Studies on the mode of action of some molluscicides on the snail, biomphalaria alexandrina

Comp. gen. Pharmac., i97 o, x, 2oi-2o8

2oI

STUDIES ON THE MODE OF ACTION OF SOME MOLLUSCICIDES ON THE SNAIL, BIOMPHALARIA ALEXANDRINA I. EFFECT OF BAYLUSCIDE,* SODIUM PENTACHLOROPHENATE, AND COPPER SULPHATE ON SUCCINATE, GLUTAMATE, AND REDUCED TMPD OXIDATION M. M. I S H A K , A. A. S H A R A F , t A. M. M O H A M E D , AN~ A. H. M O U S A Institute of Oceanography and Fisheries, Ministry of Scientific Research, Cairo, Egypt, and National Research Centre, Ministry of Scientific Research, Egypt

(Received 28 Nov., 1969) ABSTRACT I. The addition of citrate, succinate, glutamate, and reduced TMPD stimulated the oxygen consumption of tissue homogenates of the snail Biomphalaria alexandrina. 2. The rate of stimulation was increased by increasing the concentration of these substrates. Bayluscide and sodium pentachlorophenate in very low concentrations stimulated the oxidation of all tested substrates, while higher concentrations inhibited their oxidations. 3. Copper sulphate inhibited the oxidation of all the added substrates. 4. The relation between the mollnscicidal activity and the inhibitory action on the intermediary metabolism of the snail is discussed.

THE importance of molluscicides as a means of controlling the snail vectors of bilharziasis is well established (McMullen, K o m i y a m a , Ishil, Endo-Itabashi, Ozawa, Askawa, and Mitoma, t95I ; Hoffman, x953; Wright, I953; World Health Organisation, I96I). T h e basis on which chemical compounds are selected for testing as mollnscicides remains largely empirical. Any approach that enables selection to be made with a better understanding of the nature and mode of action of mollnscicides is of vital importance. Such knowledge will lead to the development of compounds of excellent molluscicidal efficacy. Devillier and McKenzie (t963) indicated that most of the commonly used molluscicide falls into two main groups: the first is characterized by interference with the osmoregularity system of snails when administered * Registered trade mark (Bayer 73), a 7° per cent formulation of the ethaaaolamine salt of 5,2'-dichloro-4'-nitrosalicylic anilide. t Faculty of Pharmacy, University of Cairo, Egypt, U.A.R.

at low concentrations. Members of this group include Bayluscide and sodium pentachlorophenate. No specific mechanism of action has been postulated as to how any m e m b e r of this group actually kills the exposed snails. T h e second group---copper sulphate, arsenic salts, acrolein, and the toxic organic derivatives of tin, lead, and m e r c u r y - - a r e all compounds which are known to poison the SH-contalning enzyme systems. T h e main objective of this investigation is to study the effect of copper sulphate, sodium pentachlorophenate, and Bayluscide on the intermediary metabolism of the snail Biomphalaria alexandrina. Such study m a y possibly locate the site of action of these compotmds, i.e., the enzyme system interfered with, and thus elucidate the mode of action of these compounds. MATERIAL AND METHODS The snails used were Biomphalaria alexandrina, 3-4 months old. They were the progeny of the second generation of laboratory reared snalh.

202

Comp. gen. Pharmac.

ISHAK ET AL.

Tissue preparations of B. alexandrina snails were prepared essentially by the method described by Kirdani and Evans (I954). Oxygen uptake was measured manometrically by the conventional

pH 7"4. Test substrates and molluscicides were added as indicated in the text. The reaction was started by introducing the tissue homogenates (equivalent to i6 mg. N) into

Table L--EFFECT

oF ADDED SUBSTRATES ON THE OXYGEN CONSUMPTION OF TISSUE PREPARATIONS OF THE SNAIL, B. alexandrina

OXYGEN CONSUMPTION (gl. O2 per g. tissuet per 15 minutes

SUBSTRATE 4~

CO~CENT~TION

(raM) Control 5 IO

I5 20

Reduced TMPD

Succinate

Citrate

Glutamate

43"3° 5= 5.03 60"385= 4"I3 IO3"36 5= 8"I 5 I27-I2 + i3.1o 169"56 5= 4"99

32'2I 5=2.60 35"3° 5=2"24 54"00 4- 2"88 77'505=3'32 I33"3 5=2"30

43'84 5=3'o7 49'32 + 2"9° 87'46 5=4" t 2 ioo.oi ±3"94

31'9o±3

61"75=II

o-i 5 * Basal incubation medium, o" 15 m M phosphate buffer, pH 7"4. t One g. wet weight tissue equivalent to 67 5=2'24 mg. nitrogen. Table ]I.--EPFECT

