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Some effects of early starvation on the survival and development of barnacle nauplii, Balanus improvisus (Darwin)
63
J. Exp. Mar. Biol. Ecol., 1982,Vol. 60, pp. 63-70
Elsevier Biomedical Press
SOME EFFECTS OF EARLY STARVATION ON THE SURVIVAL AND DEVELOPME~
BALANUS
WILLIAM EPA Environmental
OF BARNA~E
IMPROVISUS
H. LANG’
Research Laboratory,
NAUPLII,
(Darwin)’
and MARTHA MARCY
South Ferry Road, Narragansett.
RI 02882, U.S.A.
Abstract: Newly hatched stage I-II nauplii of Balanus i~pr5~j~s (Darwin) were “totally starved” (until death) or “partially starved” for the t‘irst 48 h and 96 h of their development. Daily mortality and molting were monitored throughout larval development in both starved and fed control groups. Fed control animals exhibited a largely synchronous molting pattern with instars of equal duration. Total starvation suppressed molting beyond stage II; 50% mortality occurred in -4 days at both 15 and 21 “C, while longest survival time was 7 days at 15 “C and 6 days at 21 “C. At 15 “C, partially starved nauplii retained the ability to complete naupliar development but at a slower overall rate and with increased mortality relative to controls. These effects were more pronounced in the 96-h group. Increased mortality of stage VI nauplii was evident in both partially starved groups (7.1% for 48 h, 18.8”/, for 96 h) relative to unstarved controls (3.1%). Stage II nauplii exhibited little resistance to starvation and survival potential may have decreased as soon as 24 h. INTRODUCTION
This study investigates the survival time of starved stage II Balanus improvisus (Darwin) nauphi and, more importantly, the effects of partial starvation on subsequent larval development. In addition to providing new information on the physiological ecology of cirripede larvae, results are intended to supplement baseline information for use of larvae in bioassay tests. Without food, larval development of most barnacle species will cease at stage II nauplius (Lang, 1979). In general, larvae just beginning to feed are very sensitive to starvation. The onset of feeding is recognized as a “critical period”, and extensive mortality will occur, as reported for fish and decapod larvae, if proper food is unavailable (Hunter, 1976; Paul & Paul, 1980). Nauplii of copepod species exhibit no particular resistance to starvation (Dagg, 1977), although the adults may be capable of surviving several weeks or more without food. In addition to acute mortality of larvae with total starvation, partially starved larvae, returned to conditions of abundant available food, may fail to recover to “normal” feeding rates and developmental patterns (Kon, 1979; Paul & Paul, 1980). The effects of “partial starvation” (i.e., subjection to a short period of starvation) on barnacle ’ Contribution No. 226 from the EPA Environmental Research Laboratory, Narragansett, RI. ’ Present address: Bureau of Land ~~anagement, NYOCS Offtce, Suite 32-120, 26 Federal Plaza, New York, NY 10278, U.S.A. 0022-098l/82/0000-0000/$02.75
0 1982 Elsevier Biomedical Press
WILLIAM
64
larvae
are essentially
swimming
unknown.
rates and perhaps
1980). The effects
H. LANG
AND
MARTHA
Stage II nauplii
altered photobehavior
of nutritional
stress
MARCY
starved
for 24 h exhibited
(Singarajah
on larvae
reduced
et al., 1967; Lang et al.,
may not always
be immediately
apparent; prolonged and abnormal development with delayed mortality can result. Although some boreo-arctic species (e.g., Semibalanus balanoides) appear to synchronize larval release with diatom blooms, most temperate species release nauplii irrespective of phytoplankton cues (see Lang & Ackenhusen-Johns, 1981). Hence it would be of interest to determine the relative sensitivity to starvation for nauplii of this latter temperate group, of which Balanus improvisus is a member.
