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Effect of pH, salinity and Ca2+, Mg2+ K+ and SO42+ ions on hatching and viability of Loligo vulgaris embryo
Camp. Biochem. Phwiol.
Vol. 94A, No.
3, pp. 477481,
0300-9629/89
1989
53.00 + 0.00
Pergamon Press plc
Printed in Great Bhtain
EFFECT OF pH, SALINITY AND Ca*+, Mg*+, K+ AND SO;+ IONS ON HATCHING AND VIABILITY OF LOLZGO VULGARIS EMBRYO A. D’ANIELLO,*~ G. D’ONOFRIO,* M. PISCHETOLA* and J. M. DENUCBS *Stazione Zoologica “A. Dohm”, Villa Communale, I-80121 Napoli, Italy; and $Zolilogisch La~ratorium I, Katholieke Universiteit, Nijmegen, The Netherlands (Received 7 April 1989) Abstract-l. The effect of salinity, pH, calcium, magnesium, potassium and sulphate ions on egg hatching and embryonic survival of squid embryos was studied. 2. In artificial sea-water, the embryos batched normally and lived up to S-6 days without food. In natural sea-water, they lived up to 67 days without food. 3. In sea-water with salinity values from 34 to 42% and at pH values between 7.8 and 8.4, the embryos developed and hatched no~aliy. Beyond this range, embryos were severely damaged, and some died. 4. The physiological concentrations of CaZ+, K+, M 2’ and SOi- ions, at which embryos hatched and developed normafly, were: 9-15 mM, 9-1.5 mM, 4670 mM and IS-37 mM respectively.
INTRODUCTION Despite numerous studies on the electrolyte composition of marine molluscs, and on the osmotic and ionic regulation in marine invertebrates in general (Robertson, 1949, 1953; Emanuel and Martin, 19.56; Deffner and Mafter, 1960; Deffner, 1961; Potts and Todd, 1965), very little is known about the effect of pH, salinity and calcium, and of magnesium, potassium and sulphate ions on hatching and viability of cephalopods. The eggs of many marine invertebrates are isosmotic to the sea-water environment and increase in size in response to changes in salinity. In many instances, secondary egg envelopes, e.g. jelly layers, prevent passive loss of water, or the uptake of water (Prosser, 1973). The aim of this study was to establish the rate of growth of Loligo uulgaris embryos inside strings in artificial sea-water, and the range of values of PI-I, salinity, and the concentrations of potassium, calcium, magnesium and sulphate ions within which development, hatching and survival of the embryos of L&go vulgaris proceed normally.
al. (1986), in which the concentrations of the major ions reflected those of sea-water of the Bay of Naples and buffered to 8.1 with boric acid, sodium borate and sodium bicarbonate at the final concentration of 10, 4 and 2 mM, respectively. The other physico-chemical characteristics of the artificial sea-water were: osmotic concentration, 1.292mOsm/kg water; salinity, 40 g/l; density, 1.022 and conductivity, 29 mMH0. Experimentalprocedure All the experiments were conducted from April to June under natural light conditions, at a temperature of 1%20°C. The effect of salinity and pH on the hatching and survivaf of Lo&go uulguris embryos was studied by incubating 30 isolated eggs, or eggs still in strings, or embryos previously hatched in 800 ml of natural sea-water which varied in pH and in salinity. The pH was changed by adding a few drops of 1 N HCI or 1 N NaOH to I 1 of sea-water, until the required pH value was reached. To change the salinity of sea-water, natural sea-water was diluted with distilled water, or concentrated by lyophilization. To study the effect of calcium, potassium and magnesium, CaCl,, KCl, MgSO, were reduced or added to artificial sea-water and NaCl was added or subtracted to maintain osmoiarity. The effect of sulphate was assessed in artificial sea-water containing MgCl, instead of MgSO,.
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Effect of salinity
Animals
Strings of Lo&o mlgaris eggs, either far from or near to hatching, were collected in the Bay of Naples and maintained in tanks containing circulating sea-water. and artificial sea-water Natural sea-water was taken from the Bay of Naples. Artificial sea-water was prepared according to D’Aniello el Natural
-._
.__________
tAuthor to whom all correspondence
should be addressed. 477
Salinity is one of the most important physicochemical factors to which marine embryos are exposed. Sepia o&icinaZis embryos can hatch in sea-water in which salinity has been reduced to 28% (Palmeggiano and D’Apote, 1983). Some Japanese cephalopods hatch at even lower salinity values: Sepioteuthis lessoniana, Sepiella maindroni and Sepia esculenta show 50% hatching at salinities of 16.8, 14.7 and 12.6X, respectively (Choe, 1966).
