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The influence of light on the hatching of Artemia cysts (Anostraca:Branchiopoda:Crustacea)
.I. Exp. Mar. Biol. Ecol., 1985, Vol. 92, pp. 207-214 Elsevier
207
JEM 559
THE INFLUENCE OF LIGHT ON THE HATCHING (ANOSTRACA : BRANCHIOPODA
OF ARTEMZA
CYSTS
: CRUSTACEA)’
ANNEMIE VAN DER LINDENS, RONNY BLUST and WALTER DEGLEIR StateUniversity of Antwerp (RUCA), Laboratory of 3iQc~e~~t~y and General Zoology, ~raene~~r~erlaan
171, B-XL%_? APttwerp,~e~~‘~~
(Received 19 April 1985; revision received 2 July 1985; accepted 5 July 1985) ~~~ra~~ The influence of different light intensities, exposure times and wavelengths on the hatching of curves. Hatching requires a certain amount of light energy, which can be administered under different combinations of light intensity and exposure time. The Bunsen-Roscoe law holds for the exposure times and low light intensities used in relation to hatching. Wavelengths between 400 and 600 nm are most effective in triggering hatching. All these results give preliminary information on the photoreceptor system involved. The possible role of the dark cyst shell in relation to light absorption is discussed.
Artemia cysts was studied by means of dose-response
Keywords:Atiemia; hatching; influence of tight; diapause deactivation
The effects of hght on the diapause and reproduction of branchiopods has been studied by Pancella & Stross (1963), Stross (1966, 1969), Shan (1970), Takabashi (19’7?), and Hemp&Zawitkowska (1970). The authors reported the existence of a positive correlation between light and/or photoperiod and the reactivation of diapause or dormant stages. Studies on the brine shrimp Artemiu by Sorgeloos (1973), Sorgeloos et al. (1976), Royan (19761, and Spektorova & Syomik (1979) have demonstrated tke existence of such a triggering effect of light on hatching, provided that the cysts are hydrated in aerobic conditions (Sorgeloos, 1973). This triggering effect seems to be memorized over several hydration-dehydration cycles, which may explain the observed percentage of cysts hatching in the dark (Sorgeloos, 1973). Other investiga~ons demonstrated a difference in iight sensitivity of cysts of different geographical origin (Vanhaecke et al., IQSl}. Despite the commercial interest of aquaculture farms in high hatching outputs of the brine shrimp cysts, surprisingly little is known about the effect of light on hatching. The present work deals with the influence of light intensity, wavelength, and exposure time on the hatching of Artemiu cysts. The results enable some prelimin~y conclusions to r This work is part of the F.K.F.O. project 20012.82. a To whom al! correspondence should be addressed. OO22-0981/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
208
ANNEMIEVANDERLINDEN~~~~.
be drawn about the nature of the photoreceptor
system, which mediates light-induced
hatching.
MATERIAL AND METHODS APPARATUS Blackboxesmade of PVC were used to test the intluence of different light intensities and wavelengths on Artemia cysts. Every box was subdivided into eight compartments, each provided with a light opening on the top. Within a compartment, hatching of the cysts was accomplished in four sealed plastic cylindrical test tubes (Figs. 1 and 2). These were attached to a joint bar rotating in the length axis of the system and driven with a belt by a motor-reductor system (Crouzet no. 82-334-5rpm.). The rotating bar passed through the compartment walls on bearings to prevent friction and light scattering. This rotating system provides the best interaction of the cysts with the medium, necessary for hydration and gas exchange (Sorgeloos et al., 1978). The light was supplied by a xenon lamp (Osram XBO-900 W) positioned above the apparatus. The light intensity was varied by regulating the power supply of the lamp, by changing the lamp’s position or by white Plexi filters (Rbhm GM-BH Darmstadt, Plexi 3 mm, no. 060). The final intensity reaching the cysts was measured with the quantum probe (FE * m - ’ +s - ’ ) of a lambda light sensor radiometer (Li-Cor-185) on a fixed spot between the test tubes and the filter. Different wavelengths were obtained by using coloured Plexi filters with known absorption characteristics (Rohm, GM-BH Darmstadt, Plexi 3 mm, no. 504: red, no. 303: yellow, no. 701: green, and no. 627: blue). The boxes were submerged in thermostatically controlled waterbaths (? 1 “C) and temperature fluctuations between the compartments were prevented by circulating water between them (Fig. 1).
