الفهرس 
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Abstract
Land snails and slugs are destructive agricultural pests causing economic damage to a wide variety of plants including vegetables, forage crops, tree fruits, shrubs, flowers, ground green cover and newly sown lawn grasses. Moreover they play an important role in transmitting and spreading diseases to cultivated plants.
In Egypt, the brown garden snail, Eobania vermiculata (Mollusca: Gastropoda: Stylommatophora: Helicidae) is considered one of the most predominant agricultural pests and is becoming a real threat to several vegetations including Orchard trees, vegetable crops as well as ornamental plants causing considerable damage to all plant parts. Thus it causes serious reduction in the yield production of crops and fruits as well as destroying plant seedlings.
In the current study, laboratory bioassays were carried out for evaluating the efficacy of certain plant products including Nicotine, Thymol, Menthol, Caffeine and Camphor as plant-origin molluscicides against E. vermiculata using the topical application method. The examined plant products can be arranged based on their potency against E. vermiculata snails in the following order: Nicotine > Thymol > Caffeine > Menthol > Camphor, with LD50 amounted to 204.02, 551.20, 932.37, 1209.08 and 1354.07 µg/snail, respectively.
The physiological and histological impacts of the most potent botanical materials (nicotine and thymol) were studied. Thus, snails under investigation were exposed to sublethal doses (LD50 and LD25) of nicotine and thymol using the topical application technique. Most of the estimated physiological indices were estimated along three post exposure time intervals (1, 7 and 15 days) as post treatment periods to evaluate the ability of the treated snails to eliminate the adverse and/or toxic effects of the applied materials with succession of post exposure time.
The obtained results can be summarized in the following points:
PHYSIOLOGICAL STUDIES
Clinical signs of toxicity
Behavioral signs of toxicity for each experimental group were noted directly after exposure. Most of the treated snails appeared inactive and remained retracted within their shells. Others were partially extended but their response to mechanical stimulation was slow. Also, excessive production of mucus was obvious in all cases. Haemolysis was recorded in snails treated with LD50 of both thymol and nicotine. Paralysis of the foot was recorded in some snails treated with either the low or high sublethal dose of nicotine.
Heart beat rate
1. The recorded values of heart beat rate of the control snails were 51.68±2.27, 53.04±1.03 and 49.25±3.28 beat/min at the three experimental periods, respectively.
2. Snails treated with LD50 Thymol exhibited significant decrease in their heart beat rate; while the rest of treatments caused marked elevation during the three experimental periods.
Oxygen consumption
1. The mean values of oxygen uptake of the control snails were 0.054±0.004, 0.054±0.002 and 0.056±0.003 ml/g wet. Wt/h at the three time intervals of estimation, respectively.
2. Only snails treated with LD50 Thymol showed significant decrease in their rate of oxygen uptake; while all the other treatments stimulated significant increase in the oxygen consumption along the three periods of estimation. This stimulation was most pronounced with the low sublethal doses of the both examined botanical molluscicides.
Biochemical parameters
a) Biochemical parameters of the haemolymph
1. Haemolymph of control snails contained 4.41±0.12, 5.11±0.13 and 4.91±0.19 mg/ml of total proteins, 1.88±0.09, 2.00±0.09 and 1.77±0.08 mg/ml of total lipids, 48.55±1.80, 49.65±2.04 and 43.44±1.55 mg/dl of total cholesterol and 44.51±0.99, 36.43±1.42 and 28.44±1.71 mg/ml of glucose at the three periods of estimation, respectively.
2. Exposure of snails to both examined materials caused general significant increase in levels of these parameters in most cases along the three post exposure periods.
3. The activities of ASAT, ALAT, ACP and ALP enzymes in the haemolymph of control snails were (21.22±0.86, 19.92±0.73, 20.04±0.45), (4.26±0.05, 4.26±0.05, 4.39±0.21), (2.17±0.08, 2.07±0.13, 2.48±0.14) and (57.74±2.87, 60.04±2.05 and 60.19±1.69) U/L at the three experimental periods, respectively.