478
A. Table
I. Effect of salinity
D‘ANIELLO
on egg hatching
et al.
and survival
Salinity (%I
Eggs inside strings contained embryos 12-14 days before hatching’r
Isolated eggs 2-3 days before hatchine?
19
no development
no hatchmg
28
no development
40-60%
34 38* 42 41
normal development normal development normal development no development
normal hatchmg normal hatchmg normal hatching 65-85% hatched
of embryos
culgurb
Survwal of hatched embryos$ .-. all died after fern minutes 40-60% hved up to 33 days, others died before hved S-6 da>) hved 6-7 days lived 5 6 days 65 -X5% hved up 10 3 4 days: other\ died hefiwc
hatched
no hatching
57
of Lo&o
*This value corresponds to natural sea-water from the Bay of Naples tThe results are the mean values derived from analys~c performed on three wng\. each containing about 30 eggs. $The results are the mean valur~ calculated from three analyses: each anal)& ~.a\ performed on 30 embryos. $The results are the mean values calculated from three analyses; each analy~\ was performed oo 30 embryos that were previously hatched m natural sea-water and then put m artificial sea-water at various aalimt~es.
Under our experimental conditions. Lolip rxlgrtris embryos hatched and lived up to 5-7 days without food in a salinity range of 3442%0. Outside this range severe damage occurred. In fact, as shown in Table 1, at a salinity below 28% which is 75% of the salinity of the Bay of Naples, and at a salinity higher than 47%0 (150% of normal salinity) none of the eggs at 12-14 days pre-hatching developed. Of the embryos at the 2-3 days pre-hatching stage, only 4&60% and 65-85% hatched at salinities of 28 and 47%0, respectively, and the embryos lived for about 34 days. At the extreme salinity values of below 19%0 and above 57%0, no hatching occurred and all hatched embryos exposed to these conditions died after a few minutes. These last results are in good agreement with findings of (Jecklin, 1934) that newly
Table 2. Effect of pH on egg hatching
PH
Eggs inside strings contained embryos 12-14 days before hatchingt
7.1
no development
1.6
7.x 8.1’ x.4 X.6
normal normal normal normal
development development development development
9.1
no development
hatched between EfSect
Lolip dguri.s embryos 28 and 42%, salinity.
are
viable
only
of pH
At pH values between 7.8 and 8.4, embryos developed and hatched normally. Beyond this range, disturbances occurred. In fact, as shown in Table 2. at pH 7.6 and 8.6 (8.1 is the pH value of natural sea-water), eggs developed and hatched normally, but did not survive. Whereas at pH 8.1 newly hatched embryos lived up to 6--7 days, at pH 7.6 and 8.6 only 50-70% of the newly hatched embryos survived up to 6 days. At pH values of 7.1 and 9.1, only 2630% and 3&40%, respectively, of the embryos hatched and lived up to 3 days. The others either failed to hatch
and surwval
Isolated eggs contained embryos 2 3 days before hatching1 ?O-30%
hatched
normal
hatching
normal normal normal normal
hatching hatchmg hatching hatching
30 40%
hatched
of embryos
of Lollgo &guru
Survival of hatched embryo@ 3040% lived up days: others dwd before 50 70% hved up davs: othsrs died hefore lived 6 7 da)\ lived 6 7 day\ hwd 6 7&q\ 50-70% hved up dav\: other\ died be&e 20 3O”O hved up days: others died before
to 3
to 7
to 6
to i
*This value corresponds to natural sea-water from the Bay of Naplea. tThe results are the mean values derived from analyw performed on three \trlngs, each containing about 30 eggs. $The results are the mean valueb calculated from three analyses: each analya~r was performed on 30 embryos. $The results are the mean values calculated from three analyses, each analyw wa\ performed on 30 embryos that were previously hatched in natural ~-water and then put m artkxd sea-water at var,ous pH value5
Physicochemical Table 3. Effect of calcium Embryos
Ca*+ (mM)
factors
on eggs hatching
and survival
inside choriont
6
no hatching no hatching. embryos died after wolent convulsions 7Om90% hatched
9
90-100%
0 4
influencing
hatched
12*
loo?/. hatched
I5
95m100% hatched
IX
50-70%
24
the embryos escaped from the chorion because of pamc reaction
hatched
419
squid hatching of embryos
Survival
of Loligo tulgaris
of hatched
embryosf
all died after a few minutes the motihty of embryos was reduced; they died after 2-3 days the motility of embryos was normal; they lived 3-4 days the motility of embryos was normal; they lived 54 days the motility of embryos was normal: they lived 67 days the motility of embryos was normal; they lived S-6 days the motility of embryos was normal; they lived only 2-3 days motdity lost. embryos died after I day
