Acta hydrochim. hydrobiol. 27 (1999) 1 – © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999 0323–4320/99/0101–0032 $ 17.50+.50/032 3732

Effect of Polianthes tuberosa (Amaryllidaceae)on the Reproduction and BiochemicalParameters in the Ovotestis of SnailLymnaea acuminata (Mollusca, Pulmonata)

Wirkung von Polianthes tuberosa (Amaryllidaceae) auf die Reproduktionund biochemische Parameter der Zwitterdrüsen der Schnecke Lymnaeaacuminata (Mollusca, Pulmonata)

K. Singh, V. K. Singh,and D. K. Singh*

* Dr. Keshav Singh, SRF, Dr. Vinay Kumar Singh, R. A., Dr. D. K.Singh, Senior Lecturer, Department of Zoology, University ofGorakhpur, Gorakhpur – 273 009, U.P., India

Correspondence to D. K. Singh

Keywords: Polianthes tuberosa, Lymnaea acuminata, Repro-duction, Ovotestis, Biochemical Estimation

Summary:The present study elucidates the effect of powder ofPolianthes tuberosa bulb and its sapogenins tigogenin and hec-ogenin on the reproduction of the snail Lymnaea acuminata. Ithas been reported that the two sapogenins tigogenin and heco-genin caused a significant reduction in fecundity, hatchability,and survival of young snails. Exposure to sublethal concentra-tion caused a small but significant reduction in protein, aminoacids, nucleic acids, and phosphatase activities in gonadal tis-sue of treated snails. Withdrawal after 7 days indicates that theeffect of powder of P. tuberosa bulb, tigogenin, and hecogeninon the reproduction and different biochemical parameters is re-versible.

Schlagwörter: Polianthes tuberosa, Lymnaea acuminata, Re-produktion, Gonadengewebe, biochemische Parameter

Zusammenfassung: Die Untersuchungen belegen eine deutli-che Wirkung des Ethanolauszuges aus den Knollen von Poli-anthes tuberosa (Amaryllidaceae) und der in ihm enthaltenenSapogenine Tigogenin und Hecogenin auf die Reproduktionder Schnecke Lymnaea acuminata (Mollusca, Polmonata). DerEthanolauszug wie die beiden Sapogenine verursachen einesignifikante Reduktion der Fruchtbarkeit, der Schlüpfrate undder Überlebensrate der jungen Schnecken. Außerdem ergibtsich durch die mehrtägige Exposition gegenüber subletalenKonzentrationen der drei Agenzien eine geringe, aber signifi-kante Verminderung der Gehalte an Protein, Aminosäuren,Nucleinsäuren und der Phosphataseaktivitäten im Gonadenge-webe der exponierten Schnecken. Bei Abbruch der Expositiontritt innerhalb von sieben Tagen eine vollständige Erholung derSchnecken und ihrer Reproduktion sowie der biochemischenParameter auf Normalwerte auf. Die möglichen Wirkungs-mechanismen des molluskiziden Effektes der Stoffe werdendiskutiert.

1 IntroductionThe snail Lymnaea acuminata (Mollusca : Pulmonata) is

the intermediate host of the liver flukes Fasciola hepatica andF. gigantica [1], which causes endemic fascioliasis in the cattlepopulation of Eastern Uttar Pradesh. This snail breeds all theyear round and lay eggs on the lower surface of leaves of aquaticplants.A number of synthetic molluscicides have been used forthe control of the snail Lymnaea acuminata, [2–6]. Mollusci-cides of plant origin have gained greater importance since it isbelieved that natural products are ecologically sound and cul-turally more acceptable than synthetic once. It has been ob-served by us that the powder of Polianthes tuberosa (Amarylli-daceae) is a potent molluscicide against Lymnaea acuminata.

The molluscicidal activity of P. tuberosa bulb is due to the pres-ence of the active sapogenins tigogenin and hecogenin [7].

The aim of the present study is to evaluate the effect of sub-lethal exposure of a powder from ethanolic extract of P. tubero-sa bulb and its active sapogenins tigogenin and hecogenin onthe fecundity, hatchability, and survival of young snails of L.acuminata and different biochemical changes in the gonadaltissue of these snails. Withdrawal experiments were also per-formed to study the reversibility of the effects on the snails.

