Annals of Tropical Medicine & Parasitology, Vol. 104, No. 8, 667–671 (2010)
Behavioural responses of the snail Lymnaea acuminata to carbohydrates and amino acids in bait pellets P. AGRAHARI and D. K. SINGH Malacology Laboratory, Department of Zoology, Deen Dayal Upadhyay Gorakhpur University, Gorakhpur 273-009, Uttar Pradesh, India Received 2 August 2010, Revised 30 September 2010, Accepted 5 October 2010
Snail control could play an important role in programmes against fascioliasis, especially if the methods used for molluscicide delivery could be improved, such as by the development of bait formulations containing both an effective attractant and a molluscicide, to ensure good levels of contact between the molluscicide and the target snail populations. In a recent study, the attractiveness to Lymnaea acuminata (an intermediate host of the digenean trematode Fasciola gigantica) of potential components of snail-attractant pellets was investigated. Carbohydrates (glucose, maltose, sucrose or starch, each at 10 mM) and amino acids (citrulline, tryptophan, proline or serine, each at 20 mM), were tested in aquaria, with the snails initially placed 22.5, 30 or 45 cm from an agar pellet containing the component under test. Under these conditions, starch and proline emerged as the strongest attractants for L. acuminata, followed by maltose and serine.
Fascioliasis occurs world-wide (WHO, 2007; Mas-Coma et al., 2009) and is caused by the liver flukes Fasciola hepatica and F. gigantica. Human fascioliasis is an emerging disease (Chen and Mott, 1990; Mas-Coma et al., 1999) that is characterised by hypereosinophilia, abdominal pain and, exceptionally, acute pancreatitis (Saba et al., 2004; Echenique-Elizondo et al., 2005). Bovine fascioliasis is very common in the eastern region of the Indian state of Uttar Pradesh (Shukla et al., 2006; Singh and Singh, 2009). In this area, the disease is caused by F. gigantica, with the freshwater snail Lymnaea acuminata acting as the intermediate host (Singh and Agarwal, 1981). An obvious solution to reduce the incidence of fascioliasis, in India and elsewhere, is to kill the vector snails and break the life-cycles of the causative parasites. Freshwater snails inhabit an environment containing macrophytes, algae and bacteria (Thomas, 1982) Reprint requests to: D. K. Singh. E-mail:
[email protected]. # W. S. Maney & Son Ltd 2010 DOI: 10.1179/136485910X12851868780144
and these organisms release copious amount of chemicals, such as carbohydrates and amino acids, into the surrounding water (Thomas, 1982, 1986; Kpikpi and Thomas, 1992). As snails that live in freshwater use these chemicals to locate their food sources (Kpikpi and Thomas, 1992; Tiwari and Singh, 2004a, b; Kumar and Singh, 2009, 2010), the use of a combination of some of these, or similar, chemicals (as snail attractants) and a molluscicide, in a solid bait formulation, may be an effective tool for the management of the intermediate hosts of Fasciola spp. (and those of other snail-borne parasites). This approach may also be environmentally ‘friendly’, as little molluscicide should be released into the water and the only animals hurt by the molluscicide should be those attracted to the bait. In the present, laboratory-based study, the behavioural responses of L. acuminata towards agar pellets containing a single additional carbohydrate or amino acid were investigated. The aim was to identify which of the tested chemicals, if any, might act as a
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good attractant, for inclusion, with a molluscicide, in snail-attractant bait.
