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Behavioral Responses of the Snail Lymnaea acuminata towards Photo and Chemo Attractants: A New Step in Control Program of Fasciolosis

Behavioral Responses of the Snail Lymnaea acuminata towards Photo and Chemo Attractants: A New... Hindawi Publishing Corporation International Journal of Zoology Volume 2013, Article ID 439276, 6 pages http://dx.doi.org/10.1155/2013/439276 Research Article Behavioral Responses of the Snail Lymnaea acuminata towards Photo and Chemo Attractants: A New Step in Control Program of Fasciolosis Anupam Pati Tripathi, V. K. Singh, and D. K. Singh Malacology Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh 273 009, India Correspondence should be addressed to D. K. Singh; dksingh gpu@yahoo.co.in Received 21 February 2013; Revised 13 October 2013; Accepted 25 November 2013 Academic Editor: Roger P. Croll Copyright © 2013 Anupam Pati Tripathi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fasciolosis is water and food borne disease, caused by Fasciola hepatica and F. gigantica.Snail Lymnaea acuminata is an intermediate host of these uk fl es. Snail control is one of the major methods to reduce the incidences of fasciolosis. Trapping of snails with the help of photo- and chemoattractants for treatment purposes will be a new tool in control program of fasciolosis. eTh present study shows that maximum numbers of snails were attracted (52 to 60%), when exposed to photo- and chemostimulant simultaneously, rather than when only chemo- (control) (18 to 24%) or photo- (control) (14 to 19%) stimulus was given. Maximum change in AChE activity in nervous tissue was observed when red monochromatic light was used (258.37% of white light control) as opposed to blue (243.44% of white light control) and orange (230.37% of white light control). eTh exposure of light directly stimulated the photoreceptors in eye which transmit the signals through nerves to the brain and snail response accordingly. In this signal transmission AChE is one of the important enzymes involved in this process. 1. Introduction Pradesh, India, a survey was conducted by Singh and Agarwal [5] which revealed that 94% buffaloes in local slaughter The fasciolosis is considered as water and food borne disease houses arehighlyinfectedby F. gigantica.Snail L. acuminata [1, 2]. Fasciolosis and other food borne trematodiases were acts as an intermediate host for this u fl ke . Annual economic added to the list of important helminthiases with a great losses caused by this disease are estimated to be US$2 billion, impact on human development, at the Third Global Meeting due to damaged livers, reduced milk yield, fertility disorders of the Partners for Parasite Control, held in WHO Head- and reduced meat production [1]. This disease affects the quarters, Geneva, in November 2004 [3]. Moreover, present general immune status of the animal and there is no accurate climate and global changes appeared to increasingly affect method of early diagnosis, before the time of egg deposition those snail borne helminthiases, which are heavily dependent adopted [6]. on the environment for dissemination. Fasciolosis is a good The estimates suggest that about 2.4 to 40 million people example of an emerging/reemerging parasitic disease in many are infected with Fasciola hepatica and 180 million are at the countries, as a consequence of many phenomena related to risk in the world [1, 7, 8]. The largest numbers of infected both environmental changes and man-made modifications. people have been reported from Bolivia, China, Ecuador, The Lymnaeidae and Planorbidae are families of snail Egypt, France, Islamic Republic of Iran, Peru, and Portugal that serve as intermediate hosts to a large number of species [6]. Hillyer and Apt [9] estimated that over 350,000 humans of Fasciola, distributed worldwide. Lymnaeidae serves as an are infected in the Bolivian Altiplano alone. Fasciolosis is intermediate host of at least 71 trematode species, distributed among 13 families, with the implication for cattle and human a domestic animal disease but now increasingly aeff cts the health [4]. In Gorakhpur district of eastern part of Uttar humans. Ramachandran et al. [10]has reported human 2 International Journal of Zoology Fasciolosis in Arunanchal Pradesh and West Bengal in India, ML for the rfi st time. Snail population management is a perfect tool to control fasciolosis because gastropods represent the weakest link L1 in the life cycle of trematode [11]. Pulmonates sense their foods by the tentacles [12]. Food initiates appetitive behavior, which generally includes orientation and locomotion towards it. es Th e movements may be similar to those in response L2 to other attractive stimuli, such as potential mates. Contact with food is sensed by both mechano- and chemoreceptors [13], which initiates the next phase of feeding [14]. Similarly, visible light colour is another stimulus, which oeff rs a variety of information that enables animals to orient themselves to their surroundings [15]. One specific feature of the visual information is the wavelength difference of light emitted from light source or reflected from objects which acts as an ML attractant due to the presence of different types of photore- Diameter 70 cm ceptors in the eyes of Lymnaea [16]. In water, too, direct light is relatively richer in short wave light. Neglecting the finer differences in the spectral content of light, this general distinction between direct and reflected light could be the cue that allows an animal to differentiate such basically dieff rent habitats as open spaces, shadowed