Fig. 1. Schematic drawing of the black box construction in PVC: M = motor-reductor system; B = belt; 0 = light filter opening; S = circulation system; R = rotating bar; E = Eheim pump; -+ = direction of flow.
EXPERIMENTAL PROCEDURE
Artemia (San Francisco Bay batch SFBB 2149) were used because of their high hatching efficiency. To obtain homogeneous samples of z 100 cysts (0.1 g of cysts) were
LIGHT-MEDIATED
HATCHING IN ARTEMIA
209
Fig. 2. Schematic drawing of an opened black box: R = rotating bar; B = bearing; C = test tubes; S = culation system; E = Eheim pump; 4 = indicates the direction of the water flow.
mixed with 100 ml artificial sea water (H-W Wimex) at 25 “C. Within 5 min, a period insufficient to cause any hydration, 750~~1 aliquots were taken. The samples were transferred into the cylindrical test tubes, filled with sea water (S 35x,) and left rotating for 1 h in the dark to hydrate. Experiments were performed at 25 a C, since this yielded the fastest and highest hatching results in comparison with two other previously tested temperatures of 15 and 35 ‘C (data not shown). The pH was 8, which is favourable for hatching (Jones, 1972). After 48 h incubation the test tubes were removed and the contents fixed with Lugol. Subsequently, hatching percentages were counted and the mean of four replicates calculated. ANALYSIS OF RESULTS
The obtained dose (light)-response (hatching percentage) data were log-logit transformed, with the logarithms of the light quanta being linearly related to the logits of the hatching percentages. Abbott’s formula was used to correct for hatching of controls. Chi-square values were calculated to test whether the logit lines were adequate representations of the experimental data. Duncan’s multiple-range test was used to compare slopes (Finney, 1978). RESULTS EXPERIMENTS
WITH DIFFERENT
WHITE LIGHT INTENSITIES
AND EXPOSURE TIMES
Analysis of the dose-response data (Fig. 3) reveals a definite relation between light intensity (O-5 PLE.me2 * s- ’ ) and exposure time (1 and 6 h), and the percentage of cysts hatching. At low light intensities, longer exposure times are needed to obtain the same hatching as for high light intensities. Forty-eight hours of exposure to different low light intensities gave maximal hatching. Fig. 4 shows the relation between logarithms of the light quanta and the logit transformed hatching percentages. Duncan analysis on the slopes revealed a significant difference between 1 and 6 h exposure to light (P = 0.05, B,,, = 0.6 + 0.1 and B 1h = 1.3 + 0.1). The figure shows the log(dose) to obtain 50% hatching for light exposure periods of 1 and 6 h.
210
ANNEMIE
VAN DER LINDEN
4J
1
LIGHT
Fig. 3. Hatching
results ofktemia
ETAL.
'4
QUANTA
(Feinst/d
cysts, exposed
set
1
for 6 h (A) and 1 h (0)
u-
to different
intensities
of white
. / .
O.,
l/
> .
.
-1..
-2. >
. -3.
/ -’
Fig. 4. Log-logit light intensities:
/
LOG,{ LIGHT
D
CIUANTA I
1
log Ipeinst / m2 set
)
transformation ofthe hatching results for 6 h (A) and 1 h (0) exposure to different white 6 h: f(x) = 0.19 + 0.571 x, r = 0.852, x2 = 17.005 for N = 5 SE slope = 0.149; 1 h: f(x) = - 2.038 + 1.42 x, r = 0.987, ,y2 = 3.075 for N = 5 SE slope = 0.117.