MATERIALS
AND
METHODS
Adult B. improvisus attached to small stones were collected from a subtidal site in the Pettaquamscutt River, Narragansett, Rhode Island. Salinity at the collection site averaged z 12%,, but has varied from 0 to 30x0 with extremes of tides and weather. Water temperatures have ranged seasonally from 0 to z 27 ‘C (Lang & AckenhusenJohns, 1981). Adults were maintained in finger bowls; constantly submerged in 0.45 pm filtered sea water (15x, salinity). 15x, water was made by diluting 30X0 sea water with deionized water. A 12 h light : 12 h dark cycle was employed. Adult barnacles were fed newly hatched Artemia salina (Aquafauna Brand, Macau, Brazil)’ approximately three times weekly. To obtain nauplii, adults were fed overnight and subsequently changed to clean filtered 15x, S water. Each container of ~20 to 30 adults was monitored for release of nauplii. Larvae released within a known 6- to 12-h period were concentrated using a light beam and removed to 0.45 pm filtered 15x, S sea water by pipette. Starved nauplii were transferred through two changes of 0.45 pm 15x, S filtered sea water and kept in 7 cm Carolina culture dishes with ~45 to 50 nauplii and 60 ml of filtered 15x, S sea water per dish (static culture). Fed nauplii were given a mixed algal diet of Tetraselmis suecia ( = Platymonas suecia or suecica) and Skeletonema costatum grown axenically in a modified f/2 medium. The algae were mixed in approximately equal volumes, filtered through a 44 nrn Nitex screen to remove cell clumps and diluted with 0.45 pm filtered 15x, S sea water to obtain a final density of 2 to 4 x lo4 cells . ml- ‘. Fed nauplii were maintained identically to starved nauplii in all respects other than feeding. Nauplii were changed to fresh media at 48-h intervals. Mortality and development were recorded at 24-h intervals by tallying and staging of all molts and dead (no movement and loss of normal transparent appearance). A daily percent composition of naupliar stages was determined. The few larvae apparently lost during transfer (< 3 %) were counted as dead in final calculations. Results in replicate cultures were nearly identical in all cases and were pooled for the presentation of data. The experi-
I Reference Agency.
to trade names does not imply endorsement
of the United
States Environmental
Protection
STARVATIONOFBARNACLENAUPLII
65
ments were designed to give information on general trends only and thus no statistical tests for significance were performed on the data. In one experiment, survival and development were determined at 15 & 1 “C and 20 f 2 “C with 15%, S sea water, 12L : 12D light cycle. Larvae were obtained from adults collected in August 1980, and maintained in the laboratory for 3 days. Nauplii were divided into fed (control) and totally starved groups. In another experiment, the ability of stage II nauplii starved for 48 h and 96 h (i.e., “partially starved”) (15 “C, 15x, S sea water, 12L : 12D light cycle) to complete naupliar development was tested. Larvae were obtained from adults cohected in November 1979 and maintained under laboratory conditions for over 6 months. Initially, several hundred newly hatched nauplii were kept in a 600-ml container of 0.45 pm 15%, S filtered sea water. At 0, 48 and 96 h, two groups of z 50 nauplii (47 to 5 1) were transferred to duplicate 60-ml cultures of algae and sea water. Cultures were monitored to an endpoint of 20% cyprid, based on the initial number of larvae started. RESULTS
At 15 *C, 100% mortality occurred in 7 days for starved stage II nauplii. First deaths were noted at 2 days. Fifty percent mortality was observed in ~4 days (Fig. IS). At 21 “C, little difference in time to 50% mortality was apparent but 100% mortality occurred in only 6 days (Fig. 1D). In contrast to the minor differences in times to 50% mortality, marked differences in overall developmental rates were evident in fed control groups at 15 “C and 21 “C. Excluding the rapid molt from stage I to II, the fed early stage nauplii of Balanus improvisus exhibited larval stages of nearly equal duration, i.e., “isochronal development” (Miller et al., 1977). At 15 “C, nearly all stage II-IV nauplii molted within 24 h of each other at 2-day intervals (Figs. lA, 2A). Although molting to stage VI also began 2 days following the stage V molt, the synchrony of molting observed in earlier naupliar stages was lost, as molting from stage V to VI occurred over a 6-day period (Fig. 2A). The metamorphosis from stage VI to cyprid most often required a minimum of 4 days and molting continued over a period of 7 or more days (Fig. 2A and unpubl. results). Nauplii reared at 2 1 ‘C, also exhibited isochronal development in stage II-IV but at about one-half the development time (Fig. 1C). Duration of these early stages was 1 day each, and cyprids first appeared after Day 6 in contrast to Day 13 at 15 “C. Surviving stage II nauplii, fed following starvation for 48 h and 96 h at 15 ‘C, retained an ability to complete naupliar development, but (1) at slower rates and (2) with increased mortality relative to fed controls. Forty-eight-h initial starvation increased naupliar development time by z 1 day in stages III-IV and up to 2 to 3 days for stage VI (Fig. 2B, Table I). Effects were more pronounced after 96 h starvation with the time for 50% of nauplii to reach the next developmental stage increased by z 2 days for stage III and up to 5; days at stage VI (Fig. 2B, Table I).