OF BAYLU$CIDE, SODIUM PENTACHLOROPHENATE, AND COPPER O N SUCCINATE O X I D A T I O N

SULPHATE

b OXYGEN CONSUMPTION

(gl. O2 per g. tissue t per 15 minutes) CONCENTRATION

oP MOLLUSCXCmE* (M)

Control iO-lt 5 X IO -11 iO-10 10 -9 10 -9 5 X I0 -a i0-~

5 ×IO-~ i0--6

Sodium Pentachlorphenate

Bayluscide Mean 5=S.D.

P

I92-4o + 5"20 219"26 + 9-06 272"76+ I3-75 280"36 4- 7'62 199.68 ± 7.48 i93.93 4- 2.24

(O'OOI ~O'OO1 ~O'OOI >O'I

I93'92 ± I2'25 i63.2o + 7.4° 9 2 . i 6 + 8.06

5 × IO-4

76"805= 4"79

P

173"oo ± 8"oo

>0'05 ~O'OOI ~O'OOI
176.46 5= 8.Ix 208.99 5=io-i2 260.93 5=x6.IO 207.5 ° 5=i8.io x t I ' 3 6 ± 9"oo 64"38 5= 7"oo 43"5o 5= 6.ii

Mean 4- S.D.

P

2 x8.o 9 5=4"58

>0. 5

5 × IO-6 lO-S 5 X I 0 -5 IO--4

Mean.± S.D.

Copper Sulphate

>o. 5 >O'OI ~0"00I >0"0I <(0'001 ~O'OO I ~O'OOI

176.58 5=6.63 I49"7I +5"m 131 "52 5=6"40 x22.4o 5=6-32 85"77 5=6"93 72"53 5=6'63 69"47 5=3"46

~O'OOI 0"00 I ~O'OOI ~O'OOI (0"00 I ~O'OOI (O'OOI

* Basal incubation medium, o. i m M phosphate buffer, pH 7"4, and aotassium succinate io mM, pH 7"4. t One g. wet weight tissue is equivalent to 67 ±2"24 rag. nitrogen. Warburg technique. The basal incubation medium contained o. I5 m M phosphate buffer,

the main compartment after thermo-equillbration for i o minutes at 3°o C. Final volume was 4 ml.

203

MOLLUSCICIDES AND SNAIL

I970 , x

Readings were recorded every 5 minutes and the results are expressed in pl. oxygen consumed per I5 minutes, Nitrogen content was determined by the Kjeldahl method. RESULTS EFFECT OF ADDED SUBSTRATES The substrates testedwere citrate, succinate, glutamate, and reduced tetramethyl-paraphenylene diamine ( T M P D + a s c o r b a t e ) , which are either intermediates of the tricarboxylic acid cycle or compounds whose

EFFECT OF COPPER SULPHATE, SODIUMPENTACHLOROPHENATE~ AND BAYLUSCIDE ON SUCCINATE OXIDATION

T h e data presented in Table H and Fig. I show that Bayluscide and sodium pentachlorophenate stimulate respiration when added in low concentrations but inhibit respiration at higher concentrations. Copper sulphate inhibits respiration even at very low concentrations of IO-s M (0.0o2 5 p.p.m.).

160

11oo

..'-

. . . .

.:

IL O

..... o

CONTROL o BAYLUSCIDE

&

£ SODIUM_ PCP.

. . . .

~%,

O0

~ 6o a. 40 20 t

!

I

I

I

I

I.

CONCENTRATION (M)

Fla. i.--Effect of Bayluscide, sodium pentachlorophenate, and copper sulphate on succinate oxidation. oxidation is linked to different carriers of the respiratory chain. Glutamate is linked to nicotinamide dinucleotide (NAD) and reduced T M P D is linked to cytochrome c. All the added substrates increased the oxygen consumption of the tissue preparation of B. alexandrina. T h e respiration was also increased by increasing the concentration of the substrates (Table I). This indicates that these compounds are metabolized by the experimental snail and that the enzymes involved in their metabolism are present in the tissue p r e p a r a t i o n used.