areas, space rich in food, hiding place, and so forth [15]. Photosensitive neurons in invertebrate functions independently are in integration with 22.00 cm external visual sense organ such as eye, which controls the Figure 1: Design of photoresponse experiments, where ML = behavior of snail [15]. u Th s, the present study will focus on monochromatic light was produced with the help of spectropho- the combination of photo- and chemoattractants to be an tometer behind the interference colour filter by xenon lamp, F = eecti ff ve tool in collection of the snail’s population in certain interference colour filter, and L1 and L2 = lenses. areas. eTh reafter, effective killing can be done by any of the snail control methods or larvae of the Fasciola inside the snail body canbedonebyphytotherapyofsnails[17]. Certainly, it will be an added advantage in fasciolosis control programme. Dr. Ravi Shanker Singh (Associate professor, Department of Physics, D.D.U Gorakhpur University, Gorakhpur, India). Experiments were performed in daytime between 8 AM and 2. Materials and Methods 2 PM. Six replicates of ten snails (size2.5±0.5 cm) were used for each set of experiments. No individual animal was used 2.1. Collection of Animals. Snail Lymnaea acuminata were repeatedly in any of the experiment. Each experiment was collected from Ramgarh Lake at Gorakhpur (latitude ∘ 󸀠 ∘ 󸀠 performed using the laboratory acclimatized snails. 26 46 Nand longitude83 22 E) andacclimatizedin A glass aquarium of 70 cm diameter and 15 cm height was laboratory conditions for 72 hours. eTh experimental animals filled with water at height of 10 mm. This arrangement was were kept in a glass aquaria containing dechlorinated tap made to neutralize the heat eeff ct of light. Each aquarium water at room temperature (22–25 C). Temperature was was stood on a piece of white paper to make clear visibility of regularly measured by thermometer during experiment. snail movement. Ten individually marked snails were kept at Water was changed once every 24 hour and dead animals adistanceof22cmontheperipheryofglassaquariumineach were removed to prevent the water from being contaminated replicate. Single visible monochromatic light wavelength was by decaying tissue. used in the center of aquarium as shown in Figure 1.Location (distance travelled towards light source of each marked snail) 2.2. Design of Photoresponse Experiments. Xenon arc lamp and number of snails were then noted aer ft every 15 up to (500W) wasusedasvisible lightsource. Spectral response 60 minutes in each light spectrum. Distance traveled towards from 400 nm to 650 nm were produced with the help of each vertically placed light source was measured on the spectrophotometer behind the interference colour filters. graduated path towards light source. Each observation for Lightintensity wasmeasuredbehindeachfilter andthen single monochromatic spectral range was replicated six times. output of light is adjusted to get the same intensity. Exposure In this way for each value (mean± S.E.) of attracted snails of monochromatic light at 500 ux fl for 15 up to 60 minutes was to single spectral range, 60 snails were used. During these used to study its effect on snails movement [ 18]. The pathway observations aquaria were maintained at room temperature of monochromatic light is given in Figure 1. (22–25 C) and covered with dark black cloth. Intensity of eTh protocol of the monochromatic light production different monochromatic light at the center of aquarium was device in the present study is designed with the help of measured by digital ux fl meter. In control experiment no light International Journal of Zoology 3 (negativecontrol)andwhitelight(positivecontrol)wereused acetylthiocholine. The absorbance change in optical density for snail attraction. at 412 nm was continuously observed on spectrophotometer for3minutesat25 Cwhich is ameasure of yellow product formed due to the hydrolysis of DTNB. us, Th the intensity of 2.3. FormationofAttractantFood Pellets(AFP). Pellets of the colour is directly proportional to the acetylcholinesterase carbohydrates (starch) and amino acids (proline and serine) activity. Enzyme activity has been expressed as𝜇 moles SH containing agar-agar were prepared at the concentration of hydrolyzed/minute/mg protein. 20 mM of starch and proline and serine were separately added in 100 mL of 2% agar solution by the method of Protein. Protein was estimated in the enzyme source super- Tiwari and Singh [19]. eTh se preparations were spread to a natant by the method of Lowry et al. [22]. 1.0 mL of the uniform thickness 5 mm and aer ft cooling attractant food enzyme supernatantwas takenand mixedin5.0mL of 5% pellets were cut out with the corer in 5 mm diameter. es Th e TCA and centrifuged at 6000×gfor 20 minutes. eTh precip- pellets were used for the evaluation of behavioral responses itatewas washed with 5.0mLof5%TCA andcentrifuged at of food preference against the snail L. acuminata. thesamespeed for20minutes.Theprecipitate wasdissolved in 4.0 mL of NaOH 1.0 mL of diluted supernatant was mixed 2.4. Design of Chemoresponse Experiments. Chemoresponse with 5.0 mL of reagent—C. Reagent—C was prepared by the studies of the food preference of starch, proline, and serine addition of 50.0 mL of reagent—A (2% sodium carbonate against L. acuminata were made in a clean glass aquarium in 0.1 N NaOH) and 1.0 mL of reagent—B (1% of sodium having a diameter of 70 cm. Aquarium was filled with potassium tartrate and 0.5% copper sulphate, mixed in 1 : 1 water at height of 10 mm. This arrangement was made to ratio at the time of experiment) and mixture