Aquacultural Engineering 3 (1984) 1-13

PROBIT Analysis Predicts Hatching Rates of Brine Shrimp Artemia sp. Cysts* Stephen Spotte, Gary Adams
Mystic Marinelife Aquarium, Sea Research Foundation Inc., Mystic, Connecticut 06355, USA

and Paul E. Stake

Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut 06268, USA

ABSTRA CT Thirty replicates o f San Francisco Bay brine shrimp Artemia: sp. cysts from one lot were weighed to 0.01 mg. The mean mass o f a single cyst was 2.59 7 rig (+-O. 040 tlg, 95% confidence level). Batches o f I g from the same lot were hatched in the laboratory, and fraction hatched versus time data were fitted to a nonlinear curve using PROBIT analysis. Median time o f hatch was 24.8 h with a standard error o f +3.92 h. Cysts and nauplii in 1-ml aliquots were pipetted at regular intervals and counted under a dissecting microscope. The number o f cysts pipetted at the beginning o f the experiment was 19% less than estimates based on the known number per volume o f hatching medium, indicating that hatching data derived from pipetting procedures may contain large sampling errors. PROBIT analysis allows fraction hatched to be predicted when a particular lot o f cysts is hatched under controlled conditions.

INTRODUCTION Nauplii of the brine shrimp A r t e m i a spp. are ideal food organisms for larvae o f plankton-feeding fishes and invertebrates. Nutrient assays have * Contribution no. 33 of Sea Research Foundation, Inc. and no. 977 of the Storrs Agricultural Experiment Station.
1

Aquacultural

Engineering 0144-8609/84/$03.00-Elsevier

Applied Science

Publishers Ltd, England, 1984. Printed in Great Britain

S. Spotte, G. Adams, P. E. Stake

shown that newly hatched nauplii contain full complements of amino acids (Seidel et al., 1980) and most of the co3-series polyunsaturated fatty acids considered essential to seawater fishes (Schauer et al., 1980). Benijts et al. (1976) showed that the nutritional value of a nauplius declines markedly after the first molt at ~ 24 h, suggesting that nauplii should be harvested soon after hatching. This was confirmed by Ablett and Richards (1980). Brine shrimp cysts hatch at nonlinear rates, a n d efficient hatching methods must account for the fraction of cysts hatched versus time (the number of cysts that become nauplii), in addition to the number of nauplii ultimately obtained. Because the number of cysts is an estimate based on collective mass, the fraction hatched is not predictable unless representative cysts are weighed accurately beforehand. Methods of hatching large quantities o f nauplii for use in aquaculture were given by Bossuyt and Sorgeloos (1980), Kinne (1977), Nash (1973), Persoone and Sorgeloos (1972) and Sorgeloos and Persoone (1975). None of these reports, however, provided data on cyst mass or a quantitative procedure for predicting the fraction hatched.

MATERIALS AND METHODS Experimental design Commercially packed, desiccated cysts were used (San Francisco Bay Brand Inc., Newark, California 94560, USA, lot number 2851). Thirty replicates were weighed to 0-01 mg on an analytical balance (Sartorius model 2604, distributed by Brinkmann Instruments Inc., Westbury, New York 11590, USA). The cysts in each replicate were then counted under a dissecting microscope (Table 1). To keep the cysts from absorbing moisture they were left in the vacuum-sealed container until just before weighing. Hatching was carried out in four l-liter boiling flasks, each containing 500 ml new artificial seawater (Spotte, 1979) diluted to a salinity of 20.1~6o with distilled water. The flasks were plugged with three-hole rubber stoppers and set up as illustrated in Fig. I. A thermometer was inserted through one hole, and another (8 mm I.D.) was left open to vent air. Air was injected through 27.9 cm of standard glass laboratory tubing inserted in the third hole. Flexible polyethylene airline tubing

PR OBIT analysis predicts hatching rares o: brine shrimp cysts

TABLE 1

Cyst Masses from 30 Replicates Mass ( r a g ) 0-60 0.73 0.60 0-57 0.66 0.54 0.57 0.55 0.57 0.53 0.75 0-54 0.59 0.59 0-60 0.56 O.54 0.58 0-61 0.57 0.70 0.57 0.56 0.58 0.58 O-58 0.58 0.61 0-54 0.59 Number of cysts 198 249 215 266 276 235 239 298 182 226 273 203 250 204 236 208 2O2 202 255 189 261 232 200 201 220 2OO 228 206 264 213

(4 mm I.D.) was c o n n e c t e d to the exposed end o f the glass tubing; a rigid p o l y p r o p y l e n e ' T ' was attached to the submerged end to keep it above the b o t t o m o f the flask. Air was supplied from the main compressors at Mystic Marinelife Aquarium through a splinter line to

S. Spotte, G. Adams, P. E. Stake

~VentI~e

/ C Flask

Airlinetubing

"

III
Airs o u r c e

Fig. 1.

