1 SEM analysis of surface fine topography of Diplozoon paradoxum (Monogenea,
2 Polyopisthocotylea) from the snow trout, Schizothorax richardsonii (Cyprinadae,
3 Osteoichthyes) in Aunachal Pradesh, India with comments on host fish
4
7
Stelin M. Singh
5 Laboratories of Fish Parasitology , Deparment of Zoology ,Rajiv Gandhi University ,
6 Doimukh,Rono Hills Arunachal Pradesh , India ,791112
E- mail : mairembam@gmail.com
8 Abstract
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19
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21
9 The edible freshwater fish Schizothorax richardsonii in Arunachal Pradesh , India was
10 examined for its helminth parasite spectrum . One monogenean fluke representing the genus
11 Diplozoon sp was recovered from the gills of host fish. The information on its surface fine
12 topography was studied using SEM ( Scanning Electron Microscopy ). The X shaped, fused
13 individuals were well displayed with the prominent prohaptor and opisthohaptor both containing
14 host attachment structures. The paired prohaptor is divided by a partition which separates the
15 muscular oral sucker. There are numerous muscular convolutions within the sucker. The body is
16 covered with spines and convolutions. The opisthohaptor contains four pair of laterally
17 positioned corrugated clamps and a rectangular shaped concave terminal end.
Keywords: Diplozoon sp .; Freshwater fish; Monogenea; SEM
Introduction

22 Monogeneans, which are mainly fish ectoparasites, use various types of haptoral
23 (posterior) attachment apparatus to secure their attachment onto their hosts. Diplozoon spp.-, are
24 spending their whole life attached to the gills of freshwater fish. That connection is of vital
25 importance to these invertebrates, because if they lose it, they will drift away from their hosts
26 and die. . Diplozoon uses opisthohaptor with four pairs of clamps to attach itself onto the host.
27 Prohaptor consists of paired suckers opening into the oral cavity. Genital pore is in the posterior
28 half of the body. The genital pores of the paired adults are united with complimentary pores for
29 each partner worm. Diplozoon is regarded as being completely monogamous ( Wey Lim Wong
30 and Stanislav N. Gorb , 2013).
31 Diplozoon spp are Monogeneans found in freshwater fishes in Asia and Europe . This
32 parasite is commonly found on the gills of European cyprinid fishes. Many fish host genera are
33 listed by Yamaguti (1963): Cyprinus,Esox, Abramis, Barbus, Carassius, Rutilus, Cottus,Amar,
34 Acipenser, Parabramis, Silurus etc. Its size is usually around 0.7 cm . It has several hooks at the
35 level of its mouth which are used to grab on the gills of their host fish. There it will feed on the
36 blood ( Richard A. Heckmann et al.,2012) The development of Diplozoon is unique. The
37 oncomiracidium hatching from the egg in water is completely covered by a layer of ciliated
38 epithelium. In addition , it possesses two pigmented eye – spots , a well-developed pharynx and,
39 a blind- sac intestinal caecum. This ciliated larva is free swimming and after it finds a host, it
40 becomes attached to the gills of the fish . Each larva metamorphoses , losing its eye-spots and
41 developing branched intestinal caeca . The posterior end of the body becomes elongated and
42 wider .A small sucker appears on the midventral surface , and a small conical knob appears at
43 about the same level on the dorsal surface . This larva type is designated a diporpa. .It will
44 remain in that stage unless two larvae come together. Then the two larvae will undergo

45 metamorphosis and become fused together (Kagel and Taraschewski, 1993). Only after the two
46 worms are united the two additional pairs of clamps take form, the first pair being formed prior
47 to the union .
Host fish 48
49 Schizothorax richardsonii is one of the dominant genera in Schizothoracinae
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51
, inhabiting stream and rivers of Himalayan and sub-Himalayan regions.
These streams usually have stony/rocky substratum. The species is widely distributed all
52 along the Himalayas in India, besides Pakistan, Tibet, Nepal, Sikkim ,Afghanistan etc. In central
53 Himalayas S . richardsonii, locally known as “asela” has fishery importance and constitutes
54 principal subsistence food fishery (K.D.Joshi 2004).In the recent past, it is reported that the
55 population of this species in this region has declined significantly(Sharma 1989; Das and Joshi,
56 1993; Sehgal 1999).. Decline in the stocks in uplands could be ascribed to overfishing,
57 destructive fishing and increased human pressure. The fish is very important to aquatic
58 biodiversity of the region and contributes to economy of hill fishermen. (K.D.Joshi 2004).
59 Schizotharax richardsonii is a column feeder, thus, considered to move relatively longer
60 distance as compared to other species dwelling the river bed.It is considered to perform
61 migration within same river. To cope the low temperature during peak winter season it move
62 downwards. It finds a tributary to spawn from May to September. The migratory pattern of
63 Schizothorax richardsonii is not well studied in the region but other data from the Himalayan
64 rivers show that they have no specific breeding grounds.( Hirong H.E project, Arunachal Pradesh
65 ,2009; Environmental Impact Assessment – Fish & Fisheries, ).
66 The aim of the paper is to explore the exciting attachment systems of parasitic
67 invertebrates.

