Feeding pattern and gut enzymes activity of Giant African land snail (Archachatina marginata) during growth phases

Snails are multivarious feeders that eat different kinds of food present in their habitat. The feeding pattern, occurrence of gut microbes and gut enzymes (proteinase, lipase, cellulase, glucosidase and α-glucosidase) activity of the three stages of development of the Giant African land snail, Archachatina marginata were examined in this study. Results showed that the snails at all growth phases fed at night period (19:00 to 4:00 GMT), while the juvenile stage spent more time on feeding and had significantly higher feed intake than in the other two growth phases. The activities of the gut enzymes were significantly affected by the growth phase as the adult stage recorded the highest activity level for all the five enzymes followed by the juvenile stage, while the snailet stage recorded the lowest one. The stomach region of the gut had the highest enzymes activity while the oesophagus had the lowest. There was presence of many microbial species in the gut across the stages of development but, more bacteria than fungi species were isolated. These findings suggest that the adult stage of A. marginata is better equipped to hydrolyse its numerous diets.


INTRODUCTION
The numerous benefits derived from giant African land snails have necessitated increased raising in captivity.Shells, mucin and flesh have all been isolated and researched and found to be rich raw materials for industries (Akinnusi, 2014).In captivity, three stages of development are found namely: Snailet, juvenile and adult (South, 1992).
According to Ademolu et al (2013a) the juvenile stage of Archachatina marginata laid average of 9.3 eggs followed by the adult stage that laid 4.7 eggs, while no egg was laid by the snailet stage.Similarly, Idokogi and Osinowo (1998) observed that the reproductive tract of A. marginata increased in size as the age increased.
Previous works on snails revealed that the three stages of development have different circulatory physiology.The juvenile stage has highest haemolymph biochemical values while the adult recorded the least values (Ademolu et al, 2009).In a related study, Abiona et al, (2007) observed that juvenile phase produced highest number of spermatozoa and ova suggesting their higher sexual activity than the two other developmental phases.
The success of any animal husbandry depends on how the animals can convert and utilize food substances given to it.This factor depends majorly among others on the presence and activities of the enzymes in the gut.Snails are good converter of various household wastes into good body weight (Akinnusi, 2014).They are able to do this effectively because they are equipped with necessary enzymes required to breakdown these foods (Yoloye, 1994).Lipase, proteinase and carbohydrases were earlier detected in the gut regions of A. marginata at varying levels during aestivation (Ademolu et al, 2013b).
However, unlike studies done on reproductive tract of A. marginata there has not been a comparative study on its digestive physiology across the three development phases.This knowledge will reveal its nutrients utilization and feeding behaviour which are prerequisite in rearing animals in captivity.

ExpErimEntal sitE
This study was conducted in the animal house of the Department of Pure and Applied Zoology, Federal University of Agriculture, Abeokuta, Nigeria (7 0 10'N3 0 2'E).

ExpErimEntal animals
A total of sixty snails (A.marginata) where purchased from Kuto market in Abeokuta, Nigeria (7 0 2'N 3 0 4' E).They were bought based on their weight and number of whorls (20 individuals per stage of development) as earlier described by Idokogi and Osinowo (1998).

DissEction procEDurEs
Snails were dissected following the methods described by Segun (1975) and modified by Ademolu et al (2013b).The alimentary canal was carefully separated from the common hermaphroditic duct and was totally removed using flamed forceps and scissors.The isolated alimentary canal was cut out into various sections: Oesophagus, Stomach and intestines.The gut contents of each section were then emptied into separate labelled sterile petri dishes for further analysis.

morphomEtrics of thE alimEntary canal
The length and width of the gut regions were measured using calibrated tape rule and the weight of the stomach was taken with aid of a sensitive weighing balance (PM-Mettler-K)

fEED intakE anD fEEDing pattErn
The amount of feed consumed by the experimental snails was determined by subtracting the weight of food left (uneaten) from the weight of food given.
Feed Intake= food given -food uneaten Feeding period was determined as the time spent by individual snail at the feeding place (Grimm and Schaumberger, 2002).

gut microbEs Estimation
The methods described by Idowu et al (2008) were followed for gut microbial estimation.The bacterial enumeration and identification were done by methods of Sneath et al (1986).Identification of fungi was done by Bernett and Hunter (1972) techniques.

