antibiotics in the egg fingers consisted of tying fingers to
autoclaved string attached to a weight. Plastic petri dishes covered
the sterile glass beakers. All beakers were placed in a water bath
which fluctuated between 16-18 C. Throughout the experiments,
embryos were aged according to stages rather than days.
Temperature influences the developmental schedule, the higher the
temperature the shorter the incubation period (Fields, 1965;
Strathmann, 1987). All solutions (sterile sea water, natural sea
water, fungal-rich and protease inhibition solutions) were changed
at least every third day. Natural sea water came directly from
Monterey Bay.
Terms: Finger: The finger, with its bacteria associated sheath and
embryo-chorion mass, is what is naturally laid and attached to sandy
substrates. By cutting both ends and making an incision along the
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
long axis, the finger can be divided into the sheath and embryos.
Sheath: The sheath contains bacteria and is the wrapping or case
surrounding the cluster of embryos. Once unwrapped (from the
embryos), the separated sheath contains no embryos. Embryos:
After the removal of the sheath, the embryo-chorion mass remains.
This mass consists of developing fertilized eggs. The chorion,
generally described as the secondary envelope (the primary
envelope being the vitelline membrane), is a product of the follicular
cells (Boletzky, 1986). After fertilization, the egg shrinks widening
the space between the just fertilized egg and its surrounding chorion
(Fields, 1965). In vitro fertilization: Unfertilized eggs removed from
ovaries and sperm taken from the spermatophores are the sources of
the gametes used in in vitro fertilization.
Antibiotic sensitivity testing: To determine the significance of
the symbiotic bacteria on embryological development, an effective
antibiotic solution was needed to eliminate all sheath-associated
bacteria. Thirty-six representative strains previously isolated from
the squid accessory nidamental (AN) gland and egg sheath were
streaked onto five plates each containing a separate antibiotic
(tetracycline 15 ug/ml, kanamycin 40 ug/ml, streptomycin 100
ug/ml, rifampicin 100 ug/ml, ampicillin 100 ug/ml). The plates were
then incubated overnight at room temperature. When compared to
the control of marine agar (MA), the combination of rifampicin and
streptomycin inhibited the growth of all thirty-six strains.
Rifampicin binds to bacterial subunits of RNA polymerase to inhibit
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
transcription. Bactericidal streptomycin irreversibly binds to a
ribosomal subunit thereby decreasing the rate of translation while
increasing the chance of misreading (Volk et al, 1986).
With a final pH of 8.2, the following final concentrations were used
in the streptomycin-rifampicin antibiotic solution; streptomycin: 100
ug/ 1 ml distilled water; rifampicin:100 ug/ 1 ml DMSO. A milliliter
(ml) of each 100x stock solution was diluted with 1 liter (L) of sterile
sea water (SSW). Besides being replaced daily for freshness, the
antibiotic solution was tested for efficacy in vitro. Following the egg
fingers marinade in the antibiotic solution, 100 ul of the used
antibiotic solution was plated onto marine agar plates to monitor any
bacterial growth on the nutrient-sufficient medium.
The streptomycin-rifampicin antibiotic combination demonstrated
no effect on Strongylocentrotus purpuratus fertilization or short term
(t« 2 days) development.
Assaying embryo development in antibiotic solution: Newly
laid egg fingers were placed into the antibiotic solution after several
washes in sterile sea water (Diagram 1). To monitor development,
the fingers were periodically (-3 days) removed and examined using
light microscopy. Concurrently, egg fingers developing in sterile sea
water were transferred into the antibiotic solution every three days.
Using sterile techniques, egg fingers which underwent their entire
development in sterile sea water served as controls.