2 Materials and MethodsPowder of P. tuberosa bulb

The powder was prepared by slicing the P. tuberosa bulbs into3 mm thick slices and dried at 50 °C over night and pulverizing in amortar and pestle. 1 g dried powder was extracted in 10 mL of etha-nol at normal room temperature, the solvents were removed undervacuum, and the residue was used for the treatment.

Acta hydrochim. hydrobiol. 27 (1999) 1 –32 37 33

Fecundity experiment

Adult snails ((2.6 ± 0.30) cm in length) were collected fromfresh water ponds and stored for 48 h in glass aquaria containingdechlorinated tap water. Lymnaea acuminata lay their eggs in theform of an elongated gelatinous capsule containing 5–180 eggs.Groups of 20 snails in 3 L water were exposed to sublethal concen-tration (20 % and 60 % of LC50;24 h) of P. tuberosa bulb powder, tigo-genin ([(25R]-5α-spirostan-3β-ol), and hecogenin (5α-spirostan-β-ol-12-one) (Sigma USA) (Table 1).

Table 1: Concentrations (20 % and 60 % of LC50; 24 h and 40 % and80 % of LC50; 96 h) of ethanolic powder of Polianthes tuberosa andits active components tigogenin and hecogenin used againstLymnaea acuminata.

Verwendete Konzentrationen des Ethanolauszuges der Knollen vonPolianthes tuberosa und seiner aktiven Komponenten Tigogeninund Hecogenin für die Versuche mit Lymnaea acuminata (20 % und40 % der LC50; 24 h sowie 40 % und 80 % der LC50; 96 h).

Name of molluscicide LC50; 24 h , mg/L LC50; 96 h , mg/L20 % 60 % 40 % 80 %

Ethanolic powder 18.38 55.15 18.81 37.62Tigogenin 1.96 5.88 0.71 1.42Hecogenin 1.38 4.15 0.77 1.54

Since it is difficult to detect the mother snails for a particularspawn, capsules containing eggs from each treated group were incu-bated at 30 °C in covered petridishes containing the same concentra-tion of bulb powder of P. tuberosa, tigogenin, and hecogenin as giv-en to adult snails. The development of embryos at regular intervalswas observed under a binocular microscope up to their hatching.Percent hatching was studied only in eggs obtained after 24 h expo-sure of 20 % and 60 % of LC50; 24 h of P. tuberosa bulb powder, tigo-genin, and hecogenin to adult snails. Dead eggs were removed toavoid any water contamination. Young snails were immediatelytransferred to fresh water and their survival was observed up to 72 hafter hatching.

In withdrawal experiments the snails were transferred from 96 hexposure of 20 % and 60 % of LC50; 24 h of P. tuberosa bulb powder,tigogenin, and hecogenin into the fresh water and fecundity was ob-served for 24 h, 48 h, and 72 h after withdrawal to fresh water.

Biochemical experiments

Groups of 10 snails in 3 L water were exposed to sublethal con-centration (40 % and 80 % of LC50; 96 h) of powder of P. tuberosabulb, and its active sapogenins, tigogenin, and hecogenin for 96 h(Table 1). In withdrawal experiment snails were transferred from theabove treatment to fresh water for the next 7 days. The treated snailsin both groups were removed from the aquaria and washed with wa-ter. The gonadal tissue (ovotestis) were dissected out, placed on fil-ter paper, and weighed. Protein (Lowry et al. [8]), amino acids(Spies [9]), DNA, RNA (Schneider [10]) levels and phosphataseactivity (Bergmayer [11]) were measured.

Each estimation was replicated six times (measurement in sixdifferent pools of ovotestis). The values were expressed as mean± SE. Student's `t' test was applied to determine the significant(P < 0.05) difference between treated and control animals. Productmoment correlation coefficient was applied in between exposuretime and fecundity/survival of snails [12].

