Impact of contamination on biology, biochemical, and histology of Eobania vermiculata and Monacha obstructa during different seasons in Ismailia, Egypt

Land snails are found to be an appropriate sentinel organism, and the biomarkers chosen are effective for terrestrial heavy metal biomonitoring. The study aimed to compare the biological, biochemistry, and histology of two land snails in the Ismailia governorate, Egypt during different seasons. Random snails were collected from January 2015 to December 2015 from two sites in the Ismailia Governorate, on wet days during each season’s middle month. Soft tissues were taken from the dissected snails. It was noticed that most of the morphometric parameters measured shell height (ShH), last whorl width (LWW), maximum diameter (MaxD), aperture height (AH), and aperture width (AW) were higher in Eobania vermiculata (Sp. 1) than those measured in Monacha obstructa (Sp. 2), except for shell height measurement, which was the same in both species. The growth rate of Sp. 1 and Sp. 2 changed seasonally. In the more polluted areas with heavy metals, lipid peroxidation (LPO) was higher in snails and total protein content than in the snails collected from the less polluted areas for all seasons. However, the snails displayed lower levels of glutathione (GSH) as compared to snails at a less polluted site. GSH and LPO levels, on the other hand, have a negative relationship between them. Histopathological alterations in the digestive gland were more obvious in the general architecture of the digestive gland that had lost its tubular appearance. The excretory cells showed an increase in their excretory granules’ number and size while calcium cells decreased. Also, gonad follicles have lost their normal architecture with the degeneration of some stages of spermatogenesis and oogenesis. In conclusion, There was a strong correlation between GSH levels and total protein content in the same soft tissues. GSH and LPO levels, on the other hand, have a negative relationship. The overall results display the usefulness of E. vermiculata and M. obstructa land snails as bioindicator organisms and support the application of this ecotoxicological approach for evaluating the biologic impact of toxins. E. vermiculata is more abundant than M. obstructa. The density, morphometric, biochemical, and histology of E. vermiculata and M. obstructa were different at different seasons.

3 70 the population living in thick tropical forests and nearby areas, where snail farming is a major 71 concern (Toader- Williams and Şara, 2010). 72 Due to their capacity to absorb trace metals in their tissues, land snails such as H. 73 aspersa and Arianta arbustorum have historically been used to estimate urban or industrial 74 waste (Regoli et al., 2006;Itziou and Dimitrids, 2009). E. vermiculata was found to be an

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Many investigations into the morphology and histology of the digestive gland of the 93 land snail E. vermiculata have been conducted (Hamed et al., 2007;Radwan et al., 2008; 94 Mohammadein et al., 2013). There are just a few studies that determine the number and kinds 95 of cells that make up the digestive gland epithelium of terrestrial gastropods, despite the fact 96 that the anatomy and physiology of digestive gland cells are well understood. This is mostly 97 owing to the digestive gland epithelium's multifunctional nature and the significant 98 ultrastructural changes that occur during digestion (Radwan et al., 2008). 99 The morphological details of the shell provide information regarding their real body 100 development (Roth and Mercer, 2000). Multivariate statistical studies of a collection of 101 quantitative variables, such as the length, breadth, and height of a shell, were used in reported 102 morphometrics. As the shell deteriorates, as it increases in size, shell material is added to the margins to keep its form, and at the top. The spiraling axis around which the aperture 5 136 to the key given by Radwan et al. (2008). The individual species' absolute population density 137 was calculated as follows: 138 Absolute population density = Total number of individuals of species shells with a reflected lip were used because this signals the snail's maturity as well as the end 150 of its growth cycle (Madec et al., 2003).  Tahreer St., Dokki, Giza, Egypt). The creation of colored complexes between peptide bonds 169 and cupric ions in an alkaline medium is the basis for this process.

Histological study
For histological studies, the gonad and digestive gland of snails from different stages 173 were quickly excised and immersed in formalin solutions (10%) for 48 hr. The thin sections 174 of 4µm were obtained by semiautomatic microtome (Kiernan, 1990 and M. obstructa (supplemented material, 2b), were found in the two sites described earlier  The growth rate was determined using different shell parameters. Table 2    During the autumn season, the MaxD of Sp.1 was 2.58 cm, the maximum AW 203 reached 1.58 cm, and the maximum AH was 1.52 cm. However, the greatest LWW was1.37 7 204 cm in the spring season, while, the highest Sh.H value was 1.51 cm in both the spring and 205 autumn seasons. During the spring season, Sp. 2 attained its maximum size in all 206 morphometric parameters. There was no significant difference between the two sites in terms 207 of morphometric characteristics (P > 0.05). All morphometric parameters assessed for both 208 species were likely to be strongly linked.  were significantly different (P ≤ 0.01) between the four seasons. Throughout the summer, 216 GSH reached its highest concentration for two species at the two sites. During the autumn, 217 the GSH concentration was reduced significantly at site I for the two species as compared to 218 site II (Fig. 1).