These results illustrate agreement ofthe dose-response data with the Bunsen-Roscoe law of reciprocity, which states that the response should be similar for all combinations of exposure time and intensity having equal products (Schafer et al., 1983). The calculations are given in Table I. EXPERIMENTS
WITH
DIFFERENT
WAVELENGTHS
Based upon the results from the experiments with different white light intensities, shown in Fig. 3, the selected intensity range and exposure time were respectively, 0 to
I~IGHT-MEDIATED
211
HATCHING IN ARTEMfA TABLE I
Summary of the data analysis which shows that the reciprocity law holds for the dose-response (examples of 507; hatching are presented). _.-__
~__. 6h
results
Light exposure period _.._______.. _.___ lh
.__~__
~Linear function after log-logit transfo~ation of the hatching results x value = log, (dose) to obtain 50% hatching (P = 0.5); or logit value = log, (P/l-P) = 0 Dose = light quanta in FE. rnmz. s - ’ Product of the combination: light quanta and exposure time. resulting in 50% hatching
f(x) = 0.19 + 0.571,~
f(x) = - 2.038 + 1.42.~
- 0.33
1.46 cl 46
e- 0.33
15516 pE.m-2
15552 pE,rnm2
2FE *m - * 1s - ’ and 6 h. Under these conditions the differences in hatching percentages are maximal at minimal light intensity variation. The dose-response data for different wavelengths (Figs. 5 and 6), show that exposure to green, yellow or blue light gives the best hatching results at the lowest light intensities. There was no significant difference between these light treatments (Duncan’s test, P = 0.05,Bgreen = 0.6& 0.1,
r----
-’
H~:~ ,,,: LIGHTQ”ANT4
;”
fjiO
-1
,,
,2
b
/
p
1
I )JEINS:&ECl
Fig. 5. Hatching percentages ofArtemia cysts, exposed for 6 h to different intensities ofyellow (a), blue (b), green (c), and red (d) light.
212
ANNEMIE VAN DER LINDEN ET&.
Fig. 6. Log-Iogit transformation of hatching results of Anemia cysts after 6-h exposure to different intensities of( A)red, (m) blue, (*) yellow or (0)green light: yellow light:f(x) = 1.052 t 0.805 X, I = 0.958, x2 = 11.856 for N = 8; green light: f(x) = 0.682 = 0.643 x, r = 0.905, x2 = 8.841 for N = 5; blue light: f(x) = 0.034 + 0.632x, r = 0.956, x2 = 7.432 for N= 5; red light: f(x) = - 1.85 = 1.875 X, r = 0.975, x2 = 11.17 for N= It.
B yellow = 0.8 + 0.1 and Bblue = 0.6 2 0.1). For red light, an intensity cz 7 times higher is needed to achieve a similar hatching percentage (Bred = 1.2 & 0.1, difference is significant, P = 0.05). Taking into account the absorption characteristics of the used filters, we may conclude that the most effective region for hatching is at wavelengths between 400 and 600 nm.
DISCUSSION
The important role of Artemiu in aquaculture demands a continuous effort to improve hatching and culturing conditions. Our results add some important information about the light conditions favouring hatching. Under expe~ment~ conditions, z 21600 PE 3m - * light energy is needed to achieve maximal hatching percentages for SFB (2 149) cysts. Higher energies do not result in a greater hatching percentage. The required light energy can be obtained either by the combination: low intensity, long exposure time or high intensity, short exposure time. In the first case one can expect a delayed hatching since the required energy is obtained after a longer period. This delay is not detectable within the investigated 48-h incubation period, but has been demonstrated earlier by Vanhaecke et af. (198 1). Since light of the wavelen~hs between 400 and 600 nm is most effective for hatching, the choice of the light source is of great importance (e.g. fluorescent lamps will be more appropriate than glowlamps because of the distinct spectral qualities). Yellow, green or blue light sources are also suitable for trigger hatching.
