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Effects of streptomycin on the hatching of Australorbis glabratus eggs
EXPERIMENTAL
PARASITOLOGY
Effects
9,
9-13 (1960)
of Streptomycin Eli
Department
on the Hatching gla bra tus Eggs’
Chernin
of Tropical
(Submitted
and Ann
R. Schork
Public Health, Harvard School Boston, Massachusetts
for publication,
of Austrdorbis
of Public Health,
18 December 1958)
Mature eggs of Australorbis glabratus were incubated in various inorganic salt solutions containing streptomycin, penicillin, both, or neither antibiotic. Penicillin had no adverse effect on eclosion. However, in certain salt solutions eclosion occurred except when streptomycin was present; in other eclosion occurred whether or not streptomycin was present; and in still others, eclosion was inhibited by the nature of the solution per se regardless of any effect ascribable to streptomycin. The data indicate that both the species of ion(s) and their concentration in solutions containing streptomycin are important in preventing the inhibitory effects of the antibiotic. Comparable results were obtained from experiments done under axenic and nonaxenic conditions, suggesting that the effect of streptomycin on the snail eggs is probably direct. --
Evidence that streptomycin may inhibit the growth of Australorbis glabratus (Chernin, 1957a) led to a study of the effects of this antibiotic on the hatching (eclosion) of A. glabratus eggs. Experiments are now reported which disclosed that streptomycin inhibits eclosion of these eggs in certain types of solutions. Data drawn from experiments done under non-axenic as well as axenic conditions are presented.
Experimental
Procedures
Each group of about 20 mature eggs was washed 3 times in the salt solution in which it was to be observed, and then placed in an S-cm petri dish containing about 35 ml of the corresponding solution. Four dishes were used for the simultaneous testing of each salt solution, antibiotics2 having been incorporated as follows: dish 1, streptomycin (100 pg/ml) ; dish 2, streptomycin (100 rg/ml), plus penicillin (100 MATERIALS AND METHODS units/ml) ; dish 3, penicillin (100 units/ml) ; Egg-masses were all derived from a dish 4, no antibiotics. Dishes were incuPuerto Rican strain (PR-1) of A. glabratus bated at 27-28°C and examined for hatched maintained in our laboratory since 1954. snails daily for 4 days. This period of exIndividual mature eggs (i.e., containing amination is adequate since observation snails about to hatch) were secured by dis- has shown that very few eggs hatched secting suitable egg-massesusing a method thereafter. already described (Chernin, 1957b). Eggs While most of the experiments were done were distributed in a random manner into in the foregoing manner, a smaller series experimental groups. was done under axenic conditions. For these experiments, groups of 10-20 mature eggs ‘This investigation was supported (in part) by Grant E-513-C from the National Institute of were disinfected externally as previously Allergy and Infectious Diseases, National Instidescribed (Chernin, 1957b), and washed tutes of Health, Public Health Service, and (in part) by a Senior Research Fellowship (SF-lo) from the Public Health Service to the senior author.
