Abstract 1 quantified the spatial distribution, physiological tolerances, and respiration rates of
larvae of the intertidal fly, Oedoparena glauca. O. glauca larvae consume the barnacle, Balanus
glandula, and use the shell for protection and pupation. The percent of barnacles infested with
larvae and pupae was greatest in a high-intertidal crevice (29%), moderate on upper north-facing
intertidal walls (-3%), and zero at lower tidal heights and on south-facing walls. Thermal stress,
high salinity, and submergence are physiological stresses imposed on the larvae and pupae due to
life in the intertidal zone. In laboratory tests of thermal tolerance, all larvae died following 3
hours of exposure to 39°C, but tolerated temperatures 838°C. Pupae appeared more sensitive to
thermal stress, with 0% eclosion by pupae treated at 35°C, 38°C, and 41°C for four hours.
Intertidal datalogger records and field measurements of internal B. glandula temperatures
exceeded 36°C, near the thermal limit of O. glauca larvae. In addition to this thermal stress,
during periods of aerial exposure, B. glandula was significantly more saline in vivo than the
surrounding seawater, perhaps due to evaporative cooling by the barnacle. In tests of
submergence tolerance, larvae survived 8 days of submergence in filtered seawater held at 13°C.
but all died after 10 days. The pupae were apparently more sensitive to submergence; 40% of
pupae submerged for two hours eclosed within 16 days, whereas there was only 12.5% eclosion
of pupae submerged for 24 hours. The larvae were observed not to respire during six hours of
submergence. However, the larvae respired at a rate of 0.4317 umol Oy/hr/g, over four days of
submergence. This study illustrates that O. glauca larvae are hardy and capable of tolerating
thermal stress and submergence. In contrast, the pupae may be more sensitive to these
environmental conditions, perhaps restricting their geographical distribution.
Introduction
The rocky intertidal zone experiences large temperature fluctuations and extended periods of
submergence. This environment is extremely stressful for a terrestial insect, and may explain
why insects rarely inhabit and reproduce in the intertidal zone. One little studied terrestial insect
that occupies this niche is the intertidal fly, Oedoparena glauca. It is the first and only
confirmed record of a dipterous predator of barnacles (Burger et. al 1980). O. glauca larvae prey
on the acorn barnacle Balanus glandula. O. glauca larval development consists of three instars
(Burger et. al 1980). This report focuses on the second and third larval stages and the pupae
(Figures 11 and 12). The adult flies are 5-Omm in length (Figure 10), and feed primarily on
diatoms. O. glauca inhabits the Endocladia-Balanus zone within the intertidal zone.
There are only two other reports of Diptera associated with barnacles, both from Europe.
Roubond (1903) found fly larvae among the Semibalanus balanoides. Mercier (1921) observed
larvae living within barnacles, and hypothesized that these larvae were also predaceous on
barnacles. Neither account reared larvae to adults, so the identification of the species was not
certain.
There are many unusual stresses that the O. glauca must overcome to survive in intertidal
habitats. Glynn (1965) determined that the center of the Endocladia-Balanus zone may spend
27% percent of the time submerged. For a terrestial insect, this is a tremendous amount of time
to be submerged in saline water. Furthermore, temperature may fluctuate a great deal in the
intertidal zone, and change rapidly depending on the tide. Since the adult cannot tolerate
submergence, O. glauca must coordinate behavior such as oviposition with periods of low tide
emersion.
1 quantified the physiological tolerances of the second and third instar larvae and pupae.
Although the mechanisms for the tolerance of much of the physiological stress is unknown, it is
clear that the larvae are capable of withstanding severe stress. The pupae appear much more
sensitive to thermal stress and submergence than the larvae. These tolerances may have
important effects on both the local and geographical distribution of this species.
Materials and methods
Organisms
Pupae and second and third instar larvae of Oedoparena glauca were collected from high
intertidal crevice habitats around a surge channel on the wave exposed point at Hopkins Marine
Station in Central California (Figure 13). This channel contains many wave-force meters
installed by Mark Denny and his lab group. Collected larvae and pupae were placed in petri
dishes with raised lids. A small opening was kept in the Parafilm sealing the petri dishes to
prevent anoxia. The larvae and pupae were kept moist and held in air at approximately 15°C
above a flowing seawater tank. During the treatments of the larvae, the larvae were not fed. Wet
masses of second and third instars were between 0.0020g and 0.0170g. Wet weights of the
pupae were approximately 0.0150g.