LIGHT-MEDIATED
HATCHING
IN ARTEMIA
213
Since the data are in accordance with the Bunsen-Roscoe law of reciprocity, we can postulate the existence of a photoreceptor which mediates ii~t-induced hatching. This photoreceptor is expected to absorb light between 400 and 600 nm. The present data, however, allow no speculation on the nature of the photoreceptor since this requires an action spectrum. Because light mediation takes place before the loss of the outer cyst shell (Sorgeloos et al., 1976), it is necessary to draw the attention to the remarkable dark colour of this shell, due to its high haematin content (Gilch~st & Green, 1960). Inevitably, it will play a role, either as a light filter or as a site for photoreception. Shan (1970), working on Pleuroxus denticulatus, a branchiopod with light-induced hatching, stated that the dark pigmentation in the ephippium, surrounding the resting eggs, constitutes a mechanism for retarding development until hatching can be accomplished under favourabie environmental conditions. This was based upon expe~ments comparing hatching efficiency of resting eggs remaining in and dissected out of their ephippium. He proposed that the dark colour of the ephippium is first bleached by the sunlight to allow further light penetration and development. Possibly the dark haematin-rich cyst shell plays a similar role. It is interesting to note that haematin absorbs in the region most eflective to hatching (absorption peaks at 438, 532, and 558 nm: Gilchrist & Green, 1960). Future work will focus on the construction of an action spectrum of the light-induced hatching. ACKNOWLEDGEMENTS
We wish to thank Dr P. Sorgeloos for critically reading this paper. We also wish to express our gratitude to K. Cuypers and A. Vlaeminck for their technical assistance. REFERENCES FINNEY, D.J., 1978. Sf~fjs~jcul~er~~~ in b~oIo~cu~assay. Charles GriBin, London, 3rd edition, 508 pp. GILCHRIST, B.M. & GREEN, J., 1960. The pigments of Artemiu. Proc. Sot. London, Vol. 152, pp, 118-136. HEMPEL-ZAWITKOWSKA, J., 1970. The influence of strong ultra violet radiation on hatchability of Triops canceriformis (BOX.) eggs. Pal. Arch. Hydrobiol., Vol. 17, pp. 4X3-493. JONES,A. J., 1972. An inexpensive apparatus for the large scale hatching ofArtemia salina L. J. Cons.,Cons. Int. Explor. Mer, Vol. 34, pp. 351-356. PANCELLA,J. R. & R. G. STROSS, 1963. Light induced hatching of Daphnia resting eggs. Chesapeake Sci.. Vol. 4, pp. 135-140. ROYAN,J. P., 1976. Effect ofhght on the hatching and growth ofArtemias&a. Muhasager, Vol. 9, pp. 83-85.
SCHKFER, E., L. FUKSHANSKY& W. SHROPSHIREJR., 1983. Action spectroscopy of photoreversible pigment systems. Encycl. Plant Physiol., Vol. 16(A), pp. 39-68. SHAN,R. K. C., 1970. Influence of light on hatching of resting eggs of chydorids cladocera. Int. Rev. Gesamten Hydrobiol., Vol. 55, pp. 295-302. SORGELOOS,P., 1973. First report on the triggering effect of light on the hatching mechanism of Artemiu saliva dry cysts. EAar. Biol., Vol. 22, pp. 75-76. SORGELOOS, P., M. BAEZA-MESA,F. BENIJTS& G. PERSOONE,1976. Current research on the culturing ofthe brine shrimp.&temia salina L. at the State University of Ghent, Belgium. In, Proc. 10th Eur. Symp. Mar. Biol., Vol. 1, edited by G. Persoone & E. Jaspers. Universa Press, Wetteren, Belgium, pp. 473-495.