was kept for 10 neutralize the heat eeff ct of light. The food pellets containing minutes for room temperature. eTh n 0.5 mL of reagent—D serine/proline/starch (size 5 mm × 5mm × 5 mm) were (freshly prepared phenol reagent and distilled water in ratio kept in the center of glass aquarium and ten individually 1 : 2) was added and mixed thoroughly. After 10 minutes blue marked snails in each replicatewereplacedatanequal colour developed. eTh resulting blue colour was monitored distance of 22 cm on the periphery of aquarium. Number in absorbance at 600 nm aeft r 10 minutes. Values have been of attracted snails and their distance traveled towards the expressed as𝜇 g protein/mg nervous tissue. food source were noted after every 15 up to 60 minutes. The same processisrepeatedagain with vertically givenvisible 2.6. Data Analysis. Onewayandtwowayanalysesofvariance light for the observation of the behavior of snails with the and𝑡 -tests were applied between the obtained data to observe combination of food pellets (chemoresponse) and visible any signicfi ant variation [ 23]. light (photoresponse) as shown in Figure 1.Duringthese observations aquaria were maintained at room temperature (22–25 C) and covered by dark black cloth with a ne fi hole in 3. Results the centre for light source. In control experiment (no light stimulus is given) there was no movement of snails towards the central position of 2.5. Enzyme Assays In Vivo. The adult snails were collected aquarium. Exposure of red light source for 15 minutes in locally and allowed to acclimatize at 25 Cfor 72h. Snails were the center of aquarium evoked maximum attraction of snails exposed to different spectra of visible light (violet, blue, green, (14.88%) from periphery of aquarium. Prolongation of light yellow,orange, andred)at500 ufl xofintensity for15minutes stimulus for 60 minutes caused significantly higher attraction of exposure period. of snails (19.73%) towards the center (Figure 2). Significant Acetylcholinesterase (AChE). Acetylcholinesterase (AChE) variation in the number of attracted snails between the 15 activity in the nervous tissue of L. acuminata was measured and 60 minutes in violet light stimulus is also noted. Lowest according to the method of Ellman et al. [20]asmodiefi d attraction of snails was observed in green light in 15 (6.28%) by Singh and Agarwal [21]. The substrate acetylthiocholine and 60 (8.91%) minutes. In chemoresponse experiment 15- iodide (ACThI) was hydrolyzed into acetic acid and choline minute exposure of bait caused maximum attraction of snails by the catalytic action of an enzyme acetylcholinesterase. (18.04%) when serine was used as attractant in bait. Proline During hydrolysis, free SH-group is released from the containing baits caused less attraction of snails (12.92%). thioesters of choline which hydrolyzed the dye DTNB to However, prolongation of proline chemo- (bait) stimulus for form the yellow product. One mole of dye is formed for 60 minutes caused significantly higher attraction of snails each mole of substrate hydrolyzed. The nervous tissue was (30.00%) as compared to serine (24.19%) (Figure 2). Bait homogenized (50 mg/mL) in 0.1 M phosphate buffer (pH- containing starch attracted maximum number of snails in 60 8.0) for 5 minutes in an ice bath and centrifuged at 1000×g minutes (24.09%) rather than 15 minutes (Figure 2). for 30 minutes at 4 C. The clear supernatant was taken Attraction of snails was increased significantly ( 𝐹 = (2,13) as the enzyme source. The enzyme activity was measured 15.64,13.23) in the center of aquarium when combinations of in a 10 mm path length cuvette using incubation mixture photo- (light) and chemo- (bait) stimulus were used simulta- consisting of 0.1 mL of enzyme source, 2.9 mL of 0.1 M phos- neously. The maximum attraction (52.73%) was observed in phate buffer (pH-8.0) 0.1 mL of chromogenic agent DTNB red light + baits containing serine in 15 minutes. Prolongation (5,5-dithiobis-2 nitrobenzoate), and 0.2 mL freshly prepared of photo- and chemostimulus for 60 minutes also caused 4 International Journal of Zoology Ser = serine; Pro = proline; St = starch Photo- and chemostimulus 15 minutes 60 minutes Figure 2: Percentage of attracted snail Lymnaea acuminata towards the bait and vertically exposed monochromatic light at different time intervals. Significant when one way/two way ANOVA was applied between percentage of attracted snails towards the light sources at different time intervals. Table 1: Acetylcholinesterase (AChE) activity in nervous tissue of snails L. acuminata exposed to visible monochromatic light (500 u fl x) source placed vertically in the center of aquarium at 15-minute exposure periods. Acetylcholinesterase (AChE) (𝜇 mol SH-hydrolyzed/minute/mg-protein) Monochromatic light (wavelength) Light ux fl 15-minute Percent change Violet (400) 500 0.89±0.07 148.25 Blue (450) 500 1.46±0.06 243.44 Green (510) 500 0.59±0.01 98.33 Yellow (570) 500 1.03±0.01 171.47 Orange (590) 500 1.38±0.01 230.37 Red (650) 500 1.55±0.03 258.33 Control (white light) 500 0.60±0.01 100.00 Control (no light) 500 0.57±0.01 — Each value is the mean of six replicates. Enzyme activity is expressed as𝜇 mol SH-hydrolyzed/minute/mg-protein. Percent change in activity is calculated with respect to control (white light). Significant when student 𝑡 -test was applied between the intensity of visible monochromatic light and control value. significantly higher attraction of snails in red light + bait acquisition of information with their sensory systems, with containing serine (60.00) (Figure 2). Significant variation in the photoreceptors in the retina as the main element for the thenumberofattracted snails between15minutes (44.06%) visual information. The optics of eye of gastropod molluscs and 60 minutes (51.74%) in violet light + bait containing vary greatly and range from a pigmented pit without a lens serine was noted with respect to bait containing proline (15 to sophisticatedeyeswithhardspherical lenses andimage minute 45% and 60 minute 55.75%) and starch (15 minute forming capacity [24]. 