‘Streptomycin sulfate and penicillin G (cryst.pot.), both from Eli Lilly and Company, used in all experiments. 9
10
CHERNIN
in three 5-ml volumes of the particular sterile salt solution in which they were to be maintained. Treated eggs were placed singly in 20 x 150 mm rubber-stoppered tubes containing 5 ml of the corresponding salt solution; all test solutions contained streptomycin (100 pg/ml) , penicillin (100 units/ml), and up to 1.0 ml/100 ml of 1.4% NaHC& . Tubes were incubated vertically (27-28”(Z) and were examined daily for 7 days for newly-hatched snails. Subsequent sterility tests in liquid thioglycollate medium showed that only 6% of snails or unhatched eggs were contaminated. The contaminated individuals were so scattered through the series as to have no effect on the interpretation of experiments. Salt Solutions
AND SCHORK
Presentation
of Data
Parts A-E of Table I, which correspond to sections in the text, summarize the findings. Many experiments were repeated 2 or more times and results have been combined. For purposes of presentation, hatch rates of 75-100% are described as “high,” 40-740/o as “moderate,” and below 46% as “poor.” It should be noted that since the present experiments involved eggs which had been separated from the mass and from each other, the findings may not apply to eggs in intact masses. EXPERIMENTAL
Part A. Handling
RESULTS
Solution
Uniformly high rates of eclosion (88100%) occurred among A. glabratus eggs Handling solution (HS)3 was used to in HS, HS concentrated ~2, or in HS diprovide base line information. Different concentrations of HS, and of Hanks’ solu- luted 1:2, regardless of the presence or absence of streptomycin. Similarly high tion4, were also utilized. Other tests were hatch rates were also observed in HS didone using the individual salts comprising luted 1: 4 or 1: 10 when these solutions were HS, separately or in various combinations streptomycin-free, but sharply reduced and concentrations. Stock solutions were rates of hatch (330%) occurred therein prepared in double-distilled water, steriwhen streptomycin was incorporated. The lized by filtration through sintered glass, poor hatch rates (O-26%) observed under and stored in stoppered flasks at 5°C. The all test conditions in HS concentrated x4 pH of solutions used in the axenic series were apparently due to the hypertonicity of ranged approximately from 7-8, depending the salt solution per se rather than to any on the nature of the solution and the coneffect of the antibiotics. centration of NaHC03. The same solutions used in the non-sterile studies varied Part B. Hanks’ Solution more widely in pH, particularly because This solution resembles HS in its main NaHC03 was omitted; however, for any components but differs from it mainly in one solution the differences among the 4 that it is about 4~ as concentrated. Altest dishes did not as a rule exceed 0.5 pH lowing for this difference, the findings with unit. Hanks’ solution generally parallel those with HS. As with HS concentrated x4 ’ HS (g/100 ml) : NaCI, 0.2; KCl, 0.01; (part A), uniformly poor hatch was obNa2HPOI (anh.), 0.005; MgSO,.7HzO, 0.02; tained in full-strength Hanks’. However, CaCL*2&0,0.01. under all test conditions high hatch rates ‘Hanks’ Solution (g/100 ml): NaCl, 0.8; KCl, 0.04; NazHPOA (anh.), 0.006; MgSOl.7Hz0, 0.02; were recorded for eggs maintained in CCCL.~HZO, 0.014; glucose 100 mgm%. Hanks’ diluted 1:2 or 1: 4. While further Both HS and Hanks’ solutions contained phenol dilution of Hanks’ (to 1 :lO) also yielded red (0.5 ml/100 ml of 0.4% aqueous stock) except a high rate of hatch (89%) when streptowhen used in non-sterile conditions. NaCl from mycin was omitted, the same solution susMerck (“For Biological Work”); all other salts tained only poor rates of hatch (19-30%) reagent grade. where streptomycin was incorporated.
EFFECTS
OF
STREPTOMYCIN
ON HATCHING
OF SNAIL
11
EGGS
TABLE I Effects of Streptomycin (Strep) and Penicillin (Pen) on the Hatch Rate of Mature A. glabratus Eggs (Various salt solutions were used under axenic and non-axenic conditions. Results are expressed as proportions: the number hatched in the numerator and the number of eggs used in the denominator. Manv experiments were repeated two or more times and results have been combined in each instance.) Maintenance conditions
Non-Axenic
Axenic (
Part
Antibiotics
_ Solutions tested HS HS HS HS HS HS
None I
,strep + pen Strep only
cont. X2 cont. X4 dil. 1:2 dil. 1:4 dil. 1:lO
Hanks’ Hanks’ dil. 1:2 Hanks’ dil. 1:4 Hanks’ dil. 1: 10
--
Strep f pen
Strep present
Pen only
No.
Total h,atch y& _ No.
No.
No.
