Histological Investigation of the Digestive System and SEM Study of Radula in Fusinus nicobaricus (Gastropoda: Fasciolariidae)

Specimens of Fusinus nicobaricus used in the present study were collected during low tides from the sea in their natural habitat that is intertidal zone, from reefs by divers and from trawl nets used for crab fishing from Gulf of Mannar coastal region. They were brought to the laboratory and maintained under laboratory conditions for further observations. Preserved specimens were not used for anatomical study of digestive system as they lose their original colour due to preservation. Additionally, the organs may show considerable shrinkage. Freshly killed specimens were used for the study of digestive system. Measurements were made using ocular and stage micrometers. Camera lucida drawings were made with the help of a stereo-zoom microscope.
 
Histological studies of the digestive system
 
For histological studies, the tissue samples were taken from proboscis, oesophagus, stomach, hepatopancreas, intestine and rectum. These different soft tissues were fixed in aqueous Bouins and Zenker’s solution. Sections of 6-8 mm thickness were made from different organs and stained in delafield hemotoxylin with eosin as a counter stain.
 
SEM study of radula
 
For scanning electron microscope study of radula, the anterior portion of the proboscis was excised, the radula with adjoining tissues were removed from the proboscis and boiled with alkaline iodide solution until the dissolution of all the muscle fibers attached to the radula. The radula thus obtained was then dehydrated by immersing them in increasing concentration of alcohol (50, 70, 90 and 100%). Then the dehydrated radula was prepared with coating for making it suitable for scanning electron microscopical observation.
        
Principally coating of the radula consists of placing the specimen in a vacuum evaporator pumping the chamber down to a vacuum level equivalent to that in the scanning electron microscope specimen chamber and heating the conductive metal (gold) to the point of vapourization. A small part of the cloud of metallic vapour hits the surface of the object and sticks to it so as to provide a thin metallic coating over it. Continuous tiltation and rotation of the specimen ensures that the metallic vapour covers all sides of the specimen. Then the specimens were mounted to SEM stub and the observations were made with different magnifications for making the electron micrographs by using JEOC JSM 35CF scanning electron microscope (Annamalai University, Chidambaram).

Histological studies of the digestive system of Fusinus nicobaricus
 
The digestive system of Fusinus nicobaricus contains proboscis, oesophagus, stomach, hepatopancreas, intestine, rectum and anus.The general organization of digestive system of Fusinus nicobaricus, follow the same general pattern of neogastropod. The cellular organization of the various body parts of Fusinus nicobaricus, histologically proved the same functional significance as encountered in other Fasciolarids.The digestive system in the Fasciolaria Fusinus nicobaricus begun with the pleurembolic type of proboscis and the rachiglossate type of radula (Fig 1-3) similar to that of Thais sp, Muricanthus sp, Uro sucpinx sp (Kool,1993).
 
Proboscis
 
The most conspicuous features of the digestive system was the massive proboscis. In Fusinus nicobaricus the proboscis was a large, yellowish organ and of pleurembolic type in which the basal part was invaginable (Fig 1a). When the proboscis invaginated, it retracted the distal part into a proboscis sheath. The mouth lied at the anterior end of the proboscis. The odontophore and the radula sac lied within the proboscis.The proboscis of Fusinus nicobaricus was similar to the description of the proboscis of Thais species by Tagore (1989), Rapana rapiformis by Rajakumar (1995), Babylonia spirata by Thilaga (2005) and Chicoreus virgineus by Sharmila (2009).
 
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Oesophagus
 
The oesophagus which followed the buccal mass was long in Fusinus nicobaricus. The oesophageal region was complicated and divided into anterior, mid and posterior oesophagus. A pair of salivary glands were associated with the oesophagus. Ducts of salivary glands opened into the oesophagus. Histologically the wall of the oesophagus contains ciliated columnar epithelium under lined by basement membrane and secretary cells with large nuclei (Fig 1b).
 
