Prevalence of rhabdochoniasis in snow trout of river Swat and river Panjkora , Khyber Pakhtunkhwa province , Pakistan

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT This research was aimed to explore the helminth parasitic diseases in Schizothorax plagiostomus (the snow trout) from river Swat and river Panjkora, Pakistan. Collection of 360 fish specimens have done from the lower, middle and upper reaches of both the rivers through gill nets, cast nets, dragon nets and hooks. All the samples were examined in the University of Malakand, Zoology Department for helminth parasites during the months from January 2015 to December 2016. Of the total examined fish samples 21.9% (n=79) were infected with Rhabdochona spp including 17.7% (n=32/180) in river Swat and 26.6% (n=47/180) in river Panjkora. Highest month-wise prevalence (p=0.09878,p<0.05) was reported in May (30%), then by February and October each (26.6%) while the lowest during August (13.3%). Highest prevalence (p=0.9723, p<0.05) was reported in summer season (26.6%), while lowest in the winter season (20%). Adults were highly infected (p=< 0.0001) in prevalence (63.7%) followed by sub-adults (13.2%) while no infection was found in juvenile specimens. Females fish samples had higher (p=0.0277, p>0.05) prevalence (28.8%) than males (16.6%). Fishes of the lower reaches had highest (p=0.0029, P>0.05) prevalence (31.7%) followed by middle reaches (16.5%) while the lowest prevalence was observed in samples of fish collected from upper reaches (9.87%). Present study address that Rhabdochona spp in the intestine of snow trout has a long term relationship and call as a natural infection in cyprinids and zoonotic risk to human.

trout from river Swat and river Panjkora, in order to take steps for the control.

Materials and Methods
This research was carried out on the year round infection of Rhabdochona species in snow trout of both the rivers in Swat and in Panjkora, Khyber Pakhtunkhwa, Pakistan. The fish samples were collected from the lower, middle and upper reaches of both the rivers through gill nets, cast nets, dragon nets and hooks during the months from January to December 2015 and 2016. The collected fish specimens were kept in containers containing preservatives (10% formalin) and transported University of Malakand, where they were necropsied for parasites.
Measurements of fish were made by ruler and Venire caliper. Body length of each fish sample was measured by china tape and steel scale. Each sample of fish was weighted with the help of precise balance model No.18220 Switzerland. The sexes of the fish were determined by using the procedure: (1) the abdomen of each rip fish sample was pressed as to extrude whitish milt (represent male) or eggs (represent female).
(2) Each sample of fish was dissected and ensure the presence or absence of testes or ovaries. Presence of testes evidenced maleness and presence of ovaries considered that the fish was a female. (3) During dissection gonads were removed and examined under the microscope for immature eggs or milt. Fish samples were identified by the keys provided by Talwar and Jhingran (1991), Jayaram (1999), Mirza and Sandhu (2007).
Eeach fish samples was washed with distilled water, dissected and screened for parasites. By using the surgical dissecting incisors each sample of fish was cut alongside the mid ventral line. The visceral organs were exposed and placed in separate dishes. Hand lens was used to examine the encysted larvae, parasites embedded on the visceral organs, body cavities and membranes. The recovered specimens of parasites were washed properly in sodium chloride solution and then fixed in alcohol-formol acetic acid (Olurin and Somorin, 2006). Each of the nematode were preserved in vials labeled containing 1:1 parts alcohol (70%) pure glycerin. The specimens were placed on a glass slide in a small drop of fixative and put a coverslip. Excess fixatives were removed with tissue paper, the cover slip sealed with Vaseline and observed under a microscope first under (10x4) and then (10x40) power lenses.
The nematode parasite specimens were identified up to the species level, through keys provided by Yamaguti (1963), Quentin and Petter (1976), Schmidt (1986), Baker (1987), Anderson (2000. Observation on differential morphology was noted. Differences of the number of individuals examined and the number infected regarding months, seasons, age (length and weight), sex and localities between the host S. plagiostomus and the parasite Rhabdochona spp were calculated. The statistical analysis was done with Graph pad Prism-5. A statistically significant difference was considered, if P > 0.05.

