​The Histopathology of Alimentary Canal of Stinging Catfish Heteropneustes fossilis (Bloch, 1794), Susceptible for Trematodes Incursion

Digenetic trematodes, a class of parasitic flatworms that mainly live in the intestine, stomach and gall bladder of fishes. They are important fish infestation agents that may harm their hosts by irritating, damaging and obstructing the tissue. They often induce changes in the layers of the intestine, affecting fish digestion, fish welfare and exposing them to various diseases. As a result, understanding how diseases are spread and the severity of damage is important.  
       
Heteropneustes fossilis (Bloch, 1794) (Cypriniformes: Saccobranchidae) is native to India. It contains a high amount of iron (226 mg 100 g^-1) and fairly high content of calcium compared to many other freshwater fishes (Saha and Guha, 1939), so it is considered a great source of nutrition. In India, this is high value food fish and a common host for trematode parasite M. vittatusia that inhabit the gastrointestinal tract of marine and freshwater bony fishes. Genus Masenia Chatterji, 1933 is often distinguished from the other cephalogonimids by the possession of two rows of enlarged circumoral spines and vitelline follicles located mainly within the hind body (Bray et al., 2008). Present form of Masenia vittatusia Agarwal, 1963, is distinguishable from other species of Masenia in having genital pore slightly away from oral sucker, cirrus- sac overlaps ventral sucker anteriorly, ventral sucker pre-equatorial, sub-spherical, gonads sub-equal, vitellarium confined in the lateral field and uterus post-testicular (Fig 1).
 

       
The histopathological studies not only provide an error-free diagnosis of diseases but also gives information about pathogenicity of these parasites as well. All the layers of the intestine contribute to the distinct properties such as the mucus layer covers the lumen of the digestive tract and maximizes its function (Dezfili et al., 2009; Sanil et al., 2010; Matos et al., 2017). The increase in fluke infection may be the cause of intestinal secretion, changes in the mucosa and submucosa. Though many studies have been conducted on fish trematodes, relatively few studies have focused on histopathological changes in host tissues (Bose and Sinha, 1979; Barbara, 1980; Chung Yui-tan 1981; Gupta and Agarwal, 1983). Therefore, the present study deals with the histopathological effects on fish intestine derived due to infestation of trematode parasite M. vittatusia Agarwal, 1963.

Collection of fish
 
Heteropneustes fossilis (Bloch, 1794) is a fish found throughout the year. Collection part of research work was completed in the Helminthology Laboratory, Department of Zoology, University of Lucknow, Lucknow. A two-year survey, from January 2018 to December 2019, was performed to see the parasitic load in the gastrointestinal tract of fish. A total number of 248 fish were collected from the Gomti River, Lucknow, identified with the help of the handbook “Fishes of U.P. and Bihar” (Srivastava, 2002) and dissected. The entire alimentary canal is removed, kept in physiological saline (0.9%), washed multiple times to remove tissue and fats debris and thoroughly examined to check the presence of trematode parasites. Specimen of trematode parasites fixed in A.F.A fixative and preserved in 70% alcohol for taxonomy purpose. Parasites were identified using Systema Helminthum’ by Satyu Yamaguti, Part I, Digenetic trematode of fishes, (1953).
 
Histopathological analysis
 
Histopathology part of research work conducted in Central laboratory (DST-FIST), Department of Zoology, University of Lucknow, Lucknow in September-October 2019.
       
To perform the histopathological analysis, infected and uninfected fragments of intestine were fixed in Bouin’s fixative (picric acid fixative) for 12-24 hours, washed and preserved in 70% alcohol. All the histological procedures performed using the histological protocol in accordance with Suvarna et al., (2018). For staining purposes, haematoxylin and eosin stains were used and sections were photographed using a Nikon compound microscope (Eclipse E200, Japan).

The intestine in H. fossilis starts from the end of the pyloric region of the stomach, anterior intestine, posterior intestine and end with the rectum (Fig 2). Earlier, Rashad et al., (2004) also described the four regions of the intestine in teleosts. We recovered maximum parasites from the anterior part of the intestine, hence we used this part for histological analysis purpose.
 

