Efficacy of biopesticide Protecto (Bacillus thuringiensis) (BT) on certain biochemical activities and histological structures of land snail Monacha cartusiana (Muller, 1774)

The investigation aimed to show the effect of two concentrations of (Protecto 9.4%) commercial formulation of Bacillus thuringiensis kurstaki (Bt) on some biochemical changes of the land snail Monacha cartusiana at three different experimental periods (24, 48 and 72 h). Also, some histobiological altrations of the snail at a lethal experimental period of 96 h were studied. Pathogenicity effect of two sub-lethal concentrations of biopesticide Protecto; LC20 (6.72 × 106 IU/mg) and LC40 (17.28 × 106 IU/mg) were studied on the land snail M. cartusiana at 3 different exposure periods (24, 48, and 72 h). Some biochemical parameters such as Alkaline phosphatase (ALP), Alanine amino transaminase (ALT), Aspartate amino transaminase (AST), and Total protein (TP) were investigated. These observations cleared that; a significant elevation of ALP, ALT, and AST increased with increasing the sub-lethal concentration of protecto (LC20 and LC40) against the exposed snail. Also, the activity of enzymes significantly increased with increasing the time of exposure (24, 48, and 72 h), respectively. Conversely, the level of TP in the snail was significantly decreased under pathogenic exposure for both (LC20 and LC40) concentrations of Protecto at the same three treated periods (24, 48 and 72 h). The histbiological examinations at LC20 and LC40 for the exposure period 96 h, showed that the digestive gland with vacuolated degenerated, ruptured digestive cells and hemocyte infiltration. Moreover, the foot was observed with necrotic changes, vacuolated connective tissue, as well as, deformation in muscle fiber, and rupture the outer layer. Final results showed that protecto B. thuringiensis had a pathogenic effect on land snail enzymatic activities and histbiological structures of land snail.


Background
Nowadays, the use of environmental safety microbial in pest management received more significant attention from many authors (Kumar et al. 2021). B. thuringiensis is a Gram-positive soil bacteria that do not have a cytotoxic effect on mammalian cells (Ohba et al. 2009). Instead of infecting most invertebrates pests, and secrete either toxic proteins by bacteria or soluble toxins produced by vegetative cells, which are toxic (Malovichko et al. 2019). During the last years, several studies on biological control of land snails using microbial agents such as B. thuringiensis have been carried out (Said and Ali 2018). Land

Open Access
Egyptian Journal of Biological Pest Control *Correspondence: omhashim1979@gmail.com 1 Faculty of Science, Department of Zoology, Beni-Suef University, Beni-Suef, Egypt Full list of author information is available at the end of the article Page 2 of 8 Gaber et al. Egyptian Journal of Biological Pest Control (2022) 32:36 gastropods belonging to phylum Mollusca are a wide sector of the fauna around the world (Diaz et al. 2017).
Recently, Land snails, considered the only one between the phylum Mollusca attacking most agricultural fields in Egypt, causing great damage to; greenhouses, nurseries, fruit crops, orchards, ornamentals, vegetables, medical, navel orange, and apple trees (Ali and Robinson 2020). M. cartusiana (Muller) is a widespread land snail in the Egyptian agricultural fields and caused economic losses (Rady et al. 2019). The present work studied the effect of protecto (B. thuringiensis) on some biochemical changes of M. cartusiana land snail under laboratory conditions. Also, histobiological investigations of the land snail at sub-lethal concentrations after exposure to 96 h. were studied.

Experimented snails
Adults land snails M. cartusiana with a shell size about (9-12 mm) and (weight 3.1 g) were collected manually from infested field crops of Yosef El Sedek district, El Fayoum Governorate (29 22° N: 30 51° E), middle Egypt, during the spring season of 2020. The collected adult snails were transferred to the laboratory at Fayoum Agric. Res. Station, Agricultural Research Center, Egypt, in a muslin bags, then they were washed with distal water. The experimental snails were transferred into rearing plastic boxes, each (30 × 30 × 25 cm 3 in size), used as housing, filled with moist sterilized loamy soil at 25 ± 2 °C and 75% ± 5% soil moisture. The experimental housing was covered with a muslin cloth to prevent snails from escaping. The snails were fed on leaves of lettuce daily for 14 days.