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses. Each analysis on 30 isolated eggs. :The results are the mean values calculated from three analyses; each analysis on 30 embryos that were prewously hatched in natural sea-water and then sea-water and then put in artificial sea-water at WUIOUS concentrations of
or died. Below and above pH 7.1 and 9.1, eggs did not develop. Effkct
qf Ca’+, K+
and Mg’+
To our knowledge, this is the first study of the effect of these three cations on egg hatching and embryo survival in L&go vulgaris. Interestingly, calcium and potassium exerted the same effect: when their level was less than half that of Bay of Naples sea-water, severe damage occurred (see Tables 3 and 4). For example, when Ca2+ or K+ was present at a concentration of up to 4 mM (which corresponds to 33% of their concentration in natural sea-water), hatching did not occur, and embryos transferred to these conditions after having hatched in natural seawater. died immediately. The same results were obtained when Ca2+ and K+ were at 18 mM (150% of the normal value). In this case, only 5670% of eggs hatched and the embryos lived only 2-3 days. At a concentration between 9 and 15 mM (75 and 125%, with respect to natural sea-water), Ca2+ and K ’ did not affect either eggs hatching or embryo survival.
Table 4. Effect of potassium K+ (mM) 0
Embryos
on eggs hatching
Inside choriont
6
no hatching no hatching. embryos died after violent convulsions 70-90% hatched
9
9&lOO%
4
hatched
12’
all hatched
I5 I8
9&lOO% hatched 50-70% hatched
24
the embryos escaped from the chorion because of panic reaction
was performed was performed put in artificial calcium.
The effect of magnesium on embryo hatching and survival is shown in Table 5. At a range of concentrations between 46 and 70mM (80 and 120%, respectively, compared to sea-water), all embryos hatched and survived normally. Outside these values, the embryos died and hatching did not occur. Effect of SOi-
The absence of sulphate from sea-water did not seem to affect hatching or survival, apart from causing a slight delay in hatching (Table 6). At a sulphate concentration of 45 mM, only 4&60% of Loligo eggs hatched and of all the embryos hatched only 4&60% lived 2-3 days. At a concentration of 60 mM, which corresponds to 200% with respect to sea-water, only l&20% of the eggs hatched, and the embryos lived only l-2 days. CONCLUSION
We previously found that the concentrations of Na+, K+, Ca’+, Mg2+ and SO:+ ions in the periv-
and survival Survival
of embryos of hatched
of L&go
wlgaris
embryos$
all died after a few minutes the motlhty of embryos was lost; they died after 2-3 days the motility of embryos was normal; they lived 2-4 days the motility of embryos was normal; they lived 4-S days the motility of embryos was normal; they lived 67 days the motility of embryos was normal the motility of embryos was reduced; they lived only 2-3 days all embryos died after few hours
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses; each analysis was performed on 30 isolated eggs. :The results are the mean values calculated from three analyses; each analysis was performed on 30 embryos that were previously hatched in natural sea-water and then put in artificial sea-water at various concentrations of potassium.
A.
480 Table 5. Effect of maenesium
Mg*+ (mM)
Embryos
on eees hatchine
29 46
all hatched
58’
all hatched
70
all hatched
86
3&40%
hatched
116
l&20%
hatched
4
et al.
and surwval
inside choriont
no hatching, embryos died after few minutes died immediately after hatching 6&80% hatched
0
D’ANIELLO
of embrvos
Survival
of hatched
of Lo&w oulmrrs embryos$
died after a few minutes the motility of embryos was lost; they lived only 2-3 hr the motility of embryos was reduced: they lived 2-3 days the motility of embryos was normal; they lived 67 days the motility of embryoa was normal: they lived &7 days the motility of embryos was normal they lived 54 days the motility of embryos was reduced: they lived only 2-3 days the motility was lost; the embryos lived onlv I-2 days
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses: each analysis was performed on 30 isolated eggs. $The results are the mean values calculated from three analyses; each analysts was performed on 30 embryos that were previously hatched in natural sea-water and then put in artificial sea-water at various concentrations of magnesium.