68 A scanning electron microscope will give a clear picture of what the surface structures of
69 clamps look like and this is the first report of SEM, diplozoon sp from the hillstream fishes of
70 Arunachal Pradesh , India .
Material and methods 71
72 The parasites were collected from the gills of Schizothorax richardsonii from the Tenga
73 River through the Himalayan ranges of Arunachal Pradesh within the Kameng river system
74 following examination in a saline medium under stereoscopic microscope. For light microscopy,
75 worms were fixed in 5 % formalin under slight cover-slip pressure, stained in alcoholic
76 chlorhydric carmine ( Langeron 1949), dehydrated through a gradient alcohol series, cleared in
77 beechwood creosote and mounted in Canada balsam . For Scanning Electron Microscopy (
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79
SEM), worms were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 12 hours at 4°C-. in 1% OsO4 for 2 hours at 4°C. The samples
80 were dehydrated in ascending grade of acetone, critical- point dried (Critical point dryer,
81 Polaron) and mounted on aluminium stubs . They were sputter-coated( SCD 050 Super Cool
82 Sputter System; Baltec Technology, Liechtenstein) with colloidal gold and observed under a Leo
83 435 VP scanning electron microscope (Cambridge, UK) at an operating voltage 15 Kv. Images
84 were digitally acquired by using a CCD camera attached to the microscope.
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86
Results
Figures 1 to 6 show the results of the scans completed for Diplozoon paradoxum using(
87 SEM)- Figure 1 depicts the characteristic shape of the2 fused monogeneans. Both the prohaptor
88 and opisthohaptor are prominent. The prohaptor is represented by figures 2 and 3. The oral
89 sucker of the prohaptor is prominent with a definite partition between the two parts giving it a
90 paired appearance. These openings yield entrance into the oral cavity and blind digestive

91
92 system. The opisthohaptor is rectangular in shape with a concave terminal shape. There are 4 pairs of lateral corrugated “brushes” or clamps which aid in host attachment (figure 5). For the
93 opisthohaptor note the attachment structures used by the ectoparasite (sucker, clamps and hooks).
94 The body is covered with
95 spines or tubercles from anterior to posterior(figures 2and 3 ). There are numerous
96 knobs(tubercles) surrounding the anterior region(figures 2 and 3).
97
98 Discussion
99 Diplozoon spp,, spend their whole life attached to the gills of freshwater fishes. It is
100 interesting how the parasite could maximize the use of energy for reproduction while
101 maintaining its secure and long term attachment to the host explained that in the course of its
102 evolution, the worm may have developed an efficient long term energy saving attachment device
103 for its survival. (Wey Lim Wong,2013) To date, there have been no published articles on the
104 presence of Diplozoon in Arunachal Pradesh, India. Carps are common hosts for D. paradoxum
105 and these cyprinids and close relatives are moved throughout the world (Yamaguti, 1963).
106 Surveys and check lists for all parasites including the monogenea are numerous(Yamaguti, 1963;
107 Bychovskaya-Pavlovskaya,1962; Kennedy, 1974; Bauer, 1984; Hoffman, 1970).
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109
Acknowledgement
UGC Minor Research Project
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111
SAIF, AIIMS, New Delhi for the facilities offered for the use of Electron Microscope
References

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Figure Legends
Figure 1
A pair of whole worm in union (200µ scale bar)
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148 Figure 2
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150 100µ)
View of sub terminal oral sucker showing rough texture with lack papillae. ( scale bar
Figure 3
An “en face” view showing the partitions (arrows) between the paired oral sucker.
Numerous knobs or bumps on the surface.
Figure 4

156 The terminal part of the opisthohaptor with the concave end and the comb-like brushes on
157 the lateral side
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Figure 5
Magnified single row of clamps (scale bar 10 μm)
Figure 6
Integument for Diplozoon sp showing numerous bumps and micropores.
200µ