40/40 20120 8/20 20/20 20120 20/20
40140 21/21 2/19 21/21 20120 20/20
Total - h,atch No. -- Jl95 81/85 19/20 95 o/20 0 15/17 88 6/20 30 2/17 12
41/41 !lo/40 81/81 20120 19/19 39/39 2/20 7/19 9/39 19/19 19/20 38/39 o/20 5121 5/41 o/20 l/20 l/40
l/19 18/20 14118 6/20
O/23 o/20 18/20 18/20 20121 17/20 6/20 l/17
5 90 78 30
No.
100 100 23 97 12 3
-
Strep absent Total No.
SO/SO 41/41 10/39 41/41 40/40 40140
atch % 100 100 26 100 100 100
o/43 36/40 37141 7/37
0 90 90 19
o/20 o/20 o/40 19120 19/20 38/40 22122 20120 42/42 18/21 23/25 41/46
0 95 100 89
Aquarium Hz0 Distilled Hz0 0.1% NaCl 0.2% NaCl 0.4oj, NaCl
o/30 2/25 2/55 l/24
0 8 4 4
9/22 o/40 2/20 8/40 o/19
8/21 l/40 l/19 16/40 2/18
17/43 l/80 3/39 24/80 2/37
40 1 8 30 5
20120 36/40 15/20 25/39 O/18
22/22 15/41 18/20 22/37 o/20
42/42 51/81 33/40 47/76 O/38
100 63 83 62 0
0.01% KC1 O.O05a/,NasHPOd 0.02yo MgSO, 0.01% CaCl2 4 salts above combined
O/8
17/20
0 0 0 13 85
o/20 o/19 o/20 o/20 16/19
2/20 o/20 o/19 2/19 18/20
2/40 o/39 o/39 2/39 34/39
5 0 0 5 87
16/20 17/20 18/18 20/20 20/20
17/18 18/20 18/19 12/20 20/20
33/38 35/40 36/37 32/40 40/40
87 88 97 80 100
o/g o/11 o/11 13/19
0 0 0 68
o/21 o/20 o/20 11/21
o/20 o/41 o/20 o/40 7/20 7/40 16125 27/46
0 0 18 59
o/20 4/20 18/20 16/20
o/20 o/40 o/20 4/40 7119 25/39 10/20 26/40
0 10 64 65
0.057% KC1 O.O36oj,NazHP04 0.047% MgSO, 0.038y0 CaCls
o/10 o/10
l/8
-
L
Part C. Aquarium Water, Distilled and NaCl Solutions Aquarium
water5
containing
-
Water,
streptomy-
tin sustained a hatch rate of only 40%, 5Taken from aquaria containing established populations of A. glabratus. Water filtered through sintered glass to remove microorganisms and debris.
-
-
but this rate rose to 100% in the absence of streptomycin. Poor hatch rates (O-30%) were also obtained when streptomycin was incorporated into distilled water or into 0.1 or 0.2% NaCl, but rates in these solutions rose to much higher levels (62--83%) upon the omission of streptomycin. Eclosion was uniformly poor (O-5%) in 0.4% NaCl, presumably for reasons of tonicity.
12
CHERNIN
Part D. Components of HS other than NaCl Tests done with the individual salts at their customary concentrations disclosed hatch rates of only O-13% with streptomycin incorporated. However, these salt solutions were actually capable of sustaining eclosion as shown by hatch rates of 8097% achieved when streptomycin was omitted from each in turn. When these 4 salts were combined (in effect, HS lacking NaCl), high rates of hatch occurred whether streptomycin was present (85-87%) or absent (100%).