Fig. 1. Daily percent mortality and A, fed nauplii at 15 “C, 15x, S (N 21 “C, 15x, S (N = 50); D, starved IV, VI) are indicated; odd numbered
naupliar stage distribution for stage I-II Balanus improvisus nauplii: = 48); B, starved nauplii at 15 ‘C, 15x, S (N = 47); C. fed nauplii at nauplii at 21 “C, 15x,, S (N = 48); even numbered naupliar stages (II, stages (III, V) and cyprid are cross-hatched areas in appropriate order between labelled stages
SO
Fig. 2. Daily naupliar stage distributions for Balunus improvisus larvae: A, larvae fed at hatching (control); B. larvae fed after 48 h starvation; C, larvae fed after 96 h starvation; Day 0 represents the time of initial feeding; percent distributions are based on the number of surviving larvae at each 24-h interval; see Table II for mortality and sample size; each group was terminated at an endpoint of 20% cyprid; even numbered naupliar stages (II, IV, VI) are indicated; odd numbered stages (III, V) and cyprid are cross-hatched areas in appropriate order between labelled stages.
STARVATION
OF BARNACLE
67
NAUPLII
In addition immediate
to the increased larval development times, starvation resulted in an mortality of stage II nauplii starved 96 h (Table II). Increased mortality 8%
The comparative rate of development ofBalanus imptwisus larvae starved for 0.48, or 96 h and then reared at 15 “C, IS%, salinity using a mixed algal diet (see text): time in days for 50% attainment of next naupliar stage is calculated based on the number of surviving nauplii and extrapolated as a constant rate of molting during each 24-h interval: developmeIlta1 times begin at initiation of feeding, irrespective ofinitial starvation periods. Time (days) to 50°$ naupliar Hours
starved 0 48 96
stage
III
IV
V
VI
2.5 3.6 4.6
4.5 5.2 7.4
6.5 7.5 10.8
9.5 11.3 14.1
TABLE II
The percent mortality of ~a~~ff~ j~~rov~~ larvae starved for 0,48 or 96 h and then reared at 15 “C, l5& salinity using a mixed algal diet (see text): mortality during initial starvation periods is undetermined; initial sample numbers are: control, 96; 48 h, 98; 36 h, 101. Hours
starved
0
48
96
II III IV V VI
1.0 0 1.0 4.2 3.1
1.0 1.0 3.1 2.0 7.1
7.9 1.0 4.0 2.0 18.8
Total
9.3
14.2
Naupliar
stage
_I~
of stage VI nauplii was evident controls (Table II).
33.7 ._
for both starved
groups
(48 and 96 h) relative
to fed
DISCUSSION
Temperature has a pronounced effect on cirripede larval developmental rates (Lang, 1979; Marcy, 1980). The nearly doubled developmental rate with a 6 “C rise in temperature from 15 to 21 “C was not unexpected. The loss of molting synchrony and reduction in developmental rate of later stage nauplii observed in fed groups may have been indicative of the increasingly complex structural development of the larvae and metamorphic changes in preparation for the cyprid stage (Walley, 1969). It is also
WILLIAM
6X
H. LANG
AND MARTHA
MARCY
possible that the diet used here may not have been completely adequate for later stage development. Total starvation of stage II nauplii suppressed further development. No molting to stage III occurred and all nauplii died by 7 days (Fig. 1). This starvation survival time is within the range of that reported for other temperate water crustacean larvae (Table III). TABLE III
Survival
times for starved
crustacean
larvae:
C, copepodite:
N, nauplius;
2, zoea.
Mortality
(days)
SOS,,
loo:”
11 11
11 15
18 18
Kon, 197Y Ken, 1979 Anger
Temp. Stage
Reference
z Z
f 11
Z
I
12
9
14
2
I
20
3
I
z
I I
6-9
6
10
NIII NIV
15 15
5 4
7 7
Fernandez, Fernandez,
21
-
Dagg,
1977
2 2
Dagg, Dagg,
1977 1977
7 6
This study
2
___--._.
(“C)
6.5
25
c5
15
Nl Nl
15 15
NII
15 21
4 4
& Nair,
Bigford,
1979
1978
Sulkin & Epifanio, Kurata,
1975
1962 1979 1979
--
Sublethal effects of starving stage II nauplii at 15 “C become evident at 24 to 48 h. Nauplii starved for only 24 h exhibited some loss of swimming speed and perhaps depressed sensitivity to light but no acute or delayed mortality (Lang et al., 1979, 1980). In the present work, 48-h starvation resulted in z 10% mortality (Fig. 1B) and subsequent development of surviving nauplii fed at this point lagged ~24 h behind fed controls (Fig. 2B). Effects of 96-h starvation were more pronounced with 50)1/, acute mortality (Fig. lB), increasing loss of molting synchrony, and generally prolonged intermolts (Fig. ZC). Kon (1979) observed that as the duration of initial starvation (followed by feeding) of stage I Chionectes opilio was increased, a progressive decline in percent of successful molt to stage II occurred and the intermolt period was prolonged. In contrast, low food rations or starvation of Palaernonetes vulgaris larvae had a minimal immediate effect on molting frequency but suppressed growth per instar (Knowlton, 1974). Repetition of larval stage was also observed with no development to postlarva and eventual death. These responses perhaps reflected the basic differences in the rigidly prescribed number of larval instars for B. irnprovisus and C. opilio versus the high variability in the
STARVATION
Pulaemonetes
OF BARNACLE
larvae. The net effect of limited starvation,
69
NAUPLII
however, was to prolong larval
development. Of particular
interest,
the present
results
show a delayed
increase
in mortality
at
stage VI for larvae partially starved at stage II. Death of these larvae may be a function of increased developmental time and the resulting prolonged exposure to negative artifacts of laboratory culture, such as collision with container walls and bacterial build-up on the integument. More probable, the larvae failed to “compensate” for initial lack of food and exhausted a critical nut~tion~ component by stage VI. Although beyond the scope of the present study, it is reasonable to believe the latter effect would be even more pronounced at metamorphosis to cyprid and spat. Stage II B. irnprovisus nauplii exhibited little resistance to starvation. Extrapolation of laboratory results to field situations is tenuous but it appears essential that nauplii be released into a food-rich environment. Survival potential of starving stage II nauplii may decrease as soon as 24 h. Similarly, if nauplii are used for toxicity tests or similar applications, it is recommended that only newly hatched organisms should be used. Assays > 24 h should involve a feeding regime or account for additional physiological impairment induced by starvation.