MATERIALS AND METHODS Snails Both field-collected and laboratory-reared adult L. acuminata, each measuring 22.5¡ 3.0 mm in length, were used in the experiments. Before they were employed in the assays, the field-collected snails, which came from Ramgarh Lake, which lies next to the campus of Gorakhpur University, in Gorakhpur, in eastern Uttar Pradesh, were allowed to acclimatize for 72 h, in dechlorinated tap water at 25¡1uC. Preparation of Bait Pellets The bait pellets were made as described by Madsen (1992), with nothing (as a control), a single carbohydrate (glucose, maltose, sucrose or starch) or a single amino acid (citrulline, tryptophan, proline or serine) added to the 100 ml of molten 2% (w/v) agar solution used to make each batch. Following the earlier reports of Tiwari and Singh (2004a, b), the carbohydrates and amino acids were tested at final concentrations of 10 and 20 mM, respectively. Each molten agar solution was allowed to set in a 5-mm-thick layer. Cylindrical pellets, each 5 mm in diameter and 5 mm high, were then cut from the solidified agar. Assay Apparatus and Procedure The chemo-attraction of the bait pellets to the adult L. acuminata was assayed as described by Tiwari and Singh (2004a, b), using clean cylindrical aquaria with diameters of 45, 60 or 90 cm, each holding sufficient dechlorinated tap water (1272, 2262 and 5091 ml, respectively) to give 8 mm of water depth. Each aquarium was stood on a piece of paper on which a circle of 15-cm diameter was drawn, so that the circle was centred on the centre of the aquarium base. [For other investigations,
this inner circle was surrounded by three concentric circles measuring 25, 35 and 45 cm (45-cm aquaria), 30, 45 and 60 cm (60-cm aquaria) or 30, 60 and 90 cm in diameter (90-cm aquaria).] A shaped glass block was placed over the centre of the aquarium base, creating a small annular elevation, measuring 9 mm in height and 1.5 cm in diameter, to hold a bait pellet. All the aquaria were maintained at 25¡1uC. At the start of each assay, 10 individually marked snails were placed around the periphery of an aquarium so that they were equally spaced (and 138, 185 and 279 mm apart in the 45-, 60- and 90-cm aquaria, respectively). Simultaneously, a single bait pellet was placed on the centre of the aquarium base. The location of each snail was then noted every 15 min for 2 h, with the percentage of the test snails in the central 15cm zone recorded after 1 and 2 h. Pellets containing each of the eight test chemicals were tested, in six replicates, with fieldcollected snails in aquaria of each of the three sizes. The whole series of assays was then repeated with laboratory-reared snails. Data Analysis The results (as percentages of the snails tested present in the 15-cm central zone after 1 and 2 h) were arcsine-transformed before analysis. Comparisons were made, using one- or two-way analyses of variance (Sokal and Rohlf, 1973), between each test chemical over the same test distance, between the initial test distances (i.e. 22.5, 30 and 45 cm) for each chemical, and between the results for the field-collected and laboratory-reared snails (for each chemical at each test distance). A P-value of ,0.05 was considered indicative of a statistically significant difference.
RESULTS The results recorded half-way through and at the end of each assay are summarized in
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
None (control)
Tryptophan
Serine
Proline
Citrulline
Starch
Maltose
Sucrose
Glucose
(h)
Test chemical
Time
1.00 0.33 3.50 4.33 4.00 4.66 4.83 4.83 3.66 5.33 2.16 3.00 3.33 4.00 3.50 4.33 1.66 2.50
(0.25) (0.21) (0.34) (0.61) (0.36) (0.21) (0.30) (0.30) (0.42) (0.61) (0.16) (0.44) (0.33) (0.00) (0.34) (0.21) (0.20) (0.33)
FC
[10.0] [4.2] [35.0] [44.2] [40.0] [47.9] [49.2] [49.2] [37.9] [54.2] [22.9] [30.0] [34.2] [40.0] [35.0] [44.2] [17.9] [25.0]
1.00 0.33 4.00 4.66 4.33 5.00 5.16 5.66 4.66 6.00 2.50 3.33 3.66 4.33 3.83 4.66 2.16 2.83
LR (0.25) (0.21) (0.36) (0.55) (0.20) (0.25) (0.30) (0.20) (0.41) (0.44) (0.22) (0.33) (0.20) (0.20) (0.16) (0.20) (0.16) (0.30)
45-cm aquarium
[10.0] [4.2] [40.0] [47.9] [44.2] [50.0] [52.9] [57.9] [47.9] [60.0] [25.0] [34.2] [37.9] [44.2] [39.2] [47.9] [22.9] [29.2]
0.33 0.50 2.16 2.50 2.00 2.16 2.66 3.00 2.50 3.50 2.00 2.50 2.66 3.33 2.66 2.83 1.33 2.33
(0.21) (0.22) (0.30) (0.22) (0.44) (0.30) (0.21) (0.25) (0.22) (0.22) (0.00) (0.22) (0.49) (0.49) (0.21) (0.16) (0.20) (0.20)