Part E. Solutions at Ionically Equivalent Strength The total ionic strength of the 4-salt combination used in part D was calculated (0.0077)) and an appropriate solution of this ionic strength was prepared for each of the component salts. Two of these salt solutions (KC1 and Na2HP04) were inimical to hatch (O-10%) under all test conditions. In MgS04 rate of hatch was poor in the presence of streptomycin (O--18%), but moderate (64%) in its absence. By contrast, the solution of CaClz sustained moderate rates of hatch (59-68s) whether or not streptomycin was present. DISCUSSION
Since penicillin did not adversely affect hatch rate, interest centers on the effects of streptomycin on the eclosion of A. glabratus eggs in different salt solutions. The solutions studied fell into three general groups as evidenced by the experimental results: a) those in which rates of eclosion were moderate or high whether or not streptomycin was present; b) those in which rates of eclosion were moderate or high in the absence of streptomycin, but low when the antibiotic was present; and c) those in which rates of eclosion were uniformly low under all test conditions because of the nature of the solutions per se-independent of any antibiotic effects. Disregarding solutions in the last group, it seems clear that under some conditions streptomycin may inhibit eclosion while under other circumstances this phenomenon
AND
SCHORK
may be partly or wholly blocked. This interference with streptomycin appears to be related to the concentration and species of ion(s) present in the test solutions. A few examples may be cited to illustrate these points. (1) The capacity of certain solutions to interfere with the inhibitory effects of streptomycin on eclosion was markedly altered by the simple expedient of diluting them (compare hatch rates in HS with those in HS 1: 10, or in Hanks’ 1:2 with Hanks’ 1: 10). (2) While the inhibitory effects of streptomycin were almost entirely blocked in HS (or in HS lacking NaCI), streptomycin plainly inhibited hatch in separate tests with each of the component salts (parts C and D). (3) When these component salts were tested at an increased ionic strength, only CaC$ showed a capacity for interfering with streptomycin (part E) . Manganese ions are reported to block the inhibitory effects of streptomycin on the growth of certain plants (Rosen, 1954; Gray, 1955). However, in tests done during the present studies, various concentrations of manganese (as MnSOJ were found to be ineffective in blocking the inhibition of eclosion among A. glabratus eggs exposed to streptomycin. Of further interest is that in the reports on plants, calcium ions were also shown to be effective (but less so than Mn+ +) in blocking the growth-inhibiting effects of streptomycin. The mechanisms involved in the effect of this antibiotic on plants, or the interference with this effect by metallic cations, are obscure (Gray, 1955). The mechanisms underlying the present findings are similarly obscure, but the results parallel those previously reported on the effects of streptomycin on the growth of A. glabratus (Chernin, 1957a). In the latter studies evidence was presented that the antibiotic probably affects the snails directly rather than in some intermediary fashion; similar evidence can be adduced from the present data inasmuch as the inhibition of eclosion due to streptomycin was observed under axenic as well as nonaxenic conditions. It is not known whether there may be differences among strains of
13
EFFECTS OF STREPTOMYCIN ON HATCHING OF SNAIL EGGS
A. glabratus in relation to streptomycin sensitivity, nor is it known whether the site of action of streptomycin is the egg membrane, the snails within, or both. It is evident, however, that A. glabratus is sensitive to streptomycin at several stages in its life cycle. At least one other antibiotic, nystatin, is known to inhibit eclosion (Chernin, 1957bj, and also to be toxic to adult A. glabratus (Seneca and Bergendahl, 1955; Chernin, 1959). REFERENCES E. 1957a. Studies on the biological control of schistosome-bearing snails. VII. Streptomycin-induced inhibition of growth and reproduction in the schistosome-bearing snail,
CHERNIN,
Australorbis glabratus. Am. J. Hyg. 66, 321330. CHERSIN, E. 195713.A method of securing bacteriologically sterile snails (Australorbis glabratus).
Proc.
Sot.
Exptl.
Biol.
and
Med.
96,
204-210. CHERNIN, E. 1959. Notes on the effects of various glabratus. J. Paraantibiotics on Australorbis sitol. 45, 268.
