Brazilian Journal of Medical and Biological Research (2004) 37: 587-590 Attraction to amino acids by snail 587 ISSN 0100-879X Short Communication

Attraction to amino acids by acuminata, the snail host of Fasciola species

F. Tiwari and Department of Zoology, Deen Dayal Upadhyay Gorakhpur University, D.K. Singh Gorakhpur, UP,

Abstract

Correspondence Adult Lymnaea acuminata (average length 20-22 mm) were collected Key words D.K. Singh locally from lakes and low-lying submerged fields from Gorakhpur. • Snails Malacology Laboratory The chemoattraction studies were made in round glass aquaria meas- • Lymnaea acuminata Department of Zoology uring 30 cm in diameter and filled to a depth of 10 mm with 500 ml • Fasciola DDU Gorakhpur University dechlorinated tap water. Each aquarium was divided into four concen- • Amino acids Gorakhpur 273009, UP • Attraction India tric zones. At the starting time of the assay 10 snails were placed on the E-mail: [email protected] circumference of outermost zone 0. Snail attractant pellets (SAP) were added simultaneously in the center of central zone 3. SAP of different amino acids were prepared at concentrations of 10, 20, 50, 80 and 100 mM/2% agar solution and, subsequently, spread to a uniform thick- Received October 23, 2002 ness of 5 mm. After cooling, SAP were cut in small pieces of 5 mm in Accepted January 21, 2004 diameter. Lymnaea acuminata’s attraction to amino acids was studied using different amino acid concentrations in SAP. Pellets containing amino acids with non-polar R groups (proline and tryptophan), a charged polar group (arginine) and uncharged polar R groups (serine, citrulline and asparagine) were tested. The snails were more attracted to the uncharged polar R group amino acid serine than to other groups of amino acids. The preferred amino acid concentration was 80 mM. The attraction of snails to different amino acids was concentration dependent. Snails could discriminate amongst the different amino acids at ≥50 mM.

Fascioliasis is an important live-stock containing the desired attractant and mollus- health problem (1,2). This disease is trans- cicides. Snails, like other gastropod mol- mitted by the flukes and luscs, use chemical clues to locate food F. gigantica through the vector snail Lym- sources (3-6). naea acuminata (1). Snail control with mol- In the present study, a variety of amino luscicides has been one of the effective meth- acid bioassay methods was screened to de- ods used for rapid and effective control of termine the behavioral responses of the snail this disease. Bait formulation of different L. acuminata. Amino acids were identified molluscicides would be an effective tool for in the snail modular system as chemicals selective killing of the snail with minimal diffusing from aquatic organisms and are adverse effect on the environment. It is there- probably used by the snails as indicators to fore important to identify strong attractant identify their food (7). compounds for preparing bait formulations Adult L. acuminata snails (mean length:

Braz J Med Biol Res 37(4) 2004 588 F. Tiwari and D.K. Singh

2.25 ± 0.20 cm) were collected locally from made in the center of the aquarium (zone 3). lakes and low-lying submerged fields and Each container was filled to a depth of 10 were used as test . The snails were mm with 500 ml dechlorinated water. The acclimated to laboratory conditions for 72 h. containers were maintained at 25 ± 1ºC. Ten Snail attractant pellets (SAP) of agar con- snails of the same size were first placed in taining different amino acids such as argi- zone 0 at equally spaced positions and SAP nine, proline, tryptophan, serine, citrulline, were simultaneously added to zone 3. The and asparagine were prepared according to position of each snail was recorded at 15- the method of Madsen (8). Three groups of min intervals for 2 h. A clear glass aquarium amino acids, i.e., i) non-polar R group (pro- containing agar alone was used as control for line and tryptophan), ii) charged polar R each experiment. Each set of experiments group (arginine), and iii) uncharged polar R was repeated six times. The behavioral re- group (serine, asparagine and citrulline) were sponses of snails to SAP containing different used. Each amino acid was prepared at con- amino acids were observed and two-way centrations of 10, 20, 50, 80 and 100 mM/ ANOVA was applied to determine signifi- 2% agar solution. The preparations were cant alterations in behavior (9). spread to a uniform thickness of 5 mm and, Figure 1 shows the number of snails at- after cooling, SAP were cut out using a corer tracted towards the SAP containing different measuring 5 mm in diameter. concentrations of amino acids in zone 3. The The chemoattraction studies were car- uncharged polar R group amino acid serine ried out in round glass aquaria measuring 30 caused highest attraction of snails at 80 mM. cm in diameter. Each aquarium was divided After 2 h of the experiment, the behavioral into 4 concentric zones. Zone 3 (central zone) responses of snails showed high attraction to zones 2 and 1 (middle zones) and zone 0 the SAP containing 80 mM arginine, pro- (outer zone) had diameters of 13, 18, 24 and line, tryptophan, serine, and asparagine. The 30 cm each, respectively. Zone 0 had an area citrulline-containing SAP showed the high- of 254 cm2 around the inside edge of the est attraction at 100 mM (Figure 1). There aquarium. A small annular elevation was was a significant (P < 0.05) concentration-

Figure 1. Number of snails that 5.0 reached the zone where the pel- 12 Control Arginine Proline lets containing amino acids were 12

4.5 12 located. Data are reported as Tryptophan Serine Cetrulline Asparagine mean number of snails of six 4.0 12 replicates that reached zone 3 12 which contained the pellets 2 h 12 12 12 3.5 12 12 12 after the beginning of the ex- 12 12 12 12

12 123 periment. 3.0 12 12 12 12 123

12 12 123 12 12 12 123

123 12 12 12 123 12 12 12

123 12 12 12 123

2.5 12 12 12 123 12 12 12 123

123 12 12 12 12 12 12 12 123

123 12 12 12 123

1 12 12 12 12 2.0 123 12 12 12 123

1 123 12 12 12 12 12 12 12 123

123 12 12 12 123

1 12 12 12 12

123 12 12 12 123

1.5 1 123 12 12 12 12 12 12 12 123

123 12 12 12 123 1 12 12 12 12 Number of snails in the zone 3

123 12 12 12 123

1 12 12 12 12 1.0 123 12 12 12 123 123 12 12 12 123 1 12 12 12 12

123 12 12 12 123

1 12 12 12 12 123 12 12 12 123

123 12 12 12 123 0.5 1 12 12 12 12

123 12 12 12 123

1 12 12 12 12 123 12 12 12 123 0 1 123 12 12 12 12 12 12 12 123 10 20 50 80 100 Amino acid concentrations (mM)

