r kamoto -rag
There have been relatively few reports of success¬
ful in vitro rearing of barnacle larvae to the settled
cyprid stage. Within the last decade cypris larvae of
Balanus eburneus and Balanus amphitrite denticulata
have been raised on a diet of Chlamydomonas sp. and
rbacia eggs (Costlow and Bookhout, 1957, 1958). Full
development of Balanus balanus larvae was accomplished
on the diatom Skeletonema costatum, both alone and mixed
with the diatoms Biddulohia regia, Ditylum brightwellii,
and Phaeodactylum cornutum (Barnes and Costlow, 1961).
Recently, several of 17 unicellular algae were used suc¬
cessfully in rearing cypris larvae of Balanus balanoides,
Elminius modestus, Chthamalus stellatus and Lepas anati-
fera (see Moyse, 1963). For a review of earlier work
see Costlow and Bookhout (1957). The present study re¬
presents an attempt to assess the usefulness of four spé¬
cies of unicellular green algae and two species of diatoms
as food in rearing Balanus glandula and Chthamalus dalli.
For each food species the following questions were posed.
Do the barnacles ingest it? Do they digest it? How long
do they survive on it? And does it support growth and
development of successive larval instars?
The study waspursued at the Hopkins Marine Station
of Stanford University, Pacific Grove, California between
May 1 and June 5, 1967. The algae employed as foods for
the nauplii, supplied from the culture collection at the
Marine Station, were Platymonas sp., Dunaliella viridis,
Chlorella sp., Stichococcus sp., Nitzschia closterium,
and Nitzschia closterium minutissima. Balanus glandula
and Chthamalus dalli were collected in the Monterey Penin-
sula area. Mature ovigerous lamellae were removed from
the mantle cavity and transferred immediately to a glass
container of millipore filtered sea water where time was
allowed for hatching. In collecting for experimental use
177
Eric Okamoto - Page. 3
the larvae were first swirled in the hatching beaker to
disperse them; they were then withdrawn with a Pasteur
pipette, counted, and added to the culture vessels. Fur-
ther details are given in the appropriate sections below.
Assistance in the identification of the various nau-
pliar stages was provided by John B. Snodgrass. Thanks
are extended to Dr. Donald P. Abbott for his advice dur-
ing the study.
FEEDING AND DIGESTION
Due to the wide variation in size, shape, and mo-
tility of the algae, there was a question as to whether
all six foods could be captured and ingested by the lar-
vae. In two separate preliminary experiments B. glandula
and C. dalli nauplii, taken 0-30 minutes after hatching,
were placed in individual cultures of the six foods. In
approximately 30 minutes the larvae were removed and ex-
amined microscopically for changes in digestive tract
contents. In both experiments approximately equal pro-
portions of first and second instar (stage I and II)
nauplii were observed. Several stage II nauplii that had
ingested food were found in all the algal cultures.
Feeding was confirmed by observation of intact or frag-
mented algae inside the digestive tract, or by noting a
conspicuous color change in the gut. No indication of
feeding was ever noted in a first stage nauplius of
either barnacle species. This supports the observation
of Lochhead (1936) and others that barnacle nauplii pass
into the second stage without feeding. In all later ex-
periments, larvae of both barnacle species were found to
ingest the algal or diatom species provided for them.
While the larvae were usually actively swimming when ob-
served, they appeared at times to graze on the algal cells
adhering to the walls and floors of the culture containers.
Some hints as to the digestive capabilities of Balanus
17.
Eric Okamoto - Page
glandula larvae were gained by examining naupliar fecal
pellets. Feces formed by nauplii feeding on Chlorella
were impregnated with intact green algal cells which showed
no evidences of digestion. Undigested algae were never
detected in fecal pellets produced by nauplii feeding on
latymonas, Dunaliella, and Nitzschia spp.
Whether or
not Stichococcus is digested by the larvae remains un-
certain.
SURVIVAL
Two experiments were conducted to compare the sur-
vival times of B. glandula larvae fed on different algae.
In the first experiment thirteen 50 ml. Erlenmeyer
flasks were used. To each vessel 5 ml. of millipore fil-
tered sea water and 20 nauplii were added. To each pair
of flasks one of the six algal suspensions was added.
