Pestic. Sci. 1983, 14, 123-129

Busulphan and Thiourea as Chemosterilants for the Snail Lymnaea acuminata

Ravindra Singh and Ram A. Agarwal

Department of Zoology, University of Gorakhpur, Gorakhpur 273 001, India

(Revised manuscript received 19 July 1982)

Treatment of the snail Lymnaea acuminata with an alkylating agent (busulphan) or a thiocarbamide (thiourea), for 3 days, significantly reduced the number of eggs and prevented embryonic development. Both the drugs caused a dose dependent reduction in the levels of protein, DNA, RNA and the activity of the alkaline phosphatase of the ovotestis, and a dose-dependent enhancement in the levels of amino-acids and the activity of the acid phosphatase. Discontinuation of the drug treatment for 7 days resulted in a near complete recovery in DNA and RNA levels, while there was no change in the levels of protein, amino-acids and the activities of the acid and alkaline phos- phatases.

1. Introduction

In spite of a substantial amount of work on the control of insect pests through the use of chemo- sterilants,l-3 almost no effort has been made towards the sterilisation of harmful gastropod pests and vectors. The present authors4 proposed, for the first time, the use of cyclophosphamide [2-bis(2- chloroethyl)aminoperhydro-1,3,2-oxazaphosphorinane 2-oxide1, an antitumour alkylating agent, as a snail sterilant. Alkylating agents that inhibit DNA ~ynthesis,~, 6 and thiocarbamides that reduce RNA levels,' have been extensively used as sterilising agents. The present study deals with the effects of busulphan (an alkylating agent) and thiourea (a thiocarbamide) on the egg laying, embryo- nic development, and biochemical changes in the levels of DNA, RNA, protein, total free amino- acids and the activities of the acid and alkaline phosphatases in the ovotestis of Lymnaea acuminata, the vector of the liver flukes Fasciola gigantica and F. hepatica. To study the reversibility of the effects of these drugs, the same parameters were measured a week after the drugs were discontinued.

Busulphan is the British Pharmacopoeia Commission Approved Name for tetramethylene di(methanesu1phonate). Bisulfan is the US equivalent.

2. Experimental methods

2.1. Snails and their treatment Adult L. acuminata (1.367 2 0.094 g; 2.6k 0.3 cm in length) were collected from local freshwater ponds and stored in glass aquaria containing dechlorinated tap water and aquatic vegetation as food. Busulphan (6, 15 and 21 pg per snail) or thiourea (15, 21 and 30 pg per snail), dissolved in physio- logical saline8 and divided into three equal doses, were injected into the foot of experimental snails every 24 h on three consecutive days. Controls were injected with physiological saline alone. Snails were removed 24 h after the last injection; their ovotestes were dissected, cleaned of adherent tissues, and used for biochemical estimations. For the study of recovery from drug treatment, snails were kept in fresh dechlorinated tap water for 7 days after the 3-day treatment period.

123

124 R. Sin& and R. A. Agarwal

2.2. Egg laying and the viability of embryos L. acuminata lay ribbon-like gelatinous capsules containing 10-180 eggs. The total number of eggs laid by a group of 15 snails was counted every 24 h over a period of 6 days. As it was difficult to find the mother snail for a particular spawn, capsules containing 20 eggs from each treated group were randomly selected and incubated at 30°C in Petri dishes containing aquarium water. During the embryonic development, which lasted 10-12 days, the spawn were examined under a microscope every 12 h in order to follow the progress of development. At the end of the incubation period, newly hatched snails were counted in order to determine the viability of the eggs.

2.3. Estimation of nucleic acids Estimations of DNA and RNA were made according to SchneiderO using diphenylamine and orcinol reagents. Homogenates of the ovotestes (1 mg ml-1) were prepared in 10% trichloroacetic acid at 90°C and centrifuged at 5000g. The supernatant phases were used for the estimations of DNA and RNA.