Introduction
Pakistan is one of the deficient country in protein production derived from fish. Inland fisheries can have a significant role in the propagation of fish. In Pakistan 179 species of fresh water fishes are survived. Amongst them only 31 species are economically important. In the fresh water reservoirs of Malakand division, almost 40 species of the fish fauna are found. The famous game fish: S. plagiostomus has a wide range of distribution found in all the rivers, and tributaries of Himalaya uptoborders of China, Afghanistan, Pakistan, Turkistan, Nepal, Tibet, Bhutan and north-eastern India. S. plagiostomus is the chief edible fish of the Himalayan region in all over India, including Kashmir, Himanchal Pradesh, Uttarakhand, the Uttar Pradesh foothills and Assam (Day, 1888).
Due to increasing in rate of fishing and inundation of spawning ground S. plagiostomus is becoming rare. Further this species is indigenous and is facing anthropogenic, environmental and natural hazards. Diseases free fish is the need of today and tomorrow. Parasites effect on fishes, weakening the size, fish become not attractive for selling and making them not suitable for human consumption. Parasites present in fishes can transmit to human and cause diseases, and even death. Fish is one of the aquatic animals studied for helminth infection. Fish disease is one of the leading health problem and great attention is being paid to get more and more attention. About 1000 species of fish (both marine and fresh waters) are found in Pakistan. Most of them are edible and very few are studied for helminth parasites (Cheng and Cooperman,1964).
Nematode of the genus Rhabdochona Railliet, 1916 parasitizes fish fauna in all the zoogeographical regions of the world. This genus of nematode parasitizes more than 150 known species distributed worldwide. Ninety two species of the genus Rhabdochoana were considered valid (Moravec et al., 2008 1978;Fattohy, 1975;Rahemo, 1978;Rahemo & Kasim, 1979;Ali et al., 1987;Moravec et al., 1991;Saraiva et al., 2007). However, R. mesopotamica Rahemo & Kasim, 1979 has been synonymized with R. Denudata by Moravec and Scholz (1991), whereas R. mesopotamica Fattohy, 1975 is an invalid name according to the International Code of Zoological Nomenclature (González-Solís et al., 1997). However, broad geographic range and high host specificity of Rhabdochona spp., result massive kills of fishes is perhaps the most important pathogen of fresh water fishes throughout the world.
Keeping in mind the problem in beef consumption, diseases of mad cow, foot-mouth disease in sheep and goats, bird flu in in poultry, acquired more demand today for fish than before. Healthy fish is the demand of today and tomorrow. It is therefore needed to understand and recognize the prevalence of parasitic diseases of snow 3/6 Rhabdochoniasis in snow trout Adults' fish specimens were found highly 63.7% infected followed by sub-adults 13.2% while no infection was observed in juvenile fish specimens. The relationship between the host examined and that of infected was significantly associated (P< 0.0001). Female fish were more infected than females but the infection rate was not significantly associated with the host examined (Table 3).
Regarding locality the fish collected from lower reaches were found highly 31.7% than middle 16.5% while least infection rate 9.87% was found in the fish collected from upper reaches of both the rivers. The significant difference (P= p=0.0029) was noted between the examined and the infected fish specimens collected from both the rivers (Table 4).