214
ANNEMIE VAN DER LINDEN ETAL
SORGELOOS,P.,G. PERSOONE,M.BAEZA-MESA,E.BOSSUYT&E.BRIJGGEMAN,I~~~. Theuse ofArtemia cysts in aquaculture: the concept “hatching efficiency” and description of a new method for cyst processing. In, Proc. ofthe 9th Arm. ~or~~ap ofthe W. h4. S., edited by J. W. Avault, Jr., Louisiana State University, Division of Continuing Education, Baton Rouge, Louisiana, U.S.A., pp. 715-721. SPEKTOROVA,L. V. & A.M. SYOMIK, 1979. The influence of incubation conditions upon Ar&nia hatching efftciency in three strain models. Book of Abstracts, lnternutional Symposium on the brine shrimp, Artemia salina. Corpus Christi, August 20-30, Artemia Reference Center, Ghent, p. 121. STROSS,R. G., 1966. Light and temperature requirements for diapause development and release in Daphniu. Ecology, Vol. 47, pp. 368-374. STROSS,R.G.,1969. Photoperiod control of diapause in Dophnia. III. Two stimulus control of long-day. short day induction. Biol. Bull. (Woods Ho/e. Mass.), Vol. 137, pp. 359-374. TAKAHASHI.F.,1977. Pioneer life of the tadpole shrimp Tn’opssp. Notostraca Triopsidae. Appl. EmomoL Zool., Vol. 12, pp. 104-l 17. VANHAECKE.P.,A.CO~REMAN& P.SORGELOOS,1981.Internationa~studyon Arremia. XV.Effectoflight intensity on hatching rate ofAr?emia cysts from different geographical origin. kr. Ecol. Frog. Ser., Vol. 5, pp. 1t I-1 14.
207
JEM 559
THE INFLUENCE OF LIGHT ON THE HATCHING (ANOSTRACA : BRANCHIOPODA
OF ARTEMZA
CYSTS
: CRUSTACEA)’
ANNEMIE VAN DER LINDENS, RONNY BLUST and WALTER DEGLEIR StateUniversity of Antwerp (RUCA), Laboratory of 3iQc~e~~t~y and General Zoology, ~raene~~r~erlaan
171, B-XL%_? APttwerp,~e~~‘~~
(Received 19 April 1985; revision received 2 July 1985; accepted 5 July 1985) ~~~ra~~ The influence of different light intensities, exposure times and wavelengths on the hatching of curves. Hatching requires a certain amount of light energy, which can be administered under different combinations of light intensity and exposure time. The Bunsen-Roscoe law holds for the exposure times and low light intensities used in relation to hatching. Wavelengths between 400 and 600 nm are most effective in triggering hatching. All these results give preliminary information on the photoreceptor system involved. The possible role of the dark cyst shell in relation to light absorption is discussed.
Artemia cysts was studied by means of dose-response
Keywords:Atiemia; hatching; influence of tight; diapause deactivation
The effects of hght on the diapause and reproduction of branchiopods has been studied by Pancella & Stross (1963), Stross (1966, 1969), Shan (1970), Takabashi (19’7?), and Hemp&Zawitkowska (1970). The authors reported the existence of a positive correlation between light and/or photoperiod and the reactivation of diapause or dormant stages. Studies on the brine shrimp Artemiu by Sorgeloos (1973), Sorgeloos et al. (1976), Royan (19761, and Spektorova & Syomik (1979) have demonstrated tke existence of such a triggering effect of light on hatching, provided that the cysts are hydrated in aerobic conditions (Sorgeloos, 1973). This triggering effect seems to be memorized over several hydration-dehydration cycles, which may explain the observed percentage of cysts hatching in the dark (Sorgeloos, 1973). Other investiga~ons demonstrated a difference in iight sensitivity of cysts of different geographical origin (Vanhaecke et al., IQSl}. Despite the commercial interest of aquaculture farms in high hatching outputs of the brine shrimp cysts, surprisingly little is known about the effect of light on hatching. The present work deals with the influence of light intensity, wavelength, and exposure time on the hatching of Artemiu cysts. The results enable some prelimin~y conclusions to r This work is part of the F.K.F.O. project 20012.82. a To whom al! correspondence should be addressed. OO22-0981/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
208
ANNEMIEVANDERLINDEN~~~~.
be drawn about the nature of the photoreceptor
system, which mediates light-induced
hatching.