FC [10.0] [5.0] [22.9] [25.0] [20.0] [22.9] [27.9] [30.0] [25.0] [35.0] [20.0] [25.0] [27.9] [34.2] [27.9] [29.2] [14.2] [24.2]
0.33 0.50 1.50 2.00 1.83 2.16 2.00 2.83 2.16 3.16 1.83 2.00 2.33 3.00 2.00 2.66 1.50 1.83
LR (0.21) (0.22) (0.50) (0.00) (0.59) (0.30) (0.57) (0.16) (0.54) (0.16) (0.60) (0.36) (0.71) (0.36) (0.63) (0.20) (0.33) (0.30)
60-cm aquarium
[10.0] [5.0] [15.0] [20.0] [19.2] [22.9] [20.0] [29.2] [22.9] [32.9] [19.2] [20.0] [24.2] [30.0] [20.0] [27.9] [15.0] [19.2]
0.16 0.16 1.66 2.33 1.50 2.00 1.66 2.83 2.16 3.16 1.66 2.33 2.50 2.83 2.00 2.66 1.50 2.00
(0.16) (0.16) (0.21) (0.21) (0.22) (0.25) (0.21) (0.16) (0.16) (0.16) (0.33) (0.42) (0.22) (0.16) (0.00) (0.21) (0.22) (0.25)
FC [2.9] [2.9] [17.9] [24.2] [15.0] [20.0] [17.9] [29.2] [22.9] [32.9] [17.9] [24.2] [25.0] [29.2] [20.0] [27.9] [15.0] [20.0]
0.16 0.16 1.33 1.50 1.50 1.83 1.66 2.16 2.00 2.50 1.33 1.50 2.16 2.33 1.83 2.16 1.33 1.50
LR (0.16) (0.16) (0.42) (0.55) (0.49) (0.74) (0.42) (0.72) (0.63) (0.49) (0.49) (0.42) (0.54) (0.61) (0.60) (0.64) (0.61) (0.66)
90-cm aquarium
Mean (S.E.) no. and [%] of snails in central 15-cm zone around pellet, in assays involving:
[2.9] [2.9] [14.2] [15.0] [15.0] [19.2] [17.9] [22.9] [20.0] [25.0] [14.2] [15.0] [22.9] [24.2] [19.2] [22.9] [14.2] [15.0]
TABLE. Distributions of field-collected (FC) and laboratory-reared (LR) Lymnaea acuminata around bait pellets, in assays in aquaria measuring 45, 60 or 90 cm in diameter
SNAIL RESPONSES TO BAIT PELLETS
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the Table. Of the four carbohydrates tested in the bait pellets, starch attracted the highest percentages of the test snails, whether the snails were field-collected (54.2%, 35.0% and 32.9% attracted, after 2 h, in the 45-, 60- and 90-cm aquaria, respectively) or laboratory-reared (with corresponding values of 60.0%, 32.9% and 25.0%, respectively). Of the amino acids tested, serine gave the best results in 45-cm aquaria (44.2% of field-collected snails attracted after 2 h) whereas proline attracted the highest percentages of snails in the 60-cm (34.2% of field-collected snails after 2 h) and 90-cm aquaria (29.2% of field-collected snails after 2 h). The carbohydrate or amino acid used in the bait pellet caused significant variation in the number of snails reaching the central (15-cm diameter) circle in each aquarium, after both 1 h (the field-collected snails in 45-, 60- and 90-cm aquaria giving F2,135530.24, F8,1355 12.31 and F16,13555.58, respectively, and the laboratory-reared snails giving corresponding values of F2,135551.49, F8,13558.82 and F16,13555.91, respectively; P50.01 for each) and 2 h (the field-collected snails in 45-, 60and 90-cm aquaria giving F2,135547.79, F8,135530.41 and F16,135517.56, respectively, and the laboratory-reared snails giving corresponding values of F2,135578.79, F8,135518.84 and F16,135511.75; P50.01 for each). Curiously, the results for the field-collected snails differed significantly from those of the laboratory-reared snails only when the 45-cm aquaria were used (i.e. when the initial test distance was relatively short, giving F16,85 values of 15.44 after 1 h and 17.34 after 2 h, with the laboratory-reared snails generally appearing more attracted to the test pellets; P50.01 for each) and not when the larger aquaria were employed.
DISCUSSION Starch — which, in the present study, appeared to be the most attractive of the
carbohydrates tested, irrespective of the initial test distance — is the major storage carbohydrate of the aquatic plants that, in healthy growth or in decay, constitute essential elements in the nutrition of snails (Madsen, 1992; Abd-El-Hamid, 1996; Tiwari and Singh, 2004a; Singh and Singh, 2008). Serine was only the most attractive amino acid when the initial test distance was relatively short (22.5 cm), probably because it is the smallest of the amino acids tested and diffused away from the bait pellets most quickly. It appears that adult L. acuminata are capable of differentiating between some types of carbohydrates and amino acids included in bait pellets, even when the pellets are initially 45 cm away from the snails. Further research to optimise the attractiveness of bait pellets to vector snails, especially that of pellets that contain molluscicide (which may counteract the attractiveness of other components) is clearly merited.
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