The results indicate that Baylnscide is a very potent compound. I t stimulates respiration at IO-11 M (o'ooo0o33 p.p.m.), and m a x i m u m stimulation is brought about by a concentration of IO - 1 ° M (o.oooo33 p.p.m.). Inhibition of respiration is attained by further increase of its concentration. T h e effect of Baylnscide can be summarized in the following stages : - a. I t stimulates respiration when added in small concentrations up to xo - a0 M . b. It reverses the stimulation of respiration by increasing its concentration up to IO - 9 M .

Comp. gen. Pharmac.

ISHAK ET AL.

~o4

c. It inhibits respiration up to the level of original respiration when the concentration ranges between io -8 and io -7 M. d. I t inhibits respiration below the original level when its concentration is higher than IO -7 M .

Sodium pentachlorophenate exerts its effect on respiration in a way similar to that of Bayluscide, except that it is less potent. The

trations and inhibit it at higher concentrations. Copper sulphate inhibited respiration at all tested concentrations ranging from ~o-s to 1o -4 M. It is obvious that glutamate oxidase is less sensitive to inhibition by copper sulphate than succinate oxidase. The concentration of copper sulphate which produces half maxim u m inhibition of succinate oxidase is

160

W v

.....

l&O

CONTROL o BAYLUSCIOE -" S O O l l J M _ P C P tOPeR SULPHATE

120 100 x o M. o

80 60

MJ (J

,,=, /,0 O. 20 0 1

'

1 . CONCENTRATION ( M )

;0,

10

;+,

Fro. 2.--Effect of Bayluscide, sodium pentachlorophenate, and copper sulphate on glutamate oxidation. m a x i m u m stimulation of respiration is brought about by a concentration of 5 × I OO6 M (1"45 p.p.m.). Also the shape of the curve differs somewhat from that for Bayluscide in that the inhibition of respiration is sharply increased by increasing the concentration of sodium pentachlorophenate so that the stages b and c merge into one effect (Fig. I). EFFECT OF BAYLUSCIDE~ SODIUM PENTACHLOROPHENATE~ AND COPPER SULPHATE ON GLUTAMATEOXIDATION

T h e results shown in Table 11I and Fig. 2 also indicate that these mollnscicides affect glutamate oxidation in the same way that they affect succinate oxidation. Bayluscide and sodium pentachlorophenate stimulate oxygen consumption at low concen-

2. 5 × IO-~ M while that for half m a x i m u m inhibition of glutamate oxidase is 2.5× i o - S M . O n the other hand, glutamate oxidase is more sensitive to the inhibitory action of Bayluscide and sodium pentachlorophenate than succinate oxidase (Table V). EFFECT OF BAYLUSCIDE~ SODIUM PENTACHLOROPHENATE, AND COPPER SULPHATE ON OXIDATIONOF REDUCED T M P D

( T M P D +AsCORBATE) I t is evident that both Bayluscide and sodium pentachlorophenate stimulate the oxygen consumption at low concentrations and inhibit it at higher concentrations (Table IV, Fig. 3). Baylnscide appears to be a more potent compound than sodium pentachlorophenate since the degree of stimulation

1970 , x

205

MOLLUSCICIDES AND SNAIL

with the latter is higher than that with Bayluscide. Copper sulphate inhibits the oxygen consumption at all tested concentrations ranging from Io -s to Io -4 M. The degree of inhibition of reduced T M P D oxidation by these compounds differs somewhat from that of both succinate and glutamate oxidations

(Table V).