Spatial distribution
The abundances of larvae and pupae were surveyed in seven habitats around the wave-exposed
surge channel. These seven habitats were located in a high intertidal crevice and on north and
south-facing walls at low, mid, and high tidal heights. The tidal heights were obtained by
surveying the middle of these zones (Table 1). From these seven habitats, 100-150 acorn
barnacles, Balanus glandula, were haphazardly removed from the rock with a screwdriver. To
determine percent infestation by Oedoparena glauca larvae and pupae within these habitats, the
barnacle body was removed from each B. glandula and the contents of each barnacle shell were
scrutinized in the laboratory.
Temperature loggers
Temperature dataloggers (Optic StowAway, Onset Computer Corp., Pocasset, MA) were
installed in the high and low, north and south-facing habitats from 28 May 2001 thru 1 June
2001. A smaller temperature datalogger (Tidbit, Onset Computer Corp.) was placed in the high
crevice for the same period. All of the dataloggers were set to record the ambient temperature
every ten minutes.
Barnacle Temperatures
Barnacle temperatures were taken periodically in the field from 30 May 2001 through 1 June
2001. The internal temperature of the barnacle was taken by opening the opercular plates with a
scalpel and inserting a thermocouple inside the shell. Ten barnacle temperatures were taken at
each site during each sampling. Barnacle temperatures were also taken periodically in a 10 cm
radius around one temperature datalogger to test whether barnacle temperatures were correlated
with datalogger records
Thermal tolerance
A 1.8 m long aluminum block was used to establish a thermal gradient to test the tolerances of
the larvae and pupae. On one side of the aluminum block, a heater was attached, and on the
other side a water bath. The temperature set points were manipulated until a preferred thermal
gradient was maintained. The aluminum block was drilled with holes, four across and 31
lengthwise, so that 2.0 mL Polypropylene flat top microcentrifuge tubes could be inserted within
the aluminum block. The microcentrifuge tubes were punctured on the cap to prevent anoxia.
The upper thirds of the tubes were filled with tightly fitting foam cores that had been soaked in a
solution of filtered seawater and antibiotics (15 mg/L penicillin-G and 15 mg/L streptomycin).
Three thermal tolerance experiments were performed on the second and third instar
larvae. Five different temperatures were chosen per experiment. The first of three experiments
had temperatures of 24°C, 28°C, 32°C, 36°C, and 40°C. The second had temperatures of 32°C.
33°C, 34°C, 35°C, and 36°C. In the final experiment, larvae were maintained at temperatures of
36°0, 37°C, 38°C, 39°C, and 40°C. A control tube with moistened foam was placed in the
fourth hole at each experimental temperature along the block. A thermocouple was inserted
through a hole in the top of these tubes, and the temperatures were monitored to assure stable
conditions during the experiment. Larvae were exposed to experimental temperatures for three
hours, followed by a three hour recovery period. During the recovery period, the larvae were
placed into petri dishes covered with Parafilm through which a small hole had been made and
held at 15°C above the seawater tank. Responsiveness to stimuli immediately following the
treatment and following the recovery period were scored.
In a similar experiment, pupae in microcentrifuge tubes with seawater moistened foam
cores (see above) were exposed to 35°C, 38°C, or 41°C for four hours (n = 3 pupae x 3 tubes per
temperature). Following the period of exposure, pupae were placed in petri dishes with raised
lids and held at 15°C above the flow tank. Percent eclosion was scored after 16 days.
Submergence tolerance
To test submergence tolerance of the second and third instar larvae, larvae were placed into 2 mL
screw top glass vials that were filled with a solution of filtered seawater and antibiotics (15 mg/L
penicillin-G and 15 mg/L streptomycin). A single larva was placed within each of three vials per
treatment. The vials were submerged in a 13°C water bath and maintained up to (but not
submerging) the level of the bottom of the air-permeable screw top. Larvae were submerged in
6, 12, 24, 48, 96, 144, 192, 240, and 264 hour treatments. Following submergence, the larvae
were allowed to recover for three hours. During the three hour recovery period, the larvae were
placed inside petri dishes and maintained at 15°C, as described above. As before, responsiveness
to stimuli was scored immediately following the treatment and following the recovery period.
Pupae were subjected to a similar esperiment. Pupae were placed into plastic and nylon
mesh tea strainers (PermaBrew Tea Infuser, Upton Tea Imports, Hopkinton, MA). A large
beaker was filled with the solution of filtered seawater and antibiotics (see above), and the tea
strainers were submerged at 13°C for either 2 hours or 24 hours (n = 8-10 pupae per treatment).
Following the submergence treatment, the pupae were placed inside petri dishes at 15°C and
percent eclosion was quantified after 16 days.