The odd flask served as a starved control. The cultures
were maintained at 12-14°0. under constant agitation on
a sliding platform. The number of surviving nauplii was
determined periodically by concentrating the larvae with
a strong light and counting them. The results of this
experiment are found in fig. 1.
A second experiment employed a modified version of
the culturing technique of Moyse (1960). Each of twenty-
five 600 ml. beakers containing 475ml. of millipore fil-
tered sea water received 120 nauplii in stages I and II.
Before the algae were added to the naupliar cultures, each
algal suspension was filtered through a nylon mesh screen
to segregate cell aggregations as much as possible. The
screen pore sizes utilized to filter particular algae
were as follows: Nitzschia spp., 250; Platymonas, 154;
all other species, 104. The density for each algal sus-
pension was then determined on a Coulter Electronic Par-
ticle Counter. The beakers of larvae were divided into
groups of four, each group receiving one of the six foods
17
Eric Okamoto - Page
added in an amount calculated to provide an initial con-
centration of 3000 particles per ml. One beaker was set
aside as a starved control. All cultures were maintained
between 16.5 to 17.5°0. Every two days a beaker from
each group was selected; all surviving nauplii in it were
extracted, mounted on slides, and counted. The results
are shown in fig. 2.
In both experiments mortality was highest. in nau-
plius cultures to which Stichococcus or Chlorella had
been added. Survival was best in cultures containing
Platymonas. The other microorganisms gave intermediate
results and fall in the following descending order: N.
closterium, N. closterium minutissima, Dunaliella.
An experiment to determine duration of survival of
C. dalli on each of the same six food organisms was con-
ducted similarly, except that larvae were maintained in
plastic boxes at room temperature (2112'0). In many re¬
spects the results obtained (fig. 3) were similar. Survival was
greatest in cultures containing Platymonas. Nauplii fed
with Dunaliella and Nitzschia closterium survived in
receiyi
those e
Chlorella, Sticho-
greater numbers than
coccus, or Nitzschia closterium minutissima.
GROWTH
The experiments described above were designed to
vield data on growth as well as survival times. Growth
was assessed by counting the number of living nauplii of
each instar which were presentat each observation. Nau¬
liar stages were identified using characters determined
for thesespecies by Snodgrass (1967). Primary use was
made of the setation formula of the first appendage;
when this appendage could not be seen clearly, the de¬
termination of stage was made on the basis of body size
and the shape of the tail components.
As fig. 4 indicates, the only pronounced progression
of B. glandula larvae into the later instars was found in
186
Eric Okamoto - Page 6
cultures where Platymonas formed the diet. Only a few
stage III (and one stage IV) nauplii were found in lar-
val cultures feeding on N. closterium and N. closterium
inutissima, although in a similar experiment performed
t room temperature (20-24°C) third and fourth stages
were achieved on these same nutrients in 2.5 days before
death occurred. Dead nauplii in these and all other cul-
tures were examined to see whether a substantial number
of later stage larvae were present; they were not.
Results showing growth of C. dalli maintained on
different diets are shown in fig. 5. The same criteria
were applied in differentiating the various stages, but
here distinctions as to which criterion was utilized are
indicated in the graph. The blackened areas of each bar
indicate those larvae classified on the basis of their
first appendage, whereas the stippled zones represent
arvae classified according to body size and shape of
tail. Although the latter two criteria are considered
less reliable indicators of instar than the first an-
tenna, it is felt that the error introduced through their
use is negligible.
The development of the larvae of C. dalli on the
various foods correlates closely with its survival time
on these foods (fig. 3). Larvae cultured with Platymonas
were successfully reared to the cyprid settling stage. The
majority of the cyprids had settled when they were ex-
tracted from the culture. Although larvae did not pass
beyond stage III when feeding on N. closterium and not
beyond stage IV when feeding on Dunaliella, the results
suggest that these food organisms provide at least some
of the requirements for growth.
No growth occurred in cultures fed on Chlorella or
Stichococcus. Others have reported parallel results.