R. A. 1955. Inhibition of root growth by streptomycin and reversal of the inhibition by manganese. Am. J. Botany 42,327-331. ROSEN, W. G. 1954. Plant growth inhibition by streptomycin and its prevention by manganese. GRAY,
Proc. Sot. Exptl.
Biol. and Med. 85, 385-388.
H., AND BERGENDAHL, E. 1955. Toxicity of antibiotics to snails. Antibiotics & Chemo-
SENECA,
therapy
5,737-741.
PARASITOLOGY
Effects
9,
9-13 (1960)
of Streptomycin Eli
Department
on the Hatching gla bra tus Eggs’
Chernin
of Tropical
(Submitted
and Ann
R. Schork
Public Health, Harvard School Boston, Massachusetts
for publication,
of Austrdorbis
of Public Health,
18 December 1958)
Mature eggs of Australorbis glabratus were incubated in various inorganic salt solutions containing streptomycin, penicillin, both, or neither antibiotic. Penicillin had no adverse effect on eclosion. However, in certain salt solutions eclosion occurred except when streptomycin was present; in other eclosion occurred whether or not streptomycin was present; and in still others, eclosion was inhibited by the nature of the solution per se regardless of any effect ascribable to streptomycin. The data indicate that both the species of ion(s) and their concentration in solutions containing streptomycin are important in preventing the inhibitory effects of the antibiotic. Comparable results were obtained from experiments done under axenic and nonaxenic conditions, suggesting that the effect of streptomycin on the snail eggs is probably direct. --
Evidence that streptomycin may inhibit the growth of Australorbis glabratus (Chernin, 1957a) led to a study of the effects of this antibiotic on the hatching (eclosion) of A. glabratus eggs. Experiments are now reported which disclosed that streptomycin inhibits eclosion of these eggs in certain types of solutions. Data drawn from experiments done under non-axenic as well as axenic conditions are presented.
Experimental
Procedures
Each group of about 20 mature eggs was washed 3 times in the salt solution in which it was to be observed, and then placed in an S-cm petri dish containing about 35 ml of the corresponding solution. Four dishes were used for the simultaneous testing of each salt solution, antibiotics2 having been incorporated as follows: dish 1, streptomycin (100 pg/ml) ; dish 2, streptomycin (100 rg/ml), plus penicillin (100 MATERIALS AND METHODS units/ml) ; dish 3, penicillin (100 units/ml) ; Egg-masses were all derived from a dish 4, no antibiotics. Dishes were incuPuerto Rican strain (PR-1) of A. glabratus bated at 27-28°C and examined for hatched maintained in our laboratory since 1954. snails daily for 4 days. This period of exIndividual mature eggs (i.e., containing amination is adequate since observation snails about to hatch) were secured by dis- has shown that very few eggs hatched secting suitable egg-massesusing a method thereafter. already described (Chernin, 1957b). Eggs While most of the experiments were done were distributed in a random manner into in the foregoing manner, a smaller series experimental groups. was done under axenic conditions. For these experiments, groups of 10-20 mature eggs ‘This investigation was supported (in part) by Grant E-513-C from the National Institute of were disinfected externally as previously Allergy and Infectious Diseases, National Instidescribed (Chernin, 1957b), and washed tutes of Health, Public Health Service, and (in part) by a Senior Research Fellowship (SF-lo) from the Public Health Service to the senior author.