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Development following transient antibiotic treatment: To
determine the egg fingers' susceptibility to infection in the absence
of symbionts, fingers were rinsed in SSW and placed in the
rifampicin-streptomycin antibiotic solution (Diagram 2). After five
days, the fingers were divided into four solutions: sterile sea water,
natural sea water, fungal-rich solution I (Lagenidium callinectes)
fungal-rich solution II (Rhodotorula glutinis). A scoop of L.
callinectes, transferred from a frozen glycerol and plated onto a
marine agar plate, was placed into the fungal-rich I solution; 500 ul
of an overnight culture of R. glutinis was inoculated into the 500 mls
SSW containing fingers in fungal-rich II solution. Both fungal-rich
solutions consisted of fungi added to sterile sea water. All fingers
were examined using light microscopy and sterile techniques every
third day. I specifically examined the encasing sheath surfaces for
fungal attack. Normal fingers were placed in both fungal-rich
solutions as the control after a rinse with sterile sea water. Fingers
placed in sterile sea water after a five day marinade in antibiotics
were a second control.
Development of separate tissues in sterile sea water and
sea water: To determine how the separate components (intact
fingers, sheath, embryos) of the finger developed in isolation, the
different parts of the finger were dissected (Diagram 3). Using
sterile techniques, intact fingers were longitudinally cut and their
chorion-embryo mass removed. Unwrapped from all embryos, the
separated sheaths were placed apart from the remaining embryo¬
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
chorion portion. These six (fingers, sheath, embryos in both SSW and
SW) beakers were set up simultaneously from egg fingers collected
by staff scuba divers. Unfertilized eggs and sperm were removed
from freshly dead squid and fertilized in vitro.
Embryo development was monitored in the intact fingers, isolated
embryos, and in vitro fertilizations. The sheath, separated from all
embryos, was watched for any general observations as discoloration.
thickening, or surface texture. All four parts (fingers, sheath,
embryos, in vitro fertilizations) were placed in both sterile and
natural sea water solutions. All eight beakers were checked and
their solutions changed every third day. The isolated embryos were
staged every third day.
Protease Inhibition Solution: To examine the relationship
between bacterial protease secretion and/or development, hatching,
and anti-fungal activity, the effects of protease inhibition on
bacterial proteases were examined. The experiments below
ascertained which inhibitors prevented protease activity. Five of the
representative thirty-six (including two negative controls) bacterial
strains and a concentrated protease were plated onto marine agar
plates overlaid with a known protease inhibitor. Four protease
inhibitors were individually placed on a marine agar plate; 1 uM
leupeptin, 1 um pepstatin, O.5 mM EDTA, 30 um SBTI. A plain plate
of marine agar was the control. The plates were incubated overnight
at room temperature. From these streaks a protease inhibition
solution (PIS) was created. From stock solutions of O.5 mM EDTA and
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
10 mg SBTI/ml, five milliliters (ml) EDTA and 1.5 ml SBTI were
mixed into 500 ml sterile sea water. EDTA inhibits metalloproteases
whereas SBTI inhibits trypsin (product information). The acidic (pH
4.5) solution was then brought up to -pH 7.6 with the addition of 1 M
NaoH.
Effect of Protease Inhibition On Hatching: Egg fingers rinsed
with SSW were placed into the protease inhibition solution to study
hatching. The solution was changed every third day to insure
freshness. Like the antibiotic solution, the used protease inhibition
solution was also streaked onto marine agar plates to test for
bacterial activity.
Results
Development in the antibiotic solution: The fingers immersed
in the rifampicin-streptomycin antibiotic solution were collected by
staff scuba divers. The fingers used as the sterile sea water control
were gathered from the holding tanks from captured squid.
Fingers placed in the antibiotic solution following a sterile sea
water wash developed normally, but at an unusually slow rate. At
t=17 days in the water bath, no depression separating the future
external yolk sac from the future embryonic body could be seen on
most embryos (younger than Segawa’s stage 8). A few cloudy
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
chorions were seen although most embryos appeared transparent
and healthy. A few embryos did show distinctive features
characteristic of more advanced stages. These more advanced
embryos were used as evidence for the healthy, but slow,
development of the younger embryos. The fingers transferred from
sterile sea water into the antibiotic solution initially appeared
healthy. However, as time and development progressed, the eg
fingers in the antibiotic solution failed to continue development
(t=21).
