Acra pharmacol. ei toxicol. 1983, 52, 112-120.

From the Department of Zoology, University of Gorakhpur, Gorakhpur-273001, India

Chemosterilization and its Reversal in the Snail Lymnaea acuminata BY

Ravindra S ingh and R. A. Agarwal* (Received July 23, 1982; Accepted September 15, 1982)

Absiracc Treatment of the snail Lymnaea acuminata, the vector for the liver flukes Fasciola hepatica and Fasciola giganrica, with two alkylating agents (cyclophosphamide and busulfan) and one thiocarbamide (thiourea) caused reduction in the number of eggs and production of non-viable embryos. All the three drugs caused reduction in the levels of DNA, RNA and protein and the activity of alkaline phosphatase in the gonadal tissues of this snail. There was enhancement in free amino acid levels and activity of acid phosphatase by these drugs. Discontinuation of treatment could not reverse the cytotoxic effects of these drugs. Treatment of sterilized snails with prostaglandin El (PGEI) . prostaglandin F2a(PGFza) or hydrogen peroxide not only restored the number of eggs but also caused full embryonic development. Also, there was near total recovery of RNA, DNA and protein levels and small changes in free amino acid levels as well as the activity of alkaline and acid phosphatase. It has been suggested that the prostaglandins play a modulatory role in protein synthesis. The possible therapeutic use of PGEI, PGFza and H202 has been pointed out.

Key-words: Lymnaea aruminafa - chemosterilization - prostaglandins - protein synthesis.

Although some success has been achieved in the control of harmful insects by using chemosterilants (Borkovec 1976; Richard 1977; Sukumar & Naidu 1978) this method was not used for the control of gastropod pests and vectors. The present authors (Singh & Agarwal 1981) proposed the use of cyclophosphamide, an alkylating agent, as a poten- tial chemosterilant for harmful snails.

Alkylating agents are highly reactive chemicals which transfer alkyl groups to important cell constituents by combining with nucleophilic groups such as amino, sulfhydryl, carboxyl, hy- droxyl and phosphate. These agents alkylate DNA and more specifically, the purine base guanine in which the 7 nitrogen is highly nucleophilic (Cala- bresi & Parks 1980). Because of their inhibitory effect on DNA synthesis (Lambert & Eriksson 1979), they possess remarkable sterilizing powers (Borkovec 1976). Thiourea, a thiocarbamide, also causes sterility in insects (Gouck et a/ . 1963) and is therefore used for their control.

Prostaglandins Fz alpha (PGF2a) and E I (PGEI) play an important role in the reproduction of a variety of animals. Spies & Norman (1973) and Harms et af . (1974) reported PGEl induced ovula- tion in rats. Morse et al. (1977) for the first time reported induced spermatogenesis by exogenous application of prostaglandins E and F in the snail Haliotis rufescens.

In the present investigation the chemosterilizing effects of cyclophosphamide, busulfan (both alkyl- ating agents) and thiourea (thiocarbamide) were studied in the snail Lymnaea acuminata, the vector of the giant liver flukes Fasciola hepatica and Fasciola gigantica. Effects of drugs on number and viability of eggs, the levels of DNA, RNA, protein, total free amino acids as well as activities of alkaline and acid phosphatases in the ovotestis of L. acuminata were studied. Reversal of the chemo- sterilizing effect of these drugs was studied by exogenous application of PGF2a and PGEI. Since it has been shown by Morse et al. (1977) that H202

CHEMOSTERILIZATION AND ITS REVERSAL IN SNAILS 113

can induce endogenous synthesis of prostaglandins in snails, reversal of chemosterilization was also tried with H202.

Materials and Methods

Adult Lymnaea acuminata (1.367f0.094 g; 2.6k0.3 cm length) were collected from local freshwater ponds and stored in glass aquaria containing dechlorinated tap water.