30% and 60 minute 36.26%) (Figure 2). The lowest attraction The present study clearly demonstrates that there are two of snails was observed in green light with different baits. dieff rent orientations of L. acuminata in relation to light A measurement of enzyme activity in the nervous tissue that is high photoorthokinesis and positive phototaxis. In of snails exposed to the monochromatic light source for experiments where no light stimulus is given (control) there 15 minute shows that the AChE activity was increased 2.5 was no movement of snails towards the center of aquarium. times in red light exposed snails when compared to the Earlier, this type of behavior has been reported in snail control group (Table 1). Maximum change in AChE activity Biomphalaria glabrata [25]. Although exposure of different in nervous tissue was observed when red monochromatic wavelength of light stimulus initiates the movement of snails light was exposed (258.37% of control) than blue (243.44% towards the light sources, more attraction of L. acuminata of control) and orange (230.37% of control). towards the red light indicates more sensitivity of these snails to higher spectral wavelength. It has been reported that L. stagnalis hasatwotypeofocularphotoreceptors 4. Discussion [16]. Type-A photoreceptor had more spectral sensitivity between 480 and 500 nm and Type-T photoreceptor had a Animals sustain their life by accumulating the necessary energy from their environment, requiring the continuous much broader spectral sensitivity between 450 and 600 nm. Attracted snails (%) No light White light Serine Proline Starch Violet Violet + Ser Violet + Pro Violet + St Blue Blue + Ser Blue + Pro Blue + St Green Green + Ser Green + Pro Green + St Yellow Yellow + Ser Yellow + Pro Yellow + St Orange Orange + Ser Orange + Pro Orange + St Red Red + Ser Red + Pro Red + St International Journal of Zoology 5 Type-T photoreceptor was more light sensitive than Type- affected by AChE triggers the release of 5-HT in snail L. A photoreceptor. It may be possible that higher attraction at acuminata. Interaction between cholinergic and other kinds higher wavelength, that is, red and violet may be due to the of neurons in snail is still not known. However, higher presence of more Type-T receptor in L. acuminata.Type-T activity of AChE in the light stimulated snail indicates that photoreceptor cells send processes to the cerebral ganglion it acts independently or through tryptaminergic neurons and terminate close to the end of statocyst hair cells. The hair in photoreception, which ultimately affects the behavior of cells send their terminal branches to cerebral ganglion close snails. Although there is no report on the eeff ct of photic to the termination of Type-T photoreceptor cells [16]. stimulus on AChE, yet the present study clearly demonstrates There was a significant variation in the number of that photostimulus alter the physiological status of snail attracted snails at 15- and 60-minute exposure of different towards red light is due to higher activity of AChE in exposed wavelength of light stimulus. It clearly indicates that variation snail. in wavelength of light stimulus evokes dieff rently to the The present study clearly indicates that with the help of photoreceptors of snails, so that there is a signicfi ant variation photo- and chemostimuli, trapping of snails in certain area in the number of attracted snails by them. Higher attraction is possible. In this way phytotherapy of trapped snails as of snails towards light source as exposure period prolongs suggested by the Sunita and Singh [17]for killing Fasciola from 15 to 60 minutes indicates that variation in exposure larva in snail body or trapped snails can be killed by the time has significant eeff ct on the attraction of L. acuminata treatment of eeff ctive molluscicides [ 36]. The trapping of towards light source because the conversion of rhodopsin to snails with the help of photo- and chemoattractant and there metarhodopsin and vice versa takes several minutes to hours treatment for eecti ff ve control of snails/ Fasciola larva will be depending upon the species [26, 27]. a new tool in fasciolosis control program. Similarly, feeding consists of a variable sequence of food-finding movements, followed by a series of rhythmic 5. Conclusion movements in which food is consumed. Both food-n fi ding and consummatory movements are modulated in response On the basis of the above experimental data, it can be to different foods, in response to change in internal state, or conclusively noticed that the snails have a capacity to monitor as aresultofpreviouslearningassuggested by Sunada et al. photo- and chemostimuli. This behavioral response of snail [28]. The tentacles and lip nervous contain the axon primary will be a tool in their trapping and can be used to reduce the chemosensory neurons which sense food in environments occurrence of fasciolosis by killing them. [29]. eTh study of the behavioral response of Lymnaea acumi- References nata towards photo- and chemostimulus demonstrates that the snail L. acuminata was significantly more attracted [1] M. S. Mas-Coma, J. G. Esteban, and M. D. 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Behavioral Responses of the Snail Lymnaea acuminata towards Photo and Chemo Attractants: A New Step in Control Program of Fasciolosis