3 ResultsTable 2 indicates that in control animals, a group of twenty

snails laid 160–175 eggs per day. There was a significant

(P < 0.05) reduction in the fecundity of L. acuminata exposedto 20 % and 60 % of LC50; 24 h of ethanolic powder of P.tuberosa, tigogenin, and hecogenin. There was a significantnegative correlation between the exposure period and fecundi-ty. There was no fecundity after 72 h in snails exposed to 20 %and 60 % of LC50; 24 h of tigogenin (Table 2). Treatment of 20 %and 60 % of LC50;24 h of hecogenin and 60 % of LC50;24 h of pow-der of P. tuberosa bulbs against L. acuminata caused no egg lay-ing. Withdrawal of snails after exposure to 20 % and 60 % ofLC50; 24 h of P. tuberosa bulb powder, tigogenin, and hecogeninto fresh water indicates that there was a significant positive cor-relation between the withdrawal period and fecundity of with-drawn snails (Table 2). Fecundity started even in snails withoutprior fecundity.

There was a significant (P < 0.05) reduction in percenthatching of eggs of snails exposed to 20 % and 60 % of LC50;24 hof powder of P. tuberosa bulb, tigogenin, and hecogenin for 24 h(Table 3). Maximum reduction in hatching (48.9 % of control)was observed in 60 % of LC50; 24 h of tigogenin exposed eggs.Hatching period of treated eggs (8...12 days) was prolongedwith respect to control (7...9 days) groups.There was a negativecorrelation between the survival time and survival of youngsnails from eggs laid by snails exposed to powder of P. tuberosabulb, tigogenin, and hecogenin (Table 3). Tigogenin caused100 % mortality in offspring's after 48 h hatching period(Table 3).

There was a significant trend of recovery in the hatchabilityof eggs of withdrawn snails to fresh water (Table 4). Maximumhatching was observed in the eggs exposed to 20 % of LC50; 24 hof powder of P. tuberosa bulb.The hatching period was also de-creased to 7...9 days. Significant recovery was observed in thesurvival of young snails in withdrawal group (Table 4).

There was a significant decrease in protein and total aminoacid levels in the gonadal tissue of snails exposed to 40 % and80 % of LC50; 96 h of powder of P. tuberosa bulb, tigogenin, andhecogenin. Maximum reduction in protein level (14.1 % ofcontrol) amino acid level (6.8 % of control) were observed inthe snail exposed to 80 % of LC50; 96 h of tigogenin (Table 5).

There was a significant reduction in the levels of nucleicacids in the gonadal tissue of snails exposed to 40 % and 80 %of LC50; 96 h of powder of P. tuberosa bulb, tigogenin, and heco-genin. Maximum reduction in DNA and RNA levels was ob-served (9.5 % and 5.7 % of control) in snails exposed to 80 % ofLC50; 96 h of tigogenin (Table 5).

Table 5 shows that there was a significant reduction in acid/alkaline phosphatase activity in gonadal tissue of 40 % and80 % of LC50; 96 h of P. tuberosa bulb powder, tigogenin, andhecogenin Maximum reduction in levels of acid/alkaline phos-phatase activity was observed in the gonadal tissue of snails ex-posed to 80 % of LC50; 96 h of tigogenin.

Withdrawal of snails from 40 % and 80 % of LC50; 96 h ofP. tuberosa bulb powder, tigogenin, and hecogenin for 7 days infresh water caused a significant recovery in the levels of pro-tein, amino acids, nucleic acids and phosphatase activity in thegonadal tissue of snail L. acuminata from their treated groups(Table 6).

4 DiscussionIt is evident from the results that the powder of Polianthes

tuberosa and its active sapogenins components tigogenin and

Acta hydrochim. hydrobiol. 27 (1999) 1 –32 3734

Table 2: Effect of sublethal exposure of 20 % and 60 % of LC50; 24 h of powder of Polianthes tuberosa bulb, tigogenin, and hecogenin and itswithdrawal on the fecundity of snail Lymnaea acuminata.

Wirkung der subletalen Exposition gegen 20 % und 60 % der LC50; 24 h des Ethanolauszuges von Polianthes tuberosa sowie von Tigogenin undHecogenin auf die Fruchtbarkeit von Lymnaea acuminata einschließlich Erholung nach Abbruch der Exposition.