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The total protein levels of the different species did not distinguish substantially across 220 the two locations or during the four seasons. Unless total protein levels at sites I and II 221 somewhat rise in the winter and spring seasons, respectively (Fig. 2).

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LPO levels were found to be somewhat higher in the winter for two species at site-I 223 than in the spring and summer. However, LPO levels of both species were found to be 224 somewhat greater in the autumn at site II (Fig. 3). There was a strong correlation between 225 GSH levels and total protein content in the same soft tissues. GSH and LPO levels, on the 226 other hand, have a negative relationship.  to be empty and replaced by brown granules (Fig. 5b).
The digestive glands of E. vermiculata and M. obstructa are quite big, dark brown, 239 and take up a significant portion of the snail's visceral hump. The tubules of this gland are 240 highly branching and blindly terminating tubules that are linked together by connective tissue 241 (Fig. 6a). Each tubule of the digestive gland is surrounded by a thin layer of circular muscle 242 fiber (Fig. 6b). When these cells were investigated, three types of cells were found. The cells 243 of digestion, calcium, and excretion. Digestive cells are columnar cells with numerous 244 vacuoles that accommodate yellowish brown granules and rounded or oval nuclei at the base.

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Excretory cells are spherical cells with a tiny nucleus and big dark brown excretory granules.

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Calcium cells are pyramidal in shape, with spherical calcium spherules (looking as luminous 247 bodies) piled in the center and globular nuclei.  plantations (Eshra, 2013), so they were used in the current study.

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The vermiculata land snail was the predominant species at the two studied sites in the activity of land snails is also influenced by moisture.

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The mucus of land snails consists of 98% water. As a result, water deficiency inhibits 303 high-temperature activity. Light influences the activity of land snails; they remain in their 304 hides during the day and only after dusk to search for food. Snails moved to foliage as the 305 light intensity decreased due to a drop in temperature below 21 o C and an increase in humidity due to dewfall at dusk, while in the morning and during the day they returned to the 307 upper soil or between the earth clods where it is cold and shady (Daoud, 2004;Ramzy, 2009).

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In the current study, E. vermiculata reached its maximum size ( .

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In the current study, there was no significant difference in morphometric parameters of 318 two species of land snails between the studied sites. This could be due to the close distance 319 between the two studied sites and since they are exposed to the same environmental

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The digestive gland of gastropod mollusks is the key organ of metabolism, serving as 394 the primary location for xenobiotic accumulation and biotransformation (Desouky, 2006). In 395 the current data, it was noticed that the histological structure of the gonads and digestive 396 gland of two species of land snails at the two studied sites were more or less similar to each

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In the present study, the digestive cells displayed morphofunctional changes that may 433 be explained by the fact that they were at distinct stages of activity. This follows Hamed et al.   The data presented as mean ± S.E (n= 5 samples). Site I: Ring road, Site II: El Balah road. a Significant difference as compared to the site I, b significance difference as compared to Sp. 1, c significant difference as compared to the winter season, d significant difference as compared to the spring season, e significance difference as compared to the summer season, and f significant difference as compared to the autumn season (P<0.05).

Fig. 2:
Total protein level (mg/g) in the soft tissues of two species of land snails in two sites. The data presented as mean ± S.E (n= 5samples). Site I: Ring road, Site II: El Balah road. a Significant difference as compared to the site I, b significance difference as compared to Sp. 1, c significance difference as compared to the winter season, d significant difference as compared to the spring season, e significant difference as compared to the summer season, and f significant difference as compared to the autumn season (P<0.05). The data presented as mean ± S.E (n= 5samples). Site I: Ring Road, Site II: El Balah Road. a Significant difference as compared to the site I, b significant difference as compared to Sp. 1, c significance difference as compared to the winter season, d significant difference as compared to the spring season, e significant difference as compared to the summer season, and f significant difference as compared to the autumn season (P<0.05).