To confirm that all bacteria were eliminated by the antibiotics, the
antibiotic solution that the fingers had been immersed in was plated
onto marine agar and incubated overnight. This was done several
times throughout the experiment. Finger tissue immersed in the
antibiotic solution was plated once. An orange colored bacteria
eventually grew on some of the streaked plates. Not present in a
concentration large enough to fear infection, only a handful of orange
colonies grew when 100 ul of used antibiotic solution was streaked
onto the nutrient-rich marine agar plates. The streptomycin-
rifampicin solution effectively eliminated the broad majority of
finger bacteria.
Development following a transient antibiotic treatment:
Egg fingers placed into sterile sea water following the five day
antibiotic treatment appeared normal, but slow in development. At
t=17 days in the water bath, most sterile sea water fingers showed
no beginning of organogenesis (Segawa’s stage 8). Similarly, the
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
natural sea water fingers looked like they were developing slowly
and normally. The fingers placed in the fungal-rich solution of R.
glutinis were also developing slowly (when compared to the sterile
sea water control). Besides slow development, many of the R.
glutinis embryos were peculiar in shape (snowman-like). Not
circular, the embryo shapes failed to resemble forms drawn and
described by either Arnold or Segawa. More suspicious were the
embryos removed from the L. callinectes fingers. A mist was seen in
the chorions of the L. callinectes fingers; this mist did not resemble
the particulate debris viewed in other embryos. Also, development
appeared stalled and embryos unusually large. By t-21, normal
embryos were seen (younger than Segawa’s stage 8), but no normal
development. It was undetermined whether the embryos were
stalled in development or dead. No movement within the chorion
was seen. Tissue from the L. callinectes embryos were plated onto
yeast and marine agar.
Development of separate tissues in sterile sea water and
sea water: The egg fingers used in this experiment were gathered
from staff scuba divers off of Hopkins Marine Station. To
compensate for the ambiguity about the egg fingers' exact age, the
fingers were staged before beginning (Segawa et al, 1988; Arnold,
1965).
Embryos: When separated from the sheath, embryos in both
sterile sea water and natural sea water failed to develop into
10
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
hatchlings. Isolated embryos developed eyes and chromatophores in
both the sterile and natural sea water solutions (Figure 1), but soon
after deteriorated into cloudy chorions impossible to stage (Figure 2)
The natural sea water embryos degenerated before the embryos in
the sterile sea water (Chart 1, Graph 1). By t=16 days, small, mobile
specks identified as protozoa were seen associated with the chorion
surfaces when all embryos appeared dead. Tissue samples from both
sea water solutions along with some natural sea water solution were
plated.
In vitro fertilization: Unfertilized eggs from freshly dead females
deteriorated quickly (t=2 days). Just as rapidly, both the sterile and
natural sea water in vitro fertilization eggs degenerated (Figures 3,4)
Although a percentage of the unfertilized eggs were immature and
incapable of fertilization, in both sea water solutions chorions were
seen and used as evidence for fertilization. Both in vitro solutions
had distinct smells and were cloudy two days after fertilization.
Only dead embryos and lysed material remained by the second day.
Control Fingers: Hatchlings broke through the natural sea water
finger sheath on the sixteenth day in the water bath. Although low
in viability, embryos survived and developed into the hatchling
stage.
Shortly after the hatchlings broke emerged both of the natural sea
water fingers began to disintegrate. This tissue was plated for
bacterial and fungal attack. Especially noticeable was the thinning
and weakening of the sheaths which were unable to hold the
11
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
enclosed embryos. No hatchlings emerged from the sterile sea water
control. Enclosed in the sheath, embryos developed eyes and
chromatophores before turning opaque in color. Unlike the natural
sea water fingers’ sheaths, the sheath remained intact around the
still embryos.
Sheaths: The separated sheaths in both sterile sea water and
natural sea water appeared seemed unaffected by their separation
from the embryos; no bacterial or fungal attack was seen on the
sheath surfaces. In both sea water solutions, the sheaths thickened.
Toward the end of the experiment (t-22 days), the natural sea water
sheaths appeared slightly discolored. Intact fingers held in tanks
with a constant flow of natural sea water demonstrated the same
discoloration likely to be algal settlement. Overall, the sheaths in
both sea water solutions appeared basically unaffected by their
separation from the embryo-chorion mass.
