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.B. Forward et al. J. Exp. Mar. Biol. Ecol. 248 2000 225 –238
from musculature contractions of the adult or active swimming of larvae upon perception of light Svane and Young, 1989 and depends upon aspects of light exposure
and prior dark adaptation. The duration of light exposure before the onset of larval release is termed the dormant Huus, 1939 or latency period Lampert et al., 1981 and
varies with species and lighting conditions Svane and Young, 1989. In addition, in some species the number of larvae released increases as the length of the dark adaptation
period increases Watanabe and Lampert, 1973.
The present study considered light induced larval release by the polystyelid ascidian Polyandrocarpa zorritensis, which occurs on hard substrate in shallow water areas. After
egg incubation in the atrial cavity, this species releases fully developed free swimming larvae about 1.4 mm in length Vazquez and Young, 1996. Larvae become less active
over time and settle and metamorphose a number of hours after release Vazquez and Young, 1998. Larvae have a photolith, which is a compound sensory structure
composed of a statocyst and ocellus Berrill, 1947; Torrence, 1980. Although many invertebrate larvae are positively phototactic early in development and become negative
near the end of the larval phase Thorson, 1964, phototaxis of ascidian larvae is variable throughout development Svane and Young, 1989. For example, P
. zorritensis became positively phototactic near the end of development, which would be useful for
locating suitable shallow water habitats for settlement Vazquez and Young, 1998. If larval release is induced by light, then it is reasonable to hypothesize that larvae
should not be photonegative but rather should be attracted to settlement areas where light is sufficient for inducing larval release. To evaluate this hypothesis, the present
study determined 1 the minimum light intensity necessary to initiate larval release by P
. zorritensis, 2 the preference of larvae for light and dark areas, and 3 the minimum light intensity necessary for this preference. It was assumed that a preference for light
represented attraction to lighted areas for settlement. The hypothesis was supported, as larvae were attracted to areas where the light intensity was sufficient for larval release.
2. Material and methods
Polyandrocarpa zorritensis Van Name, 1931 colonies were collected at depths of , 0.5 m from a floating dock at Ft. Pierce, Florida USA. Colonies had about 15–30
individuals. Prior to experimentation, they were maintained in aerated seawater 36 psu at a temperature of about 248C in darkness for a minimum of 16 h. The minimum length
of this dark period was constant because a dark adaptation period is necessary for light induced larval release e.g. Watanabe and Lampert, 1973; Svane and Young, 1989, and
the number of larvae released depends upon the length of the dark period in some species Watanabe and Lampert, 1973. All experiments were conducted between 10:00
and 17:00 h. Variations in larval release due to a biological rhythm was unlikely because 1 Watanabe and Lampert 1973 found that larval release could be induced at any time
after an adequate dark adaptation period, and 2 spawning by a solitary ascidian could occur at any time during the day Svane and Havenhand, 1993.
Initial experiments determined the relationship between larval release and the intensity of natural sunlight. Colonies were placed individually in finger bowls diameter 5 10.5
R .B. Forward et al. J. Exp. Mar. Biol. Ecol. 248 2000 225 –238
227
cm containing new seawater 36 psu on a white surface in sunlight. At 15-min intervals larvae were removed from each bowl and counted. Observations continued until larval
release declined to zero for two consecutive time intervals. Sunlight intensity was controlled by placing layers of black fiberglass screening over the bowls and was
measured at each 15 min interval with a scalar irradiance meter with a 4p collector Biospherical Instruments Inc.; Model QSP170B, which had similar layers of screening
over the light sensor. This screening changed the light intensity but did not alter the spectral composition of the light. Since most days had minimal clouds, variation in light
levels throughout an experiment was minimal. Light levels were averaged over similar experiments with the same number of screens. Water temperature in the bowls was
measured at the beginning and end of each experiment and was found to increase a maximum of about 48C when exposed to unscreened sunlight. After testing on the first
day, colonies were again exposed to 16 h of darkness and tested the next day at a different light intensity. Larval release remained relatively consistent over the 2 days but
declined if colonies were tested after 3 days. Thus, colonies were only tested on the first 2 days in the laboratory. A range of light intensities was tested in order to determine the
lowest sunlight intensity that induced larval release.