ACKNOWLEDGEMENTS
We wish to thank Dr. D. C. Miller, Dr. F. G. Lowman, and R. A. Voyer for their valuable comments on the text and 3. S. Gardiner for her great help in the preparation of the manuscript.
REFERENCES
NAIR, 1979. Laboratory experiments on the larval development of Hyus ~I’~IIPUS (Decapoda, Majidae). Helgol. Wiss. Meeresu~ters., Vol. 32, pp. 36-54. BIGFoRD, T. E., 1978. Effect of several diets on survival, development time, and growth of laboratory-reared spider crab, Libinia etnarginaia, larvae. Fish. Bull. Nat/. Mar. Fish. Serv. U.S., Vol. 76, pp. 59-64. DAGG, M., 1977. Some effects of patchy food environments on copepods. Limnol. Oceanogr., Vol. 22, pp. 99-107. FERNANDEZ, F., 1979. Nutritional studies in the nauplius larva of Calunuspac$cus (Copepoda : Cnlanoida). Mar. Biol., Vol. 53, pp. 131-147. HUN’I IIK, J. (ed.), 1976. Report of a colloquium on larval fish mortality studies and their relation to fishery research, January, 1975. NOAA Tech. Report NMFS Circ. 395, 5 pp. KNOWI.I-~N, R.E., 1974. Larval developmental processes and controlling factors in decapod Crustacea, with emphasis on Caridea. Thalassiu fugosl., Vol. 10, pp. 139-158. KON, T., 197Y. Ecological studies on larvae of the crabs belonging to the genus Chionectes. I. The influence of starvation on the survival and growth of the Zuwai crab. Bull. Jpn. Sot. Sci. Fish., Vol. 45, pp. 7-9. KURATA, H., 1962. Studies on the age and growth of Crustacea. Bull. Hokkaido Reg. Fish. Res. Lab., Vol. 24, pp. i-115. LANG, W. H., 1979. Larval development of shallow water barnacles of the Carolinas (Cirripedia : Thoracica) with keys to naupliar stages. NOAA Tech. Report NMFS Circ. 421, 39 pp. LAN& W.H. & A. ACKENHUSEN-JOHNS, 1981. Seasonal species composition of barnacle larvae (Cirripedia : Thoracica) in Rhode Island waters, 1977-1978. J. Plankton Res., Vol. 3, pp. 567-575. ANGER, K. di K.K.C.