Withdrawal after 96 h exposureName of Treatment Fecundity Fecundity Fecundity Fecunditymolluscicide (mg/L) after 24 h no. after 48 h no. after 72 h no. after 96 h no.

of eggs of of eggs of of eggs of of eggs ofFecundity Fecundity Fecundity

20 snails 20 snails 20 snails 20 snailsafter 24 h no. after 48 h no. after 72 h no.

of eggs of of eggs of of eggs of20 snails 20 snails 20 snails

Control 175.6 ± 2.32 169.5 ± 3.10 167.0 ± 0.96 160.6 ± 0.42 166.6 ± 1.67 167.5 ± 1.12 161.0 ± 2.24Hecogenin 20 % of LC50 +145.8 ± 2.01* 54.3 ± 2.67* 18.6 ± 1.41* – $65.0 ± 2.24* 89.6 ± 2.72* 120.0 ± 1.83*

(1.96)60 % of LC50 +111.8 ± 2.72* 35.0 ± 1.83* 11.0 ± 0.44* – $42.5 ± 1.12* 74.6 ± 1.49* 114.2 ± 1.54*

(5.88)Tigogenin 20 % of LC50 +90.5 ± 4.80* 32.5 ± 1.12* – – $63.0 ± 1.67* 90.0 ± 1.83* 116.3 ± 2.03*

(1.38)60 % of LC50 +75.0 ± 2.24* 27.5 ± 1.12* – – $43.0 ± 1.41* 72.5 ± 1.12* 108.0 ± 1.00*

(4.15)Powder 20 % of LC50 +155.8 ± 2.01* 63.3 ± 2.11* 27.6 ± 0.92* 6.5 ± 0.50* $75.8 ± 1.54* 95.0 ± 1.83* 124.2 ± 3.01*

(18.38)60 % of LC50 +135.8 ± 2.01* 37.8 ± 1.30* 22.5 ± 1.12* – $48.0 ± 1.00* 83.2 ± 1.08* 116.2 ± 1.30*

(55.15)

Each value is mean ± SE of six replicates, each replicate represents the eggs laid by a group of 20 snails.(–) No egg laying was observed.(*) (P < 0.05) significant when student's ‘t’ test was applied to treated and control groups.Product moment correlation coefficient showed that(+) there was a significant negative correlation (after treatment)($) significant (after withdrawal) positive correlation between withdrawal time and fecundity.

Table 3: Effect of sublethal exposure of 20 % and 60 % of LC50; 24 h of powder of Polianthes tuberosa bulb, hecogenin, and tigogenin on thehatchability and survival of eggs of snail Lymnaea acuminata.

Wirkung der subletalen Exposition gegen 20 % und 60 % der LC50; 24 h des Ethanolauszuges von Polianthes tuberosa sowie von Tigogenin undHecogenin auf die Schlüpf- und Überlebensrate der Eier von Lymnaea acuminata.

Name of Treatment Fecundity % Hatchabilityxx Survival after Survival after Survival aftermolluscicide (mg/L) after 24 h no. of (hatching period 24 h of hatching 48 h of hatching 72 h of hatching

eggs of 20 snails in days) % % %

Control 175.7 ± 2.32 100 100 100 100(7...9)

Hecogenin 20 % of LC50; 24 h 145.8 ± 2.01 70.40 ± 0.26 +67.40 ± 1.59* 43.5 ± 0.60* 21.7 ± 0.30*(1.96) (8...12)60 % of LC50; 24 h 111.8 ± 2.72 56.5 ± 2.96 +53.1 ± 0.00* 23.9 ± 1.19* 10.9 ± 0.29*(5.88) (8...12)

Tigogenin 20 % of LC50; 24 h 90.5 ± 4.80 59.4 ± 2.36 +40.8 ± 2.25* 16.8 ± 0.00* –(1.38) (8...12)60 % of LC50; 24 h 75.0 ± 2.24 48.9 ± 1.40 +19.8 ± 0.68* – –(4.15) (8...12)

Powder 20 % of LC50; 24 h 155.8 ± 2.01 71.8 ± 1.04 +67.4 ± 0.01* 45.5 ± 0.29* 23.2 ± 0.46*(18.38) (8...12)60 % of LC50; 24 h 135.8 ± 2.01 59.9 ± 2.96 +42.3 ± 0.35* 30.6 ± 0.55* 15.9 ± 0.37*(55.15) (8...12)

Each value is mean ± SE of six replicates, each replicate represents the eggs laid by a group of 20 snails.(*) (P < 0.05) significant when student's ‘t’ test was applied to treated and control groups.(+) Product moment correlation coefficient showed that there was a negative correlation between exposure time and survival of miniaturesnails.(xx) Hatchability was studied only in the eggs obtained from 20 % and 60 % of LC50; 24 h treatment for 24 h exposure period. Survival of the samehatched snails were observed up to 72 h.