EnzymEs activitiEs assay
Each gut region of the experimental snails was homogenized separately in 20ml of phosphate buffer (ph 7.0).The homogenate was centrifuged at 4,000 rpm for 30 minutes.The sediment was thrown away, while the supernatant was used as the enzyme extract.
Cellulase, α-glucosidase, amylase, proteinase and lipase activities were determined following methods of Adedire et al (1999).They were estimated quantitatively by Dinitrosalycyclic acid reagent (DNSA).The amount of reducing sugar (glucose) produced at the end of incubation period was determined calorimetrically at 550nm.Each reaction mixture composed of 0.2ml enzyme extract, 0.2ml of phosphate buffer (pH 7.0) and 0.4ml of the substrate.The reaction mixtures were incubated at 37 0 C for 1hour.Lipase activity was determined by adding 0.4g of sodium taurocholate to the enzyme extract and incubated at 35 0 C. The absorbance of the sample was read at 415nm.All enzymes assays were done in triplicates.

statistical analysis
All data from the above experiments were analysed by one-way analysis of variance (ANOVA) and where there were significant differences, means were separated using Student Newman-Kuel Test (SNK)

RESULTS
The measurements of the alimentary canal of A. marginata across the three growth phases are shown in table I.There was a significant difference in the measu- rements of the gut sections of A. marginata across the growth phases, with the adult stage having the highest size.
Figure 1 showed the feeding pattern of the three (3) growth phases of A. marginata.During the snailet stage, two peaks of feeding time were observed at 4:00 and 23:00 GMT, while there was no feeding between the hours of 9:00 and 17:00 GMT.The juvenile stage fed between the hours of 19:00 and 4:00 GMT.However, highest feeding activity was noticed at 3:00 GMT.No feeding occurred between 7:00 and 18:00 GMT.The adult stage of A. marginata did not feed between the hours of 5:00 and 18:00 GMT, but commenced feeding at 19:00 to 4:00 GMT.During this feeding period, juvenile stage spent more time than other two stages.
Table II showed the feed intake of the experimental snails.The juvenile stage had the highest feed intake (129.8g)followed by the adult stage while the least feed intake was recorded by the snailet stage (78.8g).
The enzyme activities in the oesophagus of the 3 growth phases of A. marginata are shown in table III.The snails in adult stage recorded the highest enzyme activities (except lipase) while the snailet stage recorded the least activity.
Table IV described the enzyme activities in the intestine of A. marginata during different growth phases.Adult stage similarly had the highest activities while the snailet stage had the least activity.Amylase activity was significantly (p<0.05)higher than other enzymes activities in the intestine.Similar observation was noticed in the stomach region of the gut (table V).Amylase and α-glucosidase recorded the highest and lowest enzyme activities respectively in the stomach region of the snails.Comparison of means showed that the stomach region had the highest enzyme activities than the other gut regions.Escherichia coli, Penicillium sp (2.0x10 6 , 1.45x10 6 )

() cfu of bacteria and fungi isolates respectively
There were presence of both bacteria and fungi species in the gut regions of the experimental snails (table 6).However, bacterial species were more present than the fungi.Also, the stomach region (juvenile and snailet stages) had highest bacterial colony forming units while the intestine region had the least.