Braz J Med Biol Res 37(4) 2004 Attraction to amino acids by snail 589 dependent variation in mean number of snails Table 1. Distribution of Lymnaea acuminata snails in the various zones around snail in the third zone around SAP of different attractant pellets (SAP) containing 50 mM amino acids 1 and 2 h after the beginning of amino acids 2 h after the beginning of the the experiment. experiment. Accumulation of snails on the serine-containing SAP was highest at all con- Attractant Time (h) Mean number of entries of snails around attractant centrations (Figure 1). There was no signifi- Z1 Z2 Z3 cant variation in the number of snails present in different zones containing SAP with dif- Agar (control) 1 0.66 ± 0.21 (33.1) 0.66 ± 0.33 (33.1) 0.50 ± 0.22 (25.1) 2 1.66 ± 0.42 (41.6) 1.50 ± 0.42 (37.5) 0.83 ± 0.30 (20.8) ferent amino acid concentrations. A signifi- Arginine 1* 1.33 ± 0.42 (27.5) 1.16 ± 0.30 (24.0) 2.33 ± 0.49 (48.3) cant variation in the number of snails on 2 2.00 ± 0.25 (32.4) 1.83 ± 0.30 (29.7) 2.33 ± 0.95 (37.8) SAP containing different amino acids was Proline 1* 1.16 ± 0.30 (33.2) 1.00 ± 0.25 (28.6) 1.33 ± 0.33 (38.1) observed only for 50 mM concentrations 2 3.00 ± 0.44 (41.8) 1.50 ± 0.22 (20.9) 2.66 ± 0.42 (37.1) after 1 h (Table 1). Tryptophan 1* 0.50 ± 0.22 (13.6) 1.33 ± 0.33 (36.3) 1.83 ± 0.54 (50.0) Except for citrulline, all amino acids at- 2 1.33 ± 0.21 (18.5) 2.33 ± 0.71 (32.5) 3.50 ± 0.96 (48.8) tracted snails most when present in SAP at Serine 1* 1.00 ± 0.25 (20.0) 1.50 ± 0.22 (30.0) 2.50 ± 0.34 (50.0) 80 mM. Among all the amino acids of the 2 2.33 ± 0.61 (27.4) 1.66 ± 0.33 (19.5) 4.50 ± 0.56 (53.0) uncharged polar group, serine was the most Citrulline 1* 2.33 ± 0.33 (42.4) 1.83 ± 0.16 (33.3) 1.33 ± 0.61 (24.2) preferred by the snails at all concentrations. 2 3.16 ± 0.54 (37.2) 2.66 ± 0.49 (31.3) 2.66 ± 1.47 (31.3) Abd El-Hamid (7) reported that proline is Asparagine 1* 1.33 ± 0.61 (38.1) 0.83 ± 0.40 (23.7) 1.33 ± 0.21 (38.1) 2 2.66 ± 0.76 (38.1) 1.66 ± 0.55 (23.7) 2.66 ± 0.21 (38.1) the preferred attractant for Biomphalaria alexandrina and suggested that this high Z3 = zone 3 (center of the container where snail attractant pellets were located); Z2 attraction of proline may be due to the fact (zone 2) and Z1 (zone 1) = the middle zone between Z0 (outer zone) and Z3. Numbers that it is released from the snails into the in parentheses are the percent of snails successfully locating snail attractant pellets, i.e., snails in Z3, compared to the snails that failed to locate them. Statistically surrounding water as a signal (10). How- significant (P < 0.05) when two-way ANOVA was applied between the number of ever, in the present study serine was found to snails present on SAP containing different amino acids (*) at 50 mM concentration. be the most preferred amino acid. It is pos- There was no significant variation in the number of snails present in different zones. sible that differences in behavioral responses between L. acuminata and other snails may of snails attracted by different amino acids at be due to the differences in the feeding be- any concentration, except 50 mM. This indi- havior and metabolism of these species. The cates that snail receptors can only differenti- higher attraction to serine by snails may be ate the amino acids when they reach a certain due to its more rapid diffusion in different threshold level (50 mM). At higher concen- zones since its molecular weight is lower trations, again the differentiation of amino than that of other amino acids. The attraction acids by snails is not significant. The gastro- of all amino acids, except asparagine, in pods are able to detect their food sources SAP was dose dependent (P < 0.05). Usu- sensing amino acids as indicators of the ally, L. acuminata snails are less responsive presence of their food (11-15). Amino acids to amino acids (63.2% maximum) than B. are released by aquatic plants and some al- alexandrina (85.7% maximum) when ex- gae. It is possible that the amino acid to posed to the SAP containing different con- which each species is sensitive is related to centrations of amino acids (7). There was no the detection of the extent of decay in the significant difference in number of snails in plant material on which it feeds. Finally, we the different zones in the presence of differ- suggest that the snail L. acuminata, an inter- ent concentrations of the various amino acids, mediate host of Fasciola species, might be indicating that within 1 and 2 h these amino controlled by using bait formulations con- acids diffused throughout all three zones. taining highly attractive compounds and spe- There was no significant variation in number cific molluscicides.

Braz J Med Biol Res 37(4) 2004 590 F. Tiwari and D.K. Singh

References

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