MATERIALS AND METHODS

Study area: Archachatina marginata is highly dispersed and ubiquitous. Thirty snail samples, used for the study, were procured in July, 2021 from peasant farmers who handpicked the snails on their farmlands around the Ile-Ife axis of Osun State, Nigeria.

Collection and handling of snails: The procured snails were immediately transferred to the snail pen in the Department of Zoology, Obafemi Awolowo University, Ile-Ife and raised on pawpaw leaves and the rind of watermelon in the laboratory till they were dissected. The snails were on an average of 257 g in weight.

Dissection of snails: The snails were carefully removed from their shell. The main body organs were washed thoroughly with distilled water to remove body slime. This will enable a proper dissection, where the various parts (head, mantle, trunk and foot) were identified.

Snails were dissected on a tray containing ice to arrest any enzymatic activity. The dissection of the snail started from the inner part of the mantle (collar region) that connects to the head. This reveals the beginning of the digestive tract which is infused into the head, the oesophagus and the salivary gland. The outer layer covering the visceral hump is cut open for a clear view of the organs and the digestive tract is traced from the head, around the mantle to the visceral mass which holds the bulk of the digestive system i.e., the digestive gland where the stomach and small intestine are embedded. The organs, carefully separated, are immediately stored in the deep freezer (-18°C) till the analysis of the enzymes.

Enzymes assay and analyses: The snail organs were buffeted and extracted into a solution. The specific activity of the enzymes was expressed as the enzyme activity per mg of protein. The analyses included the protein assay to measure the protein concentration, the lipase assay to measure the activity of lipase, the protease assay to measure the activity of protease and the glucose substrate enzyme assay (α-amylase, cellulose and sucrase). All experiments were conducted in triplicate measurements.

Assay of protein: The protein concentration was determined using the Bradford method³². Bovine serum albumin (BSA) was used as the known standard protein. The Bradford method is a typical colorimetric protein assay which is based on absorbance shift of dye Coomassie brilliant blue G-250. Six test tubes were set up with the various quantity of BSA, 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mL, respectively and distilled water (in various concentrations as well) after which the Bradford reagent (the colour developer) was added to each test tube, 0.2 mL each. The absorbance rate was taken at 595 nm (the standard for protein concentration).

Glucose substrate enzyme assays: The assay for the glucose substrate enzymes was carried out using the 3, 5 dinitrosalicylic acid method (DNSA) as described by Ademakinwa and Agboola³³. The enzymatic reaction mixtures contained 500 mL of 1% (w/v) of carboxymethyl cellulose, starch and sucrose respectively in 0.5 mM acetate (buffer pH 5.5) and 200 mL of the enzyme. The reaction mixture was incubated at 25°C and the reaction terminated using DNSA reagent after 20 minutes. The obtained mixture was boiled for 10 min and the reducing sugars present were quantified appropriately. A control experiment was set up such that the enzyme was pre-incubated with the DNSA before adding the substrate.

Protease assay: Protease activity was assayed by the method described by Cupp-Enyard³⁴ with some modifications³⁵. The substrate was prepared using 0.65% casein solution dissolved in 50 mM Tris-HCl buffer pH 7.4. The reaction mixture was made up of 5 ml of the casein substrate and 1 ml of the centrifuged enzyme supernatant. The reaction was terminated after 30 min of incubation (37°C) by adding 5 mL of 110 mM trichloroacetic acid (TCA). The 5 mL of 0.5 M sodium carbonate and 1 mL of Folin’s reagent were added to each test tube. Absorbance was measured using a UV spectrophotometer at 660 nm. The concentration of protease produced was measured using a standard graph of tyrosine obtained in the range of 10-100 μg mL^–1. One unit of protease activity was defined as the amount of the enzyme resulting in the release of 1 μg mL^–1 of tyrosine per minute under the assay conditions.

Lipase assay (kinetic assay): The activity of lipase was analysed using p-nitrophenyl laurate (p-NPL) according to the method of Vorderwulbecke et al.³⁶ with slight modification³⁷. The 1 mL of isopropanol containing 0.001 g of p-NPL was mixed with 9 mL of 0.05 M Tris-HCl buffer (pH 7.5) containing 50 μL Triton X-100 and 0.01 g gum Arabic to form an emulsion. 300 μL of the diluted enzyme solution was mixed with 700 μL of the prepared substrate solution. Using a spectrophotometer, the liberated p-nitrophenol was observed by the change in absorbance at 410 nm. One unit of enzyme activity is defined as the amount of enzyme that released 1 μ mol of p-nitrophenol from p-NPL in one minute under the assay conditions.