Respirometry
These respirometry trials were designed to test if the larvae were respiring during short and long
submergence periods. A water-jacketed microrespiration chamber was attached to a water bath
and maintained at 13°C. Data were recorded using PowerLab (model 8SP, ADInstruments,
Mountain View, CA) and the PowerLab Chart Program. Filtered seawater treated with
antibiotics (15 mg/L penicillin-G and 15 mg/L streptomycin) was aerated with an air pump prior
to setting 100% 02 saturation. Six gas-tight test tubes (-3 mL volume) were then filled with the
treated seawater. In the six hour trial, four of the six tubes had two larvae placed in each and two
tubes were used as controls. In the four day trial, three of the tubes had two larvae in each and
three tubes were used as controls. All tubes were maintained in the dark at 13°C. At the end of
the treatments, samples of water from each tube were injected into the microrespirometry unit
and percent O2 saturation was quantified.
Barnacle Osmolarity
On 31 May 2001, two B. glandula in each habitat were sampled near the time when they were
likely experiencing their maximum daily temperatures for that day. Immediately following
removal from the rock, internal fluid was extracted from the barnacle using a 1 mL syringe and
20G1 PrecisionGlide needles. As a control, a sample of untreated seawater was also collected in
the Hopkins Marine Life Refuge. The barnacle fluid and seawater were analyzed in a Wescor
5500 Vapor Pressure Ösmometer. Differences in osmolarity among the fluid samples and the
seawater were analyzed using a t-test.
Results
Spatial distribution
The highest infestation of O. glauca larvae and pupae was in barnacles of the high zone crevice
(Figure 1). Infestation was moderate (2.7%) in the north-facing high habitat and in the north-
facing mid-habitat (3.8%). There was no infestation in any of the south-facing habitats and zero
infestation in the low north-facing habitat.
Barnacle field temperatures
The dataloggers recorded temperatures above 35°C in the south-facing low and high habitats as
well as in the crevice habitat (Figures 2 and 5). The maximum recorded temperatures occurred
on 31 May 2001 (Figure 3). Highly stressful temperatures, above 35°C, were recorded by the
dataloggers only in the south-facing and the high crevice habitats. A tight correlation was found
between datalogger readings and the internal temperatures of nearby barnacles (Figure 4, R2 =
0.7576), indicating that the datalogger records were representative of the temperatures that larvae
of O. glauca would experience within these habitats.
Thermal tolerances
O. glauca's lethal thermal limits were 38-39°C, with zero larvae responding to stimulus
immediately following the 38°C treatment, and 67% responding following the three hour
recovery period (Figure 6). All larvae subjected to treatments 2 39°C were unresponsive
immediately following the treatments, and none recovered. Temperatures of 36°C also seemed
stressful to the larvae; although all larvae were able to recover, 66% were unresponsive
immediately following exposure to 36°C
The pupae of O. glauca seemed more sensitive than the second and third instar larvae.
No pupae had eclosed in the 16 days following the three treatments (35°, 38°, 41°C). Although
larvae were able to tolerate temperatures as high as 38°C, the pupae appeared unable to
withstand the 35°C treatment.
Submergence tolerances
The second and third instar larvae of O. glauca were all responsive following submergence for as
long as four days (Figure 7). After six days submergence, 33% of the larvae were unresponsive
following the treatment and were unable to recover. Following eight days of submergence, zero
larvae were responsive immediately following submergence. However, following the three hour
recovery period, 67% were responsive. All larvae were unresponsive following treatments of 10
days or longer and no larvae recovered from these treatments.
Again, pupae of O. glauca appeared more sensitive than the second or third instar larvae.
40% of pupae had eclosed in the 16 days following the 2 hour treatment, compared with 12.5%
eclosion following the 24 hour treatment.
Respirometry
No measurable respiration was found during a six hour period of submergence. The O,
saturation did not differ among control tubes and tubes with larvae. In contrast, after the four
day trial, a significant percentage of O2 was removed from the tubes containing larvae. The
average respiration rate was 0.4317 umol/hr/g.
Barnacle Ösmolarity
The osmolarity of the barnacle fluid collected from the seven habitats on 31 May 2001 was
significantly greater than that of the seawater samples collected on the same day (Figure 9, t-test.
p50.001).
