Scanning Electron Microscopic Study of Aplectana Duttaphryni Sou, Sow and Nandi, 2014 from A New Frog Host, Fejervarya limnocharis in West Bengal

Aplectana duttaphryni Sou, Sow and Nandi, 2014 was first reported in Kulti, Asansol coal-mine region, West Bengal from a toad, Duttaphrynus melanostictus (Schneider, 1899) only using light microscope. Present article describes, A. duttaphryni in details collected from the rectum of an anuran frog, Fejervarya limnocharis (Gravenhorst, 1829) in Birbhum district, West Bengal. Aplectana duttaphryni is reported for the first time from Birbhum district, West Bengal. The parasites identified by its lanceolate shaped gubernaculum, presence of 1 pair of similar and equal spicules and also with the presence of 3 pairs of preanal papillae, 2 pairs of adanal papillae, 13 pairs of postanal papillae and single papillae on the upper lip of anus. In the present study, Birbhum district recorded as new geographical locality and F. limnocharis recorded as a new host.

Fejervarya limnocharis (Gravenhorst, 1829) is a frog species (Amphibia, Anura, Dicroglossidae) and are commonly found in South East Asia and these frogs are popularly known as rice field frog or Asian grass frog. They are generally available in cultivated fields, ponds and also found around slow-flowing water streams 4 . The frogs are grey brown in colour, sometimes a V-shaped dark mark between eyes is found and a yellow vertebral stripe mostly present; throat is mottled with brown in male. These frogs are eating small insects such as flies and moths, as well as snails, slugs and worms 4 . In the field it can be identified by its relatively pointed snout and flattened head. The toes of the frog are long. The frog is typically brownish, with darker blotches, sometimes with a greenish or reddish tinge. A vertebral stripe may sometimes be present; this may be cream in colour, or sometimes greenish 1,2,3 . Dutta and Mukhopadhyay 4 , reported the presence of nine anuran species from Durgapur region, West Bengal, and F. limonocharis is one of them. But there was no such reports are available in Birbhum district on amphibians.
In this survey, seven male and nine female nematode parasites were collected from two F. limnocharis from Dubrajpur region, Birbhum, West Bengal. The recovered nematodes were identified as Aplectana duttaphryni 5 . As per our best knowledge, this is the first scanning electron microscopic work of A. duttaphryni from South Asia as well as from India. Till now, five species of Aplectana from the Indian Subcontinent are A. macintoshii 6 , A. duttaphryni 5 , A. dubrajpuri 7 , A. hoplobatrachusia 8 and A. akhrami 9 in Pakistan 5,6,7,8,9,10 . Recently Sou and Banerjee 11, reported a new species, Aplectana sriniketanensis Sou and Banerjee, 2020 from West Bengal. Thus, now 57 species of Aplectana have been reported from different biogeographical regions. Among them only five species Aplectana reported from India and seven species from Oriental region (Table: 1) are considered now valid 11 .

MATERIALS AND METHODS
Two grass frogs, F. limnocharis were brought to the laboratory of the Department of Zoology, Kulti College during March, 2021 from Dubrajpur town (23.7946° N, 87.3761° E), Birbhum, West Bengal and were subsequently dissected and examined for any parasitic infection. Seven males and nine female nematodes were collected from the two said frogs. Recovered parasites were clean using normal saline water and stored in 70% alcohol after killing with the same solution. 5% glycerine was used for light microscopic study. The specimens for scanning electron microscopic (SEM) study, were dried after post-fixation with 2% osmium tetroxide, then transferred to isoamyl acetate (100%), coated with gold and microphotographs were taken in a Hitachi S-530 (Japan) at a resolution of 50 ú and operating at an accelerating voltage of 15 kV. For helminthological records, nematode specimens have been deposited in the Parasitology Research Laboratory, The University of Burdwan, Purba Bardhaman, W.B. India (Regn. BUPL-170). During measurement, values were taken in millimetres (mm).

DISCUSSION
As per our best of knowledge, no redescription of A. duttaphryni, as such, is available from West Bengal as well as India. The original species was described by Sou et. al. 5 from Asansol region of Purba Bardhaman, West Bengal, India. Present nematodes identified as A. duttaphryni on the basis of morphological length (Table: 1) and also with presence of lateral alae, somatic papillae, three lips and with a large oesophageal bulb. It also has a short filamentous tail, well distributed caudal papillae, and a well sclerotized gubernaculum. Female reproductive system is prodelphic 12 . In the present study spicules and gubernaculum were separated by dissection of the nematode species. So the collected nematodes were identified as A. duttaphryni 5 .
Sou et. al. 5 studied the morphological structures of A. duttaphryni with the help of light microscopy. They described that mouth of nematode is triangular with three simple lips provided with cuticular fold. The scanning electron microscopic studies of present nematodes showing that cuticular fold of the mouth, structure of cephalic papillae and presence of amphids. Distribution pattern of caudal papillae in males are also clearly understandable in this studies. So the present scanning electron microscopy confirms the actual caudal papillae pattern of this species which is different from earlier descriptions of Sou et. al. 5 . The position of phasmids have also been included in the present study.

CONCLUSION
As per record, Aplectana duttaphryni has been reported only from Asansol region of West Bengal, India. Present paper dealing with the same nematode species but from a different locality with a new host record i.e. Fejervarya limnocharis. There are so many variations recorded during this study, both in morphological features and on general body measurements. Present work therefore, suggests this is due to the intraspecific variations of the nematode species. Finally, it can be concluded that Dubrajpur town of Birbhum district, West Bengal is a new locality for this parasite and at the same time F. limnocharis is a new host record since earlier description of A. duttaphryni was based on a different host, D. melanostictus.

ACKNOWLEDGEMENT
Thankful acknowledgement is due to The University of Burdwan and Kulti College for giving us laboratory facilities. The authors are thankful to Dr. Anadi Prasad Nandi, Retd. Professor of Zoology, The University of Burdwan for his guidance, encouragement and help during this work. Thanks are due to the authorities of the University Science Instrumentation Centre and Central Instrumentation Facilities (USIC & CIF), The University of Burdwan for affording Scanning Electron Microscope in studying surfacetopography of the present nematodes. The authors thankfully acknowledge the Amphibia Section, Herpetology Division, Zoological Survey of India, Kolkata, West Bengal for identification of hosts.

Conflict of interest
Authors hereby declared that there is no conflict of interest.

Funding Sources
No funding agency.

Compliance with ethical standards
Though there is no ethics committee in our Institutions, snake has been sacrificed for the present study following guidelines of The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) formed by the Act of the Indian Parliament.