Bacteria Identification via Biolog: Samples of SSW and SW
embryo tissue, along with SW embryo solution, were spread (100 ul
plate) onto marine agar plates. Bacteria isolated from the SW
embryo tissue was examined using the commercially available Biolog
identification kit (Hayward, California). The bacteria associated
within the chorion of the cloudy SW embryos was identified as
Vibrio harveyi B.
12
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Discussion
The evidence suggests a direct correlation exists between the
period and extent of embryo development and the embryos'
association with the sheath. The longer the embryos' association
with the sheath, the greater the developmental stages reached. This
connection endorses the theory that the sheath-associated bacteria
are symbionts of the developing L. opalescens embryos. Öther
experiments support this relationship. Following a transient
antibiotic treatment, egg fingers were placed into four different
media. Although the sheath remained, the sheath bacteria were
eliminated with the streptomycin-rifampicin antibiotic treatment.
These egg fingers which were stripped of their bacteria via
antibiotics then placed into fungal-rich solutions (R. glutinis, L.
callinectes) showed the greatest tendency to deviate from normal
development with unusual shapes, sizes, and chorion fluid. Although
the effects of the antibiotic, a fluctuating water bath temperature,
and the lack of circulation complicated the experiment, the uniform
treatments equalized the results. With no association with the
sheath, both in vitro fertilizations rapidly degenerated. The finger
embryos, which underwent their entire development encased in the
sheath, demonstrated the greatest development and the longest
survival time.
Note: For the development of separate tissues, the time (t=16 days)
refers to time in the water bath not time since spawning.
13
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Correlation between sheath association and embryo
survival- development: Control Fingers: Although the sterile sea
water control fingers failed to bear healthy hatchlings, the SSW
fingers followed normal development (Segawa et al, 1988; Arnold,
1965) until the eighteenth day in the fluctuating water bath.
Similarly at t=18 days, the whole fingers immersed in the natural sea
water began to deviate from the detailed accounts of normal
development. Unlike the sterile sea water fingers, the natural sea
water fingers did yield hatchlings beginning on the sixteenth day in
the water bath. The reasons for the discrepancy in hatchling results
between the sterile and natural sea water solutions may be
explained by the occasional wild temperature fluctuations in the
water bath. Except for sterile versus natural sea water, all aspects of
the experiment treated the two egg finger groups identically. Thus,
it seems unlikely that the sterile sea water fingers would succumb to
an infection when the natural sea water fingers yielded hatchlings.
Although changed every third day, perhaps the SSW fingers were
more sensitive to the lack of circulating water than the SW fingers.
In either case, both fingers developed until t=18 days before signs
of abnormality surfaced. The SSW and SW isolated embryos did not
last as long. The SSW embryos began to look irregular on t-12 days.
and the SWembryos deviated earlier at t=10 days. Without the
sheath and its associated symbionts, the isolated embryos were more
susceptible to infection than the intact fingers. The embryos
developed through several stages before deteriorating; perhaps
several layers of the sheath remained associated providing some, if
14
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
minimal, protection to the embryos. The growth of infectious
bacteria and fungi also takes time.
Embryos: Isolated embryos appeared unable to develop into the
hatchling stage without the sheath. However, the factor(s) which
prevented the SSW control fingers from hatching may also be
blocking the development of the embryos. The embryos developed
in conditions uniform with the fingers; sea water solutions were
changed every third day and they shared the same water bath.
In Vitro Fertilization: Both sterile and natural sea water in vitro
fertilizations failed to progress past the second day. Removed from
dead females' ovaries, the unfertilized eggs had no association with
the sheath and its bacteria. The rapid deterioration of both in vitro
fertilizations and unfertilized eggs support the hypothesis that the
sheath bacteria are symbionts of the squid embryos.
Identified infectant: Identified with the Biolog system, the
bacteria associated within the cloudy chorions of the SW embryos
was Vibrio harveyi B. V. harveyi B is associated with high
mortality rates of the pearl oyster, Pinctada maxima Jamson (Pass et
al, 1987). The increased occurrences of infection in the pearl oysters
were attributed to inadequate water circulation. Of the eight
beakers, none were equipped with a circulating flow. Thus, perhaps
their exists a correlation between the absence of a circulating flow
and the V. harveyi Binfection.
