Middle East Journal of Applied Volume : 07 | Issue :03 |July-Sept.| 2017 Sciences Pages: 595-612 ISSN 2077-4613

Ultrastructural alterations in cells of the digestive gland of (Muller) treated with three chemical compounds

Kamel Zaki Hemmaid1, Salem Abd-El Fattah Ahmed2 and Fatma Ibrahim El-akhrsay2

1Cytology and Histology, Zoology Department, Faculty of Science, Zagazig University, Egypt. 2Pesticides, Plant Protection, Research Institute, Agricultural Research Center Giza, Dokki, Egypt. 2Department of the Harmful to Agriculture, Plant Protection Research Institute, Agricultural Research Center, Giza, Dokki, Egypt. Received: 10 May 2017 / Accepted: 25 August 2017 / Publication date: 28 Sept. 2017

ABSTRACT

The Eobania vermiculata was treated with 1/2 LC50 and LC50 of Newmyl (insecticide), Vertimec (biocide) and Amino (fertilizer). The fine structure of cells comprising the digestive gland of control and treated snails was studied. Application of 1/2 LC50 of Newmyl resulted in cytoplasmic degeneration in digestive cells while the nucleoplasm was moderately osmiophilic and little heterochromatin clumps were seen. Mitochondria were pleomorphic and hypertrophied and showed ballooned cristae. Application of LC50 of Newmyl showed that the digestive cells were widely degenerated and harboring pleomerphic mitochondria. Nuclei of some cells were about to be pyknotic. Treatment of Eobania vermiculata with 1/2 LC50 of the fertilizer Amino led to deteriorations in cells of the digestive gland and appearance of cytoplasmic degenerations. Mitochondria showed a little hypertrophy with ballooned cristae. Nuclei revealed some enlargement with heterochromatin scattered in the nucleoplasm. Application of the LC50 of Amino resulted in higher alterations represented by wide degenerations in the cytoplasm of digestive cells. Mitochondria showed undiscrimination of their cristae. Nuclei displayed hypertrophy and harbor moderately osmiophilic nucleoplasm containing some scattered clumps of heterochromatin. Treatment with 1/2 LC50 of Vertimec biocide resulted in degenerative changes in the cytoplasm of the digestive cells. Mitochondria were crowded coalesced and pleomorphic and the matrix contained elongated darkly osmiophilic strands. The nuclei showed pale osmiophilic nucleoplasm and strongly osmiphilic heterochromatin clumps in addition to marginated aggregates of heterochromatin. Treatment with the LC50 of Vertimec revealed that the digestive cells showed wide cytoplasmic degenerative regions. Mitochondria were coalesced together and exhibited obvious pleomorphism. Nuclei were indented and the nucleaplasm was faintly osmiophilic. As for the excretory cells all treatments agreed in response but with different degrees of changes. The excretory cells which have large excretory vacuoles loaded with excretory granules displayed low to moderate osmiophilic excretory granules. The excretory vacuoles increased in size and contained variable size and shape of deeply osmiophilic granules. Due to the scarcity of calcium cells they were hardly illustrated in the digestive gland of treated snails.

Key words: Land snail, Eobania vermiculata, digestive gland, nucleoplasm, heterochromatin clumps Introduction

A lot of literature explored the abundance of studies investigating the toxicity of insecticide, biocides or fertilizers to snails (Ghamry et al., 1994; Aioub et al., 2000; Heiba et al., 2003; Hemmaid and Mohammadein, 2003; Abd El-Wahab, 2004; Ismail and Hegab, 2006; Genena and Mostafa, 2008; Ismail et al., 2010; Elshafy, 2011). Aioub et al. (2000) investigated the toxicological and pathological effects of seven pesticides on the digestive gland of the land snail Eobania vermiculata. Hemmaid and Mohammadein (2003) investigated ultrastructurally the effects of feeding the land snail, Eobania vermiculata with oxamyl in cells of the digestive gland. Salem et al. (2007) and Abd El-Aal (2007) evaluated the percent reduction of some pesticides against Eobania vermiculata. Methomyl recorded the highest reduction value. Corresponding Author: Kamel Zaki Hemmaid, Cytology and Histology, Zoology Department, Faculty of Science, Zagazig University, Egypt. 595 Middle East J. Appl. Sci., 7(3): 595-612, 2017 ISSN 2077-4613