Schedule of drug administration. Cyclophosphamide (21 pg), busulfan (15 pg)and thiourea (21 pg) dissolved in physiological saline (La1 & Agarwal 1968) and divided in 3 equal doses, were injected in the foot of the experimental snails every 24 hrs on 3 consecutive days. Controls were injected with physi- ological saline only. On the fourth day, the treated snails

t'rc divided in four groups. One group was given PGEl or PFG2, ( 5 pg/animal/day) and a third given H202 (9 pg/ animal/day for 3 days. The fourth group was used as 'treated-control' which received only saline from the fourth day onwards.

Estimation of egg laying and viability of embroys. Lymnaea acuminata lay their eggs in the form ofelongated gelatinous capsules containing 10 to 180 eggs. The total number of eggs laid by a group of 10 snails was counted every 24 hrs. Since it was difficult to detect the mother snail for a particular spawn, capsules containing 20 eggs from each treated group were randomly selected and incubated at 30" in covered petridishes. The development of the embryos was studied for 10 days during which either the embryos perished or hatched into young snails.

Biochemical estimations. Experimental snails were taken out of the water; their ovotestis were dissected, cleaned and the adherent tissues were removed before estimations of DNA, RNA, protein, free amino acids and activities of acid and alkaline phosphatases.

Estimation of nucleic acids. Estimations of DNA and RNA were made according to Schneider (1957) using diphenylamine and orcinol reagents. Homogenates of ovotestes ( I mg/ml, w/v) were prepared in 10% TCA at 90" and centrifuged at 5,OOOXg for 20 min. Supernatants were used for DNA and RNA estimations.

Estimations ofprotein and total free amino acids. Protein

EGOS a PROTEIN ACID PH03 DNA AMINO ACID ALK. PH03

m RNA

C Y CL OPHOSPH A Y KW: BUSULFAW THIOUREA Fig. 1. Histogram showing per cent change in egg laying, change in levels of DNA, RNA, protein, total free amino acids and activity of acid and alkaline phosphatase in the ovotestis of L. acuminata following treatment with cyclophos- phamide (21 pg/animal), busulfan (15 pg/animal) and thiourea (21 pg/anirnal).

114 RAVINDRA SINCH AND R. A. ACARWAL

levels were estimated according to Lowry et al. (1951). using bovine serum albumin as standard. Hoinogenates ( 1 mg/ml, w/v) were prepared in 10%) TCA.

Estimation of total free amino acids were made accord- ing to the method of Spies (1957). Ovotestes from two animals were pooled and homogenates (10 mg/ml. w/v) were prepared in 96% ethanol. centrifuged at 6OOOXg and used for amino acid estimation.

fhn.rpha/a.re ucrivi/y. Acid and alkaline phosphatase activity was measured according to the method of Andersch & Szcypinski (l947), as modified by Bergmeyer ( 1967) using p-nitrophenylphosphate as substrate. Ho- mogenates (2% w / ~ ) were prepared in ice-cold 0.9%) sodium chloride solution, centrifuged at 5000Xgat 0" for 20 min. Supernatants were used as enzyme source.

Each experiment was replicated at least six times. Values (mean *S.E.) have been expressed as pg/mg ovotestis in the case of DNA, RNA, protein and total free amino acids and pmol substrate hydrolyzed/mg protein in case of acid and alkaline phosphatase. Students' t-test was used to locate significant differences (P<0.05).

Results

In control animal, a group of 15 snails laid about 250 eggs over a period of six days. Treatment with cyclophosphamide, busulfan or thiourea reduced the numberofeggsto71%,58%and85%(fig. I)of controls, respectively. While all the eggs in the control group developed into young snails follow- ing incubation at 30" for 10 days, complete embry- onic development was lacking in eggs laid by cyclophosphamide and busulfan treated snails. In the thiourea treated group however, approximately 20% of the eggs underwent full development.

Effect of chemosterilizing agents on DNA. RNA. protein and amino acid levels. DNA levels which ranged between 67 and 70 pg/mg of ovotestes were reduced to 54%. 59% and 72% of the controls following treatment with

Table 1.