15
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Although no bacteria has yet been isolated and identified from the
intact fingers, V. harveyi B partially explains the SSW finger
anomaly. Sitting in a still sea water solution, the fingers lay prone to
attack from the V. harveyi B. The presence of this bacteria could
partly explain the sterile sea water’s lack, and natural sea water’s
low percentage, of hatchlings. The reason for the difference
separating the SSW and SW hatchling results remains unknown.
Antibiotic Development: Egg fingers placed into the rifampicin-
streptomycin antibiotic treatment began to develop normally (t=18
days in solution), but at an unusually slow rate. At t=26 it appeared
that the visibly young (before Segawa’s stage 9) embryos were
stalled in development. Although a common characteristic of the egg
fingers immersed in the antibiotic, it is unproven whether the slowed
and stalled development is linked to the absence of bacteria, the
antibiotic solution, or both. To answer this unknown more antibiotics
capable of stripping the sheath of its bacteria need to be used. If
different antibiotics (playing the same function of eliminating
bacteria) yield different results, then the antibiotics effect on
development can be monitored. If an array of antibiotics are used
with the same developmental schedule, then it is more likely that it
is the absence of bacteria which is influencing the slowed
development.
The fingers transferred from sterile sea water into the antibiotic
solution every three days displayed differences. The transferred
fingers, all gathered from the same finger cluster, all appeared slow
16
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
in developing. However, the fingers which were transferred into the
antibiotic solution earlier were much smaller than the fingers
transferred from the SSW later. In all transferred fingers (t=O, 3, 6,
9, 12 days), no features characteristic of organogenesis were visible.
The fingers transferred at different times resembled one another in
developmental stages, but differed in finger size.
Protease Inhibition Development: Like the antibiotic fingers,
the fingers immersed in the protease inhibitor solution (younger
than Segawa’s stage 9: t=16 days in solution) also displayed normal,
but slow, development initially. As of t=24, the egg fingers in the
protease inhibition solution failed to show the rudimentary optic
vesicles or primordium shell gland indicative of the beginning of
organogenesis (Segawa, 1988). Development was prominently
behind the natural sea water control; they appeared retarded in
development. It is undetermined whether the protease inhibition,
the protease inhibition solution, or both were responsible for the
slow, and eventually stalled, development of the embryos.
The effect of the protease inhibition on hatching could not be
studied since the embryos stalled in development.
Different media following antibiotic treatment: The testing of
different media following transient antibiotic treatment gave more
evidence for the necessity of the symbiotic sheath bacteria in
embryological development. The fingers immersed in sterile sea
water appeared normal, along with the fingers in the natural sea
17
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
water solution. Fungi-rich solution I (R. glutinis) finger embryos
appeared basically normal, but with strange embryo outlines. No
unusual debris or infection was seen. The second fungal-rich
solution (L. callinectes) had some strangely large embryos. Also
associated with the L. callinectes, a mist was seen within the chorions
that did not resemble the particulate matter previously seen in
healthy embryos. Embryo tissue streaked onto yeast and marine
agar produced mucoid, cream colonies. As of yet, no infectants have
been identified. The fungal-rich solutions looked a little unordinary
with unusual embryo shapes, sizes, and chorion mist. Beginning on
t=26, no circulation or movement within the chorion was seen.
Although the embryos may have been stalled in development, it is
also quite likely that they were dead.
Judging from the other experiments using the antibiotic solution, it
is quite probable that the streptomycin-rifampicin combination
caused a slowing, and ultimate stalling, in embryo development.
However, the egg fingers placed in the fungal-rich solutions following
the antibiotic treatment showed the greatest irregularities and at the
earliest time. Thus, the egg fingers stripped of the sheath bacteria
were more susceptible to the fungal-rich solutions than any of the
control egg fingers. The controls were fingers that received no
antibiotic treatment before placement in the fungal-rich solutions,
and fingers transferred from the antibiotic solution into sterile and
natural sea water solutions. The antibiotic solution may be blocking
embryo development, but still the hypothesis holds: fingers stripped
of the sheath bacteria via antibiotics showed a greater tendency to
deviate from normal development.