Ali (2007) studied the molluscicidal activities of six insecticides on Eobania vermiculata and Monacha cartusiana. Poisonous baits of six pesticides were used to declare their toxicity action on Eobania vermiculata (Genena and Mostafa 2008). Newmyl was the most effective from 3 expertized insecticides on Eobania vermiculata (El-Sayed 2010). Vertimec biocide toxicity to Eobania vermiculata was also proved by Abd El-Wahab (2004) and Ismail and Hegab (2006). Likewise, Helmy et al. (2006) evaluated the activity of Vertimec on the same snail in the form of poisonous bait technique. Vertimec came secondly between 4 tested biocides in toxicological effect on Eobania vermiculata (Gabr et al. 2006). The normal ultrastructure of cells compositing the digestive gland of some gastropods had been investigated (Rebecchi et al., 19996; Taieb and Vicente, 1999; Lobo-da-Cunha, 2000; Arrighetti et al.; Nath et al., 2015). There was scarcity in studies concerned with the ultrasturcture of the cells of digestive gland of snails after chemical compounds treatment. However, some studies related with the effects of insecticides on cells of E.vermiculata digestive gland at the ultrastructural levels (Hemmaid and Mohammadein. 2003; Hamed et al., 2007). Therefore, the present study has been planned to investigate ulrastructuraly the effect of Vertimec. Amino and Newmyl (1/2 LC50 and LC50) on the fine structure of the digestive gland cells of Eobania vermiculate. Finally, it could be concluded that Vertimec biocide in comprise with the reference insecticide Newmyl proved to be nearly of the same efficacy in controlling E. vermiculata. Hence, it could be considered in mollusks control programs if risks on the environment from hazards of insecticides are to be avoided. In addition, Amino fertilizer could be more safe to the ecosystem, but the present results proved that it of lesser effectiveness in controlling the present snail comparable to the Newmyl and Vertimec.

Materials and Methods

1-Chemicals used Table (1) shows the three chemical compounds used in the present study.

Table 1: The three compounds used The compound Chemical Chemical Formula Structure class name Amino Fertilizer Combining(Fe, 1 (Biotech) Zn, Mn, Mg)

Newmyl Carbamate 2 Methomyl C H N O S (KZ) insecticides 5 10 2 2

Vertimec Acaricid, 3 Abamectin C H O (PPRI)* insecticide 48 72 14

* PPRI=Plant Protection Research Institute

2-The Tested land snail:

Eobania vermiculata

Culture of adult land snail Eobania vermiculata (, ) were collected from untreated orchards in Anshas district, Sharkia Governorate. Chosen snails of nearly equal shell diameter were directly transferred to the laboratory. The healthy individuals were kept for acclimatization in a glass cage (50 x30 x 30 cm) containing moist clay soil to a depth of about 10 cm and were provided daily with fresh cabbage leaves for two weeks before any tests (El-Okda 1984).

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The cage was kept clean by removing left-over food when the new food was introduced and the soil was adjusted at 75 ± 5 % relative humidity. Before starting the experiment, the snails were starved for 48h. and chemical compounds 1/2 LC50 and LC50 was prepared as poisonous baits. The poisonous baits were prepared by mixing 1/2 the LC50 and the LC50 values of each pesticide with five parts of black sugarcane syrup molasses then the mixture was incorporated with wheat bran to be finally 100 parts. The bait was moistened with appropriate amount of water to form a crumbly mash mixture. The snails were divided into two groups. The first group (10 snails) served as control; where as the second group (60 snails) served as experimental one and were subjected to insecticidal treatment. Snails were housed in one liter capacity plastic jars (10/jar) tightly covered with muslin clothes netting and secured with rubber bands. Treated snails (3 replicas) fed for 72 hours on baits consisting of 1/2 LC50 and LC50 value of the three tested pesticide as in table (2).