Results
A total of 360 specimens of S. plagiostomus were screened. The rate of infection rate was calculated on the basis of months, seasons, age (length plus weight), sex and locality of the fish. The highest infection 30% was noted in the fish collected in May followed by February and October each 26.6% while minimum infection 13.3% was noted in the fish collected in the month of August. However, (P>0.005) was noted between the examined and the infected individuals of both the rivers (Table 1).
The fish samples collected in summer were highly 26.6% infected while in winter season the fish samples were found least 20% infected with Rhabdochona spp, however no significant association (P>0.005) was found between the fish samples examined and that of infected from both the rivers (Table 2).  Omeji et al. (2014) parasitic infection increase with the increasing of length and weight, they had been studied 5 species of nematodes from the fish Malapteruru selectricus. These nematodes were Camallanus sp., Capillaria sp., Contracaecum sp., Eustrongylides sp and Caenorhabditisbriggsa. The overall prevalence of infection noted was 47%. Present findings were in accordance with the studies conducted by Bichi and Yelwa (2010), they had studied the fish Clarias gariepinus from Nigeria and observed a nematode Procamallinus laevionchus with 28% rate of infection. The incidence was found to increase as the fish grew.
Present study reveals that female fish were found more infected28.8% than male individuals16.6% infection rate.

Discussion
Present study provides information regarding the prevalence of a nematode parasite, Rhabdochona spp in the population of S. plagiostomus (the snow trout) from different localities in river Swat and river Panjkora, Pakistan. The species of the genus Rhabdochona have also been reported in S. plagiostomus inhabiting rivers Panjkora and river Swat, Pakistan (Ahmad et al., 2020;Khalid et al., 2020). The natural route of this nematode transmission in snow trout remain unresolved and it may include the possible involvement of an intermediate host, Trichopteran (caddisfly) larvae of the genus Hydropsyche (H. angustipennis and H. pellucidula) as natural intermediate hosts (Moravec and Scholz, 1995) where the parasite becomes more infectious to the snow trout. This is due to the extreme flexibility in Rhabdochona species that can be induced by manipulating various hosts and culture conditions. If an intermediate host exists, then the results from this study indicate that its geographic range must be very expansive throughout the world. Even with the economic importance of snow trout, published information on its biology and fishery is scarce. Ahmad et al. (2014) studied 115 snow trout from river Swat for the prevalence of internal and monogenean parasites and find out 93% infection including with of river Panjkora were more infected than the snow trout of river Swat. Current study enlighten the issue of introduction of parasites in freshwater fishes particularly in the fish fauna of river Swat as well as river Panjkora which are the main reservoirs of edible fish in this locality. These diseased fish must be strictly check for parasitic infection as to safeguard the ecosystem and diversity.
Findings of the present research agreed with Amare et al. (2014), they were studied 412 fish from the Lago lake Nigeria and recorded species of the genus Clinostomum from nematode, further they concluded their results as females were found more infected than male fish. Omeji et al. (2014) was noted that female fish were highly infected with 57.5% infection rate and male with 42.4%.  Raissy et al. (2010). In another study conducted by Rafique et al. (2002) 37.5% of the fish Mystusvittatus was infected with R. magna. According to Leela and Rao (2014) studied goldfish in India and reported R. garuaiin with 40.6% infection rate. Their result indicates 3.14 mean intensity and 1.40 relative density.
Present study reveals that fish samples collected in summer were highly 26.6% infected while in winter season the fish samples were found least 20% infected with The seasonal occurrence of helminth parasites in the fish may also be due to the age of fish host and life cycle of the parasites because as the length of fish host increases, the prevalence of infection also increases. Data reveals that the prevalence of infection is found in almost directly proportion to the body weight in where the weight is increasing, it record to increase the infection rate caused by the parasite. In case of sex, female fish were found more infected than male and these differences in the incidence of infestation could be due to the physiological status of the females and their increased rate of food intake to meet their food requirements for the development of their egg might have exposed them to more contact with the parasites, which subsequently increased their chance of being infested. Infection between the small and big fish as related to their length and weight may be due to changes in their diet from weeds, seeds, phytoplankton and zooplankton to insect larvae, crustacean and worm. Ecological factors have been held widely responsible for the occurrence of the parasites.
Year round infection of Rhabdochona spp was noted in the S. plagiostomus (the snow trout) in river Swat and river Panjkora Pakistan for the first time. The snow trout BICHI, A. and YELWA, S., 2010