70
WILLIAM
H. LANG
AND MARTHA
MARCY
LANG, W.H., S. LAWK~~NC~ & D.C. MILLER, 1979. The effects of temperature, light and exposure to sublethal levels of copper on the swimming behavior of barnacle nauplii. In, Advances in marine environmenral research, Proceedings of a symposium, edited by F. Jacoff, Ofice of Research and Development, U.S. Environmental Protection Agency, Narragansett, Rhode Island, pp. 273-289. LANG, W.H., M. MARCY, P.J. CLEM, D.C. MILLER & M.R. RODELLI, 1980. The comparative photobehavior of laboratory-hatched and plankton-caught Balanus improvisus (Darwin) nauplii and the effects of 24-hour starvation. J. Exp. Mar. Biol. Ecol., Vol. 42, pp. 201-212. MARCY, M., 1980. Seasonal effects of temperature and salinity on the larval development of the barnacle Balanus amphitrite amphirrife (Darwin) (Cirripedia : Thoracicia). M.S. thesis, University of South Carolina, Columbia, 47 pp. MII LER, C. B., J. K. JOHNSON & D.R. HEINLE, 1977. Growth rules in the marine copepod genus Aurria. Limnol. Oceanogr., Vol. 22, pp. 326335. PA~JI , A.J. & J.M. PAUI., 1980. The effect of early starvation on later feeding success of king crab zoea. J. E.rp. Mar. Biol. Ecol., Vol. 44, pp. 247-251. SINGARAJAH, K.V., J. MOYSE & E. W. KNIGHT-JONES, 1967. The effect of feeding upon the phototactic behaviour of cirripede nauplii. J. Exp. Mar. Biol. Ecol., Vol. 1, pp. 144-153. SULKIN, S.D. & C.E. EPIFANIO, 1975. Comparison of rotifers and other diets for rearing early larvae of the blue crab. Ctr//iurcre.~ .rrrpidus Rathbun. Estuorirw Coos/a/ Mnr. Sci.. Vol. 3. pp. 109% I 13. wALl.LY. L.J., 1969. Studies on the larval structure and metamorphosis of Brdanus balonoides (L.). Phi!. Trans. R. Sot. London Ser. B, Vol. 256, pp. 237-279.
J. Exp. Mar. Biol. Ecol., 1982,Vol. 60, pp. 63-70
Elsevier Biomedical Press
SOME EFFECTS OF EARLY STARVATION ON THE SURVIVAL AND DEVELOPME~
BALANUS
WILLIAM EPA Environmental
OF BARNA~E
IMPROVISUS
H. LANG’
Research Laboratory,
NAUPLII,
(Darwin)’
and MARTHA MARCY
South Ferry Road, Narragansett.
RI 02882, U.S.A.
Abstract: Newly hatched stage I-II nauplii of Balanus i~pr5~j~s (Darwin) were “totally starved” (until death) or “partially starved” for the t‘irst 48 h and 96 h of their development. Daily mortality and molting were monitored throughout larval development in both starved and fed control groups. Fed control animals exhibited a largely synchronous molting pattern with instars of equal duration. Total starvation suppressed molting beyond stage II; 50% mortality occurred in -4 days at both 15 and 21 “C, while longest survival time was 7 days at 15 “C and 6 days at 21 “C. At 15 “C, partially starved nauplii retained the ability to complete naupliar development but at a slower overall rate and with increased mortality relative to controls. These effects were more pronounced in the 96-h group. Increased mortality of stage VI nauplii was evident in both partially starved groups (7.1% for 48 h, 18.8”/, for 96 h) relative to unstarved controls (3.1%). Stage II nauplii exhibited little resistance to starvation and survival potential may have decreased as soon as 24 h. INTRODUCTION
This study investigates the survival time of starved stage II Balanus improvisus (Darwin) nauphi and, more importantly, the effects of partial starvation on subsequent larval development. In addition to providing new information on the physiological ecology of cirripede larvae, results are intended to supplement baseline information for use of larvae in bioassay tests. Without food, larval development of most barnacle species will cease at stage II nauplius (Lang, 1979). In general, larvae just beginning to feed are very sensitive to starvation. The onset of feeding is recognized as a “critical period”, and extensive mortality will occur, as reported for fish and decapod larvae, if proper food is unavailable (Hunter, 1976; Paul & Paul, 1980). Nauplii of copepod species exhibit no particular resistance to starvation (Dagg, 1977), although the adults may be capable of surviving several weeks or more without food. In addition to acute mortality of larvae with total starvation, partially starved larvae, returned to conditions of abundant available food, may fail to recover to “normal” feeding rates and developmental patterns (Kon, 1979; Paul & Paul, 1980). The effects of “partial starvation” (i.e., subjection to a short period of starvation) on barnacle ’ Contribution No. 226 from the EPA Environmental Research Laboratory, Narragansett, RI. ’ Present address: Bureau of Land ~~anagement, NYOCS Offtce, Suite 32-120, 26 Federal Plaza, New York, NY 10278, U.S.A. 0022-098l/82/0000-0000/$02.75
0 1982 Elsevier Biomedical Press
WILLIAM
64
larvae
are essentially
swimming
unknown.
rates and perhaps
1980). The effects
H. LANG
AND
MARTHA
Stage II nauplii
altered photobehavior
of nutritional
stress
MARCY
starved
for 24 h exhibited
(Singarajah
on larvae
reduced
et al., 1967; Lang et al.,
may not always
be immediately
apparent; prolonged and abnormal development with delayed mortality can result. Although some boreo-arctic species (e.g., Semibalanus balanoides) appear to synchronize larval release with diatom blooms, most temperate species release nauplii irrespective of phytoplankton cues (see Lang & Ackenhusen-Johns, 1981). Hence it would be of interest to determine the relative sensitivity to starvation for nauplii of this latter temperate group, of which Balanus improvisus is a member.