‘Streptomycin sulfate and penicillin G (cryst.pot.), both from Eli Lilly and Company, used in all experiments. 9
10
CHERNIN
in three 5-ml volumes of the particular sterile salt solution in which they were to be maintained. Treated eggs were placed singly in 20 x 150 mm rubber-stoppered tubes containing 5 ml of the corresponding salt solution; all test solutions contained streptomycin (100 pg/ml) , penicillin (100 units/ml), and up to 1.0 ml/100 ml of 1.4% NaHC& . Tubes were incubated vertically (27-28”(Z) and were examined daily for 7 days for newly-hatched snails. Subsequent sterility tests in liquid thioglycollate medium showed that only 6% of snails or unhatched eggs were contaminated. The contaminated individuals were so scattered through the series as to have no effect on the interpretation of experiments. Salt Solutions
AND SCHORK
Presentation
of Data
Parts A-E of Table I, which correspond to sections in the text, summarize the findings. Many experiments were repeated 2 or more times and results have been combined. For purposes of presentation, hatch rates of 75-100% are described as “high,” 40-740/o as “moderate,” and below 46% as “poor.” It should be noted that since the present experiments involved eggs which had been separated from the mass and from each other, the findings may not apply to eggs in intact masses. EXPERIMENTAL
Part A. Handling
RESULTS
Solution
Uniformly high rates of eclosion (88100%) occurred among A. glabratus eggs Handling solution (HS)3 was used to in HS, HS concentrated ~2, or in HS diprovide base line information. Different concentrations of HS, and of Hanks’ solu- luted 1:2, regardless of the presence or absence of streptomycin. Similarly high tion4, were also utilized. Other tests were hatch rates were also observed in HS didone using the individual salts comprising luted 1: 4 or 1: 10 when these solutions were HS, separately or in various combinations streptomycin-free, but sharply reduced and concentrations. Stock solutions were rates of hatch (330%) occurred therein prepared in double-distilled water, steriwhen streptomycin was incorporated. The lized by filtration through sintered glass, poor hatch rates (O-26%) observed under and stored in stoppered flasks at 5°C. The all test conditions in HS concentrated x4 pH of solutions used in the axenic series were apparently due to the hypertonicity of ranged approximately from 7-8, depending the salt solution per se rather than to any on the nature of the solution and the coneffect of the antibiotics. centration of NaHC03. The same solutions used in the non-sterile studies varied Part B. Hanks’ Solution more widely in pH, particularly because This solution resembles HS in its main NaHC03 was omitted; however, for any components but differs from it mainly in one solution the differences among the 4 that it is about 4~ as concentrated. Altest dishes did not as a rule exceed 0.5 pH lowing for this difference, the findings with unit. Hanks’ solution generally parallel those with HS. As with HS concentrated x4 ’ HS (g/100 ml) : NaCI, 0.2; KCl, 0.01; (part A), uniformly poor hatch was obNa2HPOI (anh.), 0.005; MgSO,.7HzO, 0.02; tained in full-strength Hanks’. However, CaCL*2&0,0.01. under all test conditions high hatch rates ‘Hanks’ Solution (g/100 ml): NaCl, 0.8; KCl, 0.04; NazHPOA (anh.), 0.006; MgSOl.7Hz0, 0.02; were recorded for eggs maintained in CCCL.~HZO, 0.014; glucose 100 mgm%. Hanks’ diluted 1:2 or 1: 4. While further Both HS and Hanks’ solutions contained phenol dilution of Hanks’ (to 1 :lO) also yielded red (0.5 ml/100 ml of 0.4% aqueous stock) except a high rate of hatch (89%) when streptowhen used in non-sterile conditions. NaCl from mycin was omitted, the same solution susMerck (“For Biological Work”); all other salts tained only poor rates of hatch (19-30%) reagent grade. where streptomycin was incorporated.
EFFECTS
OF
STREPTOMYCIN
ON HATCHING
OF SNAIL
11
EGGS
TABLE I Effects of Streptomycin (Strep) and Penicillin (Pen) on the Hatch Rate of Mature A. glabratus Eggs (Various salt solutions were used under axenic and non-axenic conditions. Results are expressed as proportions: the number hatched in the numerator and the number of eggs used in the denominator. Manv experiments were repeated two or more times and results have been combined in each instance.) Maintenance conditions
Non-Axenic
Axenic (
Part
Antibiotics
_ Solutions tested HS HS HS HS HS HS
None I
,strep + pen Strep only
cont. X2 cont. X4 dil. 1:2 dil. 1:4 dil. 1:lO
Hanks’ Hanks’ dil. 1:2 Hanks’ dil. 1:4 Hanks’ dil. 1: 10
--
Strep f pen
Strep present
Pen only
No.