18
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Problems and Suggestions: The following suggestions may be
successful in overcoming some of the experiments’ ambiguities. First,
all egg fingers should come from a single source at a simultaneous
spawning time. For this, mature squid need to be captured and
induced to mate in the holding tanks. Unfortunately, due to time
constraints, egg fingers were collected from Monterey Bay by staff
scuba divers and later from the holding tanks. A second suggestion
eliminates worries concerning the effects of stationary solutions;
paddles should be used to provide a consistent, continual circulation
over the egg fingers and finger parts. Next, a larger-sized container
might possibly decrease complications associated with a limited
volume solution. Fourth, more antibiotic testing needs to be done to
eliminate the experimental results seen as a reaction to the antibiotic
influence (as opposed to absence of bacteria). Finally, the
temperature needs to remain stable to insure consistent conditions.
The isolated embryos and in vitro fertilized eggs were just as
susceptible to the effects of the uncirculating sea water solutions as
the control fingers. But, the difference in days of normal
development hold. The lack of circulation may have stimulated the
growth of a bacterial infection in all beakers, but the separate tissue
sections responded differently. The in vitro fertilized eggs died
within two days; the absence of an association with the sheath and
its bacteria explain the quick deterioration. The embryos associated
and separated from the sheath lasted longer (SSW t=12, SW t=10)
than the in vitro fertilized eggs, but shorter than the whole fingers.
19
Nakashima: Loligo opalescens Embryo Development and Sheath Bacteria
Again, the association with the sheath and its bacteria comply with
the normal development time of the different tissue types. With no
sheath association, the in vitro fertilized and unfertilized eggs rapidly
worsened. The embryos, with an initial association with the sheath,
lasted longer but still failed to yield hatchlings. Never separated
from the sheath, the intact fingers’ embryos in both sterile and
natural sea water solutions developed and survived the longest.
When the sheath remained but the bacteria were removed, the
fingers which were transferred from the antibiotic solution to the
fungal-rich solutions demonstrated the largest irregularities in
development. Either by removing the sheath with forceps, or
stripping the bacteria with antibiotics, the unprotected embryos
appeared more susceptible to infection and developmental
irregularities when compared to the embryos with the sheath
bacteria. Thus, the sheath associated bacteria appear to be
symbionts of the developing L. opalescens embryos.
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Embryo Development in SSW and SW Solutions
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e: isolated embryos
iv: in vitro fertilization
SSW: sterile sea water
SW: natural sea water
Segawa’s (1988) stages used
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Figure 1: An isolated embryo developing normally in the natural
sea water solution.
Figure 2: An isolated embryo in the natural sea water solution.
Note the clouded chorion and littered chorion surface.
Figure 3: In vitro fertilization in the sterile sea water solution.
Within two days of fertilization only dead, lysed debris
remained.
Figure 4: In vitro fertilization in the natural sea water solution.
Like the SSW in vitro fertilization, only broken chorions
and debris remained two days after fertilization.
Acknowledgments
ThankYou... I extend an especially big thankyou to Dr. Melissa Xaufmann with her
limitless curiousitu, endless patience, and constant grin. Thankyou to Dr. Epel for the
quidance and care. To Chris Patton, Emile, and Paul Sund, I appreciate all of the
energu. And to the Epel lab, Fenrik, Barney, Beth, and Barb, thankyou for
accomodatinq this undergraduate. I enjoyed and appreciated it all.
ARNOLD, J. M., 1965. Normal embryonic stages of the squid, Loligo
pealii (Lesueur). Biological Bulletin. 128: 24-32.
BIGGS, J. and D. EPEL, 1991. Egg capsule sheath of Loligo opalescens
Berry: structure and association with bacteria. The Journal of
Experimental Zoology. 259: 263-267.
BLOÖDGOÖD, R. A., 1977. The squid accessory nidamental gland:
ultrastructure and association with bacteria. Tissue & Cell. 9 (2)
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