Table 2: The pesticides concentration The compound 1/2 LC50 LC50 Newmyl 1.493 2.986 Vertimec 1.375 2.750 Amino 2.1035 4.207

3-Electron Microscopy

The electron microscope prouder:

For electron microscopically studies, small pieces of fresh specimens of digestive gland were removed from the tested land snail after 3 days of treatment. The tissues were fixed by immersing them immediately in formalin-glutaraldehyde fixative (4F:1G) then in phosphate buffer solution (pH 7.2) at 4°C for 3 h. Specimens were then postfixed in 2 % OsO4 in the same buffer at 4°C for 2 h. Samples were washed in the buffer and dehydrated at 4°C through a graded series of ethanol. Specimens were embedded in Epon-araldite mixture in labeled beam capsules. Semi thin sections (1μm thick) were stained with toluidine blue. Ultrathin sections (60-70 nm thick) were picked upon 200 mesh naked copper grids and double stained with uranyl acetate (for ½ hr.) and lead citrate for 20-30 min (Reynolds, 1963). Specimens were examined under Jeol 100 CX TEM at the department of Histology, Faculty of medicine, Zagzig University.

Results

1- Cells of control digestive gland of Eobania vermiculata:-

Investigation of electron micrograph obtained from the digestive gland of control Eobania vermiculata revealed the presence of 3 cell types:-

Digestive cells:-

These are long columnar cells appear in various shapes owing to the plane of sectioning. The cells are surrounded with clearly demarcated cell membranes. The free surface of these cells is endowed with a huge number of microvilli. The cytoplasm is filled with a lot of rounded to oval mitochondria with transverse cristae. The huge numbers of small mitochondria are scattered throughout the whole cytoplasm of digestive cells. In some sections these mitochondria may appear concentrated in the apical part of the cytoplasm (Fig. 1). Golgi apparatus and cisternae of the rough endoplasmic reticulum are hardly detected in the digestive cells. The nuclei of digestive cells are large spherical containing scattered euchromatin and clumps of deeply osmiophilic heterochromatin and large nucleoli (Figs 2, 3 and 4).

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Fig. 1: An electron micrograph passing through the digestive gland of control Eobania vermiculate showing microvillus (Mv), digestive cell (DC), mitochondria (M) and execratory cell (Ex C). X10000

Fig. 2: Very enlarged electron micrograph passing through one control cell of digestive gland in Eobania vermiculate show nucleus(N), nucleolus (nu), rough endoplasmic reticulum (RER), Golgi apparatus (Go), and (M) mitochondria. X15000

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Fig. 3: An electron micrograph passing through control digestive gland tubules of Eobania vermiculate showing mitochondria(M), execratory cell (ExC), execratory granule (ExG), execratory vacuole (ExV), nucleus(N), nucleolus (Nu) and cell membrane(CM).X 6000

Fig. 4: An electron micrograph passing through control excretory cell of digestive gland in Eobania vermiculate showing nucleus (N), nucleolus (Nu), execratory cell (ExC), rough endoplasmic reticulum (RER), Golgi apparatus (Go), and (M) mitochondria, execratory granule (ExG) lysosomes (Ly). X10000

2- Excretory cells:-

These cells are fewer in number than the digestive cells. These cells are outlined by a distinct cell membrane and characterized by the presence of a large vacuole nearly occupied the whole cell and surrounded by a thin rim of cytoplasm. This layer of thin cytoplasm is pressed around the nucleus at one side of the cell. The nuclei of excretory cells are oval to elongated and contain moderately osmiophilic nucleoplasm in which clumps of highly osmiophilic heterochromation and conspicuous nucleoli are scattered (Figs. 4 and 5). Excretory cells have numerous excretory vacuoles filled with darkly osmiophilic excretory granules of variable shapes (Figs. 5 and 6 ).

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Fig. 5: An electron micrograph passing through digestive cell and excretory cell of digestive gland in control Eobania vermiculate show execratory cell (ExC), execratory granule (ExG), mitochondria (M), digestive cell (DC), cell membrane (CM)and microvillus (MV) . X8000

Fig. 6: An electron micrograph passing through excretory cell of digestive gland in control Eobania vermiculate showing execratory cell (ExC), execratory granule (ExG), execratory vacuole (ExV), nucleus (N), digestive cell (DC) and cell membrane (CM). X8000 Calcium cells:-

These are large spherical to oval cells of lowest distribution in the mucosa of digestive gland. They have free cell surface endowed with brush border of microvilli (Fig. 7). The cytoplasm of calcium cells is filled with calcium spherules localized inside membrane bound vacuoles (Figs 7 and 8). These spherule either completely darkly osmiophilic or interrupted with pale lucent vacuoles of variable size (Fig 7). The spherules are seen in different degrees of growth and numerous elongated mitochondria could be demonstrated in the cytoplasm (Fig. 8).