2.4. Estimations of protein and total free amino-acids Protein estimations were made according to Lowry et a1.,10 using bovine serum albumin as a standard. Homogenates were prepared in 10 % trichloroacetic acid. Total free amino-acids were estimated according to the method of Spies.ll Ovotestes from two animals were pooled, homogenised and extracted in 96% ethanol (10 mg ml-1) for amino-acids determination.

2.5. Phosphatase activity The activites of acid and alkaline phosphatases were measured according to Andersch and Szcypinski,lZ as modified by Bergmeyerlz using 4-nitrophenyl phosphate as the substrate. Homo- genates of the ovotestes (20 mg rn-l) were prepared in ice-cold 0.9% sodium chloride, centrifuged at 0°C at 5000g and the supernatant phases used as the enzyme source.

Each experiment was replicated at least six times. Values (mean k s.e.) were expressed as pg mg-l of the ovotestis for DNA, RNA, protein and total free amino-acids, and as pmol of substrate hydrolysed in 30 min mg protein-1 for the acid and alkaline phosphatases.

2.6. Statistical analysis Significant differences between treatment groups were calculated by one-way analysis of variance (P < 0.05) in order to determine dose dependence. Newman-Keul's test then determined which treatment groups were significantly different from controls ( P < 0.05).13

3. Results

3.1. Egg laying and embryonic development The reduction in the number of eggs following treatment with busulphan and thiourea are shown in Table 1. Both drugs caused dose-dependent reductions in the total number of eggs laid by the treated snails. Treatment with 6, 15 or 21 pg of busulphan reduced the number of eggs to 60%, 35% and 16% of the controls, respectively, while treatment with 15, 21 or 30 pg of thiourea reduced the number of eggs to 69%, 52% and 35% of controls, respectively. Although complete embryonic development was lacking in eggs laid by busulphan-treated snails, 20% of the eggs, laid by the snails that were given the lowest dose of thiourea (15 pg), developed completely.

3.2. Protein and total free amino-acids Changes induced by busulphan and thiourea in the levels of protein and total free amino-acids in the ovotestes of L. acuminata are presented in Table 2. Both drugs caused a dose-dependent reduction in protein levels and a dose-dependent similar enhancement in total free amino-acid levels. The maximum decrease in protein levels (59 % of the control value) was observed in snails treated with

Busulphan and thiourea as chemosterilants 125

Table 1. Changes in the number of eggs laid in a 6-day period by Lymnaea acuminata treated with

busulphan or thiourea

Drugs injected (pg per snail) Number of eggsb

Control (saline only) 257 (100)

Busulphan 6 15 21

Thiourea 15 21 30

155 (60) 90 (35) 42 (16)

179 (69) 135 (52) 89 (35)

a The dose was divided into three equal portions, injected at 24 h intervals on three consecutive days.

Q Egg counts were taken during the 3-day treat- ment period and 3 days following drug admini- stration, and are the totals for I5 snails; the values in parentheses indicate the percentage change, with control taken as 100%.

Table 2. Changes in the protein and free amino-acid levels in the ovotestes of Lymnaea acuminata treated with busulphan or thiourea

Dose (pg per snail)

Control (untreated)

Busulphan 6

I5

21

Thiourea 15

21

30

Treated snails

Proteina (pg mg-1) Total free amino-acidsa (pg mg-l)

81.43 f 2.64 ( 100)

67.61 + 1.9gb

59.19f0.72b

47.98 f0.74b

(83)

(73)

(59)

73.98 _+ 1 .09b (91)

63.74_+ I S b (78)

58.60f 1.07b (72)

Recovery after Treated Recovery after 7-day treatment snails 7-day treatment

71.66& 1.35b 47.45 f 2.34b 47.37 f 1.740 (92) (183) (177)

64.02 f 1.16b 48.51 k 1.236 47.93 _+ 1 .04b (82) (187) (180) Died 57.13f1.55b Died

(220)

a Values are expressed as the mean f s.e. of six replicates; the values in parentheses indicate the percent- age change, with control values taken as 100%. The dose was divided into three equal portions, injected at 24-h intervals on three consecutive days. Recovery was measured 7 days after the last injection.

b Significantly different (P < 0.05) compared with controls. Analysis of variance showed that the response to the drugs was dose dependent (Pc0.01).