Statistical analysis: Descriptive statistics were employed to compare the mean values for the enzyme activities in each organ. One-Way ANOVA with Tukey’s HSD test was used to determine statistical difference at (p<0.005) level of significance using IBM SPSS Statistics 25 software.

RESULTS

Enzyme activities: α-amylase, cellulase, sucrase, protease and lipase activities in the digestive tract viz: The salivary gland, crop juice, digestive gland, stomach and small intestine of Archachatina marginata were summarized in Table 1.

α-amylase: In the digestive tract of A. marginata, α-amylase activity (590.20±17.326 μmol min^–1) in the salivary gland was the highest, followed closely by in the digestive gland (562.19±24.988 μmol min^–1) as shown in Table 1. α-amylase in the crop has the highest specific enzyme activity (112.01 μ mol^–1). The digestive gland recorded the lowest specific enzyme reaction (42.61 μ mol^–1) as shown in Table 2. There was a significant difference (p<0.05) between α-amylase activities across the organs. However, α-amylase activity was not significantly different between the salivary gland and the digestive gland (Table 1).

Cellulase: Cellulase activity in the crop (594.20±41.583 μmol min^–1) was the highest, this was followed by cellulase activity in the small intestine (532.18±28.364 μmol min^–1) as shown in Table 1. The digestive gland recorded the lowest cellulase activity (98.03±17.326 μmol min^–1). The specific enzyme activity of cellulase in the other organs is relatively low compared to the crop and small intestine (232.63 and 208.35 μ mol^–1) respectively as shown in Table 2. There was a significant difference between cellulase activities (p<0.05) across the organs.

However, cellulase activity was not significantly different between the salivary gland and the digestive gland and between the small intestine and the crop (Table 1).

Sucrase: Sucrase activity (190.06±30.209 μmol min^–1) was highest in the stomach of A. marginata. The salivary gland recorded the lowest sucrase activity (34.01±3.465 μmol min^–1) as shown in Table 1. The specific enzyme activity of sucrase was lowest in the salivary gland (4.74 μ mol^–1) as shown in Table 2. There was no significant difference in sucrase activity across the organs except in the salivary gland (Table 1).

Protease: The highest level of protease activity (28.00±0.560 μmol min^–1) was recorded in the stomach followed by the salivary gland (16.80±1.482 μmol min^–1). Protease activity in the digestive gland was the lowest (Table 1). The specific enzyme activity of protease in the crop was the highest and the lowest in the digestive gland as shown in Table 2. There was a significant difference between protease activities across the organs (p<0.05). However, protease activity was not significantly different between the salivary gland and the crop and between the small intestine and digestive gland (Table 1).

Lipase: Lipase activity was highest in the salivary gland (1.74±0.150 μmol min^–1), followed by the crop. The stomach recorded the lowest enzyme activity (0.46±0.023 μmol min^–1) as shown in Table 1. The specific enzyme activity of lipase is the lowest among other enzyme activities. There was a significant difference between lipase activities across the organs (p<0.05). However, lipase activity was not significantly different between the salivary gland, the crop and the digestive gland (Table 1).

There was a significant difference between the enzyme activities across all the organs. However, protease and lipase activities in each organ were not significantly different. The activities of sucrase and protease in the salivary gland, cellulase and sucrase in the digestive gland and α-amylase and sucrase in the small intestine were not significantly different.

DISCUSSION

The presence of enzymatic activities of α-amylase, cellulase, sucrase, protease and lipase in the gut regions of A. marginata affirms that snails are omnivores equipped with adequate enzymes necessary for the digestion of their multivarious food items. α-amylase aids the hydrolysis of starch into simple sugars while cellulase catalyses the

breakdown of cellulose polysaccharides into glucose which is essential for digesting fruits and vegetables. Sucrase catalyses the hydrolysis of sucrose to simple sugars (glucose and fructose). These three enzymes have the glucose substrate in common. However, lipase catalyses the breakdown of lipids by hydrolysing the esters of fatty acids and protease catalyses the breakdown of proteins into smaller polypeptides or single amino acids.

This study revealed that the highest enzymatic activities were found in the glucosidases (α-amylase, cellulase and sucrase) across all the organs suggesting that A. marginata would prefer a plant-based diet. Lipase has the lowest enzyme activity across all the organs, however, the stomach has the lowest lipase activity.