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Hindawi Publishing Corporation International Journal of Zoology Volume 2013, Article ID 439276, 6 pages http://dx.doi.org/10.1155/2013/439276 Research Article Behavioral Responses of the Snail Lymnaea acuminata towards Photo and Chemo Attractants: A New Step in Control Program of Fasciolosis Anupam Pati Tripathi, V. K. Singh, and D. K. Singh Malacology Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh 273 009, India Correspondence should be addressed to D. K. Singh; dksingh gpu@yahoo.co.in Received 21 February 2013; Revised 13 October 2013; Accepted 25 November 2013 Academic Editor: Roger P. Croll Copyright © 2013 Anupam Pati Tripathi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fasciolosis is water and food borne disease, caused by Fasciola hepatica and F. gigantica.Snail Lymnaea acuminata is an intermediate host of these uk fl es. Snail control is one of the major methods to reduce the incidences of fasciolosis. Trapping of snails with the help of photo- and chemoattractants for treatment purposes will be a new tool in control program of fasciolosis. eTh present study shows that maximum numbers of snails were attracted (52 to 60%), when exposed to photo- and chemostimulant simultaneously, rather than when only chemo- (control) (18 to 24%) or photo- (control) (14 to 19%) stimulus was given. Maximum change in AChE activity in nervous tissue was observed when red monochromatic light was used (258.37% of white light control) as opposed to blue (243.44% of white light control) and orange (230.37% of white light control). eTh exposure of light directly stimulated the photoreceptors in eye which transmit the signals through nerves to the brain and snail response accordingly. In this signal transmission AChE is one of the important enzymes involved in this process. 1. Introduction Pradesh, India, a survey was conducted by Singh and Agarwal [5] which revealed that 94% buffaloes in local slaughter The fasciolosis is considered as water and food borne disease houses arehighlyinfectedby F. gigantica.Snail L. acuminata [1, 2]. Fasciolosis and other food borne trematodiases were acts as an intermediate host for this u fl ke . Annual economic added to the list of important helminthiases with a great losses caused by this disease are estimated to be US$2 billion, impact on human development, at the Third Global Meeting due to damaged livers, reduced milk yield, fertility disorders of the Partners for Parasite Control, held in WHO Head- and reduced meat production [1]. This disease affects the quarters, Geneva, in November 2004 [3]. Moreover, present general immune status of the animal and there is no accurate climate and global changes appeared to increasingly affect method of early diagnosis, before the time of egg deposition those snail borne helminthiases, which are heavily dependent adopted [6]. on the environment for dissemination. Fasciolosis is a good The estimates suggest that about 2.4 to 40 million people example of an emerging/reemerging parasitic disease in many are infected with Fasciola hepatica and 180 million are at the countries, as a consequence of many phenomena related to risk in the world [1, 7, 8]. The largest numbers of infected both environmental changes and man-made modifications. people have been reported from Bolivia, China, Ecuador, The Lymnaeidae and Planorbidae are families of snail Egypt, France, Islamic Republic of Iran, Peru, and Portugal that serve as intermediate hosts to a large number of species [6]. Hillyer and Apt [9] estimated that over 350,000 humans of Fasciola, distributed worldwide. Lymnaeidae serves as an are infected in the Bolivian Altiplano alone. Fasciolosis is intermediate host of at least 71 trematode species, distributed among 13 families, with the implication for cattle and human a domestic animal disease but now increasingly aeff cts the health [4]. In Gorakhpur district of eastern part of Uttar humans. Ramachandran et al. [10]has reported human 2 International Journal of Zoology Fasciolosis in Arunanchal Pradesh and West Bengal in India, ML for the rfi st time. Snail population management is a perfect tool to control fasciolosis because gastropods represent the weakest link L1 in the life cycle of trematode [11]. Pulmonates sense their foods by the tentacles [12]. Food initiates appetitive behavior, which generally includes orientation and locomotion towards it. es Th e movements may be similar to those in response L2 to other attractive stimuli, such as potential mates. Contact with food is sensed by both mechano- and chemoreceptors [13], which initiates the next phase of feeding [14]. Similarly, visible light colour is another stimulus, which oeff rs a variety of information that enables animals to orient themselves to their surroundings [15]. One specific feature of the visual information is the wavelength difference of light emitted from light source or reflected from objects which acts as an ML attractant due to the presence of different types of photore- Diameter 70 cm ceptors in the eyes of Lymnaea [16]. In water, too, direct light is relatively richer in short wave light. Neglecting the finer differences in the spectral content of light, this general distinction between direct and reflected light could be the cue that allows an animal to differentiate such basically dieff rent habitats as open spaces, shadowed areas, space rich in food, hiding place, and