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Fig. 7: An electron micrograph passing through calcium cell of digestive gland in control Eobania vermiculate showing digestive cell (DC), cell membrane (CM), calcium cell (Ca C), and microvellis (MV). X10000

Fig. 8: An electron micrograph passing through digestive gland of control Eobania vermiculate showing execratory cell (EX), digestive cell (DC), calcium cell (Ca C) and nucleus (N). X6000

Cells of the digestive gland of treated Eobania vermiculata:

Since calcium cells were scarce and hardly observed in digestive glands of treated snails, the investigation here below is concentrated on other two cell types digestive cells and excretory cells. a-Newmyl changes

Effects of 1/2 LC50 of Newmyl

Application of 1/2 LC50 of Newmyl resulted in perturbations in the cellular organelles. Digestive cells suffered from cytoplasmic degeneration while the nucleoplasm was moderately osmiophilic and

601 Middle East J. Appl. Sci., 7(3): 595-612, 2017 ISSN 2077-4613 little heterochromatin clumps were seen. Mitochondria were pleomorphic and others were hypertrophied showing ballooned cristae. Cisternae of RER and Golgi saccules were undistinguishable (Figs. 9 and 10). The excretory cells displayed large and numerous excretory vacuoles loaded with abundant deeply osmiophilic scattered or clumped excretory granules. Some cytoplasmic regions were widely suffering from degenerative changes (Fig. 10).

Fig. 9: Electron micrograph passing through digestive gland of Eobania vermiculate treated with Newmyl 1/2 LC50 showing execratory cell (ExC), execratory granule (ExG), nucleus (N) and cytoplasmic degeneration (Dg). X5000

Fig. 10: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Newmyl 1/2 LC50 showing execratory cell (ExC), execratory granule (EXG), nucleus (N), nucleolus (Nu), degeneration (Dg), execratory vacuole(ExV) and numerous mitochondria (M). X8000

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Effects of LC50 of newmyl

This high concentration of Newmyl exhibited drastic cytoplasmic alterations. Digestive cells displayed wide degenerative regions and pleomerphic mitochondria. Less clumps of heterochromatin were detected in nuclei and the nucleoplasm was more homogeneous. As for the excretory cells they were obviously occupied with large excretory vacuoles containing variable shapes of granules. These were of different maturation stages and exhibited variable osmiophilia. Nuclei of some of these cells showed some indentations where others were about to be pyknotic (Figs 11 and 12).

Fig. 11: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Newmyl LC50 showing execratory cell (ExC), execratory granule (ExG), nucleus (N) and very large execratory vacuole (EXV). X5000

Fig. 12: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Newmyl LC50 showing execratory cell (ExC), execratory granule (ExG), nucleus (N) and degeneration (Dg) start to appear. X5000

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Effects of 1/2 LC50 of Amino

Treatment of Eobania vermiculata with Amino led to deteriorations in cells of the digestive gland. Digestive cells exhibited the appearance of cytoplasmic degenerations. These degenerative regions sometimes seen neighboring to the nucleus (Fig. 13). Mitochondria showed a little hypertrophy with ballooned cristae. The rough endoplasmic reticulum appeared normal with its cisternae are abundant and arranged in parallelism (Figs. 13 and 14). Nuclei of digestive cells revealed some enlargement with conspicuous nucleoli and patches of heterochromatin scattered in the nucleplasm (Fig. 14). The excretory cells displayed the presence of excretory vacuoles filled with low to moderately osmiophilic excretory granules (Fig 14).

Fig. 13: An electron micrograph passing through digestive cell of Eobania vermiculate treated with Amino 1/2 LC50 showing mitochondria (M), enlarged nucleus (N), nucleolus (nu), obvious rough endoplasmic reticulum (RER) and cytoplasmic degeneration (Dg). X6000

Fig. 14: An enlarged electron micrograph passing through the digestive gland of Eobania vermiculate treated with Amino 1/2 LC50 showing nucleus (N), nucleolus (nu), rough endoplasmic reticulum (RER), execratory cell (ExC). X12000

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Effects of LC50 of Amino

Application of this high concentration of Amino resulted in occurrence of higher alterations. Degenerations in the cytoplasm of digestive cells were numerous and larger in size. Mitochondria showed undiscrimination of their cristae. Nuclei displayed hypertrophy and moderately osmiophilic nucleoplasm and some scattered clumps of heterochromatin (Figs. 15 and 16). The excretory cells were highly loaded with excretory vacuoles harboring abundant deeply osmiophilic excretory granules (Fig.17) .