The presence of substrates stimulates the secretion of digestive enzymes¹⁸. The presence but relatively low enzyme activity of protease and lipase suggests that A. marginata feeds more on a carbohydrate diet. The high activity of α-amylase was anticipated as it is needed to digest starch which is the major component of foliage and rind consumed by the snail. The present study agrees with the findings of Ademolu et al.¹⁷ who reported higher α-glucosidase activity in the gut regions of A. marginata.

The highest specific enzyme activity was recorded in the crop of A. marginata. The crop is the portion of the alimentary canal used to store food before digestion. In a related study by Charrier and Rouland¹⁶, the bulk of carbohydrate digesting activity was concentrated in the crop juice of Helix aspersa. The high enzymatic activities recorded in the crop region might be associated with the presence of symbiotic microbes that have been reported²⁷ to aid in the snail’s digestive processes. However, Ademolu et al.²¹ reported that the bulk of enzyme activity was found in the stomach of the snail while Garcia-Esquivel and Felbeck et al.²⁰ recorded the highest enzymatic activities in the hepatopancreas of Haliotis rufescens. Valarmathi and Asokan¹⁵ reported the highest enzyme activity in the digestive gland and the lowest activity in the stomach of Cryptozona bistrialis. According to Vineetha et al.¹⁸, the intestine of A. fulica showed the highest quantities of enzymes than other regions of the gut.

The activity of lipase was generally very low and almost not present in the stomach region. Vineetha et al.¹⁸ also reported very low lipid digestion in Achatina fulica. This could suggest that snails rarely feed on a diet rich in lipids. Since A. marginata feeds more on vegetative and decaying matter, it is not surprising that cellulase and α-amylase activities are found in large amounts in their tracts. This was similar to Ozioko et al.³⁸ who reported high cellulase activity in the digestive tracts of Achatina achatina.

The cellulose digestion in the gut of snails depends on the metabolic activities of the gastro-intestinal microbiota³⁹. The highest enzyme activity for cellulase and α-amylase was found in the crop and salivary gland respectively. Thus, the crop of A. marginata serves as the active site for cellulose digestion. Cellulase activity recorded in the crop (594.198±41.583 μmol min^–1) and small intestine (532.180±28.364 μmol min^–1) can be associated with the high microbial flora in the two regions. Cardoso et al.⁴⁰ reported a complex holobiont system containing diverse and abundant microbial communities in the gut of Achatina fulica. However, Pinheiro et al.⁴¹ observed that the gut microbes only assist (but are not essential for) the digestion of cellulose since snails treated with antibiotics (to eliminate bacteria) retained the capacity to digest polysaccharides.

The enormous economic and nutritional benefits derived from giant African land snails have necessitated increased cultivation. Shells, mucin and flesh have all been investigated and found to be rich raw materials for industries^24,42,43. Cellulase from the digestive tracts of A. achatina possesses high thermostability and acid/alkali stability which indicate the prospective commercial significance of cellulase in the degradation of cellulose-containing materials³⁸. Successful and profitable cultivation of A. marginata relies on adequate knowledge of the digestive physiology of snail.

The current study recommended that due to the high activity of cellulase recorded in the gut of A. marginata, cellulase secreting microbes can be isolated from the gut of A. marginata as a natural biocatalyst for the bioconversion of renewable energy resources, biofuel etc. to meet the demands of the future and sustainable world. Snails are highly nutritious gastropods that can be used as animal protein, Heliciculture is a lucrative business. A correctly formulated feed for snails can rapidly increase their growth size and greatly reduce the cost and time of rearing. Snails can be easily cultured on budget with domesticated tools and environmental resources because of their ability to feed on various food items including some of our domestic wastes.

CONCLUSION

This study revealed the high preference for a glucose substrate-based diet (carbohydrate) over any other feed type considering the high level of α-amylase, cellulase and sucrase activity found in the gut of the snail. This study showed that the activity of enzymes in A. marginata is dependent on their feeding habits. Revitalization of some wild species like snails would assist in combating the insufficiency of animal protein in many developing countries. An adequate study of the digestive enzymes in snails will aid in the proper recommendation of feeds, feeding methods and nutrition for snails. The study of digestive enzymes can also provide useful information for the feed formulation of snails to relate it to their feeding method and developmental stage.

SIGNIFICANCE STATEMENT

The study identified the digestive enzymes in Archachatina marginata. The activity of the enzymes and the functional properties were discussed. This study will guide the appropriate choice/formulation of feed for successful snail rearing in Nigeria.