Total h,atch y& _ No.
No.
No.
40/40 20120 8/20 20/20 20120 20/20
40140 21/21 2/19 21/21 20120 20/20
Total - h,atch No. -- Jl95 81/85 19/20 95 o/20 0 15/17 88 6/20 30 2/17 12
41/41 !lo/40 81/81 20120 19/19 39/39 2/20 7/19 9/39 19/19 19/20 38/39 o/20 5121 5/41 o/20 l/20 l/40
l/19 18/20 14118 6/20
O/23 o/20 18/20 18/20 20121 17/20 6/20 l/17
5 90 78 30
No.
100 100 23 97 12 3
-
Strep absent Total No.
SO/SO 41/41 10/39 41/41 40/40 40140
atch % 100 100 26 100 100 100
o/43 36/40 37141 7/37
0 90 90 19
o/20 o/20 o/40 19120 19/20 38/40 22122 20120 42/42 18/21 23/25 41/46
0 95 100 89
Aquarium Hz0 Distilled Hz0 0.1% NaCl 0.2% NaCl 0.4oj, NaCl
o/30 2/25 2/55 l/24
0 8 4 4
9/22 o/40 2/20 8/40 o/19
8/21 l/40 l/19 16/40 2/18
17/43 l/80 3/39 24/80 2/37
40 1 8 30 5
20120 36/40 15/20 25/39 O/18
22/22 15/41 18/20 22/37 o/20
42/42 51/81 33/40 47/76 O/38
100 63 83 62 0
0.01% KC1 O.O05a/,NasHPOd 0.02yo MgSO, 0.01% CaCl2 4 salts above combined
O/8
17/20
0 0 0 13 85
o/20 o/19 o/20 o/20 16/19
2/20 o/20 o/19 2/19 18/20
2/40 o/39 o/39 2/39 34/39
5 0 0 5 87
16/20 17/20 18/18 20/20 20/20
17/18 18/20 18/19 12/20 20/20
33/38 35/40 36/37 32/40 40/40
87 88 97 80 100
o/g o/11 o/11 13/19
0 0 0 68
o/21 o/20 o/20 11/21
o/20 o/41 o/20 o/40 7/20 7/40 16125 27/46
0 0 18 59
o/20 4/20 18/20 16/20
o/20 o/40 o/20 4/40 7119 25/39 10/20 26/40
0 10 64 65
0.057% KC1 O.O36oj,NazHP04 0.047% MgSO, 0.038y0 CaCls
o/10 o/10
l/8
-
L
Part C. Aquarium Water, Distilled and NaCl Solutions Aquarium
water5
containing
-
Water,
streptomy-
tin sustained a hatch rate of only 40%, 5Taken from aquaria containing established populations of A. glabratus. Water filtered through sintered glass to remove microorganisms and debris.
-
-
but this rate rose to 100% in the absence of streptomycin. Poor hatch rates (O-30%) were also obtained when streptomycin was incorporated into distilled water or into 0.1 or 0.2% NaCl, but rates in these solutions rose to much higher levels (62--83%) upon the omission of streptomycin. Eclosion was uniformly poor (O-5%) in 0.4% NaCl, presumably for reasons of tonicity.
12
CHERNIN
Part D. Components of HS other than NaCl Tests done with the individual salts at their customary concentrations disclosed hatch rates of only O-13% with streptomycin incorporated. However, these salt solutions were actually capable of sustaining eclosion as shown by hatch rates of 8097% achieved when streptomycin was omitted from each in turn. When these 4 salts were combined (in effect, HS lacking NaCl), high rates of hatch occurred whether streptomycin was present (85-87%) or absent (100%).