Laboratory experiment Bioassay
The preliminary studies were conducted to estimate the lethal concentrations of protecto on land snails M. cartusiana. 4 concentrations of the tested coimpound were prepared using dechlorinated tap water based on weight/ volume (Osman et al. 2011;Osman and Mohamed 1991) as follows; (8 × 10 6 , 16 × 10 6 , 32 × 10 6 , and 48 × 10 6 IU/ mg. The experiments took place by the leaf-dipping technique. About 30 snails were placed into rearing plastic boxes and starved for 2 days (Souza 2003) then feeding the snails by dipping the leaves of lettuce in the tested concentrations for 30 s (Ghamry 1994) then outside left it for 1 min for dryness before treatment. Fresh lettuce was used as control, five replicates each of contained 30 snails were used for each concentration. The mortality percentages of snails were recorded and computed for 48, 72, and 96 h.

Biochemical parameters
Snails were treated with calculated LC 20 and LC 40 of protecto at the periods achieved mortality between 20-80% then the tested snails from each group of control and both sub-lethal concentrations were removed and dissected out from the shell and quickly weighed, homogenized in saline (0. 9%) at a ratio of 1:9 (w:v) (Yang et al. 2015), then centrifuged at 8000×g for 10 min at 4 °C in the refrigerated centrifuge (El-Gohary and Genena 2011). The deposits were discarded, and the supernatants were used to determine the levels of biochemical parameters; ALP (U/L) was determined by commercially available diagnostic kits supplied by Biosystems company, Egypt, ALT (U/L) and AST (U/L) were estimated kinetically by Biomed using the method of (Young 2001), and TP (mg/ dl) was determined by using Diamond company according to the method reported by (Burtis and Ashwood 1999).

Histological studies
Adult snails of M. cartusiana were tested as control; another 2 groups were exposed to 2 sub-lethal concentrations of B. thuringiensis LC 20 and LC 40 . After 96 h of exposure, the tested snails were dissected out carefully and fixed in Boun's fluid, dehydrated in 70% ethyl alcohol, cleared in xylene, and embedded in paraffin wax. Histological Sections (4 µm) were cut and were stained with hematoxylin and eosin (H&E) stain (Banchroft et al. 1996). Finally, they were investigated by a light microscope and photographed.

Statistical analysis
Statistical analysis was performed using IBM SPSS (Statistical Package for the Social Sciences, SPSS, version 20). ANOVA was applied to compare the activity of enzymes at the effect of 2 sub-lethal concentrations of B. thuringiensis concerning the corresponding control at each experimental period. Lethal concentration values (LC 20 & LC 40 ) were determined according to probit statistical analysis (Finney 1971).

Lethal concentrations
Effect of different concentrations of B. thuringiensis on the mortality percentages of the adult snail M. cartusiana at intervals times (48, 72 and 96 h) were determined according to probit statistical analysis. The results  presented in Table 1 cleared that the mortality percentage of the snail increased with increasing the concentration of B. thuringiensis and the time of exposure. Also, the results indicated that the LC 20 and LC 40 values were 6.72 × 10 6 IU/mg and 17.28 × 10 6 IU/mg, respectively, at the experimental exposure period of 96 h.

Biochemical parameters
In order to investigate the biochemical activities of (AlP, U/l), (ALT, U/l), (AST, U/l), and (TP, mg/dl) of M. cartusiana exposed to 2 sub-lethal concentrations of B. thuringiensis, LC 20 and LC 40 were analyzed and computed by one-way ANOVA for the 2 concentrations comparing to the tested control at each corresponding exposure period. A significant difference at (P < 0.05) was confirmed as shown in Table 2,, at 24 h, where the activity of ALP has non-significant difference between LC 20 and the tested control but it was significantly increased at the tested concentration LC 40 than the control. After 48 and 72 h, the activity of the enzyme was significantly increased at the LC 20 value (48.38 ± 0.82, and 51.5 ± 0.82, respectively) and at LC 40 value (56.5 ± 1.42 and 66.4 ± 1.81, respectively) than the corresponding control (38.08 ± 1.18 and 38.28 ± 0.66). In addition, the activity of ALP was not only computed by one-way ANOVA but also by Post hoc analysis which compared the activity of the enzyme at the 3 different periods; which increased at 48 and 72 h in relation to 24 h. Furthermore, ALTactivity was significantly high at exposure the snail to LC 20 and LC 40 comparing to control at the different tested periods (Table 2). Additionally, the activity of the AST enzyme increased significantly after exposing the snail to LC 40 value as compared those of control at the 3 exposure times. As well as, after exposing to LC 20 values, the enzyme activity significantly increased only at the exposure periods (