Acta hydrochim. hydrobiol. 27 (1999) 1 –32 37 35

Table 4: Effect of withdrawal to fresh water from sublethal 96 h exposure of 20 % and 60 % of LC50; 24 h of Polianthes tuberosa powder,hecogenin, and tigogenin on the hatchability and survival of young snail Lymnaea acuminata.

Wirkung des Abbruchs der subletalen Exposition gegen 20 % und 60 % der LC50; 24 h des Ethanolauszuges von Polianthes tuberosa sowie vonTigogenin und Hecogenin auf die Schlüpf- und Überlebensrate der Eier von Lymnaea acuminata.

Name of Treatment Fecundity % Hatchability** Survival after Survival after Survival aftermolluscicide (mg/L) after 24 h no. of (hatching period 24 h of hatching 48 h of hatching 72 h of hatching

eggs of 20 snails in days) % % %

Control 166.7 ± 1.67 100 100 100 100(7...9)

Hecogenin 20 % of LC50; 24 h 65.0 ± 2.24* 91.4 ± 0.40* 95.0 ± 0.69 95.0 ± 0.69 95.0 ± 0.69(1.96) (7...9)60 % of LC50; 24 h 42.5 ± 1.12* 82.3 ± 0.73* 91.9 ± 0.47 91.9 ± 0.47 91.9 ± 0.47(5.88) (7...10)

Tigogenin 20 % of LC50; 24 h 63.0 ± 1.67* 87.4 ± 0.67* 93.3 ± 0.38 93.3 ± 0.38 93.3 ± 0.38(1.38) (7...9)60 % of LC50; 24 h 43.0 ± 1.14* 82.3 ± 0.48* 90.4 ± 0.47 90.4 ± 0.47 90.4 ± 0.47(4.15) (7...10)

Powder 20 % of LC50; 24 h 75.8 ± 1.54* 94.3 ± 0.73* 97.1 ± 0.22 97.1 ± 0.22 97.1 ± 0.22(18.38) (7...9)60 % of LC50; 24 h 48.0 ± 1.00* 90.6 ± 1.04* 90.5 ± 0.35 90.5 ± 0.35 90.5 ± 0.35(55.15) (7...9)

Each value is mean ± SE of six replicates.(*) (P < 0.05) significant when student's ‘t’ test was applied to treated and control group.(**) Hatchability was studied only in eggs obtained after withdrawal from 96 h exposure of 20 % and 60 % of LC50; 24 h and survival ofthe same hatched snails were observed up to 72 h.

Table 5: Effect of 96 h exposure of sublethal concentration (40 % and 80 %) of LC50; 96 h of Polianthes tuberosa bulb powder and its activecomponent hecogenin and tigogenin on the levels of protein, amino acids, DNA and RNA, and phosphatase in the ovotestis of snail Lymnaeaacuminata.

Wirkung einer 96stündigen subletalen Exposition gegen 40 % und 80 % der LC50; 96 h des Ethanolauszuges von Polianthes tuberosa sowie vonTigogenin und Hecogenin auf die Gehalte an Protein, Aminosäuren, Nucleinsäuren und auf die Phosphataseaktivitäten in den Zwitterdrüsenvon Lymnaea acuminata.