MATERIAL AND METHODS APPARATUS Blackboxesmade of PVC were used to test the intluence of different light intensities and wavelengths on Artemia cysts. Every box was subdivided into eight compartments, each provided with a light opening on the top. Within a compartment, hatching of the cysts was accomplished in four sealed plastic cylindrical test tubes (Figs. 1 and 2). These were attached to a joint bar rotating in the length axis of the system and driven with a belt by a motor-reductor system (Crouzet no. 82-334-5rpm.). The rotating bar passed through the compartment walls on bearings to prevent friction and light scattering. This rotating system provides the best interaction of the cysts with the medium, necessary for hydration and gas exchange (Sorgeloos et al., 1978). The light was supplied by a xenon lamp (Osram XBO-900 W) positioned above the apparatus. The light intensity was varied by regulating the power supply of the lamp, by changing the lamp’s position or by white Plexi filters (Rbhm GM-BH Darmstadt, Plexi 3 mm, no. 060). The final intensity reaching the cysts was measured with the quantum probe (FE * m - ’ +s - ’ ) of a lambda light sensor radiometer (Li-Cor-185) on a fixed spot between the test tubes and the filter. Different wavelengths were obtained by using coloured Plexi filters with known absorption characteristics (Rohm, GM-BH Darmstadt, Plexi 3 mm, no. 504: red, no. 303: yellow, no. 701: green, and no. 627: blue). The boxes were submerged in thermostatically controlled waterbaths (? 1 “C) and temperature fluctuations between the compartments were prevented by circulating water between them (Fig. 1).
Fig. 1. Schematic drawing of the black box construction in PVC: M = motor-reductor system; B = belt; 0 = light filter opening; S = circulation system; R = rotating bar; E = Eheim pump; -+ = direction of flow.
EXPERIMENTAL PROCEDURE
Artemia (San Francisco Bay batch SFBB 2149) were used because of their high hatching efficiency. To obtain homogeneous samples of z 100 cysts (0.1 g of cysts) were
LIGHT-MEDIATED
HATCHING IN ARTEMIA
209
Fig. 2. Schematic drawing of an opened black box: R = rotating bar; B = bearing; C = test tubes; S = culation system; E = Eheim pump; 4 = indicates the direction of the water flow.
mixed with 100 ml artificial sea water (H-W Wimex) at 25 “C. Within 5 min, a period insufficient to cause any hydration, 750~~1 aliquots were taken. The samples were transferred into the cylindrical test tubes, filled with sea water (S 35x,) and left rotating for 1 h in the dark to hydrate. Experiments were performed at 25 a C, since this yielded the fastest and highest hatching results in comparison with two other previously tested temperatures of 15 and 35 ‘C (data not shown). The pH was 8, which is favourable for hatching (Jones, 1972). After 48 h incubation the test tubes were removed and the contents fixed with Lugol. Subsequently, hatching percentages were counted and the mean of four replicates calculated. ANALYSIS OF RESULTS
The obtained dose (light)-response (hatching percentage) data were log-logit transformed, with the logarithms of the light quanta being linearly related to the logits of the hatching percentages. Abbott’s formula was used to correct for hatching of controls. Chi-square values were calculated to test whether the logit lines were adequate representations of the experimental data. Duncan’s multiple-range test was used to compare slopes (Finney, 1978). RESULTS EXPERIMENTS
WITH DIFFERENT
WHITE LIGHT INTENSITIES
AND EXPOSURE TIMES
Analysis of the dose-response data (Fig. 3) reveals a definite relation between light intensity (O-5 PLE.me2 * s- ’ ) and exposure time (1 and 6 h), and the percentage of cysts hatching. At low light intensities, longer exposure times are needed to obtain the same hatching as for high light intensities. Forty-eight hours of exposure to different low light intensities gave maximal hatching. Fig. 4 shows the relation between logarithms of the light quanta and the logit transformed hatching percentages. Duncan analysis on the slopes revealed a significant difference between 1 and 6 h exposure to light (P = 0.05, B,,, = 0.6 + 0.1 and B 1h = 1.3 + 0.1). The figure shows the log(dose) to obtain 50% hatching for light exposure periods of 1 and 6 h.