Histological studies Digestive gland
In the present study, the digestive gland of the control M. cartusiana snail showed a normal structures without histopathological alterations under a light microscope (Fig. 1a). It consists of highly-branched numerous, compressed digestive tubules (DT) separated by lost connective tissues. Each tubule has a narrow lumen (L), all surrounded by a circular thin muscle layer consisting of 3 different cells; Digestive cells (DC) with columnar shape and containing basally nuclei, Calcium cells, Excretory cells (EC). Histological alterations were observed in M. cartusiana treated with LC 20 of B. thuringiensis after 96 h of exposure observed in (Fig. 1b), the lumen of the digestive tubule enlarged and increased, the cells lining the tubules were irregularly arranged and extremely indistinguishable.The excretory cells become vacuolated (V), rupture and degeneration of digestive cells were reported. Additionally, histopathological observations were observed at exposure to LC 40 , hemocyte infiltration (HI), the digestive tubule, and their constituents lining cells; digestive, calcium, and execratory cells showed complete rupture (Fig. 1c).

Foot
The foot tissue of control M. cartusiana snail was found composed of a simple columnar epithelium layer (E), followed by a layer of connective tissue containing mucous gland (MG). The innermost layer was formed of muscle fiber (MF), as illustrated in (Fig. 2a). The foot tissue of snail exposed to LC 20 was observed with necrotic changes in the mucous gland (N), the connective tissue containing empty spaces vacuoles (V). Additionally, the muscle fiber layer suffered from the cells'deformation (Fig. 2b). Meanwhile, at snail exposure to LC 40 , the foot tissue contained vacuoles in the muscle fiber layer as long as deformation occurs in this layer. Also, the outer epithelial layer contained undifferentiated necrotic epithelial cells and ruptured the outer layer (Fig. 2c).