MATERIALS
AND
METHODS
Adult B. improvisus attached to small stones were collected from a subtidal site in the Pettaquamscutt River, Narragansett, Rhode Island. Salinity at the collection site averaged z 12%,, but has varied from 0 to 30x0 with extremes of tides and weather. Water temperatures have ranged seasonally from 0 to z 27 ‘C (Lang & AckenhusenJohns, 1981). Adults were maintained in finger bowls; constantly submerged in 0.45 pm filtered sea water (15x, salinity). 15x, water was made by diluting 30X0 sea water with deionized water. A 12 h light : 12 h dark cycle was employed. Adult barnacles were fed newly hatched Artemia salina (Aquafauna Brand, Macau, Brazil)’ approximately three times weekly. To obtain nauplii, adults were fed overnight and subsequently changed to clean filtered 15x, S water. Each container of ~20 to 30 adults was monitored for release of nauplii. Larvae released within a known 6- to 12-h period were concentrated using a light beam and removed to 0.45 pm filtered 15x, S sea water by pipette. Starved nauplii were transferred through two changes of 0.45 pm 15x, S filtered sea water and kept in 7 cm Carolina culture dishes with ~45 to 50 nauplii and 60 ml of filtered 15x, S sea water per dish (static culture). Fed nauplii were given a mixed algal diet of Tetraselmis suecia ( = Platymonas suecia or suecica) and Skeletonema costatum grown axenically in a modified f/2 medium. The algae were mixed in approximately equal volumes, filtered through a 44 nrn Nitex screen to remove cell clumps and diluted with 0.45 pm filtered 15x, S sea water to obtain a final density of 2 to 4 x lo4 cells . ml- ‘. Fed nauplii were maintained identically to starved nauplii in all respects other than feeding. Nauplii were changed to fresh media at 48-h intervals. Mortality and development were recorded at 24-h intervals by tallying and staging of all molts and dead (no movement and loss of normal transparent appearance). A daily percent composition of naupliar stages was determined. The few larvae apparently lost during transfer (< 3 %) were counted as dead in final calculations. Results in replicate cultures were nearly identical in all cases and were pooled for the presentation of data. The experi-
I Reference Agency.
to trade names does not imply endorsement
of the United
States Environmental
Protection
STARVATIONOFBARNACLENAUPLII
65
ments were designed to give information on general trends only and thus no statistical tests for significance were performed on the data. In one experiment, survival and development were determined at 15 & 1 “C and 20 f 2 “C with 15%, S sea water, 12L : 12D light cycle. Larvae were obtained from adults collected in August 1980, and maintained in the laboratory for 3 days. Nauplii were divided into fed (control) and totally starved groups. In another experiment, the ability of stage II nauplii starved for 48 h and 96 h (i.e., “partially starved”) (15 “C, 15x, S sea water, 12L : 12D light cycle) to complete naupliar development was tested. Larvae were obtained from adults cohected in November 1979 and maintained under laboratory conditions for over 6 months. Initially, several hundred newly hatched nauplii were kept in a 600-ml container of 0.45 pm 15%, S filtered sea water. At 0, 48 and 96 h, two groups of z 50 nauplii (47 to 5 1) were transferred to duplicate 60-ml cultures of algae and sea water. Cultures were monitored to an endpoint of 20% cyprid, based on the initial number of larvae started. RESULTS
At 15 *C, 100% mortality occurred in 7 days for starved stage II nauplii. First deaths were noted at 2 days. Fifty percent mortality was observed in ~4 days (Fig. IS). At 21 “C, little difference in time to 50% mortality was apparent but 100% mortality occurred in only 6 days (Fig. 1D). In contrast to the minor differences in times to 50% mortality, marked differences in overall developmental rates were evident in fed control groups at 15 “C and 21 “C. Excluding the rapid molt from stage I to II, the fed early stage nauplii of Balanus improvisus exhibited larval stages of nearly equal duration, i.e., “isochronal development” (Miller et al., 1977). At 15 “C, nearly all stage II-IV nauplii molted within 24 h of each other at 2-day intervals (Figs. lA, 2A). Although molting to stage VI also began 2 days following the stage V molt, the synchrony of molting observed in earlier naupliar stages was lost, as molting from stage V to VI occurred over a 6-day period (Fig. 2A). The metamorphosis from stage VI to cyprid most often required a minimum of 4 days and molting continued over a period of 7 or more days (Fig. 2A and unpubl. results). Nauplii reared at 2 1 ‘C, also exhibited isochronal development in stage II-IV but at about one-half the development time (Fig. 1C). Duration of these early stages was 1 day each, and cyprids first appeared after Day 6 in contrast to Day 13 at 15 “C. Surviving stage II nauplii, fed following starvation for 48 h and 96 h at 15 ‘C, retained an ability to complete naupliar development, but (1) at slower rates and (2) with increased mortality relative to fed controls. Forty-eight-h initial starvation increased naupliar development time by z 1 day in stages III-IV and up to 2 to 3 days for stage VI (Fig. 2B, Table I). Effects were more pronounced after 96 h starvation with the time for 50% of nauplii to reach the next developmental stage increased by z 2 days for stage III and up to 5; days at stage VI (Fig. 2B, Table I).