Bah'on, Sights, and Wilder, t953; Bryant, Hines, and Smith, i964; Bryant, I965). I n the present work the increase in oxygen consumption on addition of citrate, succinate, glutamate, and reduced T M P D to tissue homogenates of the snail Biomphalaria alexandrina presents evidence for the presence of Krebs cycle activity. This also demonstrates that a more or less efficient respiratory chain

Table III.--EFFECT OF BAYLUSCIDE,SODIUMPENTACHLOROFHENATE, AND COPPER SULPHATE ON GLUTAMATE OXIDATION OXYGEN CONSUMPTION

(lal. O2 per g. tissue1' per ; 5 minutes) CONCENTRATION oF

MOLLUSCI-

CIDE* (M)

Mean 4- S.D. Control iO--10 5 X I O- t 0 i0-9 5 × I0-9 io-S 5 × Io-S i0-~ 5 X I 0 -v iO-S

5 × 'o-6 i 0 -5

5 x t o -5 10-5 5 X I0 -a

Sodium

Bayluscide

166-46 4-,o-2o 168"75 4- 9-20 i95.i6 4- 8.oo 2 I I ' I 5 4 , 6.30 I95-85 + 7"I4 i81.2o 4- 6.80 I65"25 4- 7"9° 98"45 4- 4"00 95"20 4- 5"20 49"85 4- 3"36

Pentachlorophenate P

Mean -4-S.D.

P

I63"374- 6"78

Copper Sulphate Mean 4- S.D.

P

I6o'71 4-7"21I

>0- 5 KO'O01 KO'OOI


>0. 5 "~0"001 KO'OOI
35"65 4- 3"87


27"65 4- 2"24

~0"001

I97"oi 4- I4"25 254"94 4- I3"93 228-2o4- 7"35 208.644- i.x 3 179.3o 4- 6-I2 I53.22 4- 6"I2 149"23 ~ I3"oo I09-2I 4- i6.oo 78"244- 6"5o 66.83 4-13.oo

~0"001 ~O'OOI 0"I >0"I ~O'OOI ~0"001 ~0"001

I5O'87 4-6"63 149"o4 4-7'I 4 14I'24 4-5'65 I38"o3 4-7'34 121"98 4-6"08

I I3"I9 4-4'80 75"75 :t=3'46 6I'77 4-2"45 49"7O4-2'5 °

>0"I ~0"001 0"001 ~0"001 <0"001 ~0"001 <0"001

~0"00I KO'OOI

* Basal incubation medium o. 15 m M phosphate buffer, pH 7"4, tnd sodium glutamate I o mM, pH 7"4. t One g. wet weight tissue is equivalent to 67 4-2"24 mg. nitrogen. DISCUSSION T h e presence of the tricarboxylic acid cycle has been demonstrated in most m a m malian tissues investigated (Krebs and Lowenstein, I96o ). I t appears to be the major pathway of carbohydrate, fat, a n d protein intermediary metabolism. In molluscs m u c h information on the intermediates and enzymes involved in the cycle has been demonstrated (Goddard and Martin, i966 ). Several workers have investigated the effect of added substrates and inMbitors to indicate the presence of the Krebs cycle in gastropods (Baldwin, x938; Cleland, 195o;

is functioning for catalysing the aerobic oxidation of the added substrates. Copper sulphate, at a concentration of I'2 p.p.m. (5 × Io-S M ) , inhibits succinate, glutamate, and reduced T M P D oxidation up to 7° , 3 ° , and 48 per cent respectively. Therefore it is concluded that succinate oxidase is more susceptible to the inhibitory action of copper sulphate. O n the contrary, Bayluscide, at a concentration of 0"3 p.p.m., inhibits succinate oxidation by 45 per cent, glutamate oxidation by 70 per cent, and reduced T M P D oxidation by 15 per cent, i.e., glutamate oxidase is more susceptible to inhibition by Bayluscide than cytochrome

~o6

ISHAK ET AL.

oxidase. However, sodium pentachlorophenate, at a concentration of 3 p.p.m. (I × x o - S M ) , which is lethal to the snail, did not inhibit the oxidation of either succinate or reduced T M P D , but stimulated their oxidations. Glutamate oxidase was inhibited by io per cent. Weinbach (i954) suggested that the molluscicidal activity of sodium

Comp. gen. Pharmac.

the inhibition of the glycolytic pathway (Weinbach and Nolan, I956 ). The molluscicidal activity of Bayluscide could be explained on the basis of its powerful inhibitory action on the oxidative processes of the snail organism. The results of this investigation show that it uncouples oxidative phosphorylation at very low concentrations;

z,~0i 220 ..... o

200, W X

~.100 i

--..