so forth [15]. Photosensitive neurons in invertebrate functions independently are in integration with 22.00 cm external visual sense organ such as eye, which controls the Figure 1: Design of photoresponse experiments, where ML = behavior of snail [15]. u Th s, the present study will focus on monochromatic light was produced with the help of spectropho- the combination of photo- and chemoattractants to be an tometer behind the interference colour filter by xenon lamp, F = eecti ff ve tool in collection of the snail’s population in certain interference colour filter, and L1 and L2 = lenses. areas. eTh reafter, effective killing can be done by any of the snail control methods or larvae of the Fasciola inside the snail body canbedonebyphytotherapyofsnails[17]. Certainly, it will be an added advantage in fasciolosis control programme. Dr. Ravi Shanker Singh (Associate professor, Department of Physics, D.D.U Gorakhpur University, Gorakhpur, India). Experiments were performed in daytime between 8 AM and 2. Materials and Methods 2 PM. Six replicates of ten snails (size2.5±0.5 cm) were used for each set of experiments. No individual animal was used 2.1. Collection of Animals. Snail Lymnaea acuminata were repeatedly in any of the experiment. Each experiment was collected from Ramgarh Lake at Gorakhpur (latitude ∘ 󸀠 ∘ 󸀠 performed using the laboratory acclimatized snails. 26 46 Nand longitude83 22 E) andacclimatizedin A glass aquarium of 70 cm diameter and 15 cm height was laboratory conditions for 72 hours. eTh experimental animals filled with water at height of 10 mm. This arrangement was were kept in a glass aquaria containing dechlorinated tap made to neutralize the heat eeff ct of light. Each aquarium water at room temperature (22–25 C). Temperature was was stood on a piece of white paper to make clear visibility of regularly measured by thermometer during experiment. snail movement. Ten individually marked snails were kept at Water was changed once every 24 hour and dead animals adistanceof22cmontheperipheryofglassaquariumineach were removed to prevent the water from being contaminated replicate. Single visible monochromatic light wavelength was by decaying tissue. used in the center of aquarium as shown in Figure 1.Location (distance travelled towards light source of each marked snail) 2.2. Design of Photoresponse Experiments. Xenon arc lamp and number of snails were then noted aer ft every 15 up to (500W) wasusedasvisible lightsource. Spectral response 60 minutes in each light spectrum. Distance traveled towards from 400 nm to 650 nm were produced with the help of each vertically placed light source was measured on the spectrophotometer behind the interference colour filters. graduated path towards light source. Each observation for Lightintensity wasmeasuredbehindeachfilter andthen single monochromatic spectral range was replicated six times. output of light is adjusted to get the same intensity. Exposure In this way for each value (mean± S.E.) of attracted snails of monochromatic light at 500 ux fl for 15 up to 60 minutes was to single spectral range, 60 snails were used. During these used to study its effect on snails movement [ 18]. The pathway observations aquaria were maintained at room temperature of monochromatic light is given in Figure 1. (22–25 C) and covered with dark black cloth. Intensity of eTh protocol of the monochromatic light production different monochromatic light at the center of aquarium was device in the present study is designed with the help of measured by digital ux fl meter. In control experiment no light International Journal of Zoology 3 (negativecontrol)andwhitelight(positivecontrol)wereused acetylthiocholine. The absorbance change in optical density for snail attraction. at 412 nm was continuously observed on spectrophotometer for3minutesat25 Cwhich is ameasure of yellow product formed due to the hydrolysis of DTNB. us, Th the intensity of 2.3. FormationofAttractantFood Pellets(AFP). Pellets of the colour is directly proportional to the acetylcholinesterase carbohydrates (starch) and amino acids (proline and serine) activity. Enzyme activity has been expressed as𝜇 moles SH containing agar-agar were prepared at the concentration of hydrolyzed/minute/mg protein. 20 mM of starch and proline and serine were separately added in 100 mL of 2% agar solution by the method of Protein. Protein was estimated in the enzyme source super- Tiwari and Singh [19]. eTh se preparations were spread to a natant by the method of Lowry et al. [22]. 1.0 mL of the uniform thickness 5 mm and aer ft cooling attractant food enzyme supernatantwas takenand mixedin5.0mL of 5% pellets were cut out with the corer in 5 mm diameter. es Th e TCA and centrifuged at 6000×gfor 20 minutes. eTh precip- pellets were used for the evaluation of behavioral responses itatewas washed with 5.0mLof5%TCA andcentrifuged at of food preference against the snail L. acuminata. thesamespeed for20minutes.Theprecipitate wasdissolved in 4.0 mL of NaOH 1.0 mL of diluted supernatant was mixed 2.4. Design of Chemoresponse Experiments. Chemoresponse with 5.0 mL of reagent—C. Reagent—C was prepared by the studies of the food preference of starch, proline, and serine addition of 50.0 mL of reagent—A (2% sodium carbonate against L. acuminata were made in a clean glass aquarium in 0.1 N NaOH) and 1.0 mL of reagent—B (1% of sodium having a diameter of 70 cm. Aquarium was filled with potassium tartrate and 0.5% copper sulphate, mixed in 1 : 1 water at height of 10 mm. This arrangement was made to ratio at the time of experiment) and mixture was kept for 10 neutralize the heat eeff ct of