126 R. Singh and R. A. Agarwal

21 pg of busulphan. Protein levels were reduced to 83 %, 73 % and 59 % of the controls following treatment with 6, 15 or 21 pg of busulphan, respectively, and to 91 %, 78 % and 72% of the control values following treatment with 15, 21 or 30 pg of thiourea.

Total free amino-acid levels increased to 185 %, 223 % and 265 % of the controls, respectively, following treatment with the three doses of busulphan, and to 183 %, 187% and 220%, respectively, after treatment with the three doses of thiourea (Table 2). Seven days after discontinuation of the treatments with 6 and 15 pg of busulphan, or 15 and 21 pg of thiourea, there was no significant change in the protein and total free amino-acid levels. Snails treated with 21 pg of busulphan or 30 pg of thiourea died during the 7-day recovery period.

3.3. DNA and RNA Changes in the levels of DNA and RNA in the ovotestes of L. ucuminutu, following treatment with busulphan and thiourea, are given in Table 3. Analysis of variance showed that both busulphan and thiourea had a dose-dependent (Pc0.01) action on DNA and RNA levels of the ovotestes. DNA levels were reduced to 81 %, 64% and 43 %, and RNA levels to 84%, 66% and 36% of the controls,

Table 3. Changes in the DNA and RNA levels in the ovotestes of Lymnaea acuminata following treatment with busulphan or thiourea

DNA4 (pg mg-l) RNAa (pg mg-l)

Dose Treated Recovery after Treated Recovery after (pg per snail) snails 7-day treatment snails 7-day treatment

Controls

Busulphan (untreated)

6

15

21

Thiourea I5

21

30

12.35 rt 1.79 (100)

58.765 1.980 (81)

46.125 1.03* (64)

31.2221.06b (43)

(97) 45.462 1.580

(62) 26.79 & 1.000

(37)

70.18 k0.99

69.61 50.85 ( 100)

(97) 67.635 1.1OC

63.01 f 1 . 3 1 0 ~ ~ (91) Died

62.01 50.63 (100)

51.95 +0.80b (84)

40.83 k0.530 (66)

22.55 f0.13b (36)

57.15+0.71b

42.72 f 0.500

26.33 5 0.770

(92)

(69)

(42)

59.16k0.54 (100)

(96)

(94) Died

56.85 +_0.43bre

55.68 +0.54b,c

57.15 k 0.44

53.75+0.460,c

Died

(97)

(91)

a Values are expressed as the mean+s.e. of six replicates: the values in parentheses indicate the percent- age change, with controls taken as 100%. The dose was divided into three equal portions, injected at 24-h intervals on three consecutive days.

Significantly different (Pc 0.05) compared with controls. Analysis of variance showed that the response to the drug was dose dependent (P<O.OI).

C Significantly different (P<O.Ol) from the corresponding treated group when the t-test was applied.

following administration of a 6, 15 or 21 pg dose respectively, of busulphan. Treatment with 15, 21 or 30 pg of thiourea reduced DNA levels to 97 %, 62% and 37 %, and RNA levels to 92%, 69% and 42% of the controls, respectively. The small reduction in DNA levels after the treatment with 15 pg of thiourea was, however, statistically insignificant.

Seven days without the drug resulted in near complete recovery in the levels of DNA and RNA in snails treated with 6 or 15 pg of busulphan, or 21 pg of thiourea.