Experimental apparatus used to hatch brine shrimp cysts

the laboratory and distributed from a single source to the flasks through three rigid polypropylene 'Y' connections with attached airline tubing. Aeration rate was set at 1.9 liters min -~ (0.47 liter min -I to each flask) and monitored by an air flow meter (Laboratory Supplies, Hicksville, New York 11801, USA). Constant temperature (30C) was maintained by immersing the flasks in an uncovered, thermostatic, circulating water bath. The aerated flasks containing hatching medium but not cysts were placed in the water bath several hours before starting the experiment to allow the temperature to equilibrate. The experiment described below was performed four times. The first trial was terminated after 24 h; the next three were continued through

PROBIT analysis predicts hatching rates of brine shrimp cysts

30 h. Flasks were removed from the water bath and 1 g of cysts were added to each. These cysts were not the ones weighed in the 30 replicates, but were taken from the same lot. Light intensity and duration are known to affect hatching rate (Sorgeloos, 1973; Vanhaecke et al., 1981). The overhead lights were turned o f f and cyst development was initiated by illuminating the flasks for 10 rain with 2000 lux provided by one 40-W, cool-white fluorescent lamp placed 21-25 cm away. Simultaneous aeration prevented stratification, which would have shielded some cysts from the light. The level of illumination was measured with a light meter (Photo-Meter 1, Quantum Instruments Inc., Garden City, New York 11530, USA). After this initial illumination the ceiling lights were turned on, and the flasks were returned to the water bath. The light level at the surface o f the water remained constant at 1100 lux. Results of preliminary trials showed that no cysts hatched before 8 h, and that median time o f hatch occurred at "~ 24 h. Samples of 1 ml therefore were taken at 2-h intervals starting at 8 h until termination of a trial at 24 or 30 h. Before sampling, flasks were swirled to distribute cysts and nauplii evenly in the hatching medium, and to free cysts trapped against the glass above the water line. Samples were collected in 1-ml serological pipettes inserted through the vent holes in the stoppers, transferred to labeled vials, fixed with 0.5 ml formaldehyde solution and refrigerated. Pipettes were flushed after each use with distilled water to collect cysts and nauplii trapped inside. A vial was shaken gently, and 0-1 ml was pipetted into each o f 10 wells o f a Boerner slide. Nauplii were then counted under a dissecting microscope. The last step was repeated until all nauplii in the vial were counted. Afterward the walls o f the vial and pipette were examined by microscope for any remaining nauplii.

Computational procedures
A important operational problem in brine shrimp culture is predicting when a particular fraction will hatch. Finney (1971, p. 21) wrote: 'An interesting possibility is the use o f quantal response techniques in the study of some p h e n o m e n o n in a maturing animal that cannot be dated exactly b u t can readily be recorded as having occurred or not occurred . . . . ' To estimate the fraction o f cysts hatched at a given time, the experimental data must be fitted to an appropriate nonlinear curve.

S. Spotte, G. Adams, P. E. Stake

2.

0.5 . . . . . . . . . . . . . 0
Time

--" P- -1 t~

<
(b)

In(time)

(a)

Fig. 2. (a) Typical plot of fraction hatched versus time. (b) Typical plot of the
PROBIT of fraction hatched versus the natural logarithm of time. Because the PROBIT of 0-5 is zero, the intercept with the horizontal axis represents median time of hatch.