Discussion
O. glauca, a terrestrial insect, is highly unusual in that it has been able to establish a niche in a
marine environment by having larvae and pupae tolerant of physiological stresses associated
with the intertidal zone. The results of this study suggest that the second and third instar larvae
of O. glauca are more tolerant to these physiological stresses than are the pupae. This difference
is most evident in the thermal tolerance responses of the larvae and pupae. Although larvae
showed significant signs of stress, they were able to tolerate temperatures as high as 38°C. In
contrast, pupae did not eclose following exposure to either 35°C or 38°C. Pupae may take 28
days to eclose (Burger et al. 1980) so the results may not have shown all pupae that would have
eventually eclosed (age of pupae used in the study was unknown). However, many pupae did
eclose (40%) in the 2hr submergence treatment, indicating the lack of eclosion was a real effect,
It is possible that the rapid development within the pupae leading up to eclosion makes the pupae
more susceptible to thermal stress.
Pupae were most abundant in the crevice habitat that also had the longest exposure to
temperatures that appear lethal to a pupa in the laboratory. It appears that on these warm days,
the larvae are also living in a hypersaline barnacle, perhaps due to evaporative cooling by the
barnacle. However, the temperatures recorded by the datalogger may not be indicative of the
internal barnacle temperature in the high crevice. The thermocouple on the datalogger may have
been too close to the rock and may not have accurately recorded the ambient temperature. In
addition, a different datalogger model was required for the narrow crevice, and this smaller
instrument may have had different thermal properties than the other four dataloggers. If this is
not the case, it is unclear how the pupae deal with these elevated temperatures in the field,
because they were unable to do so in the lab. It is also uncertain why these sites with high
temperature exposure are chosen by the reproductive female.
When submerged, larvae of O. glauca do not appear to respire initially, but do respire
with extended submergence. It is unclear whether the onset of respiration is delayed, or whether
the initial respiration was too small to be detected above the noise of the system. The
mechanism by which respiration occurs during submergence is unknown in these larvae. Further
work is needed to elucidate how these larvae respire in both air and water.
With larvae and pupae of O. glauca able to tolerate long durations of submergence, one
might predict that high numbers of these individuals would be found in the low zone. This
seems even more likely based on the sensitivity of larvae and pupae to periods of thermal stress,
which are generally more severe at higher tidal heights. However, although there appears to be a
preference for northern exposure over southern exposure, both low habitats showed zero
infestation by O. glauca. Newly hatched O. glauca flies may require time to harden their wings.
to become competent to fly. This phase of their maturation may be difficult in the low zone due
to shorter periods of aerial exposure. Furthermore, pupae are more sensitive to submergence,
making distribution higher in the intertidal zone favorable. These facts seem consistent with
Burger et al.’s (1980) observation that pupae in WA state were distributed higher in the intertidal
zone than the larvae. Pupal distribution may also be decreased in the low zone due to decreased
barnacle density, resulting in a less attractive target for ovipositing females.
The southern geographical limit of O. glauca is reported to be in Central California
(Burger et al. 1980). This boundary could arise from the pupae living near their lethal limit,
especially on unseasonably warm days. This southern geographical limit may be established by
the availability of reproductive sites that are high enough in the intertidal zone to minimize
submergence, yet are not exposed to temperatures above the lethal limit of the pupae.
Acknowledgements
1 would like to thank Eric Sanford for all of his generous help, time, attitude, and teaching. I
would also like to especially thank George Somero and Jim Watanabe for all of their advice,
expertise, and criticism. Jason Podrabsky and Peter Fields also added valuable assistance in the
lab, and Chris Harley (University of Washington) provided valuable references and insight
during the inception of this project. Finally, I would also like to thank all of the 175H instructors
for making this a tremendous experience.
Literature Cited
Burger, JF, JR Anderson, MF Knudsen. 1980. The habits and life history of Oedoparena glauca
(Diptera: Dryomyzidae), a predator of barnacles. Proc. Entomol. Soc. Wash. 82(3): 360-
37.
Glynn, PW. 1965. Community composition, structure and interrelationships in the marine
intertidal Endocladia muricata - Balanus glandula association in Monterey Bay,
California. Beaufortia 12: 1-198.
Mercier, L. 1921. La larve de Limnophoro aesruum Villen., Diptere marin. Compt. Rend. Acad.
Sci. Paris 173: 1410-1413.
Rouboud, M. 1903. Sur les larves marines de Dolichapodes attribueés au genre Aphrosylus
(Walker). Bul. Mas. Nat. 9: 338-340.
Tables
Table 1: Tidal heights in feet above Mean Low Low Water for the seven intertidal habitats
sampled for fly larvae and pupae.
Habitat
Tidal Height (ft. above MLLV)

5.49, 4.79
North-facing low wal
North-facing mid wall
6.61
North-facing high wall
765
Crevice
7.10
430, 5.03
South-facing low wal
South-facing mid wall
5.33
South-facing high wall
5.91, 5.82
14
Figure Legends
Figure 1. Infestation of barnacles (B. glandula) in the seven intertidal habitats: a high crevice,
low, mid, and high tidal heights on north and south-facing walls.