Changes in DNA and RNA levels (pg/mg ovotestis) in L. acurninam following treatment with cyclophosphamide, busulfan. thiourea. PGEI. PGFm and H.02.

Dose DNA ( d m g ) (pg/animaI) Treated Treated control PGEl PGFra Hz02

Controls 69.90f1.16 67.63f1.10 67.07 f0 .94 67.35f I . 12 69.42f0.97 (untrcated) (100) (100) ( 100) (100) (100) Cyclophosphamide 37.82&1.82* 42.73f1.15" 58.29f1.937 64.05f 1.20t 67.73f 1.40t (21 1%) (54) (63) (87) (95) (98) Busulfan 41.31+1.12* 46.78f1.22* 56.50f1.96t 61.41f1.31t 63.01+2.05t ( 1 5 pg) (59) (69) (84) (91) (91) Thiourea 50.37+0.85* 59.05f1.03* 59.99f1.06t 63.29f 1.28t 69.42f 1.84t (21 1 4 ) (72) (87) (89) (94) (100)

Dose RNA (ps/mg) (pg/an i maI) Treated Treated control PGEi P G F ~ u H20z

Controls 49.93f0.96 48.38 f 0 . 7 0 5 I .27 f 0.68 47.50 f 0.89 5 I .90 f 0.57 (untreated) (100) (100) ( 1 00) (100) (100) Cyclophosphamide 25.70f0.48* 35.47f0.67* 50.9810.82t 47.50f0.68t 41.7610.531- (21 pg) (51) (73) (99) (100) (80) Busulfan 29.39+0.37* 38.07*0.38* 48.13f0.90t 48.21f0.69t 45.99f0.68t (15 (59) (79) (94) (101) (89) Thiourea 36.31 f0.45* 42.85*0.61* 49.68fI.OSt 47.33fO.88t 47 .2 l f0 .43t (21 clg) (73) (88) (97) (99) (91)

Values have been expressed as MeanfS.E. of six replicates. Values in parentheses indicate per cent change with controls taken as 100%. Snails were treated with cyclophosphamide, busulfan and thiourea for 3 days (treated) following which one group of six (treated controls) received saline only for the next 3 days: the other three groups were given either PGEl(15 pg), PGF2u (15 pg) or Hz02 (27 pg/animal) divided in three equal doses for the next 3 days. * - Significantly different when compared with controls (P<O.Ol). t - PGE,, PCFra and H202 treated groups significantly different when compared with treated controls (P<O.OI).

CHEMOSTERILIZATION AND ITS REVERSAL IN SNAILS 115

cyclophosphamide, busulfan and thiourea, respec- tively (table I ; fig. 1). RNA levels which ranged from 47.50 to 52.00 pg/mg in controls were simil- arly reduced to 51 96, 59% and 73%, respectively, after the same treatment (table I ) .

Protein levels (83.49k2.12 pg/mg ovotestis in controls) were reduced to 66%. 67% and 78% following cyclophosphamide, busulfan and thi- ourea treatment, respectively (fig. 1). There was a significant (P<O.Ol) increase in amino acid levels in the ovotestis after treatment with the three chemosterilizing agents. Thus free amino acid levels increased to 208%. 205% and 198% of the controls (27.73k0.61 pg/mg) following treatment with cyclophosphamide, busulfan and thiourea, respectively (fig. 1).

Changes in acid and alkaline phosphatase levels. Table 2 demonstrates that treatment with the three drugs caused significant (P<O.Ol) increase in acid phosphatase and significant decrease in alkaline phosphatase activity. Acid phosphatase activity

increased to 19996, 199% and 187% while alkaline phosphatase activity decreased to 67%, 62% and 71% of the controls after treatment with 21 pg cyclophosphamide, 15 pg busulfan and 21 pg thiourea, respectively (fig. 1).

Reversal of the effects of chemosterilants by prostaglandins. Animals which had been treated with cyclophos- phamide, busulfan or thiourea for 3 days were administered 5 pg/animal/day of PGEl or PGFza for the next 3 days.