Table 6. Effect of sulphate SO:- (mM) 0
on eggs hatching
Embryos inside choriont
15
all hatched, but with delayed 2-3 days all hatched
30.
all hatched
37
all hatched
45
4&60%
hatched
60
l&20%
hatched
and survival
of embryos
Survival
of hatched
the motility of they lived &5 the motility of they lived 5d the motility of they lived 67 the motility of they lived 5-6 the motility of they lived 2-3 the motility of they lived IL2
embryos days embryos days embryos days embryos days embryos days embryos days
of Loligo dgaris emhryosf was normal; was normal; was normal; was normal; was normal; was normal
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses; each analysis was performed on 30 isolated eggs. fThe results are the mean values calculated from three analyses; each analysis was performed on 30 embryos that were previously hatched in natural sea-water and then put in artificial sea-water at various concentrations of sulphate ions.
itelline fluid is in osmotic equilibrium with the surrounding natural or artificial sea-water and that they reflect the changing concentrations of these ions in the external medium (D’Aniello et al., 1987). The present study demonstrates that Loligo vulgaris eggs develop normally and embryos hatch only when the external fluids have a salinity between 34 and 42% and pH values between 7.8 and 8.4. In addition, each of the above-mentioned ions must be in a certain concentration range for egg development and hatching to occur: calcium and potassium between 9 and 15 mM, magnesium between 48 and 68 mM and sulphate ions between 15 and 37 mM. Eggs at the 2-3 days pre-hatching stage, placed in artificial sea-water, contained the same concentration of major ions as sea-water of the Bay of Naples, hatch normally, while eggs at the stage of 12-14 days pre-hatching, did not develop normally and the embryos did not hatch. Since the artificial sea-water prepared by us contained only the major ions that are present in natural sea-water (Na+, K+, Ca*+, Mg2+, SOi- and Cl-), and since the youngest eggs failed to survive in this water, it appears that normal embryonic development requires some substances that are
present in natural sea-water, in extremely low amounts, e.g. oligoelements or organic molecules. REFERENCES S. (1966) On the eggs, rearing, habits of the fry, and growth of some Cephalopoda. Bull. Mar. Sri. 16, 33c-348. D’Aniello A., Strazzullo L., D’Onofrio G. and Pischetola M. (1986) Electrolytes and nitrogen compounds of body fluids and tissues of Octopus culgaris Lam. J. Camp. Physiol. 156B, 503-509. D’Aniello A., D’Onofrio G.. Pischetola M. and Denuck J. M. (1987) Effect of the ionic concentration of calcium and potassium on hatching of the embryo of the squid, Leligo vulgaris. A preliminary study. Arch. Int. Phvsiol. Biochim. 95, B66. D’Apote M. P. and Palmeggiano G. B. (1982) Inseminazione della Laguna di Lessina con uova di Sepia ojicinalis L. Studio dell’ effetto di parametri ambientali sulla schiusa: temperatura e salinita’. Ric. I/. Pixie. Iftiop. 17, 112-l 19. Deffner G. G. T. and Mafter R. S. (1960) Chemical investigation of the giant nerve fiber of the squid. IV. Acid--base balance in axoplasm. Biochim. biophyx Acta 42, 200-205. Deffner G. G. T. (1961) The dialyzable free organic con-
Choe
Physicochemical
factors
stituents of squid blood; a comparison with nerve axoplasm. Biochim. biophys. Acta 47, 378-388. Emanuel C. F. and Martin A. W. (1956) The composition of octopus renal fluid. 2. Vergl. Physiol. 39, 22&234. Jecklin L. (1934) Beitrag zur kenntnis der Laichgallerten und der Biologic der Embryonen decapoder Cephalopoden. Rev. Suisse Zool. 41, 593-673. Palmeggiano G. B. and D’Apote M. P. (1983) Combined effects of temperature and salinity on cuttlefish (Sepia ojicinalis L.) hatching. Aquaculture 35, 259-264.
influencing
squid hatching
481
Potts W. T. W. and Todd M. (1965) Kidney function in the Octopus. Comp. Biochem. Physiol. 16, 479489. Prosser C. L. ed. (1973) Comparative Animal Physiology (3rd edn). W.B. Saunders Company, Philadelphia. Robertson J. D. (1949) Ionic regulation in some marine invertebrates. J. exp. Biol. 26, 181-200. Robertson J. D. (1953) Further studies on ionic regulation in marine invertebrates. J. exp. Biol. 30, 277-296.