Part E. Solutions at Ionically Equivalent Strength The total ionic strength of the 4-salt combination used in part D was calculated (0.0077)) and an appropriate solution of this ionic strength was prepared for each of the component salts. Two of these salt solutions (KC1 and Na2HP04) were inimical to hatch (O-10%) under all test conditions. In MgS04 rate of hatch was poor in the presence of streptomycin (O--18%), but moderate (64%) in its absence. By contrast, the solution of CaClz sustained moderate rates of hatch (59-68s) whether or not streptomycin was present. DISCUSSION
Since penicillin did not adversely affect hatch rate, interest centers on the effects of streptomycin on the eclosion of A. glabratus eggs in different salt solutions. The solutions studied fell into three general groups as evidenced by the experimental results: a) those in which rates of eclosion were moderate or high whether or not streptomycin was present; b) those in which rates of eclosion were moderate or high in the absence of streptomycin, but low when the antibiotic was present; and c) those in which rates of eclosion were uniformly low under all test conditions because of the nature of the solutions per se-independent of any antibiotic effects. Disregarding solutions in the last group, it seems clear that under some conditions streptomycin may inhibit eclosion while under other circumstances this phenomenon
AND
SCHORK
may be partly or wholly blocked. This interference with streptomycin appears to be related to the concentration and species of ion(s) present in the test solutions. A few examples may be cited to illustrate these points. (1) The capacity of certain solutions to interfere with the inhibitory effects of streptomycin on eclosion was markedly altered by the simple expedient of diluting them (compare hatch rates in HS with those in HS 1: 10, or in Hanks’ 1:2 with Hanks’ 1: 10). (2) While the inhibitory effects of streptomycin were almost entirely blocked in HS (or in HS lacking NaCI), streptomycin plainly inhibited hatch in separate tests with each of the component salts (parts C and D). (3) When these component salts were tested at an increased ionic strength, only CaC$ showed a capacity for interfering with streptomycin (part E) . Manganese ions are reported to block the inhibitory effects of streptomycin on the growth of certain plants (Rosen, 1954; Gray, 1955). However, in tests done during the present studies, various concentrations of manganese (as MnSOJ were found to be ineffective in blocking the inhibition of eclosion among A. glabratus eggs exposed to streptomycin. Of further interest is that in the reports on plants, calcium ions were also shown to be effective (but less so than Mn+ +) in blocking the growth-inhibiting effects of streptomycin. The mechanisms involved in the effect of this antibiotic on plants, or the interference with this effect by metallic cations, are obscure (Gray, 1955). The mechanisms underlying the present findings are similarly obscure, but the results parallel those previously reported on the effects of streptomycin on the growth of A. glabratus (Chernin, 1957a). In the latter studies evidence was presented that the antibiotic probably affects the snails directly rather than in some intermediary fashion; similar evidence can be adduced from the present data inasmuch as the inhibition of eclosion due to streptomycin was observed under axenic as well as nonaxenic conditions. It is not known whether there may be differences among strains of
13
EFFECTS OF STREPTOMYCIN ON HATCHING OF SNAIL EGGS
A. glabratus in relation to streptomycin sensitivity, nor is it known whether the site of action of streptomycin is the egg membrane, the snails within, or both. It is evident, however, that A. glabratus is sensitive to streptomycin at several stages in its life cycle. At least one other antibiotic, nystatin, is known to inhibit eclosion (Chernin, 1957bj, and also to be toxic to adult A. glabratus (Seneca and Bergendahl, 1955; Chernin, 1959). REFERENCES E. 1957a. Studies on the biological control of schistosome-bearing snails. VII. Streptomycin-induced inhibition of growth and reproduction in the schistosome-bearing snail,
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R. A. 1955. Inhibition of root growth by streptomycin and reversal of the inhibition by manganese. Am. J. Botany 42,327-331. ROSEN, W. G. 1954. Plant growth inhibition by streptomycin and its prevention by manganese. GRAY,
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