210
ANNEMIE
VAN DER LINDEN
4J
1
LIGHT
Fig. 3. Hatching
results ofktemia
ETAL.
'4
QUANTA
(Feinst/d
cysts, exposed
set
1
for 6 h (A) and 1 h (0)
u-
to different
intensities
of white
. / .
O.,
l/
> .
.
-1..
-2. >
. -3.
/ -’
Fig. 4. Log-logit light intensities:
/
LOG,{ LIGHT
D
CIUANTA I
1
log Ipeinst / m2 set
)
transformation ofthe hatching results for 6 h (A) and 1 h (0) exposure to different white 6 h: f(x) = 0.19 + 0.571 x, r = 0.852, x2 = 17.005 for N = 5 SE slope = 0.149; 1 h: f(x) = - 2.038 + 1.42 x, r = 0.987, ,y2 = 3.075 for N = 5 SE slope = 0.117.
These results illustrate agreement ofthe dose-response data with the Bunsen-Roscoe law of reciprocity, which states that the response should be similar for all combinations of exposure time and intensity having equal products (Schafer et al., 1983). The calculations are given in Table I. EXPERIMENTS
WITH
DIFFERENT
WAVELENGTHS
Based upon the results from the experiments with different white light intensities, shown in Fig. 3, the selected intensity range and exposure time were respectively, 0 to
I~IGHT-MEDIATED
211
HATCHING IN ARTEMfA TABLE I
Summary of the data analysis which shows that the reciprocity law holds for the dose-response (examples of 507; hatching are presented). _.-__
~__. 6h
results
Light exposure period _.._______.. _.___ lh
.__~__
~Linear function after log-logit transfo~ation of the hatching results x value = log, (dose) to obtain 50% hatching (P = 0.5); or logit value = log, (P/l-P) = 0 Dose = light quanta in FE. rnmz. s - ’ Product of the combination: light quanta and exposure time. resulting in 50% hatching
f(x) = 0.19 + 0.571,~
f(x) = - 2.038 + 1.42.~
- 0.33
1.46 cl 46
e- 0.33
15516 pE.m-2
15552 pE,rnm2
2FE *m - * 1s - ’ and 6 h. Under these conditions the differences in hatching percentages are maximal at minimal light intensity variation. The dose-response data for different wavelengths (Figs. 5 and 6), show that exposure to green, yellow or blue light gives the best hatching results at the lowest light intensities. There was no significant difference between these light treatments (Duncan’s test, P = 0.05,Bgreen = 0.6& 0.1,
r----
-’
H~:~ ,,,: LIGHTQ”ANT4
;”
fjiO
-1
,,
,2
b
/
p
1
I )JEINS:&ECl
Fig. 5. Hatching percentages ofArtemia cysts, exposed for 6 h to different intensities ofyellow (a), blue (b), green (c), and red (d) light.
212
ANNEMIE VAN DER LINDEN ET&.
Fig. 6. Log-Iogit transformation of hatching results of Anemia cysts after 6-h exposure to different intensities of( A)red, (m) blue, (*) yellow or (0)green light: yellow light:f(x) = 1.052 t 0.805 X, I = 0.958, x2 = 11.856 for N = 8; green light: f(x) = 0.682 = 0.643 x, r = 0.905, x2 = 8.841 for N = 5; blue light: f(x) = 0.034 + 0.632x, r = 0.956, x2 = 7.432 for N= 5; red light: f(x) = - 1.85 = 1.875 X, r = 0.975, x2 = 11.17 for N= It.