Vol. 94A, No.
3, pp. 477481,
0300-9629/89
1989
53.00 + 0.00
Pergamon Press plc
Printed in Great Bhtain
EFFECT OF pH, SALINITY AND Ca*+, Mg*+, K+ AND SO;+ IONS ON HATCHING AND VIABILITY OF LOLZGO VULGARIS EMBRYO A. D’ANIELLO,*~ G. D’ONOFRIO,* M. PISCHETOLA* and J. M. DENUCBS *Stazione Zoologica “A. Dohm”, Villa Communale, I-80121 Napoli, Italy; and $Zolilogisch La~ratorium I, Katholieke Universiteit, Nijmegen, The Netherlands (Received 7 April 1989) Abstract-l. The effect of salinity, pH, calcium, magnesium, potassium and sulphate ions on egg hatching and embryonic survival of squid embryos was studied. 2. In artificial sea-water, the embryos batched normally and lived up to S-6 days without food. In natural sea-water, they lived up to 67 days without food. 3. In sea-water with salinity values from 34 to 42% and at pH values between 7.8 and 8.4, the embryos developed and hatched no~aliy. Beyond this range, embryos were severely damaged, and some died. 4. The physiological concentrations of CaZ+, K+, M 2’ and SOi- ions, at which embryos hatched and developed normafly, were: 9-15 mM, 9-1.5 mM, 4670 mM and IS-37 mM respectively.
INTRODUCTION Despite numerous studies on the electrolyte composition of marine molluscs, and on the osmotic and ionic regulation in marine invertebrates in general (Robertson, 1949, 1953; Emanuel and Martin, 19.56; Deffner and Mafter, 1960; Deffner, 1961; Potts and Todd, 1965), very little is known about the effect of pH, salinity and calcium, and of magnesium, potassium and sulphate ions on hatching and viability of cephalopods. The eggs of many marine invertebrates are isosmotic to the sea-water environment and increase in size in response to changes in salinity. In many instances, secondary egg envelopes, e.g. jelly layers, prevent passive loss of water, or the uptake of water (Prosser, 1973). The aim of this study was to establish the rate of growth of Loligo uulgaris embryos inside strings in artificial sea-water, and the range of values of PI-I, salinity, and the concentrations of potassium, calcium, magnesium and sulphate ions within which development, hatching and survival of the embryos of L&go vulgaris proceed normally.
al. (1986), in which the concentrations of the major ions reflected those of sea-water of the Bay of Naples and buffered to 8.1 with boric acid, sodium borate and sodium bicarbonate at the final concentration of 10, 4 and 2 mM, respectively. The other physico-chemical characteristics of the artificial sea-water were: osmotic concentration, 1.292mOsm/kg water; salinity, 40 g/l; density, 1.022 and conductivity, 29 mMH0. Experimentalprocedure All the experiments were conducted from April to June under natural light conditions, at a temperature of 1%20°C. The effect of salinity and pH on the hatching and survivaf of Lo&go uulguris embryos was studied by incubating 30 isolated eggs, or eggs still in strings, or embryos previously hatched in 800 ml of natural sea-water which varied in pH and in salinity. The pH was changed by adding a few drops of 1 N HCI or 1 N NaOH to I 1 of sea-water, until the required pH value was reached. To change the salinity of sea-water, natural sea-water was diluted with distilled water, or concentrated by lyophilization. To study the effect of calcium, potassium and magnesium, CaCl,, KCl, MgSO, were reduced or added to artificial sea-water and NaCl was added or subtracted to maintain osmoiarity. The effect of sulphate was assessed in artificial sea-water containing MgCl, instead of MgSO,.
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Effect of salinity
Animals
Strings of Lo&o mlgaris eggs, either far from or near to hatching, were collected in the Bay of Naples and maintained in tanks containing circulating sea-water. and artificial sea-water Natural sea-water was taken from the Bay of Naples. Artificial sea-water was prepared according to D’Aniello el Natural
-._
.__________
tAuthor to whom all correspondence
should be addressed. 477
Salinity is one of the most important physicochemical factors to which marine embryos are exposed. Sepia o&icinaZis embryos can hatch in sea-water in which salinity has been reduced to 28% (Palmeggiano and D’Apote, 1983). Some Japanese cephalopods hatch at even lower salinity values: Sepioteuthis lessoniana, Sepiella maindroni and Sepia esculenta show 50% hatching at salinities of 16.8, 14.7 and 12.6X, respectively (Choe, 1966).