Gibor (1956) has demonstrated that the same species of
191
Eric Okamoto - Page
tichococous used here is not digested by Artemia salina,
and Moyse (1963) found that Chlorella stigmatophora will
not promote growth in nauplii of Balanus balanoides,
Elminius modestus and Chthamalus stellatus beyond the
second instar.
SUMMARY
(1) It was found that Balanus glandula and Chthamalus dalli
ingested four unicellular green algae (Platymonas sp.
Sti¬
chococcus sp., Chlorella sp., and Dunaliella viridis)
and two diatoms (Nitzschia closterium and Nitzschia
closterium minutissima).
(2) Nauplii of both barnacle species survive longest on
Piatymonas.
(3) Greatest growth and development for both naupliar
species was provided by a Platymonas diet. Chlorella and
tichococcus do not promote larval growth.
16
age
ri Okamote
REFERENCES CITED
Barnes, H., & Costlow, J. K. Jr., 1961. The larval stages
of Balanus balanus (L.) da Costa. J. mar. biol. Ass.
U.K., 41:59-68.
Costlow, J. D. Jr., & Bookhout, C. G., 1957. Larval de¬
velopment of Balanus eburneus in the laboratory.
324
Biol. Bull. Woods Hole, 112:313-324.
Costlow, J. D. Jr., & Bookhout C. G., 1958. Larval devel¬
opment of Balanus amphitrite var. denticulata Broch
reared in the laboratory. Biol. Bull. Woods Hole,
29
114:284-295.
Gibor, A.. 1956. Some ecological relationships between
phyto- and zooplankton. Biol. Bull. Woods Hole,
111:230-234.
Lochhead, J. H., 1936. On the feeding mechanism of the
nauplius of Balanus perforatus Bruguière. Linn. Soc.
Lond. Jour. Zool. 39:429-442.
Moyse, J., 1960. Mass rearing of barnacle cyprids in
the laboratory. Nature, Lond., 185:120.
Moyse, J., 1963. A comparison of the value of various
flagellates and diatoms as food for barnacle larvae.
J. Conseil Internat. l'exploration de la Mer. 28(2):
175-187.
Snodgrass, J. B., 1967. Studies of the larval stages of
the barnacles Balanus glandula, Balanus tintinnabulum,
Chthamalus dalli, and Pollicipes polymerus. Unpublished
manuscript.
11
Eric Okamoto - Page 9
CAPTIONS TO FIGURES
Survival of Balanus glandula nauplii on six dif-
Fig. 1
ferent food organisms. All cultures were started
with 40 larvae and maintained at 12-14°0. Key
to the seven different lines: a - Platymonas sp.,
b = Nitzschia closterium, c = Nitzschia closterium
minutissima, d - Dunaliella viridis, e - Sticho-
coccus sp., f = Chlorella sp., g - starved control.
Survival of Balanus glandula nauplii on six dif-
Fig. 2
ferent food organisms. All cultures were started
with 120 larvae and maintained at 16.5-17.5°0.
For identification of the lines refer to fig. 1
caption. In a starved control two stage II larvae
were living after 7.9 days.
Survival of Cthamalus dalli nauplii on six dif-
Fig.
ferent food organisms. All cultures were started
with 80 larvae and maintained at 2112'0. For
identification of the lines refer to fig. 1 caption.
Starved controls were dead by the 4- day.
Development of Balanus glandula nauplii on six
Fig. 4
different food organisms. All cultures were
started with 120 larvae. Percent of the surviv-
ing population (N) at each instar (1-4) is shown.
Development of Chtamalus dalli nauplii on six
Fig. 5
different food organisms. The first three sets
of cultures (i.e. those observed after 2.3 days,
4.1 days, and 6.5 days) were started with 80 lar-
vae each and were maintained at room temperature
1t2'C). The cultures observed after 11.7 days
and 14.8 days were started with 120 larvae each
and were maintained at room temperature for two
days and then at 16.5-17.5°0 for the remainder
1P
0
Eric Okamoto - Page 10
CAPTIONS (cont.).
of the experiment. The black sections of the
histogram bars indicate positive identification
of theinstars on the basis of the first antenna.
The stippled sections represent identifications
on the basis of size and tail morphology. Percent
of the surviving population (N) at each instar
(1,2,3...) is shown.
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