Fig. 15: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Amino LC50 showing nucleus (N), nucleolus (nu), rough endoplasmic reticulum (RER), mitochondria (M), digestive cell (DC) and degeneration (Dg) increased. X8000

Fig. 16: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Amino LC50 showing nucleus (N), rough endoplasmic reticulum (RER), digestive cell (DC) and degeneration (Dg). X8000

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Fig. 17: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Vertimec 1/2 LC50 showing nucleus (N), digestive cell (DC), mitochondria (M), cell membrane (CM) and degeneration (Dg). X6000 c-Vertimec changes

Effects of 1/2 LC50 of vertimec

Treatment of Eobania vermiculata with 1/2 LC50 of Vertimec biocide resulted in pathological alterations at the ultrastructural level. Digestive cells suffered conspicuous degenerative changes in the cytoplasm. Mitochondria were coalesced and pleomorphic. The matrix of mitochondria contained elongated darkly osmiophilic strands (Figs 18 and19). The nuclei showed pale osmiophilic nucleoplasm with less clumped heterochromatin. Nuclei of other neighboring cells were deeply indented with obvious clefts and wavy contour, such nuclei were characterized by darkly osmiophilic nucleoplasm and strongly osmiphilic heterochromatin clumps in addition to marginated aggregates of heterochromatin (Fig 19). Excretory cells displayed the presence of large excretory vacuoles contained darkly osmiophilic excretory granules of variable shape and size (Fig. 20). Mitochondria of these cells were crowded coalesced and pleomorphic.

Effects of LC50 of vertimec

Treatment with the high dose of Vertimec revealed the occurrence of advanced pathological deteriorations, where the digestive cells showed wide and numerous cytoplasmic degenerative regions. The superficial microvilli were short and disorganized (Fig. 21). Mitochondria were coalesced and obviously pleomorphic (Fig. 21). Nuclei were indented and the nucleaplasm was faintly to moderately osmiophilic. Nuclei of many cells acquired abnormal shape and the nucleolus had a bizarre architecture and coalesced with heterochromatin (Figs 21 and 22). Excretory cells suffered from degenerations and their mitochondria were pleomorphic and coalesced together (Figs. 22 and 23). The excretory vacuoles increased in number and size and harbored strongly to moderately osmiophilic excretory granules of variable size and shape (Fig.23).

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Fig. 18: Enlarged electron micrograph passing through digestive gland of Eobania vermiculate treated with Vertimec 1/2 LC50 showing indented nucleus (N), mitochondria (M) and degeneration (Dg). X10000

Fig. 19: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Vertimec 1/2 LC50 showing execratory cell (ExC), fragmented execratory granule (ExG) and execratory vacuole (ExV). X 8000

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Fig. 20: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Vertimec 1/2 LC50 showing nucleus(N) with heterochromatin, mitochondria (M), digestive cell (DC), execratory cell (ExC), execratory granule (ExG), execratory vacuole(ExV) and degeneration (Dg). X6000

Fig. 21: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Vertimec LC50 showing nucleus (N), mitochondria (M), digestive cell (DC), microvellis (MV), nucleolus (nu) and degeneration (Dg). X4000

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Fig. 22: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Vertimec LC50 showing a bizarr nucleus (N), execratory cell (EXC), digestive cell (DC), nucleolus (nu) coalesced with heterochromatin and obvious degeneration (Dg). X 8000

Fig. 23: An electron micrograph passing through digestive gland of Eobania vermiculate treated with Vertimec LC50 showing indeted nucleus (N), execratory cell (ExC), execratory granule (ExG), execratory vacuole (ExV), mitochondria (M) and degeneration (Dg). X8000