478
A. Table
I. Effect of salinity
D‘ANIELLO
on egg hatching
et al.
and survival
Salinity (%I
Eggs inside strings contained embryos 12-14 days before hatching’r
Isolated eggs 2-3 days before hatchine?
19
no development
no hatchmg
28
no development
40-60%
34 38* 42 41
normal development normal development normal development no development
normal hatchmg normal hatchmg normal hatching 65-85% hatched
of embryos
culgurb
Survwal of hatched embryos$ .-. all died after fern minutes 40-60% hved up to 33 days, others died before hved S-6 da>) hved 6-7 days lived 5 6 days 65 -X5% hved up 10 3 4 days: other\ died hefiwc
hatched
no hatching
57
of Lo&o
*This value corresponds to natural sea-water from the Bay of Naples tThe results are the mean values derived from analys~c performed on three wng\. each containing about 30 eggs. $The results are the mean valur~ calculated from three analyses: each anal)& ~.a\ performed on 30 embryos. $The results are the mean values calculated from three analyses; each analy~\ was performed oo 30 embryos that were previously hatched m natural sea-water and then put m artificial sea-water at various aalimt~es.
Under our experimental conditions. Lolip rxlgrtris embryos hatched and lived up to 5-7 days without food in a salinity range of 3442%0. Outside this range severe damage occurred. In fact, as shown in Table 1, at a salinity below 28% which is 75% of the salinity of the Bay of Naples, and at a salinity higher than 47%0 (150% of normal salinity) none of the eggs at 12-14 days pre-hatching developed. Of the embryos at the 2-3 days pre-hatching stage, only 4&60% and 65-85% hatched at salinities of 28 and 47%0, respectively, and the embryos lived for about 34 days. At the extreme salinity values of below 19%0 and above 57%0, no hatching occurred and all hatched embryos exposed to these conditions died after a few minutes. These last results are in good agreement with findings of (Jecklin, 1934) that newly
Table 2. Effect of pH on egg hatching
PH
Eggs inside strings contained embryos 12-14 days before hatchingt
7.1
no development
1.6
7.x 8.1’ x.4 X.6
normal normal normal normal
development development development development
9.1
no development
hatched between EfSect
Lolip dguri.s embryos 28 and 42%, salinity.
are
viable
only
of pH
At pH values between 7.8 and 8.4, embryos developed and hatched normally. Beyond this range, disturbances occurred. In fact, as shown in Table 2. at pH 7.6 and 8.6 (8.1 is the pH value of natural sea-water), eggs developed and hatched normally, but did not survive. Whereas at pH 8.1 newly hatched embryos lived up to 6--7 days, at pH 7.6 and 8.6 only 50-70% of the newly hatched embryos survived up to 6 days. At pH values of 7.1 and 9.1, only 2630% and 3&40%, respectively, of the embryos hatched and lived up to 3 days. The others either failed to hatch
and surwval
Isolated eggs contained embryos 2 3 days before hatching1 ?O-30%
hatched
normal
hatching
normal normal normal normal
hatching hatchmg hatching hatching
30 40%
hatched
of embryos
of Lollgo &guru
Survival of hatched embryo@ 3040% lived up days: others dwd before 50 70% hved up davs: othsrs died hefore lived 6 7 da)\ lived 6 7 day\ hwd 6 7&q\ 50-70% hved up dav\: other\ died be&e 20 3O”O hved up days: others died before
to 3
to 7
to 6
to i
*This value corresponds to natural sea-water from the Bay of Naplea. tThe results are the mean values derived from analyw performed on three \trlngs, each containing about 30 eggs. $The results are the mean valueb calculated from three analyses: each analya~r was performed on 30 embryos. $The results are the mean values calculated from three analyses, each analyw wa\ performed on 30 embryos that were previously hatched in natural ~-water and then put m artkxd sea-water at var,ous pH value5
Physicochemical Table 3. Effect of calcium Embryos
Ca*+ (mM)
factors
on eggs hatching
and survival
inside choriont
6
no hatching no hatching. embryos died after wolent convulsions 7Om90% hatched
9
90-100%
0 4
influencing
hatched
12*
loo?/. hatched
I5
95m100% hatched
IX
50-70%
24
the embryos escaped from the chorion because of pamc reaction
hatched
419
squid hatching of embryos
Survival
of Loligo tulgaris
of hatched
embryosf
all died after a few minutes the motihty of embryos was reduced; they died after 2-3 days the motility of embryos was normal; they lived 3-4 days the motility of embryos was normal; they lived 54 days the motility of embryos was normal: they lived 67 days the motility of embryos was normal; they lived S-6 days the motility of embryos was normal; they lived only 2-3 days motdity lost. embryos died after I day