Fig. 1. Daily percent mortality and A, fed nauplii at 15 “C, 15x, S (N 21 “C, 15x, S (N = 50); D, starved IV, VI) are indicated; odd numbered
naupliar stage distribution for stage I-II Balanus improvisus nauplii: = 48); B, starved nauplii at 15 ‘C, 15x, S (N = 47); C. fed nauplii at nauplii at 21 “C, 15x,, S (N = 48); even numbered naupliar stages (II, stages (III, V) and cyprid are cross-hatched areas in appropriate order between labelled stages
SO
Fig. 2. Daily naupliar stage distributions for Balunus improvisus larvae: A, larvae fed at hatching (control); B. larvae fed after 48 h starvation; C, larvae fed after 96 h starvation; Day 0 represents the time of initial feeding; percent distributions are based on the number of surviving larvae at each 24-h interval; see Table II for mortality and sample size; each group was terminated at an endpoint of 20% cyprid; even numbered naupliar stages (II, IV, VI) are indicated; odd numbered stages (III, V) and cyprid are cross-hatched areas in appropriate order between labelled stages.
STARVATION
OF BARNACLE
67
NAUPLII
In addition immediate
to the increased larval development times, starvation resulted in an mortality of stage II nauplii starved 96 h (Table II). Increased mortality 8%
The comparative rate of development ofBalanus imptwisus larvae starved for 0.48, or 96 h and then reared at 15 “C, IS%, salinity using a mixed algal diet (see text): time in days for 50% attainment of next naupliar stage is calculated based on the number of surviving nauplii and extrapolated as a constant rate of molting during each 24-h interval: developmeIlta1 times begin at initiation of feeding, irrespective ofinitial starvation periods. Time (days) to 50°$ naupliar Hours
starved 0 48 96
stage
III
IV
V
VI
2.5 3.6 4.6
4.5 5.2 7.4
6.5 7.5 10.8
9.5 11.3 14.1
TABLE II
The percent mortality of ~a~~ff~ j~~rov~~ larvae starved for 0,48 or 96 h and then reared at 15 “C, l5& salinity using a mixed algal diet (see text): mortality during initial starvation periods is undetermined; initial sample numbers are: control, 96; 48 h, 98; 36 h, 101. Hours
starved
0
48
96
II III IV V VI
1.0 0 1.0 4.2 3.1
1.0 1.0 3.1 2.0 7.1
7.9 1.0 4.0 2.0 18.8
Total
9.3
14.2
Naupliar
stage
_I~
of stage VI nauplii was evident controls (Table II).
33.7 ._
for both starved
groups
(48 and 96 h) relative
to fed
DISCUSSION
Temperature has a pronounced effect on cirripede larval developmental rates (Lang, 1979; Marcy, 1980). The nearly doubled developmental rate with a 6 “C rise in temperature from 15 to 21 “C was not unexpected. The loss of molting synchrony and reduction in developmental rate of later stage nauplii observed in fed groups may have been indicative of the increasingly complex structural development of the larvae and metamorphic changes in preparation for the cyprid stage (Walley, 1969). It is also
WILLIAM
6X
H. LANG
AND MARTHA
MARCY
possible that the diet used here may not have been completely adequate for later stage development. Total starvation of stage II nauplii suppressed further development. No molting to stage III occurred and all nauplii died by 7 days (Fig. 1). This starvation survival time is within the range of that reported for other temperate water crustacean larvae (Table III). TABLE III
Survival
times for starved
crustacean
larvae:
C, copepodite:
N, nauplius;
2, zoea.
Mortality
(days)
SOS,,
loo:”