Parameters LC50; 96 h Control Hecogenin Tigogenin Bulb powder

40 % 95.5 ± 0.00 25.9 ± 0.24* 18.7 ± 0.24* 33.4 ± 0.92*Protein(100) (27.1) (19.5) (35.9)

80 % 15.5 ± 0.61* 13.5 ± 0.61* 21.1 ± 0.31*(16.2) (14.1) (20.0)

40 % 30.7 ± 0.32 5.0 ± 0.12* 3.6 ± 0.20* 5.6 ± 0.08*Amino acids(100) (16.2) (11.7) (18.2)

80 % 3.2 ± 0.06* 2.1 ± 0.16 3.7 ± 0.08*(10.4) (6.8) (12.0)

40 % 91.4 ± 0.85 15.8 ± 0.33* 14.8 ± 0.67* 22.8 ± 0.85*DNA(100) (17.2) (16.1) (24.9)

80 % 12.1 ± 0.00* 8.7 ± 0.42* 14.8 ± 0.85*(13.2) (9.5) (16.1)

40 % 53.8 ± 1.73 7.3 ± 0.00* 4.1 ± 0.24* 13.8 ± 0.40*RNA(100) (13.5) (7.6) (25.6)

80 % 4.3 ± 0.17* 3.1 ± 0.00* 9.0 ± 0.43*(7.9) (5.7) (16.7)

40 % 2.5 ± 0.05 0.6 ± 0.07* 0.3 ± 0.03* 1.1 ± 0.03*Alkaline phosphatase(100) (24.0) (12.0) (44.0)

80 % 0.4 ± 0.01* 0.2 ± 0.02* 0.9 ± 0.02*(16.0) (8.0) (36.0)

40 % 1.5 ± 0.03 0.3 ± 0.02* 0.2 ± 0.04* 0.4 ± 0.01*Acid phosphatase(100) (20.0) (13.3) (26.6)

80 % 0.2 ± 0.02* 0.1 ± 0.01* 0.3 ± 0.00*(13.3) (6.6) (20.0)

Each value is mean ± SE (protein; amino acids; DNA and RNA given in µg/mg; acid and alkaline phosphatase given in µm per mg protein) of sixreplicates. Value in parenthesis are percent change with control taken as 100 %.(*) Significant (P < 0.05) ‘t’ test difference between treated (40 % and 80 % of LC50; 96 h) and control groups.

Acta hydrochim. hydrobiol. 27 (1999) 1 –32 3736

Table 6: Effect of withdrawal (after 96 h exposure for next 7 days in fresh water) of 40 % and 80 % of LC50; 96 h of Polianthes tuberosa powderand its active component hecogenin and tigogenin on the levels of protein amino acids, DNA and RNA, and phosphatase in the ovotestis ofsnail Lymnaea acuminata.

Wirkung des Abbruchs einer 96stündigen subletalen Exposition gegen 40 % und 80 % der LC50; 96 h des Ethanolauszuges von Polianthestuberosa sowie von Tigogenin und Hecogenin innerhalb von sieben Tagen auf die Gehalte von Protein, Aminosäuren Nucleinsäuren undPhosphataseaktivitäten in den Zwitterdrüsen von Lymnaea acuminata.

Parameters LC50; 96 h Control Hecogenin Tigogenin Bulb powder

40 % 93.3 ± 1.21 92.1 ± 1.53* 89.7 ± 1.53* 88.5 ± 1.21*Protein(100) (98.7) (96.1) (94.8)

80 % 90.9 ± 1.63* 90.9 ± 1.63* 87.3 ± 0.00*(97.4) (97.4) (93.5)

40 % 30.1 ± 0.00 29.3 ± 0.16* 28.1 ± 0.25* 29.4 ± 0.32*Amino acids(100) (97.3) (93.3) (97.6)

80 % 27.7 ± 0.21* 27.2 ± 0.00* 27.5 ± 0.32*(92.0) (90.3) (91.3)

40 % 90.0 ± 0.85 85.0 ± 0.33* 84.7 ± 0.00* 86.7 ± 0.00*DNA(100) (94.4) (94.1) (96.3)

80 % 82.7 ± 0.90* 82.0 ± 0.85* 85.3 ± 0.42*(91.8) (91.1) (94.7)

40 % 52.1 ± 0.00 49.3 ± 0.43* 48.9 ± 0.46* 49.3 ± 0.88*RNA(100) (94.6) (93.8) (94.6)