When fraction hatched is plotted against time the result is a graph like Fig. 2(a). Cumulative percentage data for which there is a threshold value, but no m a x i m u m value, are often fitted to a curve using PROBIT analysis. The PROBIT technique makes such nonlinear data linear by replacing the independent variable, time, with the logarithm o f time and the fraction hatched with the n u m b e r o f standard deviations from the mean (i.e. 0-50 fraction hatched). This transform is the PROBIT (PROBability uniT) o f the fraction, or the Normal Equivalent Deviation (NED), and is represented by the limit y in the integral P=
Vz~J-

---~_I'

e''/2 dr

(1)

where p is fraction hatched. Plotting the PROBIT o f fraction hatched against the logarithm o f time produces a graph like the one in Fig. 2(b). Because the integral has no analytical solution, y can be found using series in Abramowitz and Stegun ( 1964, sect. 26.2.23). First find r from r =~p2) (2)

where p is fraction hatched. The PROBIT o f fraction hatched is then given by

y = t -CO -[- c i r q- C2 r2

1 +dlr+d2r 2+d3r 3

(3)

PR OBIT analysis predicts hatching rares of brine shrimp cysts

where t=time, Co = 2 . 5 1 5 5 1 7 , c 1 = 0 - 8 0 2 8 5 3 , cz=0-010328, all= 1 . 4 3 2 7 8 8 , d2 = 0 . 1 8 9 2 6 9 , a n d d3 = 0 - 0 0 1 3 0 8 . This a p p r o x i m a t i o n is limited to p ~< 0.5. I f the value is > 0 - 5 replace p b y 1 - - p , calculate y , and reverse the sign. T h e a b s o l u t e value o f the m a x i m u m e r r o r in this a p p r o x i m a t i o n is 4-5 10 -4. Discussions o f P R O B I T analysis are available in F i n n e y ( 1971 ) a n d Sokal and R o h l f ( 1 9 6 9 ) .

RESULTS Data f r o m weighing 30 r e p l i c a t e s o f c y s t s are s h o w n in T a b l e 1. Values ranged f r o m 0.53 to 0.75 rag, a n d the n u m b e r o f cysts in e a c h replicate varied f r o m 182 to 298. T h e m e a n m a s s o f a single c y s t was 2 . 5 9 7 tag (-+ 0-008 tag, 95% c o n f i d e n c e level). N u m b e r s o f c y s t s h a t c h e d versus t i m e are s h o w n in T a b l e 2. F r a c t i o n h a t c h e d was t r a n s f o r m e d to P R O B I T s , a n d t i m e was t r a n s f o r m e d to the

TABLE 2 Nauplii per ml Versus Incubation Time

Elapsed time (h) 0 8 9 10 12 14 16 18 20 22 24 26 28 30

Trial I

Trial 2

Trial 3

Trial 4

Mean

~o hatched

0a 0 _a 18 56 186 190 224 301 248 346 -

0 0 _ 0 18 84 132 193 238 296 318 366 345 363

0 0 1 27 87 132 158 198 250 307 312 354 316 -

0 0 33 109 177 194 247 267 276 293 348 336

0 0 0 8.5 33-5 116-5 157-7 192.3 246.0 274.3 311-8 323.7 349-0 338.3

0 0 0 1.35 5.35 18-6 25-2 30.7 39.3 43-8 49.8 51-7 55.8 54-0

a Dashes and zeros indicate no data and no nauplii, respectivety.

8 0.7 0.6
0.5

S. Spotre, G. Adams, P. E. Stake

.S
~e/* (

u 0.4

"~ 0.3 0.2 ? 0.1~5

/
10 15 20 25 30 35
Time (h)

Fig. 3.

Fraction hatched versus time. The curve is a least squares fit using PROBIT analysis. Horizontal bar is the standard error of estimate at 50% hatch.

logarithm o f time. Transformed data were fitted to a least squares curve, and the standard error was calculated. The curve and original data are shown in Fig. 3. Data are shown numerically in Table 2. The best estimate o f the median time o f hatch, as indicated by the PROBIT technique, is 24.8 h with a standard error of + 3.92 h. The original fraction hatched versus time data are tabulated in Table 3, columns 1 and ,. " Column 3 is the natural logarithm of time, t. Column 4 is the PROBIT of fraction hatched. Column 5 is the square o f column 3. Column 6 is the product o f columns 3 and 4. The sums of columns 3 through 6 can then be calculated. The slope, m, o f the linear curve fit is given by m = ( n Z x y - - Z y Z x ) / ( n Z x 2 - (Y.x) 2) where n is the number of data points. The intercept, b, is given by (4)

b = (Zy-- mZx)/n
Median time o f hatch, tl/2, is calculated from

(5)

tl/2 = e- ~/"