Prostaglandin administration increased the total number of eggs laid over the 6 day period. Prosta- glandin Fza e.g. increased the number of eggs to 109%, 110% and 106% of the controls (or 154%, 190%) and 124% of the cyclophosphamide, busul- fan and thiourea treated animals, respectively). Furthermore, eggs laid after treatment with the two prostaglandins underwent complete embryonic de- velopment and hatched into young snails.

Fig. 2 shows the effect of PGEI and PGFZa

Table 2. Changes in acid and alkaline phosphatase activity (pmol substrate hydrolysed/30 min/mg protein) in the ovotestis of I - . acuminnra following treatment with cyclophosphamide, busulfan, thiourea, PGEI, PGF2a and H202.

Dose Acid phosphatase (pg/an imal) Treated Treated control PGEI PGF2a H202

Controls I .42fO.OI 1.41 f 0 . 0 2 1.49f0.02 1.40f0.02 1.49f0.02 (untreated) (100) ( 100) (100) ( 100) (100) Cyclophosphamide 2.82 i 0.0 I * 2.86 f 0.0 I* 2.79 f 0.05 2.60f0.02t 2.94 f 0 . 0 2 t (21 1%) (199) (203) (187) ( 1 86) (197) Busulfan 2.83 f 0.0 I* 2.74 f O . O I * 2.65 f 0 . 0 2 t 2.59+0.02t 2.79k0.008 (15 Pi?) (199) (194) (178) (185) (187) Thiourea 2.65 f O . O l * 2.49 f O . 0 I * 2.63 fO.0 It 2.65 k0 .02t 2.59f 0 .04t t (21 l E ) (187) (177) (177) (189) (174)

Dose (pg/animaI) Treated

Controls (untreated) Cyclophosphamide (21 pg) Busulfan (15 Pg) Thiourea (21 N?)

I .86f0.02 (100)

1.24+0.03* (67)

1.16*0.03* (62)

I .32*0.04* (71)

Alkaline phosphatase Treated control PGEi

1.91 f 0 . 0 2 1.91 f 0 . 0 3 (100) (100)

1.16+0.02* 1 .24f0 .02t t (61) (65)

I .24f0.02* 1 .35f0 .03t t (65) (71)

1.28 k 0.02* 1.41 f 0 . 0 3 t (67) (74)

PGF2a

1.87f.0.03 (100)

1.32f0 .05t t

1.36f0.03t (73)

1.42f0.02t (76)

(71)

H202

1.95+0.02 (100)

1.01 f 0 . 0 4 t (52)

I . I 1 f 0 . 0 2 t (57)

(64) 1.25 f 0.02

Values expressed as Mean +S.E. of six replicates. Values in parentheses are the per cent change with controls taken as 100%. Other symbols and experimental details as in table I . t t - P<0.05 when PGEI, PGF2a and H202 treated groups were compared with 'treated controls'.

116 RAVINDRA SlNGH AND R. A. AGARWAL

treatment on animals affected by the three chemo- sterilants. Significant and near complete recovery was observed in the protein levels ofthe ovotestis of L. acuminata following treatment with PGEI or PGF2a. Thus, in snails treated with cyclophos- phamide, PGEl and PGF2a treatment increased the protein levels from 68% to 99% and 96% respectively. Protein levels of the ovotestis, which were reduced to 72% and 74% after treatment with busulfan and thiourea increased to 100% and 99% of controls in case of the former and 97% and 95% in case of the latter following PGEI and PGF2a treatment, respectively (fig. 2).

As mentioned earlier the level of free amino acids nearly doubled following treatment of snails with cyclophosphamide, busulfan or thiourea. PGEI administration for the subsequent 3 days further significantly (P<0.05) enhanced the amino acid levels from 193% to 225%, 197% to 229% and 196% to 224% of the controls, respectively (fig. 3); administration of PGF2a. caused significant en- hancement in the amino acid levels of cyclophos- phamide treated snails only. In case of busulfan and thiourea treated snails although there was an increase in amino acid levels following PGF2a

PGF2a treatment, this was found to be statistically insignificant (fig. 3).