80 % 48.3 ± 0.34* 47.9 ± 0.00* 47.6 ± 0.34*(92.7) (91.9) (91.3)

40 % 2.6 ± 0.07 2.2 ± 0.06* 2.1 ± 0.00* 2.4 ± 0.03*Alkaline phosphatase(100) (84.6) (80.7) (92.3)

80 % 2.2 ± 0.03* 2.0 ± 0.03* 2.3 ± 0.04*(84.6) (76.9) (88.4)

40 % 1.5 ± 0.01 1.5 ± 0.03* 1.4 ± 0.04* 1.5 ± 0.03*Acid phosphatase(100) (100) (93.3) (100)

80 % 1.4 ± 0.00* 1.3 ± 0.01* 1.4 ± 0.01*(93.3) (86.6) (93.3)

Each value is mean ± SE (Protein; Amino acids; DNA and RNA given in µg/mg; acid and alkaline phosphatase given in µm per mg protein) ofsix replicates. Value in parenthesis are percent change with control taken as 100 %.(*) Significant (P < 0.05) ‘t’ test difference between treated (40 % and 80 % of LC50; 96 h) and its withdrawal groups.

hecogenin caused a time dependent significant reduction in thefecundity of the snail L. acuminata. Studies on the effect of P.tuberosa bulb powder and the sapogenins tigogenin and heco-genin on reproduction of snails is lacking. It has been reportedthat saponins present in different plant families prevent the up-take of oxygen by fish and leading to slow death by oxygen de-privation [13]. It may be possible that the sapogenins (aglyconsof saponins) tigogenin and hecogenin cause reduced uptake ofoxygen in snails which affect the metabolism of snail leading tolow fecundity. The CDC cells (caudo-dorsal cells) in the brainof snail Lymnaea release the ovulation hormone [14]. It is alsopossible that the sapogenin present in P. tuberosa affects theCDC, which reduces the release of ovulation hormone. Tigo-genin and hecogenin are steroidal sapogenins [15]. It has beenreported that steroidal sapogenin diosgenin used for the partialsynthesis of oral contraceptives [16]. However, exact mode ofaction of sapogenins on reproduction is yet not reported.This isthe first report on the effect of sapogenins on snail reproduc-tion.

The reduced hatchability of L. acuminata exposed to pow-der of P. tuberosa, tigogenin, hecogenin may be due to the inter-ference with embryonic growth and development of the snails.Reduction in survival of newly hatched snails may be due to thereduced intake of oxygen due to low surface tension betweenwater and respiratory surface [13]. Use of P. tuberosa againstL. acuminata is advantageous because instead of killing adultsnails, it also kills the eggs and young snails.

It is clear from the results that the powder of P. tuberosabulb, tigogenin, and hecogenin significantly alter the differentbiochemical parameters, viz. protein, amino acids, nucleicacids and acid/alkaline phosphatase activity in the gonadal tis-sue of L. acuminata. The significant reduction in the levels ofprotein, amino acids, nucleic acids and acid/alkaline phos-phatase activity indicates that the sapogenins present in the P.tuberosa bulb affect the gonadal tissue of exposed snails direct-ly or indirectly through neurohormones. Reduction of the pro-tein level in gonadal tissue of treated snails shows that these sa-pogenins act on the steps involved in protein biosynthesis. Re-duction rather than increase in free amino acid level indicatesthat sapogenin also interferes with the biosynthesis of aminoacids in the cells. These changes in the gonadal tissue may bethe cause of the reduction in egg laying, hatchability, and sur-vival of young snails of L. acuminata.

Alkaline phosphatase plays a critical role in protein synthe-sis [17] and other secretory activities [18] in gastropods, its in-hibition may result in reduction of the protein level [6]. Acidphosphatase activity was reduced significantly although its in-creased concentration, may cause breakdown of existing pro-tein [6]. In the present study, inhibition of acid phosphatase ac-tivity indicates that it is not used in breakdown of cellular pro-tein.