(6)

TABLE 3 CaLcuLationof Sums for Least-Squares Curve Fit

Time,

Fraction hatched, P

In (t), x

PROBIT,

x 2

xy

( x - X~st)2

10 10 12 12 12 12 14 14 14 14 16 16 16 16 18 18 18 18 20 20 20 20 22 22 22 22 24 24 24 24 26 26 26 28 28 28 30 30 30

0432875 0-0016 0-05271 0434313 0-02875 0438945 0.17412 0.13897 0-13418 0-29712 0.28274 0-21086 0"21086 0.30351 0.30990 0.25239 0-30830 0.35782 0-39456 0-31629 0-38019 0-48083 0-45367 0-47284 0-39936 0-42651 0.44089 0.49041 0-50798 0.55271 0.58466 0-49840 0-46805 0.55591 0-56549 0-55111 0-42102 0.50479 0.53674

2-30258 2.30258 2-48490 2.48490 2-48490 2"48490 2"63905 2-63905 2"63905 2'63905 2-77258 2.77258 2"77258 2-77258 2.89037 2-89037 2-89037 2"89037 2.99573 2"99573 2.99573 2.99573 3439104 3-09104 3-09104 3.09104 3.17805 3-17805 3.17805 3.17805 3.25809 3.25809 3.25809 3.33220 3-33220 3-33220 3-40119 3-40119 3-40119 114-79

-1-89985 -2-94866 -1-61942 -1-71583 - 1 "89985 - 1-34432 -0.93792 -1438496 -1-10687 -0"53229 -0.57432 -0.80323 -0"80323 -0.51391 -0-49570 -0-66666 -0.50024 -0.36382 -0-26700 -0"47766 -0-30454 -0-04794 -0.11611 -0.6795 -0-25458 -0.18488 -0.14838 -0-2395 0-01996 0.13223 0.21345 -0-01600 -0-07997 0.14030 0.16458 0-12820 -0.20119 0431197 0.09200 -21-09

5-30189 5.30189 6-17476 6-17476 6-17476 6.17476 6-96462 6.96462 6"96462 6-96462 7-68724 7-68724 7.68724 7.68724 8-35424 8.35424 8.35424 8-35424 8-97441 8.97441 8-97441 8.97441 9-55454 9.55454 9-55454 9-55454 10-10002 10.10002 10.10002 10.10002 10.61519 10.61519 10.61519 11.10358 11.10358 11.10358 11.56814 11.56814 11.56814 341-7

-4-37457 -6.78954 -4432411 -4.26368 -4.72095 - 3-34051 -2.47522 -2.86327 -2.92110 -1-40475 - 1-59237 -2-22704 -2.22704 -142487 - 143278 - 1-92690 - 1-44589 -1-05159 -0-79988 - 1-43096 -0"91232 -0-14361 -0-35892 -0-21003 -0.78693 -0.57149 -0-47158 -0-07614 0-06345 0-42024 0.69545 -0.01300 -0.26055 0-46752 0.54841 042719 -0-68429 0-04073 0.31292 54-25

0-00093 0.30082 0.00553 0-01488 0.04531 0.00378 0431167 0430125 0-00060 0-09507 0432373 0-00168 0-00168 0-03381 0-00564 0-00009 0-00531 0-01966 0-00683 0-00046 0-00411 0-03642 0-00383 0-00734 0-00004 0.00078 0-00169 0430042 0-00179 0-00951 0430331 0.00248 0-00763 0430277 0-00165 0.00344 0438427 0-03424 0.02117 0-8056

~x =

Y.y =

Y..x 2 =

Y.xy =

Y~(x-x~a) 2 =

10

S. Spotte, G. Adams, P. E. Stake

The elements of column 7 are calculated from

(X - - Xest) 2 = [ X -

y / m + b / m ]2

(7)

The sum of column 7 can now be calculated. The standard error of estimate, Sx,y , is calculated from

=,,/z ( x

2)

(8)

To convert standard error to the original time scale find

St,y = eltn(r")+Sx,y 1 - - tl/2

(9)

The linear curve fit results in the equation y = m In(t) + b (10)

At time, t, the expected hatch, p, can be predicted by first obtaining y from eqn (10) and then using eqn (1 l ) p= I--0-5(1 +y(el+y(e2+Y2(e3+ye4))))
-4

(11)

where et = 0.196854, e2 = 0.115194, e3= 0-000344 and e4 = 0.019527. This approximation (Abramowitz and Stegun, 1964, sect. 26.2.18)has a maximum error o f -+ 2-5 l0 -4.