There was significant recovery (P<O.O1) in the DNA levels when animals treated with any of the three chemosterilants were administered PGEl or PGFza (table 1). PGF2a caused complete recovery in RNA levels of snails treated with any ofthe three compounds. In comparison to PGEI, the effect of PGF2a was generally more pronounced, but there was no significant difference between the effects introduced by the two prostaglandins. It should be noted that prostaglandin administration did not raise the levels of DNA, RNA or proteins above those found in normal snails.

Table 2 shows the changes in acid and alkaline phosphatase activity in snails treated with prosta- glandins for 3 days after a previous treatment of cyclophosphamide, busulfan or thiourea. Except for snails which were given PGEl after 3 days of cyclophosphamide the other groups registered a significant fall in the activity of acid phosphatase enzyme following administration of the prosta- glandins (table 2). The decrease, however, was rather small and complete recovery never took place. Administration of the two prostaglandins

0 CONTROL mTREATED CONTR-TREATED+PGFz O(

TREATED $ga TREATED+PGEl m T R E A T E D + H 2 0 2

CYCLOPHOSPHAM ID€ BUS ULFAN T HlOUREA Fig. 2. Histogram showing changes in the levels of protein (pg/mg ovotestis) in L. arumitiota. Snails were treated with cyclophosphamide (7 pg), busulfan (5 pg) and thiourea (7 pg) for three days (treated) following this one group (treated control) received only saline for the next 3 days; the other 3 groups were given PGE, (15 pg), PCF2a( 15 pg)or H ~ O I (27 pg/animal) divided in 3 equal doses for the subsequent 3 days. Values in parentheses indicate per cent change with controls taken as 100%. *, Significantly different when compared with controls (P<O.Ol). t, PGEI, PGF2a and H202 treated groups significantly different when compared with ‘treated-controls’ (P<O.Ol).

CHEMOSTERILIZATION AND ITS REVERSAL IN SNAILS 117

caused partial recovery, in alkaline phosphatase activity, which had decreased during chemosteri- lant treatment.

Reversal of biochemical changes b,v hydrogen peroxide. Morse et al. (1977) demonstrated increase in pro- staglandin endoperoxide synthetase following ad- ministration of H202 to the snail Haliofisrufescens. H202 was, therefore, administered to snails treated with the three chemosterilants in order to verify whether the effect of exogenously applied prosta- glandins could be replicated by prostaglandins synthesized in the snail in vivo. Indeed, exogenously applied H202 brought about exactly similar re- covery in the levels of protein, DNA, RNA, free amino acid and the activity of alkaline and acid phosphatases (table 1 and 2; fig. 2 and 3). Thus protein levels recovered from 68% to 99%, 72% to 100% and 74% to 100% in cyclophosphamide, busulfan and thiourea treated snails following

H202 administration (fig. 2). Similar changes were observed in amino acid levels (fig. 3), DNA and RNA levels (table I ) and acid and alkaline phos- phatase activity (table 2).

Discussion The present authors (Singh & Agarwal 1981) suggested the use of cyclophosphamide as a chemo- sterilant against harmful snails. Now our study has further been extended to two popular chemosteri- lants, busulfan (alkylating agent) and thiourea (thiocarbamide).

Data from the present study demonstrate that all the three chemicals caused conspicuous reduction in the number of eggs and prevented the normal development of embryos arising out of eggs from treated snails. Burke & Schiechl (1979) reported that both busulfan and cyclophosphamide inhibit the normal course of ovarian follicular growth in

0 CONTROL

a TREATED TREATED+ PGEI m TREATED+H2 02

TREATEU CON- TREATED f PGF2 o<

70 *

60 - Y

W

50- c

\ L O - a a - 30-

3 a g 2 0 -

3 10 9 B a

LL

CYCLOPHOSPWIDE BUSULFAN THIOUREA

Fig. 3. Diagram showing changes in the levels of total free amino acid (pg/mg ovotestis) in L. acuminala following treatment with the 3 chemosterilants, PGEl, PGF2u and HzOr. Symbols, dose regimen and other experimental details as in figs. 1 & 2. tt, (P<0.05) significantly different from corresponding ‘treated-control’ when t-test was applied.