11 11
11 15
18 18
Kon, 197Y Ken, 1979 Anger
Temp. Stage
Reference
z Z
f 11
Z
I
12
9
14
2
I
20
3
I
z
I I
6-9
6
10
NIII NIV
15 15
5 4
7 7
Fernandez, Fernandez,
21
-
Dagg,
1977
2 2
Dagg, Dagg,
1977 1977
7 6
This study
2
___--._.
(“C)
6.5
25
c5
15
Nl Nl
15 15
NII
15 21
4 4
& Nair,
Bigford,
1979
1978
Sulkin & Epifanio, Kurata,
1975
1962 1979 1979
--
Sublethal effects of starving stage II nauplii at 15 “C become evident at 24 to 48 h. Nauplii starved for only 24 h exhibited some loss of swimming speed and perhaps depressed sensitivity to light but no acute or delayed mortality (Lang et al., 1979, 1980). In the present work, 48-h starvation resulted in z 10% mortality (Fig. 1B) and subsequent development of surviving nauplii fed at this point lagged ~24 h behind fed controls (Fig. 2B). Effects of 96-h starvation were more pronounced with 50)1/, acute mortality (Fig. lB), increasing loss of molting synchrony, and generally prolonged intermolts (Fig. ZC). Kon (1979) observed that as the duration of initial starvation (followed by feeding) of stage I Chionectes opilio was increased, a progressive decline in percent of successful molt to stage II occurred and the intermolt period was prolonged. In contrast, low food rations or starvation of Palaernonetes vulgaris larvae had a minimal immediate effect on molting frequency but suppressed growth per instar (Knowlton, 1974). Repetition of larval stage was also observed with no development to postlarva and eventual death. These responses perhaps reflected the basic differences in the rigidly prescribed number of larval instars for B. irnprovisus and C. opilio versus the high variability in the
STARVATION
Pulaemonetes
OF BARNACLE
larvae. The net effect of limited starvation,
69
NAUPLII
however, was to prolong larval
development. Of particular
interest,
the present
results
show a delayed
increase
in mortality
at
stage VI for larvae partially starved at stage II. Death of these larvae may be a function of increased developmental time and the resulting prolonged exposure to negative artifacts of laboratory culture, such as collision with container walls and bacterial build-up on the integument. More probable, the larvae failed to “compensate” for initial lack of food and exhausted a critical nut~tion~ component by stage VI. Although beyond the scope of the present study, it is reasonable to believe the latter effect would be even more pronounced at metamorphosis to cyprid and spat. Stage II B. irnprovisus nauplii exhibited little resistance to starvation. Extrapolation of laboratory results to field situations is tenuous but it appears essential that nauplii be released into a food-rich environment. Survival potential of starving stage II nauplii may decrease as soon as 24 h. Similarly, if nauplii are used for toxicity tests or similar applications, it is recommended that only newly hatched organisms should be used. Assays > 24 h should involve a feeding regime or account for additional physiological impairment induced by starvation.
ACKNOWLEDGEMENTS
We wish to thank Dr. D. C. Miller, Dr. F. G. Lowman, and R. A. Voyer for their valuable comments on the text and 3. S. Gardiner for her great help in the preparation of the manuscript.
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WILLIAM
H. LANG
AND MARTHA
MARCY
LANG, W.H., S. LAWK~~NC~ & D.C. MILLER, 1979. The effects of temperature, light and exposure to sublethal levels of copper on the swimming behavior of barnacle nauplii. In, Advances in marine environmenral research, Proceedings of a symposium, edited by F. Jacoff, Ofice of Research and Development, U.S. Environmental Protection Agency, Narragansett, Rhode Island, pp. 273-289. LANG, W.H., M. MARCY, P.J. CLEM, D.C. MILLER & M.R. RODELLI, 1980. The comparative photobehavior of laboratory-hatched and plankton-caught Balanus improvisus (Darwin) nauplii and the effects of 24-hour starvation. J. Exp. Mar. Biol. Ecol., Vol. 42, pp. 201-212. MARCY, M., 1980. Seasonal effects of temperature and salinity on the larval development of the barnacle Balanus amphitrite amphirrife (Darwin) (Cirripedia : Thoracicia). M.S. thesis, University of South Carolina, Columbia, 47 pp. MII LER, C. B., J. K. JOHNSON & D.R. HEINLE, 1977. Growth rules in the marine copepod genus Aurria. Limnol. Oceanogr., Vol. 22, pp. 326335. PA~JI , A.J. & J.M. PAUI., 1980. The effect of early starvation on later feeding success of king crab zoea. J. E.rp. Mar. Biol. Ecol., Vol. 44, pp. 247-251. SINGARAJAH, K.V., J. MOYSE & E. W. KNIGHT-JONES, 1967. The effect of feeding upon the phototactic behaviour of cirripede nauplii. J. Exp. Mar. Biol. Ecol., Vol. 1, pp. 144-153. SULKIN, S.D. & C.E. EPIFANIO, 1975. Comparison of rotifers and other diets for rearing early larvae of the blue crab. Ctr//iurcre.~ .rrrpidus Rathbun. Estuorirw Coos/a/ Mnr. Sci.. Vol. 3. pp. 109% I 13. wALl.LY. L.J., 1969. Studies on the larval structure and metamorphosis of Brdanus balonoides (L.). Phi!. Trans. R. Sot. London Ser. B, Vol. 256, pp. 237-279.