light. The food pellets containing minutes for room temperature. eTh n 0.5 mL of reagent—D serine/proline/starch (size 5 mm × 5mm × 5 mm) were (freshly prepared phenol reagent and distilled water in ratio kept in the center of glass aquarium and ten individually 1 : 2) was added and mixed thoroughly. After 10 minutes blue marked snails in each replicatewereplacedatanequal colour developed. eTh resulting blue colour was monitored distance of 22 cm on the periphery of aquarium. Number in absorbance at 600 nm aeft r 10 minutes. Values have been of attracted snails and their distance traveled towards the expressed as𝜇 g protein/mg nervous tissue. food source were noted after every 15 up to 60 minutes. The same processisrepeatedagain with vertically givenvisible 2.6. Data Analysis. Onewayandtwowayanalysesofvariance light for the observation of the behavior of snails with the and𝑡 -tests were applied between the obtained data to observe combination of food pellets (chemoresponse) and visible any signicfi ant variation [ 23]. light (photoresponse) as shown in Figure 1.Duringthese observations aquaria were maintained at room temperature (22–25 C) and covered by dark black cloth with a ne fi hole in 3. Results the centre for light source. In control experiment (no light stimulus is given) there was no movement of snails towards the central position of 2.5. Enzyme Assays In Vivo. The adult snails were collected aquarium. Exposure of red light source for 15 minutes in locally and allowed to acclimatize at 25 Cfor 72h. Snails were the center of aquarium evoked maximum attraction of snails exposed to different spectra of visible light (violet, blue, green, (14.88%) from periphery of aquarium. Prolongation of light yellow,orange, andred)at500 ufl xofintensity for15minutes stimulus for 60 minutes caused significantly higher attraction of exposure period. of snails (19.73%) towards the center (Figure 2). Significant Acetylcholinesterase (AChE). Acetylcholinesterase (AChE) variation in the number of attracted snails between the 15 activity in the nervous tissue of L. acuminata was measured and 60 minutes in violet light stimulus is also noted. Lowest according to the method of Ellman et al. [20]asmodiefi d attraction of snails was observed in green light in 15 (6.28%) by Singh and Agarwal [21]. The substrate acetylthiocholine and 60 (8.91%) minutes. In chemoresponse experiment 15- iodide (ACThI) was hydrolyzed into acetic acid and choline minute exposure of bait caused maximum attraction of snails by the catalytic action of an enzyme acetylcholinesterase. (18.04%) when serine was used as attractant in bait. Proline During hydrolysis, free SH-group is released from the containing baits caused less attraction of snails (12.92%). thioesters of choline which hydrolyzed the dye DTNB to However, prolongation of proline chemo- (bait) stimulus for form the yellow product. One mole of dye is formed for 60 minutes caused significantly higher attraction of snails each mole of substrate hydrolyzed. The nervous tissue was (30.00%) as compared to serine (24.19%) (Figure 2). Bait homogenized (50 mg/mL) in 0.1 M phosphate buffer (pH- containing starch attracted maximum number of snails in 60 8.0) for 5 minutes in an ice bath and centrifuged at 1000×g minutes (24.09%) rather than 15 minutes (Figure 2). for 30 minutes at 4 C. The clear supernatant was taken Attraction of snails was increased significantly ( 𝐹 = (2,13) as the enzyme source. The enzyme activity was measured 15.64,13.23) in the center of aquarium when combinations of in a 10 mm path length cuvette using incubation mixture photo- (light) and chemo- (bait) stimulus were used simulta- consisting of 0.1 mL of enzyme source, 2.9 mL of 0.1 M phos- neously. The maximum attraction (52.73%) was observed in phate buffer (pH-8.0) 0.1 mL of chromogenic agent DTNB red light + baits containing serine in 15 minutes. Prolongation (5,5-dithiobis-2 nitrobenzoate), and 0.2 mL freshly prepared of photo- and chemostimulus for 60 minutes also caused 4 International Journal of Zoology Ser = serine; Pro = proline; St = starch Photo- and chemostimulus 15 minutes 60 minutes Figure 2: Percentage of attracted snail Lymnaea acuminata towards the bait and vertically exposed monochromatic light at different time intervals. Significant when one way/two way ANOVA was applied between percentage of attracted snails towards the light sources at different time intervals. Table 1: Acetylcholinesterase (AChE) activity in nervous tissue of snails L. acuminata exposed to visible monochromatic light (500 u fl x) source placed vertically in the center of aquarium at 15-minute exposure periods. Acetylcholinesterase (AChE) (𝜇 mol SH-hydrolyzed/minute/mg-protein) Monochromatic light (wavelength) Light ux fl 15-minute Percent change Violet (400) 500 0.89±0.07 148.25 Blue (450) 500 1.46±0.06 243.44 Green (510) 500 0.59±0.01 98.33 Yellow (570) 500 1.03±0.01 171.47 Orange (590) 500 1.38±0.01 230.37 Red (650) 500 1.55±0.03 258.33 Control (white light) 500 0.60±0.01 100.00 Control (no light) 500 0.57±0.01 — Each value is the mean of six replicates. Enzyme activity is expressed as𝜇 mol SH-hydrolyzed/minute/mg-protein. Percent change in activity is calculated with respect to control (white light). Significant when student 𝑡 -test was applied between the intensity of visible monochromatic light and control value. significantly higher attraction of snails in red light + bait acquisition of information with their sensory systems, with containing serine (60.00) (Figure 2). Significant variation in the photoreceptors in the retina as the main element for the thenumberofattracted snails between15minutes (44.06%) visual information. The optics of eye of gastropod molluscs and 60 minutes (51.74%) in violet light + bait containing vary greatly and range from a pigmented pit without a lens serine was noted with respect to bait containing proline (15 to sophisticatedeyeswithhardspherical lenses andimage minute 45% and 60 minute 55.75%) and starch (15 minute forming capacity [24]. 