B yellow = 0.8 + 0.1 and Bblue = 0.6 2 0.1). For red light, an intensity cz 7 times higher is needed to achieve a similar hatching percentage (Bred = 1.2 & 0.1, difference is significant, P = 0.05). Taking into account the absorption characteristics of the used filters, we may conclude that the most effective region for hatching is at wavelengths between 400 and 600 nm.
DISCUSSION
The important role of Artemiu in aquaculture demands a continuous effort to improve hatching and culturing conditions. Our results add some important information about the light conditions favouring hatching. Under expe~ment~ conditions, z 21600 PE 3m - * light energy is needed to achieve maximal hatching percentages for SFB (2 149) cysts. Higher energies do not result in a greater hatching percentage. The required light energy can be obtained either by the combination: low intensity, long exposure time or high intensity, short exposure time. In the first case one can expect a delayed hatching since the required energy is obtained after a longer period. This delay is not detectable within the investigated 48-h incubation period, but has been demonstrated earlier by Vanhaecke et af. (198 1). Since light of the wavelen~hs between 400 and 600 nm is most effective for hatching, the choice of the light source is of great importance (e.g. fluorescent lamps will be more appropriate than glowlamps because of the distinct spectral qualities). Yellow, green or blue light sources are also suitable for trigger hatching.
LIGHT-MEDIATED
HATCHING
IN ARTEMIA
213
Since the data are in accordance with the Bunsen-Roscoe law of reciprocity, we can postulate the existence of a photoreceptor which mediates ii~t-induced hatching. This photoreceptor is expected to absorb light between 400 and 600 nm. The present data, however, allow no speculation on the nature of the photoreceptor since this requires an action spectrum. Because light mediation takes place before the loss of the outer cyst shell (Sorgeloos et al., 1976), it is necessary to draw the attention to the remarkable dark colour of this shell, due to its high haematin content (Gilch~st & Green, 1960). Inevitably, it will play a role, either as a light filter or as a site for photoreception. Shan (1970), working on Pleuroxus denticulatus, a branchiopod with light-induced hatching, stated that the dark pigmentation in the ephippium, surrounding the resting eggs, constitutes a mechanism for retarding development until hatching can be accomplished under favourabie environmental conditions. This was based upon expe~ments comparing hatching efficiency of resting eggs remaining in and dissected out of their ephippium. He proposed that the dark colour of the ephippium is first bleached by the sunlight to allow further light penetration and development. Possibly the dark haematin-rich cyst shell plays a similar role. It is interesting to note that haematin absorbs in the region most eflective to hatching (absorption peaks at 438, 532, and 558 nm: Gilchrist & Green, 1960). Future work will focus on the construction of an action spectrum of the light-induced hatching. ACKNOWLEDGEMENTS
We wish to thank Dr P. Sorgeloos for critically reading this paper. We also wish to express our gratitude to K. Cuypers and A. Vlaeminck for their technical assistance. REFERENCES FINNEY, D.J., 1978. Sf~fjs~jcul~er~~~ in b~oIo~cu~assay. Charles GriBin, London, 3rd edition, 508 pp. GILCHRIST, B.M. & GREEN, J., 1960. The pigments of Artemiu. Proc. Sot. London, Vol. 152, pp, 118-136. HEMPEL-ZAWITKOWSKA, J., 1970. The influence of strong ultra violet radiation on hatchability of Triops canceriformis (BOX.) eggs. Pal. Arch. Hydrobiol., Vol. 17, pp. 4X3-493. JONES,A. J., 1972. An inexpensive apparatus for the large scale hatching ofArtemia salina L. J. Cons.,Cons. Int. Explor. Mer, Vol. 34, pp. 351-356. PANCELLA,J. R. & R. G. STROSS, 1963. Light induced hatching of Daphnia resting eggs. Chesapeake Sci.. Vol. 4, pp. 135-140. ROYAN,J. P., 1976. Effect ofhght on the hatching and growth ofArtemias&a. Muhasager, Vol. 9, pp. 83-85.
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