Busulphan and thiourea as chemosterilants 127

3.4. Alkaline and acid phosphatases Both busulphan and thiourea caused dose-dependent increases in acid phosphatase activity and similar reductions in alkaline phosphatase activity in the ovotestes of L. acuminata (Table 4). Following injection of 6, 15 or 21 pg of busulphan, acid phosphatase activity increased to 134%, 182% and 202% (of the controls), and alkaline phosphatase activity decreased to 78%, 56% and 49% of the controls, respectively. Acid phosphatase activity increased to 125%, 194% and 230%, and alkaline phosphatase activity decreased to 90%, 66% and 45 % of the controls in snails treated with 15,21 or 30 pg of thiourea, respectively.

Table 4. Changes in the acid and alkaline phosphatase activities in the ovotestes of Lymnaea acuminara following treatment with busulphan or thiourea

Acid phosphatasen Alkaline phosphatase"

Dose Treated Recovery after Treated Recovery after (pg per snail) snails 7-day treatment snails 7-day treatment

Controls

Busulphan

- 1.46 k 0.03 1.47+0.01 2.21 k0.03 2.44+0.03b

(untreated) ( 100) (100) ( 100) ( 100)

(134) (128) (78) (72) 6 1.96 k0.03b 1.89kO.02 1.74f0.06b 1.75 f 0.040

15 2.66 f0.06b 2.58 + 0.06b 1.24 f0.02b 1.31 +0.04b

21 2.96 k 0.04b Died 1.09 k 0.04b Died (182) (175) (56) (54)

(202) (49)

(125) (114) (90) (93)

Thiourea 15 1.83+0.04b 1.67+0.06b.C 1.99 t 0.04b 2.27f0.01b

21 2.84f0.04b 2.45 +0.03'J*c 1.46 f 0.05b 1.70 f 0.06b

30 3.36 +0.03b Died 0.98 f 0.03b Died (194) (167) (66) (70)

(230) (45)

a The activities are given as pmol of substrate hydrolysed in 30 min mg-1 of protein and expressed as the meanks.e. of six replicates; the values in parentheses are the percentage change, with controls taken as 100%. The dose was divided into three equal portions, injected at 24 h intervals on three consecutive days.

b Significantly different (P < 0.05) compared with controls. Analysis of variance showed that the response to the drug was dose dependent (P < 0.01).

C Significantly different ( P < 0.01) from the corresponding treated group when the r-test was applied.

It is clear from Table 4 that there was no significant change in the acid and alkaline phosphatase activities of the ovotestes, 7 days after termination of treatment with 6 or 15 pg of busulphan. Though snails treated with 30 pg of thiourea died within 7 days after the last injection, snails admini- stered 15 or 21 pg of thiourea showed a significant reduction of the acid phosphatase activity.

4. Discussion

The data presented here demonstrate that both busulphan and thiourea caused a considerable reduction in the number of eggs and prevented embryonic development. Richard-Mercierl reported the destruction of the secondary spermatogonia of the Colorado beetle Leptinotarsa decemlineata following busulphan treatment. Busulphan inhibited the normal course of ovarian follicular growth in rats,l4 while cyclophosphamide caused a dose-dependent reduction in the number of eggs laid by L. acuminata.4 Thiourea induced a dose-dependent reduction in the number of eggs laid by the Azuki bean weevil Callosobruchus chinensis, as reported by Shukla and Pandey.15 Production of non-viable eggs in Sarcophaga ruficornis was reported by Chaudhry and Tripathi.16

128 R. Sin& and R. A. Agarwal

The present data show that both busulphan and thiourea caused a significant reduction in protein levels, and a significant enhancement in the total free amino-acid levels, in the ovotestes of L. acuminata. Although data on protein levels following treatment with busulphan or thiourea are not available, other chemosterilisation studies have shown that protein synthesis is reduced by cytotoxic chemicals. Chaudhry and Tripathil‘j reported that thiourea treatment reduces the nutrition of developing ova in Sarcophaga ruficornis. This could result from reduced protein synthesis as shown in the present investigation. Thiotepa [tri(aziridin-I-y1)phosphine sulphide] induced protein in- hibition in the testes of albino rats,17 and a similar inhibition of protein synthesis in house fly eggs was reported after treatment with thiotepa.18

Lack of significant changes in either the protein or total free amino-acid levels of the ovotestes, following withdrawal from the drugs for 7 days, demonstrates that both busulphan and thiourea have a rather prolonged inhibitory effect on the protein levels of this snail.