Preliminary experiments showed that there was no relationship between the number of larvae released and number of individuals in a test colony. Because some colonies
released many larvae while others failed to release any, only colonies that released larvae were considered in the data. Colonies that failed to release larvae under lighting
conditions that should have induced larval release were considered to lack any mature larvae. The mean time of larval release was calculated for each colony. The overall
mean time and standard error were calculated for each light intensity. In addition, the number of larvae released by all colonies were summed for each 15-min interval and
then normalized as the percent released in that time interval. These data were used to construct the time course for larval release at each light intensity. Latency was defined as
the time between placement in light and release of the first larvae by any colony at each test light intensity. The width of the larval release time interval was defined as the
central-time interval during which 90 of the larvae from all colonies were released. 90 was used because it encompassed most of the larvae released and releases tended to
be erratic outside of this interval.
2.1. Release of larvae in current flow It was hypothesized that larval release should be inhibited as current flow increased, if
larvae were to be retained in the areas of the adult population. To test this hypothesis
15
individual colonies were placed in a flow tank under full sunlight 110.5 3 10 photons
22 21
21
cm s
and subjected to current flows of 5 and 15 cm s as generated by a motor
driven propeller. The flow tank was designed after Vogel and LaBarbera 1978 and had a working section approximately 10 3 10 cm in cross-section and 50 cm long. Current
21
speeds were measured by an acoustic doppler velocimeter Sontek, and 15 cm s was
the fastest consistent speed possible in the flow tank. To prevent colonies from being swept along with the current, they were attached to a weighted plate on the bottom of the
flow trough. At 15-min interval a screen of plankton netting was placed in the tank for 1
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.B. Forward et al. J. Exp. Mar. Biol. Ecol. 248 2000 225 –238
min to collect the released larvae. Temperature in the flow tank increased a maximum of 48C throughout the observation period. Water was removed from the flow tank after each
test and new seawater added. Data were analyzed as described above and results compared to those in the finger bowls no current flow to assess the effect of current
flow on larval release.
2.2. Release of larvae in the laboratory The larval release experiment was repeated in the laboratory to determine the relations
to light intensity under more controlled conditions. The room was maintained at 248C and larvae irradiated with light from a 500-W tungsten filament bulb filtered to the
blue-green 480–540 nm spectral region Corning [ 4-96 filter; Kopp Glass Inc.. Wavelengths in this region encompass the major spectral sensitivity maximum of most
invertebrate zooplankton Forward, 1976, 1988 and the action spectrum peak at 520–550 nm for spawning of a solitary tunicate Lampert and Brandt, 1967. Light
intensity was controlled by fixed neutral density filters Oriel Corp. and measured with the above irradiance meter.
Colonies were irradiated in 5.5 cm diameter finger bowls and released larvae counted at 15-min intervals. When irradiated at low light levels, larvae were counted by
illumination with wavelengths greater than 650 nm. Most invertebrates are insensitive to light in this region e.g. Forward and Cronin, 1979. A range of light intensities was
tested and the data were analyzed as described above for the sunlight experiments.
2.3. Light dark preference of larvae in sunlight The light dark preference of larvae was tested by placing groups of ten swimming
larvae in new seawater in a plastic Petri dish diameter 5 8.7 cm diameter. Half of the dish was painted flat black on the outside and the other half was clear. The dishes were
placed on a white surface and overall light intensity controlled by covering them with layers of black fiberglass screening. The distribution of larvae in the light and dark sides
was determined after exposure to light for 1 h.
In the initial experiments, larvae were tested 1, 7 and 24 h after release to determine whether the lighting preference changed with age. Most of the larvae settle within 24 h
but enough swimming larvae were available for experimentation after this time interval.
15
The average light intensity was about 10 of unscreened sunlight 13.8 3 10 photons
22 21
cm s
. Since there was no change in preference with age, all subsequent experiments tested larvae between 1 and 6 h after release. Larvae were tested only once.
To determine the minimal light intensity larvae could perceive for distinguishing between light and dark, the stimulus light was decreased until the distribution within the
light dark sectors became about equal 50, which indicated no preference. For each test, the percentage of larvae in the light section was determined. Means and standard
errors were calculated from arcsine transformed data and back transformed values are plotted on the figures.
R .B. Forward et al. J. Exp. Mar. Biol. Ecol. 248 2000 225 –238
229
2.4. Light dark preference of larvae in the laboratory The foregoing experiment was repeated in the laboratory using the blue-green
stimulus light system described for the larval release experiments. Light intensity ranged
12 14
22 21
from 10 to10
photons cm s
to determine the lowest intensity to evoke a significant light dark preference. The data were analyzed as described for experiments in
sunlight and presented as the mean percentage of larvae 6standard error found in the light section.
3. Results