Discussion

The ultrastructure of cells in the digestive glands of gastropod mollusks were studied (Walker, 1970; Franchini and Oltaviani, 1993; Rebecchi et al., 1996; Taieb and Vicent, 1999; Lobo-da-cunha, 2000; Chaki and Misra, 2004; Arrighetti et al., 2015; Nath et al., 2015; Ojeda et al., 2015). The ultrastructural characteristics of the digestive cells of E. vermiculata are nearly similar to those of other gastropods. Earlier, Boghen and Farley (1974) described similar fine structure in cells

609 Middle East J. Appl. Sci., 7(3): 595-612, 2017 ISSN 2077-4613 of Littorina saxatilis. Such similarity was also reported by many investigators in Helix sp. ( Dimitriadis and Hondros, 1992), Planorbis corneus (Franchini and oltaviani, 1993), Aplysia depilans (Lobo-da-cunha, 2000), Achatima fulica (Chaki and Misra, 2004) and Laevicaulis alte (Nath et al., 2015). Recently, Arrighetti et al. (2015) and Ojeda et al. (2015) suggested multiple roles for digestive cells of snails and pointed out that data on digestive cells functions are incomplete. Under the effects of any of the three applied compounds (Newmyl, Vertimec and Amino) the cytoplasm of the digestive cells responded by degenerations. Such degenerative reaction was variable according to the applied dose (1/2 LC50 or LC50) and the compound expertized. Application of the LC50 exhibited wider cytoplasmic degenerations than in case of 1/2 LC50. The most drastic cytoplasmic degenerations were observed in case of Newmyl followed by Vertimec and lastly came Amino. Severe cytoplasmic vacuolization were observed in digestive cells of E. vermiculata after 14 days of topical application of methomyl (Hamed et al., 2007). Oxford and fish (1979) considered that the release of hydrolytic enzymes from the cytoplasm of digestive cells was related to an increase in the rate of cell autolysis. The digestive cells of E. vermiculata contained a huge number of mitochondria distributed throughout the cytoplasm and many mitochondria were located in the apical part of the cytoplasm. Nath et al., (2015) postulated that the number and position of mitochondria may reflect the activity of the cell. In many studies on digestive cells of gastropods, mitochondria were localized around the basal nucleus (Walker, 1970; Boghen and Farley, 1974; Dimitriadis and Hondros, 1992). Mitochondria were also demonstrated in the apical portion of digestive cells (Chaki and Misra, 2004; Nath et al., 2015). Mitochondria of the digestive cells revealed coalescence and acquired pleaomorphism without discrimination of their cristae following treatment with either of the three chemical compounds under experimentation. However, Hamed et al., (2007) demonstrated hyperdenisty of the mitochondrial matrix (mitochondria pyknosis) and the appearance of ballooned cristae in digestive cells of E. vermiculata due to methomyl treatment. Goel and Dhawan (2001) attributed such alterations to decrease in the mitochondrial energy production in addition to interaction of the toxin with membrane components. This interaction may lead to changes in ion transport along the mitochondrial membrane. After molluscicidal treatment with the present compounds, nuclei of digestive cells displayed hypertrophy and were obvious indented or exhibiting deep clefts or wavy contours. The nucleoplasm was deeply osmiophilic and harboring strongly osmiophilic scattered or marginated heterochromatin clumps. At the ultrastructureal level Hamed et al., (2007) following treatment of E. vermiculata with methomyl described gradual degenerative changes in the nuclei of digestive cells ranged from karyolysis to severe kryorrhexis and complete pyknosis. Bayne et al. (1985) considered karyolysis as a late reaction to intoxication in both vertebrates and invertebrates. At the same time, Tribskorn (1989) pointed out that the reaction of nuclei is a primary cell response that leads to cell death. This was also confirmed in many studies (Triebskorn and Kunast, 1990; Tribskorn et al. 1996). As for the excretory cells of the digestive gland the action occurring following the present bating treatment was the appearance of large excretory vacuoles filled with abundant deeply osmiophilic excretory granules. The excretory vacuoles number and size were dose-dependent as these were larger when the LC50 was applied than after 1/2 LC50. However, the contained granules clumped inside such vacuoles were variable in size and osmiophilia in different treatment chemical compounds (Newmyl, Vertimec and Amino). Methomyl application topically or by toxic baiting technique to E. vermiculata resulted in the enlargement of yellow granules or residual bodies (Hamed et al., 2007). These authors pointed out that the apparent rise in the number of residual bodies suggests accumulation of the tested molluscicide in these granules due to intoxication.

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