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses. Each analysis on 30 isolated eggs. :The results are the mean values calculated from three analyses; each analysis on 30 embryos that were prewously hatched in natural sea-water and then sea-water and then put in artificial sea-water at WUIOUS concentrations of
or died. Below and above pH 7.1 and 9.1, eggs did not develop. Effkct
qf Ca’+, K+
and Mg’+
To our knowledge, this is the first study of the effect of these three cations on egg hatching and embryo survival in L&go vulgaris. Interestingly, calcium and potassium exerted the same effect: when their level was less than half that of Bay of Naples sea-water, severe damage occurred (see Tables 3 and 4). For example, when Ca2+ or K+ was present at a concentration of up to 4 mM (which corresponds to 33% of their concentration in natural sea-water), hatching did not occur, and embryos transferred to these conditions after having hatched in natural seawater. died immediately. The same results were obtained when Ca2+ and K+ were at 18 mM (150% of the normal value). In this case, only 5670% of eggs hatched and the embryos lived only 2-3 days. At a concentration between 9 and 15 mM (75 and 125%, with respect to natural sea-water), Ca2+ and K ’ did not affect either eggs hatching or embryo survival.
Table 4. Effect of potassium K+ (mM) 0
Embryos
on eggs hatching
Inside choriont
6
no hatching no hatching. embryos died after violent convulsions 70-90% hatched
9
9&lOO%
4
hatched
12’
all hatched
I5 I8
9&lOO% hatched 50-70% hatched
24
the embryos escaped from the chorion because of panic reaction
was performed was performed put in artificial calcium.
The effect of magnesium on embryo hatching and survival is shown in Table 5. At a range of concentrations between 46 and 70mM (80 and 120%, respectively, compared to sea-water), all embryos hatched and survived normally. Outside these values, the embryos died and hatching did not occur. Effect of SOi-
The absence of sulphate from sea-water did not seem to affect hatching or survival, apart from causing a slight delay in hatching (Table 6). At a sulphate concentration of 45 mM, only 4&60% of Loligo eggs hatched and of all the embryos hatched only 4&60% lived 2-3 days. At a concentration of 60 mM, which corresponds to 200% with respect to sea-water, only l&20% of the eggs hatched, and the embryos lived only l-2 days. CONCLUSION
We previously found that the concentrations of Na+, K+, Ca’+, Mg2+ and SO:+ ions in the periv-
and survival Survival
of embryos of hatched
of L&go
wlgaris
embryos$
all died after a few minutes the motlhty of embryos was lost; they died after 2-3 days the motility of embryos was normal; they lived 2-4 days the motility of embryos was normal; they lived 4-S days the motility of embryos was normal; they lived 67 days the motility of embryos was normal the motility of embryos was reduced; they lived only 2-3 days all embryos died after few hours
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses; each analysis was performed on 30 isolated eggs. :The results are the mean values calculated from three analyses; each analysis was performed on 30 embryos that were previously hatched in natural sea-water and then put in artificial sea-water at various concentrations of potassium.
A.
480 Table 5. Effect of maenesium
Mg*+ (mM)
Embryos
on eees hatchine
29 46
all hatched
58’
all hatched
70
all hatched
86
3&40%
hatched
116
l&20%
hatched
4
et al.
and surwval
inside choriont
no hatching, embryos died after few minutes died immediately after hatching 6&80% hatched
0
D’ANIELLO
of embrvos
Survival
of hatched
of Lo&w oulmrrs embryos$
died after a few minutes the motility of embryos was lost; they lived only 2-3 hr the motility of embryos was reduced: they lived 2-3 days the motility of embryos was normal; they lived 67 days the motility of embryoa was normal: they lived &7 days the motility of embryos was normal they lived 54 days the motility of embryos was reduced: they lived only 2-3 days the motility was lost; the embryos lived onlv I-2 days
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses: each analysis was performed on 30 isolated eggs. $The results are the mean values calculated from three analyses; each analysts was performed on 30 embryos that were previously hatched in natural sea-water and then put in artificial sea-water at various concentrations of magnesium.