DISCUSSION As o f 1979 brine shrimp had been described from 164 locations around the world (Simpson, 1979). Cyst mass varies even within the same lot collected from a single strain. Obtaining the accurate mass o f a known number of cysts is therefore a prerequisite to predicting how many nauplii can be produced in a given time. Hatching characteristics are strain-dependent, in addition to being affected by environmental factors (e.g. light, temperature, salinity, dissolved oxygen concentration, environmental pH). A change in one factor must be compensated by a shift in others to maintain optimum hatching rate. Ideal temperature, for example, must be re-established with each change in salinity (Ivanovskii et al., 1980). The shift from dormancy to resumed development o f the gastrula is affected profoundly by internal pH. The extreme alkalinization that occurs during transition (>1 pH unit) exceeds that o f any organism yet studied (Busa et al., 1982). The importance of

PR OBIT analysis predicts hatching rares o f brine shrimp cysts

11

internal alkalinization on arousal from cryptobiosis was demonstrated convincingly by Busa and Crowe (1983). When they added 40 mM NH4C1 to the hatching medium (160 mM NaC1 and 50 mM tricine at pH 8-5 and 23C), the percent mean hatch at 68 h increased more than sixfold. According to Spotte and Adams (1983), under these conditions 13% of the total ammonia would exist in the free state (NH3). Free ammonia is a weak base capable o f penetrating the cyst membranes. According to Sorgetoos (1980) hatching rate can depend on processing methods used on the cysts prior to packaging them (e.g. level and duration of illumination, removal of the cyst chorion with a strong oxidizing solution). As shown in Fig. 3 some cysts had hatched by 10 h, whereas 40% still had not hatched after 30 h. Our experiments established that PROBIT analysis is a useful tool for predicting fraction hatched of a known number of cysts in a particular lot under given conditions. Numbers of cysts are traditionally estimated by mass. We therefore started by determining the mean cyst mass o f the lot selected for the experiment. The total number o f cysts added to each flask was 3-85 x l0 s (mean cyst mass extrapolated to 1 g). Afterwards cysts and nauplii were removed regularly in t-ml aliquots and counted. We sometimes found it difficult to distinguish an e m p t y cyst shell from an unhatched cyst, so cyst count data were not used, except in aliquots taken at 8 h. These gave true counts because no nauplii had hatched. A 1-ml aliquot therefore should have contained 770 cysts, but the average count of the 16 aliquots was 626 (- 61, 95% confidence level), or 81% of the estimated number present. In these experiments the fraction hatched data plotted in Fig. 2 are based on the counts o f pipetted samples, and the discrepancy did not affect the results. The removal of cysts by pipetting apparently underestimated the number actually present, thus accounting for the 19% difference between aliquot counts and the number known to have been added to the flasks. To our knowledge there are no published reports of the accuracy of pipetting techniques used to count either cysts or nauplii. A mixture of cysts or nauplii in water has traditionally been treated as a true solution instead o f a suspension of large particles. It seems logical that the distribution of such particles would be uneven because of stranding on the glass and vortex effects, and perhaps not represented accurately by procedures that rely on pipetting.

12

S. Spotte, G. Adams, P. E. Stake

ACKNOWLEDGEMENTS Patricia M. Bubucis and Paul Gaj provided laboratory and artistic assistance, respectively, Louis Mercuri helped with the mathematics, and Carl J. Berg Jr., Carol E. Bower and John D. Buck reviewed the manuscript. Their contributions are gratefully acknowledged.