118 R A V I N D R A S I N G H A N D R . A . A G A R W A L

rats. Inhibition of ovarian development following treatment with alkylating agents has also been reported in insects (Mathew & Rai 1975; Gelbic & Socha 1976; Beattie 1979). Richard (1977) has reported that the secondary spermatogonia of the colorado beetle Leptinotarsa decemlineata are de- stroyed after busulfan treatment.

Thiourea treatment was shown to cause dose dependent reduction in the number of eggs laid by azuki bean weevil, Callosobruchus chinensis (Shukla & Pandey 1978), abnormalities in the pupae of house flies (Naqvi et a/. 1978) and production of non-viable eggs in Sarcophaga rufi- cornis (Chaudhry & Tripathi 1976). These reports are in agreement with results obtained by us in the present study.

The present study demonstrated that cyclophos- phamide, busulfan or thiourea reduced DNA and RNA levels in the ovotestis of L. acuminata. Alkylating agents have been shown to reduce protein levels by inhibiting DNA synthesis (Lam- bert & Eriksson 1979; Calabresi & Parks 1980). In the present study it was demonstrated that there was a close paralellism between protein levels on the one hand and DNA/RNA levels on the other indicating that reduction in nucleic acid levels might be the primary cause of reduction in the protein levels.

Although data on the effect of the three sterilants used in this study on the protein levels in other animals are not available, chemosterilization stu- dies have shown that protein synthesis is reduced by cytotoxic sterilants. Inhibition of protein syn- thesis in house fly eggs (Gadallah eta/ . 1971) and testes of albino rats (Tarig et a/. 1977) after treatment with thiotepa has been demonstrated.

Cyclophosphamide, busulfan and thiourea caused significant enhancement in the total free amino acid of the ovotestes of L. acuminata. It appears that with impaired protein synthesis the amino acid pool gets richer. The increased activity of the lysosomal enzyme, acid phosphatase, re- ported in this study, may also cause reduction of protein and enhancement of free amino acid levels.

The present study demonstrated that cyclophos- phamide, busulfan and thiourea caused remark- able increase in the activity of acid phosphatase and similar reduction in alkaline phosphatase in the ovotestis of L. acuminata. Pavlikova & Repas

(1975) have shown that increase in acid phospha- tase activity is associated with degenerative changes in human testis. Alkaline phosphatase has been shown to play a variety of roles in different groups of animals. Pilo et a/. (1972) demonstrated that alkaline phosphatase is associated with protein synthesis; it has also been shown that thisenzyme is involved in the synthesis of certain proteinaceous digestive enzymes (Ibrahim et a/. 1974). Inhibition of alkaline phosphatase activity following treat- ment with tepa or thiotepa in the pupae of house flies has been demonstrated by Naqvi er a/. (1978).

The present investigation showed that although discontinuation of any of the three drugs, for three days, did not produce significant recovery in DNA, RNA, protein, free amino acids or acid and alkaline phosphatases, treatment with PGEl or PGF2a brought about near complete restoration of the levels of DNA, RNA and protein and partial recovery in free amino acid levels and phosphatase activity of the ovotestis.

Pausescu et a/. (1979) reported that prosta- glandin administration increased the protein levels in rat erythrocyte membrane, indicating the pos- sible role of the former in the promotion of protein synthesis. It is very likely that prostaglandins may be playing a similar role in snails, especially since the presence of prostaglandins has now been demonstrated (Nomura et a/. 1979) in bivalve molluscs.