CONTROL o BAYLUSCIDE SODIUM_ PCP ~ COPPER SULPHATE

~1 60, x

o140 b. O

120 Z I.b

ul00, M4

u. e0

&0 20 0 CONCENTRATION (M)

FIO. 3.--Effect of Bayluscide, sodium pentachlorophenate, and copper sulphate on TMPD +ascorbate oxidation. pentachlorophenate is due to the interference with the oxidative phosphorylation process. He indicated that it is a powerful uncoupler of oxidative phosphorylation in both snail and rat tissues. The present results show that the molluscicidal activity of sodium pentachlorophenate is due to its uncoupling action when used in low concentrations (3 p.p.m.). But at higher concentrations, above 3 p.p.m., this could be attributed to

0"000033 p.p.m (z × lo -10 M), which is not lethal to snails, inhibits glutamate, succinate, and reduced T M P D oxidations. SUMMARY I. The effect of added substrates, citrate, succinate, glutamate, and reduced T M P D on the oxygen consumption of tissue homogenates of the snail Biomphalaria alexandrina was studied. The added substrates

MOLLUSCIGIDES A N D SNAIL

197o , x

stimulated the oxygen uptake, and the rate o f stimulation was increased b y increasing the concentration of substrates. 2. T h e effect of Bayluscide, sodium pentachlorophenate, a n d copper sulphate on the oxidation ofsuccinate, glutamate, a n d reduced

2o7

inhibited their oxidations. Copper sulphate inhibited the oxidation of all a d d e d sub-

strates. 3. The corrclation bctwcen the molluscicidal activity and thc inhibitory action of thcse compounds on the intcrmcdiary metabolism of the snail is discussed.

Table [V.--EFFECT OF BAYLUSCIDE, SODIUM PENTACHLOROPHENATE:, AND COPPER SULPHATE

ON REDUCEDT M P D +AscoXBAT~ OXIDATION OXYGEN CONSUMPTION

(ttl. Os per g. tissuet per 15 minutes) CONCENTRATION OF MOLLUSCI-

CIDE* (M)

Mean 4- S.D. Control io-tO

i0-~ lO-S 5 X IO-s

i0-7 5 XIO-7 10-6 5 X IO - 6 iO-S 5 X IO - s

10-4

Sodium

Bayluscide

165.oo -4-17"06 168.oo -4-17'29 195.72 + I I "58 236"43 -4- 8"66 254"73 -4- 8.82 222.oo + 7"o6 184.8o4- 8.66 I43"154- 9'22

Pentachlorophenate

Mean ± S . D .

159. I2 -4-I 1.23

P

149"75 ± I3'71


~O'OOI <0'001

<0"001 ~O'OOI

I o6"56 -4-14'25


lO-a 5 X IO - s i0-|

P

0"I--0'001 <0"001

I28"I3 ± 4"89

5 × lO-6

Mean + S.D.

P

Copper Sulphate

177"31 4-17.oo 218.67±1o.1 i 26o.58 + 18.39 360"00 -4-I9"77 337.6o ± 8-i 5 020.80 -4- 7"79 192"oo ,4,2I "86 159.72 ± lO.iO I o8"OO.4. O'OO IOo'OO+ 4"47 83.20 4- 4.4°

>0"02
147.974-2o.oo 147"694- I9"02 146"02 ,±2~'00 IO4"46 ± 15"o7 93"44-4- 6"71 82"oo -4- 4"47 68.624- 3.16

>O"I
59"85 -4- 5"08

"<0"001

:>0" 5

>0. 5

>0. 5 ~O'OO1 ~0"00I ~O'OO1

* Basal incubation medium o. 15 m M phosphate buffer, pH 7"4. Concentration o f T M P D o" 15 m M + ascorbate. t One g. wet weight tissue is equivalent to 67 -4-2'24 mg. nitrogen. Table V . - - C O N C E N T R A T I O N OF MOLLUSCICIDE REQUIRED FOR H A L F M A X I M U M INHIBITION OF RESPIRATION SUBSTRATE CONCENTRATION

(M)

MOLLUSCICIDE

Succinate Bayluscide Sodium pentachlorophenate Copper sulphate

Glutamate

Reduced T M P D

8XIO -?