Discussion
Obtained data indicated that the mortality of the snail M.cartusiana after protecto treatments increased with increasing the concentration and the exposure time. The sub-lethal concentrations (LC 0 , LC 10 , and LC 25 ) reduced the survival and growth rates of S. mansoni snails after 12 weeks of B. thuringiensis treatment in relation to the corresponding control (Osman et al. 2011). The snail Physa marmorata was found more sensitive to the lethal concentration of the biocide B. thuringiensis (LC 50 ) at 24, 48, and 72 h (Mansouri et al. 2013). This finding was in accordance with that obtained by Genena and Mostafa (2008), who found out that B. thuringiensis showed high pathogenecity against land snail M. cantina, with mortality increasing with an increase in the concentration of bacteria.
The gained results in this study indicated that the commercial formulations of tested biopesticide protecto increased the activity of enzymes ALT, AST, and ALP at the two sub-lethal concentrations LC 20 and LC 40 at the 3 different tested periods 24, 48 and 72 h in comparison to the control. Also, the results showed a significant decrease in the level of total protein in the snail at both concentrations for 3 lethal times. Those enzymes are located in hepatocytes and in many organs, lungs, and hurt (Abd-El-Haleem et al. 2021). The commercial formulations of B. thuringiensis var kurstaki "Agerin, Dipel 2X and Dipel DF" significantly affected activities of AlT and AST enzymes of larvae of cotton leafworm Spodoptera littoralis (Kamel et al. 2010). Also, (Kramarz et al. 2007) showed that Bt toxin does not affect the survival rates of Helix aspersa snails. The obtained results are incorporated with those cleared by (Abdel-Halim et al. 2006), who showed that the activities of AST and ALT were decreased in M. cartusiana snail exposed to LC 50 and 0.5 LC 50 of B. thuringiensis with different intervals time. Also, (El-Gohary and Genena 2011) revealed that the molluscicides, Gastrotox, Molotov, and Mesurol caused a significant decrease in ALT activity. At the same time, they have an insignificant increase in AST activity and TP level tested on land snail E. vermiculata. Also, they increased the AST and Alt activities but decreased TP level in land snail M. cantina. The results agree with (Banaee et al. 2016), who cleared that ALT, AST, and ALP activities increased significantly in carp exposed to sublethal concentrations of paraquat but decreased the level of total protein of the carp at the same conditions. The activities of AST, ALT, and ALP of C. punctatus increased during toxic exposure to Triazophos concentrations. The total protein level decreased during toxic exposure to sub-lethal concentrations at time intervals may be attributed to involving the proteins in the metabolic purposes of the cell, which led to the breakdown of protein for utilization in metabolism (Naveed et al. 2010). The enhanced activities of AST and ALT in land snails caused by the toxic stress of B. thuringiensis may be attributed to insufficient additional energy that resulted from the deprivation of snails from food (Samanta et al. 2014). The activities of hemolymph enzymes AST and ALT of E.vermiculta when exposed the snail to sub-lethal concentrations of Lannet were increased significantly than control, but ALP activity and TP level were decreased at all tested concentrations (Khalil 2016). These results support the finding of (Awadalla et al. 2017) which showed that the total protein decreased highly in the larvae of S. littoralis treated with Protecto. The sub-lethal concentration LC 25 of Ginger extract caused AST and ALT enzymes elevation in M. cartusiana land snail (Abd El-Atti et al. 2019).
Obtained results cleared that B.thuringiensis caused histological alterations in M. cartusiana tissues; digestive glands, foot, and mantle at both sub-lethal concentrations LC 20 (6.72 × 10 6 IU/mg) and LC 40 (17.28 × 10 6 IU/mg) . The digestive cells lining the tubule are irregular and indistinguishable. The cell contained vacuoles, degenerated and ruptured. Those observations agree with that cleared by (Said and Ali (2018), who stated that the digestive tubule has fragmentation (Attia et al. 2021) exposed E. vermiculata to inorganic fertilizer and observed histological alterations in the digestive gland as; distinguished the epithelium lining and increased in a number of excretory cells. Caselio (plant fertilizer) has a pathological effect on the freshwater snail, Lanistes carinatus, which causes necrosis for the basement membrane of the digestive tubule, degenerated, and disintegration for connective tissue in the digestive gland (Sheir 2015). Ali and Said (2019) stated that the epithelial cells in the digestive tubule of Monacha obstructa land snail lost their normal shape after exposure to UV A radiation. Cofone et al. (2020) observed that the outer epithelial layer in treated E. vermiculata foot showed darker in color, a significant decrease in mucus gland, and an increase in vacuoles number within a sub-epithelial layer of the tissue. The foot of B.alexandrina snails occurring in pollutants aquatic environment was observed with shrinkage in unicellular glands, atrophy, and increased vacuoles.
Also, the digestive gland shows histological alterations (El-Khayat et al. 2018). The histological alterations in the digestive gland were necrotic changes of digestive cells and loss of cell organizations. The alterations in the foot were disruption of muscle fibers and decreased in Mucous cells and protein cells (Cengiz et al. 2005). Furthermore, the foot of M. cartusiana study showed histological changes as; deformed in Mussel fiber, necrosis, and increased in vacuoles numbers. These observations were in congruence with the results of (Parvate and Thayil 2017), who recognized that the foot in Achatina fulica land snail with increasing the vacuoles and infiltration in myoepithelial cells layer at exposed to clove oil. Furthermore, the foot of snails had histological alterations under toxic exposure, shrinkage, rupture of muscular tissue, distortion of epithelial covering, and accumulation of the pigment cells (Abdel-Rahman 2020).

Conclusions
It was concluded that Protecto had a significant pathogenic effect on mortality and biochemical activities of land snail M. cartusiana at both sublethal concentrations of exposure, LC 20 and LC 40 values at 3 different experimental periods (24, 48, and 72 h). Also, the bactericide had a lethal effect on the histological structures of the snail.
Abbreviations BT: Bacillus thuringiensis; LC 20 : Lethal concentration of protecto required to kill 20% of exposed snails.; LC 40 : Lethal concentration of protecto required to kill 40% of exposed snails.