Morse et n/. (1977) reported that exogenous application of H202 induced spawning and sperma- togenesis in the snail Haliotis rufescens by activat- ing the endogenous synthesis of prostaglandin endoperoxide synthetase which in turn increased the levels of prostaglandins in snails. Encouraged by the results obtained from the administration of PGEl and PGF2a and in view of the findings of Morse et a/. (1977), H202 was administered to L. acuminata sterilized with cyclophosphamide, bu- sulfan or thiourea. Indeed, exogenously applied H202 brought about recovery patterns exactly similar to those of PGEl and PGF2a. These findings confirm the earlier report and show that H202 may cause recovery in sterilized L. acuminata through endogenous production of prostaglandins.

It appears that H202, an ordinary laboratory chemical, may prove to be of immense practical value as the toxic effects of alkylating agents can

CHEMOSTERILIZATION AND ITS REVERSAL IN SNAILS 119

not only be countered by prostaglandins but also by hydrogen peroxide.

Recently, Robert ef a/. (1979) have given evi- dence that prostaglandins can prevent the forma- tion of gastric lesions and mucosal necrosis caused by various agents in rats without reducing gastric acid secretion. These authors suggested the term ‘cytoprotection’ to highlight this property of pro- staglandins. Even though the exact mechanism of ‘cytoprotection’ is not yet clear, the modulatory role of prostaglaninds in protein synthesis and the cytoprotection given by PGEI, PGF2a and H2Or may well be of clinical importance.

The present findings give hopeful pointers for the use of cyclophosphamide, busulfan and thiourea in the control of Lwnnaru acutninnfo. The positive effect of prostaglandins on protein synthesis and the ‘cytoprotection’ by prostaglandins and hydro- gen peroxide open up various possibilities tor their applied usage in chemotherapy of neoplastic di- seases where the use of these cytotoxic drugs is sometimes unavoidable.

References Beattie, G. A. C.: Oogenesis in Lucilin cuprina. 11. The

effect of agiridinyl chemosterilants on oogenesis. Aust. J. Zool. 1979, 21, 349-362.

Bergmeyer, U. H.: Methods of enzymatic analysis. Aca- demic Press, New York, 1967, p. 1129.

Borkovec. A. B.: Control and management of insect populations by c hemosterilants. En viron. Health Per- spect. 1976, 14, 103-107.

Burke, W. 19 H. Schiechl: The growth of follicles in the rat ovary under the influence of busulfan and endoxan. Cell Tissue Res. 1979, 186, 351-359.

Calabresi, P. & R. E. Parks Jr.: Antiproliferative agents and drugs used for immunosuppression. In: Thephar- macological bnsis o/’ therapeutics. Eds.: A. Goodman- Gilman. L. S. Goodman and A. Gilman. MacMillan,

Chaudhry. H. S. & C. P. M. Tripathi: Histopathological effect of thiourea on the ovarion tissues of Sarcophqa rufi‘c0rnI.c. Experirntia 1976. 32, 103-104.

Gadallah, A. I . , W. W. Kilgore & R. R. Painter: Protein synthesis by ribosomes from eggs of normal and thio- tepa chemosterilized house flies. J. Econ. Entomol. 1971, 64, 819-821.

Gelbic, 1 . & R . Socha: The effects of the chemosterilant nietepa on the development of ovaries of Di.rippus tnoi‘osus. Actu. En/. Rohcwroslo~’. 1976, 73, 224-233.

Gouck, H. K . , M. M. Crystal, A. B. Borkovec& D. W.

1980, p. 1256-1270.

Meifert: A comparison of techniques for screening chemosterilants of house flies and screw-worm flies. J. Econ. Entomol. 1963, 56, 506-509.

Harms, P. G., S. R. Ojeda & S. M. McCann: Prosta- glandin induced release of pituitary gonadotropins: Central nervous system and pituitary sites of action. Endocrinology 1974, 94, 1456-1464.

Ibrahim. A. M., M. G. Higazi & E. S . Demian: Histo- chemical localization of alkaline phosphatase activity in the alimentary tract of the snail, Marisa canuarietis. Zool. Soc. Egypt. Bull. 1974, 26, 94-105.

Lal, M. B. & R. A. Agarwal: A saline medium for maintaining isolated hearts of Pila globosa. Proc.. Intl.