The experiments reported here demonstrate that busulphan and thiourea reduced the levels of DNA and RNA in the ovotestes of L. acuminata. Tripathi and Chaudhry7 reported that treatment with thiourea not only caused considerable inhibition of RNA in the ovarioles of Sarcophagu ruficornis but also that this carbamide prevented the transport of RNA from nurse and follicle cells to the oocyte. Al-Adil et aZ.l9 have shown that DNA synthesis in the house fly was significantly reduced after treatment with the alkylating agents apholate [2,2,4,4,6,6-hexa(aziridin-l -y1)-2,2,4,4,6,6- hexahydro-1,3,5,2,4,6-triazatriphosphorine], thiotepa, and metepa [tris(2-methylaziridin-l-yl)- phosphine oxide]. Nordenskjold et aL20 observed breaks in the strands of both DNA and RNA in human fibroblasts following treatment with the alkylating agent cyclophosphamide. A similar inhibition of DNA synthesis in rat testicular tissues was demonstrated after cyclophosphamide treatment .5

The present findings show that even though discontinuation of busulphan or thiourea treatment for 7 days produced near complete recovery in both DNA and RNA levels of the ovotestes, there was no recovery in the protein and amino-acid levels. Calabresi and Parks6 have described this as the ‘delayed death syndrome’ and have expressed the view that cytotoxicity of these drugs extends to cellular functions unrelated to proliferative activity, and that important metabolic processes con- cerned with maintenance of the cell are disturbed. This view is further confirmed by the data on acid and alkaline phosphatases reported here.

Busulphan and thiourea were shown to reduce the activity of the alkaline phosphatase in the ovotestes of L. acuminata. Alkaline phosphatase has been credited with a variety of roles in animals. Vorbrodtzl has shown that it plays an important role in the transport of metabolites across the membrane. This enzyme has been shown to be associated with protein synthesis22 and is thus involved in the synthesis of certain enzymes,23 secretory and ~permatogenesis.2~ Timmer- mans26 has shown that it plays an active role in the formation of the shell in molluscs.

The present study demonstrated that busulphan and thiourea caused a remarkable increase in the acid phosphatase activity in the ovotestes of L. acuminata. Acid phosphatase, the lysosomal enzyme, has a proteolytic action27 and plays a significant role in catabolism, autolysis and phagocytosis.28

It appears that the initial decrease in DNA and RNA levels causes a reduction in protein synthesis and prevents egg formation, but the effect of these drugs stays, even when the DNA and RNA levels have recovered. It is likely that the decreased protein levels may be due to a reduction in alkaline phosphatase activity, and an increase in the lysosomal enzyme acid phosphatase. Decreased protein synthesis, following treatment with the drugs and their breakdown, could be causing the enormous increase in free amino-acid levels. These drugs by inhibiting alkaline phosphatase may also be preventing shell formation in the developing embryos.

From the present study it becomes clear that busulphan and thiourea have the ability to sterilise L. acuminatu. This activity persists even after treatment with the drugs has been discontinued. It is possible that chemosterilisation can be successfully used for other harmful snails as well. The authors are aware that chemosterilants are highly toxic and thus have to be used with great care so that they do not contaminate the water resources of grazing animals and man. Luckily, the kind of small ponds and dirty waters, in which these snails usually live, are hardly ever used as water sources by man. However, in order to reduce the contamination of water, it might be advisable to

Busulphan and thiourea as chemosterilants 129

sterilise groups of snails and release them in their habitat, as in the sterile-male release technique recommended for insects.29

References 1. 2. 3. 4. 5 . 6.