Figure 2. Datalogger temperatures for 30 May 2001 through 1 June 2001 in the high zone
crevice, and low and high zones on north and south-facing intertidal walls at the Hopkins Marine
Life Refuge.
Figure 3. Temperatures for 31 May 2001, the day with the highest peak temperatures for the
period 30 May 2001 thru 1 June 2001.
Figure 4. Calibration curve for datalogger temperatures versus the measured internal
temperatures of barnacles (B. glandula) within 10 cm of the south-facing high zone datalogger.
Figure 5. Time of exposure in the high zone crevice, low and high zones of north and south-
facing walls to temperatures above 30°C, 33°C, and 35°C for the period 30 May 2001 through 1
June 2001.
Figure 6. Thermal tolerance of O. glauca larvae. Percentages of larvae responsive immediately
after temperature treatment (dotted line), and final survival after a three hour recovery period
(solid line) (n = 3-9 larvae per termperature)
Figure 7. Submergence tolerance of O. glauca larvae. Percentages of larvae responsive
immediately after submergence treatment (dotted line), and final survival after a three hour
recovery period (solid line) (n = 3 larvae per submergence treatment).
Figure 8. Percentage of pupae eclosed after 16 days following submergence treatments of 24
hours and 2 hours (n = 8-10 pupae per treatment).
Figure 9. Difference in osmolarity between barnacle fluid and seawater samples. Error bars are
standard errors of the mean.
Figure 10. View of an Oedoparena glauca fly that eclosed in the laboratory
Figure 11. View of a third instar Oedoparena glauca larva.
Figure 12. View of a pupa of Oedoparena glauca.
Figure 13. Map and picture of the study site located within the Hopkins Marine Life Refuge,
Pacific Grove, CA.
Figures
Figure 1
Infestation of Barnacles on North vs. South-facing Wave Exposed
Vertical Walls
SW high vertical wall
%larvae
%pupae
SW mid vertical wall
SW low vertical wall
High crevice
NW high vertical wall
N mid vertical wall

Nlow vertical wall
5
20
25 30 35
Percent Barnacles Infested
16
Figure 2
Field Temperatures at Hopkins Marine Life Refuge
— Crevice
- North-facing High
35.00
North-facing Low
-South-facing High
30.00
- South-facing Low
25.00
20.00
15.00

10.00
5/28/01 12:00 5/29/01 12:00 5/30/01 12:00 5/31/01 12:00 6/1/01 12:00 6/2/01 12:00
AM
AM
AM
AM
AM
AM
Time
17
Figure 3
Field Temperatures on 31 May 2001
-Crevice
35.00
North-facing High
— North-facing Low
30.00
-South-facing High
- South-facing Low
25.00
20.00

15.00


S
10.00
5/31/01 12:00 5/31/01 4:48 5/31/01 9:36 5/31/01 2:24 5/31/01 7:12 6/1/01 12:00
PM
AM
PM
AM
AM
AM
Time
18
Figure 4
34.0
29.0
24.0
19.0
14.0
10.00
Datalogger Temperatures vs. Internal Barnacle Temperatures
V=0.9353x + 1.9106
R2 = 0.7576
15.00
25.00
20.00
30.00
Datalogger Temps (°C)
35.00
Figure 5
Crevice
Variation in Thermal Exposure Among Intertidal Habitats
EAbove 30°C
HAbove 33°0
EAbove 35°C
North-facing Low North-facing High South-facing Low South-facing
High
Habitats
Figure 6
100
75
25
Thermal Tolerance of Larvae in Laboratory Gradient
= - % Responsive after thermal stres
% Survival after recovery

36 38 40
32 34
24 26 28 30
Temperature (?C)
Figure 7
Submergence Tolerance of Larvae
100-—
% Survival after recovery
90
- % Responsive after submergence
80
60
40
30
20
k
50
100
150
250
200
Time (hrs)
22
Figure 8
Percentage of Pupae Eclosed following Submergence Treatments
15
35 40 45
O 5 10
20
30
% Eclosion after 16 days
23
Figure 9
850
900
Barnacle Salinity vs. Sea Water Salinity
□Sea Water Osmolarity
EBarnacle Fluid Ösmolarity
1050
1000
1100
950
Ösmolarity (mmollkg +SE)
24
1150
1200
igure 10
Figure 11
2 mm
Figure 12
2 mm
Figure 13





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