4. Nicotine and thymol caused marked enhancement in the activities of ASAT, ALAT and ACP; while the activity of ALP exhibited a significant suppression in comparison with the equivalent untreated snails. In most cases, the levels of elevation or suppression were decreased gradually until recording their lowest values at the end of the post treatment period.
Biochemical parameters of the digestive gland
1. The values of total proteins, total lipids and glycogen content in the digestive gland of control snails were (71.46±1.20, 68.59±0.80, 70.26±0.46), (17.57±0.57, 16.80±0.24, 17.14±0.24) and (3.51±0.10, 3.35±0.05, 3.54±0.09) mg/g wet tissue at the three periods of estimation, respectively.
2. There was a very highly significant reduction in the mean value of total proteins, total lipids and glycogen content in the digestive gland of the snails exposed to either thymol or nicotine when compared with the equivalent controls at the three post exposure periods.
Electrophoretic banding patterns of digestive gland proteins:
The effect of sublethal doses (LD50 and LD25) of nicotine and thymol on the digestive gland proteins was observed by electrophoresis using SDS-PAGE after 15 days post exposure.
1. A total number of 27 protein fractions were separated from the digestive gland of the control snails with mol. Wt. ranging from 13.99 to 288.71 kDa.
2. The digestive gland proteins of LD50 thymol-treated snails were electrophoretically separated into 30 bands with mol. Wt. ranged between 12.33 and 291.94 kDa. Among them were 25 bands observed in the control and the rest, 5 bands, were newly formed bands.
3. The pattern of digestive gland proteins of snails treated with LD25 thymol showed 28 bands with mol. Wt. ranged between 12.05 and 290.32 kDa. Only 23 bands of the 27 present in the control could be detected, while the rest 4 bands characterizing the control disappeared. The pattern revealed appearance of 5 newly formed protein bands.
4. The digestive gland proteins of the snails treated with LD50 nicotine were electrophoretically separated into 27 bands with mol. Wt. ranged from 13.99 to 298.39 kDa. Only 24 bands of those found in the control remained, while the rest 3 bands disappeared. At the same time, 4 newly formed bands were detected.
5. Thirty bands were separated electrophoretically from the digestive gland homogenate proteins of snails treated with LD25 nicotine with mol. Wt. ranged between 12.42 and 291.94 kDa. Among them were 25 bands observed in the control and the rest, 5 bands, were newly formed.
6. Comparing the 4 electropherograms of digestive gland proteins of treated snails and that of the control revealed presence of 21 non-affected bands although there were variations in the densities of these bands. Also one band was disappeared completely as a result of treatment. Three newly formed bands were detected, in the control range.
HISTOLOGICAL STUDIES
1. Digestive gland
The normal digestive gland of E. vermiculata snails is a brownish-yellow bilobed voluminous organ. Each lobe appears surrounded with a thin coat and is formed of numerous branched and blind-ending tubules joined together by loose connective tissue containing haemolymphatic spaces. The lining epithelium of each tubule is made up of three types of cells: digestive cells, calcium cells and excretory cells.
The digestive gland of snails treated with sublethal doses of either thymol or nicotine showed many changes and alterations. These alterations included, shredding of the apical parts of lining epithelium of the digestive tubules, cytoplasmic vacuolation, some tubules became thinner than the normal ones and having dilated lumina, deformation of the nuclei of most cells, destroyed connective tissue, intertubular haemolymphatic infiltration, accumulation of massive excretory granules inside excretory cells, breakdown of the architecture of the digestive tubules, some tubules have undergone lysis and some of the degenerated tubules appeared encapsulated with fibrous tissue.
2. Ovotestis
Histological inspection of the ovotestis of the untreated E. vermiculata snails revealed that it is composed of numerous closely packed sacs, acini, in which male and female gametes and their supporting cells (Sertoli cells and follicular cells, respectively) develop.
The present results showed severe damages in the hermaphrodite gland of E. vermiculata snails post exposure to the sublethal doses of both tested materials. Hermaphrodite follicles lost their normal architecture with degeneration of all stages of gametogenesis.