 
General morphology of alimentary canal
 
The anterior part of the intestine consisted of three layers, the mucosal layer is innermost layer of the digestive tract; its luminal face is directly in contact with ingested food and is highly specialised for the digestion, absorption, secretion and immune processes that occur along the digestive tube (Ross and Pawlina, 2016). The layer contains numerous folds of finger-like projections called microvilli that increase surface area for proper absorption of food (Fig 3A). The mucosa layer consists of two layers, one is lamina epithelium that contributes to absorptive and secretory function and another one is lamina propria, which consists of lose connective tissues. The columnar epithelium of mucosa rich in specialized goblet cells (Fig 3D) that may secrete mucus throughout the gastrointestinal tract. The presence of lacteal was also observed within villi where capillaries absorb the nutrients from villi. The second layer of the intestine is muscularis that comprises an inner circular layer and another outer longitudinal layer of smooth muscle cells. The outermost layer is the serosa where terminal alimentary canal may open into the anus.
 

 
Pathological manifestations
 
In H. fossilis maximum changes were observed in the mucosal layer; the columnar epithelium is broken at several places, high burden of parasites conjointly affected the lamina propria that significantly peeled off into the lumen (Fig 4A). Shahin et al., (2013) observed shortening of intestinal villi or compressions of mucosal folds in Clarias batrachus. On the other hand, we observed hyperplasia of villi along with fused microvilli (Fig 4A and B). Reddy et al., (2013), had seen fibrosis, hyperplasia and metaplasia in the intestine of Channa striatus which might be associated with changes in organ morphology, physiology, metabolism and secretory efficacy as well. We found increased goblet cells number in epithelium that are somewhat similar to K Cinar et al., (2006) (Fig 4B) but here goblet cells were found flattened than routine shape. Other mucosal glands were observed with changes in their regular shape. In the mucosa and muscularis layer, we noticed hypertrophy in blood vessels that might cause haemorrhage inside the layers (Fig 4B and 4C). Our findings also reveal the prevalence of mast cell and lymphocytes within the mucosa (Fig 4D).
 

       
M. vittatusia Agarwal, 1963 is a predominant trematode parasite of the GI tract of freshwater fish H. fossilis. Two suckers of this endo-parasite may adapt to maintain attachment with internal organs of the host such as the intestine and blood vessel where it not only gets nutrition to survive but also completes its definitive phase of life cycle. Collectively, this pathogen caused severe inflammation to fish tissue which can be identified by hyperplasia in villi, hypertrophy or swelling in blood vessels. Aggregation of mast cells and lymphocytes is evidence showing an immune response against the pathogenic agent. However, inflammation is a protective mechanism posed by the host H. fossilis but parasite may have caused considerable harm to its host tissue, as a result, damaged connective tissue shaded into the lumen and epithelial cell number increased. The current histopathological status of the tissue suggests that the worm possibly disrupted the host’s routine physiological process and impaired the metabolism.

Heteropneustes fossilis (Bloch, 1794) is a very nutritious and economically important food fish in India, serves as a host for digenean flatworm belongs to the family Maseniidae Yamaguti, 1954. M. vittatusia adheres to the intestine and blood vessels of the host by an oral and ventral sucker and cause extensive damage to these parts. Such damages not only affect the intestine but also impair food absorption that may be associated with the malnutrition of fish. The reduced surface area in the lumen, hyperplasia in villi, damage of blood vessels and change in routine shape of glands can affect the whole digestion of nutrients and spoil the food quality as well. Increased parasitism may be responsible for increased mortality and reduced commercialization of fishes. Histopathology of the internal organ of fish gives an insight into the lesions and also an opportunity to seek out parasites borne diseases which provide a concept to prevent them.

We are grateful to the University Grants Commission (U.G.C.), New Delhi, India (CSIR-UGC, NET/ JRF), Government of India, for financial support. We would also like to thank Department of Zoology, University of Lucknow, for providing all necessary laboratory facility during research.