Withdrawal experiments clearly demonstrate that the effectof the powder of P. tuberosa bulb, tigogenin, and hecogenin isreversible, as the fecundity, hatchability and survival of young

Acta hydrochim. hydrobiol. 27 (1999) 1 –32 37 37

snails and levels of protein, amino acids, nucleic acids and acid/alkaline phosphatase activity in the gonadal tissue of L. acumi-nata recovered significantly with respect to treated observa-tion. Thus, the reversibility of sapogenins would be an addedadvantage in their use against aquatic snails as they wouldcause only short-lived environmental toxicity.

AcknowledgementAuthors are thankful to the Indian Council of Agricultural

Research (ICAR) (Project No. 1-20/94 pp) for financialassistance.

References[1] Singh, O., Agarwal, R. A.: Toxicity of certain pesticides to two

economic species of snail in northern India. J. Econ. Entomol.74, 568–571 (1981).

[2] Sahay, N., Singh, D. K., Agarwal, R. A.: Synergistic effect ofpiperonyl butoxide on the toxicity of synthetic pyrethroids inthe snail Lymnaea (Radix) acuminata. J. Med. Appl. Malacol.3, 107–111 (1991).

[3] Singh,A., Singh, D. K.,Agarwal, R.A.: Effect of cypermethrin,mexacarbate and phorate on the phospholipid and lipid perox-idation in the snail Lymnaea acuminata. Bull. Environ. Con-tam. Toxicol. 51, 68–71 (1993).

[4] Singh, D. K., Agarwal, R. A.: Piperonyl butoxide synergismwith two synthetic pyrethroids against Lymnaea acuminata.Chemosphere 15, 493–498 (1986).

[5] Singh, D. K., Agarwal, R. A.: Effect of synthetic pyrethroidspermethrin on the snail Lymnaea acuminata. Sci. TotalEnviron. 67, 263–267 (1987).

[6] Singh, D. K., Agarwal, R. A.: Toxicity of piperonyl butoxidecarbaryl synergism on the snail Lymnaea (Radix) acuminata.Int. Rev. Gesamten Hydrobiol. 74, 689–699 (1989).

[7] Singh, K., Singh,V. K., Singh, D. K.: Investigation of mollusci-cidal activity of Polianthes tuberosa. Int. J. Pharmacog. (inpress) (1998).

[8] Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J.:Protein measurements with folinphenol reagent. J. Biol.Chem. 193, 265–275 (1951).

[9] Spies, J. R.: Colorometric procedure for amino acids. In: Colo-wick, S. P., Kaplan, N. O. (Eds.): Methods in Enzymology.Academic Press, New York, 1957, 464–471.

[10] Schneider, W. C.: Determination of nucleic acids in tissues bypentose analysis. In: Colowick, S. P., Kaplan, N. O. (Eds.):Methods in Enzymology. Academic Press, New York, 1957,680–684.

[11] Bergmayer, U. H.: Methods of Enzymatic Analysis. AcademicPress, New York, 1967.

[12] Sokal, R. R., Rohlf, R. J.: Introduction to Biostatistics. Free-man W. H., San Francisco, 1973.

[13] Lamba, S. S.: Indian piscicidal plants. Econ. Bot. 24, 134–135(1970).

[14] Roubos, E.W., Boer, H. H., Schot, L. P. C.: Peptidergic neuronsand the control of neuroendocrine activity in the fresh watersnail Lymnaea stagnalis. Proc. Int. Symp. Neurosecretion 8th,119–127 (1981).

[15] Finar, I. L.: Organic Chemistry, Volume 2. Longman House,Burnt Mill, Harlow, Essex, U. K., 1980.

[16] Fazli, F. R. Y., Hardman, R.: The spice fenugreek (Trigonellafoenum-graecum L.) its commercial varieties of seed as asource of disogenin. Trop. Sci. 10, 66–78 (1968).

[17] Pilo, B., Ansari, M. B., Shah, R. B.: Studies on wound healingand repair in pegion liver: III. Histochemical studies on acidand alkaline phosphatase activity during the process. J. Anim.Morphol. Physiol. 19, 205–212 (1972).

[18] Ibrahim, A. M., Migazi, M. G., Dexian, E. S.: Histochemicallocalization of alkaline phosphatase activity in the alimentarytract of the snail Marisa cornuarietis (L.). Zool. Soc. EgyptBull. 26, 94–105 (1977).

received 26 February 1997accepted 9 February 1998