4XlO -7

4 × IO-4

0" 5 × IO -6

I X 10 -5 2" 5 x xo - ~

2" 5 × i O -v

2. 5 × 1o-5

611o-6

T M P D was investigated. L o w concentratiom o f Bayluscide a n d sodium pentachlorophenate stimulated the oxidation o f all tested substrates, b u t higher concentrations

4. T h e concentration of the experimental molluscieides required to p r o d u c e half maxim u m inhibition o f respiration with the different substratcs is reported.

208

ISHAK ET AL.

ACKNOWLEDGEMENT

The senior author is grateful to Dr. A. EI-Kholys Director, Institute of Oceanography and Fisheries, for the facilities and encouragement he offered to complete this work.

HOFFMAN, D. O. (1953), ' Chemical structure and moUuscicidal activity ', in Congrks Internationaux de M~dedne Tropicale et du Paludisme, vol. 2, PP. 359-373. Istanbul: Gelikcilt Mothbassi. Kx~Am, R., and EVANS, G. I. (I954) , ' Studies on molluscicidal action. II. The inhibition of snail succinoxidase by some potent molluscicides ',

aVAMRU- 3 Cairo Res. Report .~f. M. REFERENCES BALDWIN, E. (I938), ' O n the respiratory metabolism of Helix pomatia ', Biochem. J., 32, x225I237. BAa~RON, E. S. G., S m i l e , W. P., and WILDER, V. (x953), ' T h e carbohydrate metabolism of heart slices ', Arch. exp. Path. Pharmac., 2x9, 338-348 • BRYANT, C. (X965), ' T h e metabolism of the digestive glands of two species of two marine gastropods (Melanerita melanotragus and Austro-

cochlea obtusa)', Comp. Biochem. Physiol., x4, 223-23o. W. J. W., and SMITH, M. J. H. (x964), ' I n t e r m e d i a r y metabolism in some terrestrial molluscs (Pomatia, Helix, and Gepaea) ', Ibid., xx, t47-I53. CLE'.AND, K. W. ('95o), ' T h e intermediary metabolism of unfertilized oyster eggs ', Proc. Linn. Soc. N.S.W., 75~ 296-319 • DEW,.LmR, J. P., and McKENzm, J. G. (i963), ' Structure and activity in molluscicides: The phenacyl halide, a group of potentially useful molluscicides ', Bull. Wld Hhh Org., 29, 424-427. GODDARD, C. K., and MAR~N, A. W. (x966), ' C a r b o h y d r a t e metabolism ', in Physiology of MoUusca (ed. WmBUR, K. M., and YONGE, C. M.), vol. I I, chap. 9, PP. 275-3 °8. New York and London: Academic Press.

----HINES,

Km~BS, H. A., and LOWENSTmN, J. M. (x96o), ' Tricarboxlyic acid cycle ', in Metabolic Pathways (ed. Gm~ENBEt~G, D. M.), vol. I, chap. 4. New York: Academic Press. MoMuLLEN, D. B., K o r u n A S , S., IsmL, N., ENDO-ITAa3~.Sm, T., OZAWA, K., ASKAWA,T., and MXTO~tA, Y. (195 x), ' Results obtained in testing molluscicides in field plots containing Oncomelania nosophora, an intermediate host of

Schistosoma japonicum ', Am..7. trop. Med. Hyg., 3 x , 583-598. WEXNBACH, E. G. ('954), ' The effect of pentachlorophenate on oxidation phosphorylation ', aT. biol. Chem., ~xo, 545-55 o. - - and NOLAN, M. O. (x956), ' T h e effect of pentachlorophenate on the metabolism of the snail Australorbis glabratus ', Expl Parasit., 5, 276-284 . WORLD H~ALTH ORGANmATmN (I96I), Mollusci-

tides: Second Report of the Expert Committee on Bilharziasis. Tech. Rep. Set. Wld Hlth Org., No. 2 x4, p. 50. WmOHT, W. H. (x953), ' Chemical control of the molluscan intermediate hosts of the human schistosomes ', in Congrks Internationaux de Mklecine Tropicale et du Palndisme, vol. 2, pp. 433-438. Istanbul: Gelikcilt Mothbassi.

Key Word Index: Molluscicidcs, Bayluscide, sodium pentachlorophenate, Biomphalaria alexandrina, tissue respiration.