REFERENCES Ablett, R. F. & Richards, R. H. (1980). Suitability of twenty-four-hour and fortyeight-hour unfed Artemia as an early foodstuff for 'O' group Dover sole Solea solea L. production. Aquaculture, 19, 371-7. Abramowitz, M. & Stegun, I. (1964). Handbook of Mathematical Functions with Formulas, Graphs and Mathematical Tables. US Government Printing Office, Washington DC. Benijts, F., van Voorden, E. & Sorgeloos, P. (1976). Changes in the biochemical composition of the early larval stages of the brine shrimp Artemia salina L. In: lOth European Symposium on Marine Biology, Vol. 1, eds G. Persoone and E. Jaspers, University Press, Wetteren, pp. 1-9. Bossuyt, E. & Sorgeloos, P. (1980). Technological aspects of the batch culturing of Artemia in high densities. In: The Brine Shrimp Artemia, Vol. 3, eds G. Persoone, P. Sorgeloos, O. Roels and E. Jaspers, University Press, Wetteren, pp. 133-52. Busa, W. B. & Crowe, J. H. (1983). lntraceUular pH regulates transitions between dormancy and development of brine shrimp Artemia salina embryos. Science, 221,366-8. Busa, W. B., Crowe, J. H. & Matson, G. B. (1982). lntraceUular pH and the metabolic status of dormant and developing Artemia embryos. Arch. Biochem. Biophys., 216, 711-!8. Finney, D. J. (1971). PROBITAnalysis, 3rd edn, Cambridge University Press, New York. lvanovskii, Yu. A., Mitrofanov, Yu. A. & Chaga, 1. L. (1980). Hatching of nauplii of Artemia salina at different salinity and temperature regimes. Soy. J. Mar. Biol., 6,202-7. Kinne, O. (1977). Marine Ecology: A Comprehensive, Integrated Treatise on Life in Oceans and Coastal Waters, Vol. 3, Part 2, Wiley, Chichester. Nash, C. E. (1973). Automated mass-production of Artemia salina nauptii for hatcheries. Aquaculture, 2,289-98.

PR OBIT analysis predicts hatching rates of brine shrimp cysts

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Persoone, G. & Sorgeloos, P. (1972). An improved separator box for Artemia nauplii and other phototactic invertebrates. Helgoliind. wiss. Meeresunters., 23, 243-7. Schauer, P. S., Johns, D. M., Olney, C. E. & Simpson, K. L. (1980). International study on Artemia. IX. Lipid level, energy content and fatty acid composition of the cysts and newly hatched nauplii from five geographical strains of Artemia. In: The Brine Shrimp Artemia, Vol. 3, eds G. Persoone, P. Sorgeloos, O. Roels and E. Jaspers, University Press, Wetteren, pp. 365-71. Seidel, C. R., Kryznowek, J. & Simpson, K. L. (1980). International study on Artemia. XI. Amino acid composition and electrophoretic protein patterns of Artemia from five geographic locations. In: The Brine Shrimp Artemia, Vol. 3, eds G. Persoone, P. Sorgeloos, O. Roels and E. Jaspers, University Press, Wetteren, pp. 375-82. Simpson, K. L. (1979). Focusing on the modest and minute brine shrimp. Marltimes, 23 (4), 9-11. Sokal, R. R. & Rohlf, F. J. (1969). Biometry, Freeman, San Francisco. Sorgeloos, P. (1973). First report of the triggering effect of light on the hatching mechanism ofArtemia salina dry cysts. Mar. Biol., 22, 75-6. Sorgeloos, P. (1980). The use of the brine shrimp Artemia in aquaculture. In: The Brine Shrimp Artemia, Vol. 3, eds G. Persoone, P. Sorgeloos, O. Roels and E. Jaspers, University Press, Wetteren, pp. 25-46. Sorgeloos, P. & Persoone, G. (1975). Technological improvements for the cultivation of invertebrates as food for fishes and crustaceans. II. Hatching and culturing of the brine shrimp Artemia saline L. Aquaculture, 6,303-17. Spotte, S. (1979). Fish and Invertebrate Culture: Water Management in Closed Systems, 2nd edn, Wiley, New York. Spotte, S. & Adams, G. (1983). Estimation of the allowable upper limit of ammonia in saline waters. Mar. Ecol. Prog. Ser., 10,207-10. Vanhaecke, P., Cooreman, A. & Sorgeloos, P. (1981). International study on Artemia. XV. Effects of light intensity on hatching rate of Artemia cysts from different geographical origin.Mar. Ecol. Prog. Ser., 5, 111-14.