7. 8. 9.

10. 11.

12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Richard-Mercier, N. Ann. Biol. Anim. Biochim. Biophys. 1977, 17, 661-672. Richmond, J. A.; DeMilo, A. B.; Thomus, H. A.; Borkovec, A. B. J. Ga. Entomol. Soc. 1978, 13, 237-240. Matolin, S. Acta Entomol. Bohemoslov. 1979, 76, 93-98. Singh, R.; Agarwal, R. A. Acta Pharmacol. Toxicol. 1981, 49, 195-199. Lambert, B.; Eriksson, G. Mutat. Res. 1979, 68, 275-289. Calabresi, P.; Parks, R. E. Antiproliferative Agents and Drugs Used for Immunosuppression. In The Pharma- cological Basis of Therapeutics (Goodman, L. S . ; Gilman, A., Eds.), MacMillan, London, 1980, pp. 1256-1270. Tripathi, C. P. M.; Chaudhry, H. S. Indian J . Exp. Biol. 1979, 17, 510-512. Lal, M. B.; Agarwal, R. A. Proc. Indian Acad. Sci. 1968, 67, 1-7. Schneider, W. C. Determination of Nucleic Acids in Tissues by Pentose Analysis. In Methods in Enzymology (Colowick, S . P.; Kaplan, N. O., Eds.), Academic Press, New York, pp. 680-684. Lowry, 0. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951,193,265-275. Spies, J. R. Colorimetric Procedures for Amino-acids. In Merhods in Enzymology (Colowick, S. P.; Kaplan, N. O., Eds.), Academic Press, New York, pp. 464471. Andersch, M. A.; Szcypinski, A. J. Am. J. Clin. Pathol. 1947, 17, 571, and in Methods of Enzymatic Analysis (Bergmeycr, U. H., Ed.), Academic Press, New York, 1967. Sokal, R. R.; Rohlf, F. J. Inrroduciion to Biostaristics W. H. Freeman & Co., San Francisco, 1969. Burkl, W.; Schiechl, H. Cell Tissue Res. 1979, 186, 351-359. Shukla, G. S.; Pandey, S. K. Z. Angew. Zool. 1978,65,237-242. Chaudhry, H. S.; Tripathi, C. P. M. Experientia 1976, 32, 103-104. Tarig, S. S.; Haqqi, M.; Adharni, U. M. Indian J. Exp. Biol. 1977, 15, 804-805. Gadallah, A. I.; Kilgore, W. W.; Painter, R. R. J. Econ. Entomol. 1971, 64, 819-821. Al-Adil, K. M.; Kilgore, W. W.; Gadallah, A. I.; Painter, R. R. J. Econ. Entomol. 1972, 65, 321-324. Nordenskjold, M. ; Soderhall, J.; Moldeus, P. Mutat. Res. 1979, 63, 393-400. Vorbrodt, A. Postepy Hig. Med. Dosw. 1959, 13, 200-206. Pilo, B.; Ansari, M. V.; Shah, R. V. J. Anim. Morphol. Physiol. 1972, 19, 205-212, Sumner, A. T. Q. J. Microsc. Sci. 1965, 106, 173-192. Ibrahim, A. M.; Higazi, M. G.; Demian, E. S. 2001. SOC. Egypt Bull. 1974, 26, 94-105. Pavlikova, D.; Repas, S. Biologiu (Bratisl.) 1975, 30, 889-895. Timrnermans, L. P. M. Netk. J. Zool. 1969, 19, 17-36. Moczon, T. Bull. Acad. Pol. Sci. Ser. Sci. Biol. 1976, 24, 289-292. Abou-Donia, M. B. Toxicol. Lett. 1978, 2, 199-203. Borkovec, A. B. Environ. Health Perspect. 1976, 14, 103-107.