DISCUSSION
Age has significant effects on the size of giant African land snails.The older the snails the bigger their sizes (Okon and Ibom, 2012).In this study, the adult stage recorded the highest measurements of the gut regions.This parallels the report of Idokogi and Osinowo (1998) that as the snail age increased the reproductive tract size also increased, thus the adult stage had the biggest size.
Juvenile stage is the most active phase in the life of snails (South, 1992).Juvenile stage in this study recorded the highest feed intake while the snailet recorded the least.The juvenile phase requires high energy substrate for its growth and activities like oviposition and movement which are highly reduced in the adult phase due to big size and age.Ademolu et al (2009) had earlier observed that snails in juvenile phase had significantly higher glucose and lipids concentration in their haemolymph than other two growth phases.
Results showed that the experimental snails across the three growth phases fed in the dark period of the day (19:00 and 4:00 GMT), while no feeding occurred in the day time.Snails are nocturnal animals that feed, walk and mate during the night hours (Hodasi, 1982;Okon and Ibom, 2012).Ademolu et al (2011) and Ogbu et al (2014) had earlier reported that A. marginata and A. achatina fed during the night from 20:30 to 2:00 GMT.According to Yoloye (1994), the nocturnal nature or behaviour of snails is adaptive in function as there are fewer FEEDING PATTERN AND GUT ENZYMES ACTIVITY OF GIANT AFRICAN LAND SNAIL DURING GROWTH PHASES Archivos de zootecnia vol.66, núm. 253, p. 33.predators to attack them in the night.Similarly, the cool environment helps to reduce water loss due to evaporation.
Juvenile stage spent comparatively more time feeding than other stages of development.This likely relates to their active nature and need for more nutrients.The adult stage on the other hand has reached their peak growth stage thus requiring less food.
Snails are equipped with adequate enzymes necessary for the digestion or hydrolysis of their multi various food items (Adedire et al 1999).Lipase, α-glucosidase, amylase, cellulose and proteinase were detected in the gut regions of the experimental snails which enabled them to hydrolyse various food items in their diet.The presence of lipase and proteinase in the gut confirmed the omnivorous nature of snails as earlier suggested by Ademosun and Imevbore (1988).Snails not only feed on plant based food but on earthworms, ants and mushroom (Amusan and Omidiji, 1999).
Phase of growth had a significant effect on the gut enzymes activities as the adult stage had the highest enzymes activities followed by the juvenile phase while the snailet had the least.Maha et al (2009) reported that steroids hormones were more abundant in the tissues of adult water snail Biomphalaria alexandrina than juvenile snails.The size of the snail gut might be responsible for this observation.Big snails (adults) have more cells in the gut which release or synthesize more enzyme than the young snails with fewer cells.Bundit (2000) observed that as the gut size of Nile Tilapia, Oreochromis niloticus increased, the enzymes activities of the intestinal tract increased.Le Francois et al (2000) similarly observed that the activities of glycolytic enzymes increased with the fish mass.The adult stage of snails had a bigger size and thus contained more food mass which consequently will stimulate secretion of relevant enzymes Amylase is needed for the hydrolysis of starch; hence, the presence of amylase in the gut of the experimental snails at a significantly higher activity than other enzymes is not unexpected.The main constituent of pawpaw leaves consumed by these snails is starch.Hildalgo et al, (1999) reported that herbivorous and omnivorous fishes have high amylase activity because it is used to break down the polysaccharide in the diet.
Comparison of means revealed that the stomach region of the snail gut recorded the highest enzyme activities.This conforms with the report of Ademolu et al (2013b) and Adedire et al (1999) that the stomach and hepato pancrease are the main sites of food digestion as earlier mentioned by Segun (1975).
Many microorganisms were found in the gut regions of the snails, however, there were more bacteria species than fungi species.Adedire et al (1999) similarly detected no fungi in the gut of A. marginata while various bacteria species were isolated.The nature of food consumed by the snails might account for this occurrence.Snails prefer moist cool environment and feed on decaying plant materials which support the growth of bacterial species more than fungi species.

Figure 1 .
Figure 1.Feeding pattern of the three (3) growth phases of A. marginata (Modelo alimenticio de A. marginata durante tres fases de crecimiento)

Table I .
Measurements of the gut regions of A.marginata at the three growth phases (Dimensiones de las regions del tubo digestivo de A.marginata en varias etapas de crecimiento)* * Mean values in each column with the same superscripts are not significantly different (p>0.5).FEEDING PATTERN AND GUT ENZYMES ACTIVITY OF GIANT AFRICAN LAND SNAIL DURING GROWTH PHASESArchivos de zootecnia vol.66, núm.253, p. 31.

Table II .
Total feed intake of A.marginata at the three growth phases (Ingesta total de alimento por A.marginata du- *Mean values in the column with the same superscripts are not significantly different (p>0.5)

Table IV .
Enzymes activity in the intestine of A.marginata at the three growth phases (mg/g) (Actividades enzimáticas en el intestino de A.marginata durante tres fases de crecimiento (mg/g))*

Table V .
Enzymes activity in the stomach of A.marginata at the three growth phases (mg/g) (Actividades enzimáti- *mean values in the same column with different superscript are significantly different (p<0.05).

Table VI .
Microflora and load (cfu)of the gut regions of A. marginata at the three growth phases (Microflora y carga microbiana (ufc) en las regiones del tracto digestivo de A. marginata durante tres fases de crecimiento)