30% and 60 minute 36.26%) (Figure 2). The lowest attraction The present study clearly demonstrates that there are two of snails was observed in green light with different baits. dieff rent orientations of L. acuminata in relation to light A measurement of enzyme activity in the nervous tissue that is high photoorthokinesis and positive phototaxis. In of snails exposed to the monochromatic light source for experiments where no light stimulus is given (control) there 15 minute shows that the AChE activity was increased 2.5 was no movement of snails towards the center of aquarium. times in red light exposed snails when compared to the Earlier, this type of behavior has been reported in snail control group (Table 1). Maximum change in AChE activity Biomphalaria glabrata [25]. Although exposure of different in nervous tissue was observed when red monochromatic wavelength of light stimulus initiates the movement of snails light was exposed (258.37% of control) than blue (243.44% towards the light sources, more attraction of L. acuminata of control) and orange (230.37% of control). towards the red light indicates more sensitivity of these snails to higher spectral wavelength. It has been reported that L. stagnalis hasatwotypeofocularphotoreceptors 4. Discussion [16]. Type-A photoreceptor had more spectral sensitivity between 480 and 500 nm and Type-T photoreceptor had a Animals sustain their life by accumulating the necessary energy from their environment, requiring the continuous much broader spectral sensitivity between 450 and 600 nm. Attracted snails (%) No light White light Serine Proline Starch Violet Violet + Ser Violet + Pro Violet + St Blue Blue + Ser Blue + Pro Blue + St Green Green + Ser Green + Pro Green + St Yellow Yellow + Ser Yellow + Pro Yellow + St Orange Orange + Ser Orange + Pro Orange + St Red Red + Ser Red + Pro Red + St International Journal of Zoology 5 Type-T photoreceptor was more light sensitive than Type- affected by AChE triggers the release of 5-HT in snail L. A photoreceptor. It may be possible that higher attraction at acuminata. Interaction between cholinergic and other kinds higher wavelength, that is, red and violet may be due to the of neurons in snail is still not known. However, higher presence of more Type-T receptor in L. acuminata.Type-T activity of AChE in the light stimulated snail indicates that photoreceptor cells send processes to the cerebral ganglion it acts independently or through tryptaminergic neurons and terminate close to the end of statocyst hair cells. The hair in photoreception, which ultimately affects the behavior of cells send their terminal branches to cerebral ganglion close snails. Although there is no report on the eeff ct of photic to the termination of Type-T photoreceptor cells [16]. stimulus on AChE, yet the present study clearly demonstrates There was a significant variation in the number of that photostimulus alter the physiological status of snail attracted snails at 15- and 60-minute exposure of different towards red light is due to higher activity of AChE in exposed wavelength of light stimulus. It clearly indicates that variation snail. in wavelength of light stimulus evokes dieff rently to the The present study clearly indicates that with the help of photoreceptors of snails, so that there is a signicfi ant variation photo- and chemostimuli, trapping of snails in certain area in the number of attracted snails by them. Higher attraction is possible. In this way phytotherapy of trapped snails as of snails towards light source as exposure period prolongs suggested by the Sunita and Singh [17]for killing Fasciola from 15 to 60 minutes indicates that variation in exposure larva in snail body or trapped snails can be killed by the time has significant eeff ct on the attraction of L. acuminata treatment of eeff ctive molluscicides [ 36]. The trapping of towards light source because the conversion of rhodopsin to snails with the help of photo- and chemoattractant and there metarhodopsin and vice versa takes several minutes to hours treatment for eecti ff ve control of snails/ Fasciola larva will be depending upon the species [26, 27]. a new tool in fasciolosis control program. Similarly, feeding consists of a variable sequence of food-finding movements, followed by a series of rhythmic 5. Conclusion movements in which food is consumed. Both food-n fi ding and consummatory movements are modulated in response On the basis of the above experimental data, it can be to different foods, in response to change in internal state, or conclusively noticed that the snails have a capacity to monitor as aresultofpreviouslearningassuggested by Sunada et al. photo- and chemostimuli. This behavioral response of snail [28]. The tentacles and lip nervous contain the axon primary will be a tool in their trapping and can be used to reduce the chemosensory neurons which sense food in environments occurrence of fasciolosis by killing them. [29]. eTh study of the behavioral response of Lymnaea acumi- References nata towards photo- and chemostimulus demonstrates that the snail L. acuminata was significantly more attracted [1] M. S. Mas-Coma, J. G. Esteban, and M. D. 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