Table 6. Effect of sulphate SO:- (mM) 0
on eggs hatching
Embryos inside choriont
15
all hatched, but with delayed 2-3 days all hatched
30.
all hatched
37
all hatched
45
4&60%
hatched
60
l&20%
hatched
and survival
of embryos
Survival
of hatched
the motility of they lived &5 the motility of they lived 5d the motility of they lived 67 the motility of they lived 5-6 the motility of they lived 2-3 the motility of they lived IL2
embryos days embryos days embryos days embryos days embryos days embryos days
of Loligo dgaris emhryosf was normal; was normal; was normal; was normal; was normal; was normal
*This value corresponds to natural sea-water from the Bay of Naples. tThe results are the mean values calculated from three analyses; each analysis was performed on 30 isolated eggs. fThe results are the mean values calculated from three analyses; each analysis was performed on 30 embryos that were previously hatched in natural sea-water and then put in artificial sea-water at various concentrations of sulphate ions.
itelline fluid is in osmotic equilibrium with the surrounding natural or artificial sea-water and that they reflect the changing concentrations of these ions in the external medium (D’Aniello et al., 1987). The present study demonstrates that Loligo vulgaris eggs develop normally and embryos hatch only when the external fluids have a salinity between 34 and 42% and pH values between 7.8 and 8.4. In addition, each of the above-mentioned ions must be in a certain concentration range for egg development and hatching to occur: calcium and potassium between 9 and 15 mM, magnesium between 48 and 68 mM and sulphate ions between 15 and 37 mM. Eggs at the 2-3 days pre-hatching stage, placed in artificial sea-water, contained the same concentration of major ions as sea-water of the Bay of Naples, hatch normally, while eggs at the stage of 12-14 days pre-hatching, did not develop normally and the embryos did not hatch. Since the artificial sea-water prepared by us contained only the major ions that are present in natural sea-water (Na+, K+, Ca*+, Mg2+, SOi- and Cl-), and since the youngest eggs failed to survive in this water, it appears that normal embryonic development requires some substances that are
present in natural sea-water, in extremely low amounts, e.g. oligoelements or organic molecules. REFERENCES S. (1966) On the eggs, rearing, habits of the fry, and growth of some Cephalopoda. Bull. Mar. Sri. 16, 33c-348. D’Aniello A., Strazzullo L., D’Onofrio G. and Pischetola M. (1986) Electrolytes and nitrogen compounds of body fluids and tissues of Octopus culgaris Lam. J. Camp. Physiol. 156B, 503-509. D’Aniello A., D’Onofrio G.. Pischetola M. and Denuck J. M. (1987) Effect of the ionic concentration of calcium and potassium on hatching of the embryo of the squid, Leligo vulgaris. A preliminary study. Arch. Int. Phvsiol. Biochim. 95, B66. D’Apote M. P. and Palmeggiano G. B. (1982) Inseminazione della Laguna di Lessina con uova di Sepia ojicinalis L. Studio dell’ effetto di parametri ambientali sulla schiusa: temperatura e salinita’. Ric. I/. Pixie. Iftiop. 17, 112-l 19. Deffner G. G. T. and Mafter R. S. (1960) Chemical investigation of the giant nerve fiber of the squid. IV. Acid--base balance in axoplasm. Biochim. biophyx Acta 42, 200-205. Deffner G. G. T. (1961) The dialyzable free organic con-
Choe
Physicochemical
factors
stituents of squid blood; a comparison with nerve axoplasm. Biochim. biophys. Acta 47, 378-388. Emanuel C. F. and Martin A. W. (1956) The composition of octopus renal fluid. 2. Vergl. Physiol. 39, 22&234. Jecklin L. (1934) Beitrag zur kenntnis der Laichgallerten und der Biologic der Embryonen decapoder Cephalopoden. Rev. Suisse Zool. 41, 593-673. Palmeggiano G. B. and D’Apote M. P. (1983) Combined effects of temperature and salinity on cuttlefish (Sepia ojicinalis L.) hatching. Aquaculture 35, 259-264.
influencing
squid hatching
481
Potts W. T. W. and Todd M. (1965) Kidney function in the Octopus. Comp. Biochem. Physiol. 16, 479489. Prosser C. L. ed. (1973) Comparative Animal Physiology (3rd edn). W.B. Saunders Company, Philadelphia. Robertson J. D. (1949) Ionic regulation in some marine invertebrates. J. exp. Biol. 26, 181-200. Robertson J. D. (1953) Further studies on ionic regulation in marine invertebrates. J. exp. Biol. 30, 277-296.