Stomach
 
Stomach was pale yellow in colour and an elongated sac. In the surface view the dorsal side of the stomach was visible and at its posterior side had a caecum. Ventrally and laterally the stomach was enclosed by hepatopancreas. The distal portion of the stomach gave rise to intestine at its anterior extremity. The stomach was lined with ciliated columnar epithelium. In all the epithelial cells, the elongated nuclei were centrally located. Mucous cells were scattered among the columnar epithelial cells. Phagocytes were seen at the base of the epithelial cells possessing oval or round nuclei. Below the epithelium dense connective tissue was present where blood cells, fibroblasts and muscle fibres were embedded in the stomach of Fusinus nicobaricus (Fig 1c).
 
Intestine
 
The intestine was a tubular structure arised from the distal part of the stomach. The intestine had a wall of its own, till it enters the mantlle cavity. It ran forward adjacent to the kidney. It was attached to the mantle as it nears the mantle cavity and was surrounded by connective tissue. The intestine was smooth leading out from stomach. Histologically the intestine showed finger-like projections inside the intestinal wall lined by ciliated cells and glandular cells (Fig 2a).
 

 
Hepatopancreas
 
The hepatopancreas was present in the upper whorls of the animal. It was single lobed, brownish yellow coloured gland and found covering the whole intestine except on the ventral side and was also enclosing the stomach. It opened through a pair of openings into the anterior part of the intestine. Histologically it contains mucous and secretory cells (Fig 2b).
 
Rectum
 
Rectum begun where the intestine was dorsoventrally flattened and the hind region of the intestine was distinguished by its size and it was differentiated as rectum. It received faecal matters from the intestine. The wall of the rectum was longitudinally folded. The rectum terminated in a conical tube which in turn opens outside through the anus. Histologically the rectal walls were made up of epithelial cells. The ciliated cells were responsible for the movement of the content of the rectum (Fig 2c).
 
Scanning electron microscopical study of radula in Fusinus nicobaricus
 
The scanning electron microscopical study of radula showed the structural parts of feeding apparatus. The shape and arrangement of the radular teeth were species – specific often important in classification and identification.The radula had been used as a main tool in the systematic studies of gastropods (Reid and Mak, 1999). Gastropods replace their radular teeth throughout their entire life cycle (Runham and Thornton, 1967). SEM observation on the radula of Fusinus nicobaricus elucidated several morphological details.
        
In Fusinus nicobaricus, the type of radula was typical rachiglossate and the radular formula was 1+R+1.  This type of radula was highly evolved one. The central or rachidian tooth was tricuspid had three large pointed sharp cusps. The median cusp was sharp and pointed straight towards the anterior end. The lateral cusps were also sharp but slightly bent and pointed outwards. The outer, hooked lateral teeth collect torn or detached particles and bring them into the center when the radula was retracted.The central tooth was elongated and broader at the base (Fig 3a and 3b). The posterior ends of all teeth, especially lateral ones were sharp pointed, curved inwards (Venkatesan et al., 2016). The radular ribbon was longer (Isarunkura and Runham, 1968). The reduction on the size of radular teeth seems to correlate with its environmental condition, food and feeding habits. The radular apparatus of Fusinus nicobaricus provided an excellent model for study of form and integration of morphology and function.
 

        
According to Carlos et al., (2003), the carnivorous animals have smaller radulae than herbivorous ones and Bandel (1984) also observed smaller radula in N.marochiensis that has a carnivore diet. Nybakken (1990) observed rachiglossate radula in Fasciolaria and Thais a morphologic ontogenic radular change, related not only to the size of the animal but also the changes in the diet during the animal development.It is evident from the literature and from the scanning electron microscope study, the structure of the radula of the study organism Fusinus nicobaricus revealed its carnivorous feeding habits respectively.

Fusinus nicobaricus is an important gastropod from Southeast Coast of India. An integrated study of histological and scanning electron microscope study of radula enables one to gain a sound knowledge about the functional morphology of digestive system of Fusinus nicobaricus. The organization of the system and its efficient functioning are understood better.