Lambert, B. & G. Eriksson: Effect of cancer chemo- theraprutic agents on testicular DNA synthesis in the rat. Mittut. Rex 1979, 68. 275-289.

Lowry. 0. H., N. J. Rosenbrough. A. L. Farr & R. J. Randall: Protein measurements with Folin Phenol reagent. J. Biol. Chem. 1951, 193, 265-275.

Mathew, G. & K . S . Rai: Chemosterilant (apholate) induced ultrastructural changes during oogenesis in Aetles oegvpti. Cytobio.r. 1975, 12, 45-56.

Morse, D. E., H. Duncan. N. Hooky & A. Morse: Hydrogen peroxide induces spawning in molluscs, with activation of prostaglandin endoperoxide synthe- tase. Science 1977, 196, 298-300.

Naqvi, S. N. H., M. Qudrat, E. Khuda, S. M. Amir & I . Ahmad: Enzymes levels in the chemosterilized pupae of house fly, Musca doinestica. Pak. J. Zool. 1978. 8. 177-182.

Nomura. T., H. Ogata & A. Lucas: Mise in evidence de prostaglandines chez des mollusques bivalves. Rev. Int. Oceanogr. Med. 1979, LIII-L. IV. . 87-94.

Pausescu, E., R. Chirvasie, R. Olinescu, S . Nita, A. Me- disan, M. V. Popescu & 1. Rusa: Effect of some prostaglandins on the main phosphorylating, electron transporting and peroxidative systems of the erythro- cyte membrane. Rev. Roum. Morphol. Embryol. Physiol. Physiologie. 1979, 16, 63-69.

Pavlikova, D. & S. Repas: Comparative histochemical studies of changes in spermatogenesis and intertubular tissue at male sterility. Biologia Brarid. 1975. 30,

Pilo. B.. M. V. Ansari & R. V. Shah: Studies on wound healing and repair in pigeon liver I l l . Histochemical studies on acid and alkaline phosphatase during the processes. .I. Anim. Morphol. Physiol. 1972.19.205-212.

Richard, M.: Action of busulfan on larval gonads of the colorado beetle Leptinotarsa clecemlineata. Ann. B i d . Anim. Bioch. Bioph.vs. 1977, 17, 661-672.

Robert, A,, J. E. Nezamis, C. Lancaster & A. J. Hanchar: Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCI. NaOH. hypertonic NaCl and thermal injury. Gastroentero/o,p,~. 1979, 17, 433443.

Schneider, W . C.: Determination of nucleic acids in tissues by pentose analysis. In: Enzymologj~. Eds.: S. P. Colowick and N. 0. Kaplan. Academic Press, 1957,

~ c a ~ ~ sci. 1968, 61. 1-7.

889-895.

120 RAVINDRA SINGH AND R. A. AGARWAL

p. 680-684. Shukla, G. S. & S. K. Pandey: Effect of chemosterilant

(thiourea) on the oviposition of Callosobruchus chinen- sis. 2. Angew. 2001. 1978, 65, 237-242.

Singh, R. & R. A. Agarwal: Cyclophosphamide as a potential chemosterilant for harmful snails. Actaphar- macol. et rowicol. 1981, 49, 195-199.

Spies, H. G. & R. L. Norman: Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E L into pentobarbital- blocked rats. Prostaglandins 1973, 4, 131-141.

Spies, J. R.: Colorimetric procedures for amino acids. In: Methods in enzymology. Eds.: S . P. Colowick and N. 0. Kaplan. Academic Press, 1957, p. 464-471.

Sukumar, K. & M. B. Naidu: Effect of chemosterilants on the male reproductive system of the caster semi- looper moth Achoea janata. Ind. J. Exp. Biol. 1978,

Tarig, S . S., N. Haqqi & U. M. Adhami: Effect of thiotepa on RNA and total protein synthesis content in the testes of albino rats. Ind. J. Exp. Biol. 1977, 15,

16. 1190-1 191.

8 04-805.