Post on 30-Apr-2020
Molecular Analysis of Monogenoidean Parasites in some Cyprinids
A THESIS SUBMITTED TO
UNIVERSITY OF LUCKNOW
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ZOOLOGY
BY RITIKA RAJ SHRIVASTAVA M.Sc.
Department of Zoology University of Lucknow
Lucknow-226007 2013
CERTIFICATE
This is certified that RITIKA RAJ
SHRIVASTAVA has worked under my supervision and
guidance for the award of the degree of DOCTOR OF
PHILOSOPHY IN ZOOLOGY. The work incorporated
in the thesis entitled “MOLECULAR ANALYSIS OF
MONOGENOIDEAN PARASITES IN SOME
CYPRINIDS” has been carried out in the
DEPARTMENT OF ZOOLOGY, UNIVERSITY OF
LUCKNOW, LUCKNOW.
Prof. Madhu Tripathi Prof. Nirupama Agrawal Head Supervisor Department of Zoology, Department of Zoology, University of Lucknow, University of Lucknow, Lucknow Lucknow
CONTENTS
Acknowledgements
Introduction 1-3
A Brief History of Work Done 4-5
Materials and Methods 6-15
Chapter –I Dactylogyrus Deising, 1850 16-41
1. D. longiacus Gusev, 1976 2. D. subtilis Gusev, 1976 3. D. sp.1 4. D. sp.2 5. D. sp.3 6. D. sp.4 7. D. sp.5 8. Molecular Phylogenetics
Chapter –II Dactylogyroides Gusev, 1976 42-63
1. D. tripathii (Tripathi, 1959) Gusev, 1963 2. D. longicirrus (Tripathi, 1959) Gusev, 1976 3. D. mahecoli (Gusev, 1976) Agrawal et al., 2002 4. D. dorsali (Gusev, 1976) Agrawal et al., 2002 5. Molecular Phylogenetics
Chapter-III Esomocleidus n. g. 64-86
1. E. esomi (Gusev, 1963) n. comb. 2. E. chakrabartii (Gusev, 1976) n. comb.
3. E. lucknowensis n. sp.
4. Molecular Phylogenetics
CHAPTER- IV Molecular analysis of 14 Dactylogyrids,
included in Chapters I, II and III 87-96
References 97-111
Summary 112-118
Acknowledgements
A research work not only involves coordinated and guided
job between a scholar and the guide, but it is also a result of
teamwork where many backstage persons play vital roles. Likewise,
my research work too carries a strong guidance, support, coordination
and untiring efforts from all concerned persons of my life.
First and foremost, I express my deepest and sincere thanks
and gratitude towards my supervisor and mentor Prof. Nirupama
Agrawal, Former Head, Department of Zoology, University of
Lucknow, a hardworking and dedicated lady and a tough task master
with a humane heart. She not only took pains in facilitating and
monitoring my research work but also keen interest in research work
and allowed me to go through her laboratory and rich personal
library. She provided the much needed constant guidance,
constructive criticism towards improving my work and developing a
broader perspective to my thesis.
I gratefully acknowledge the help received from Prof. K.C.
Pandey of the Department of Zoology, University of Lucknow and
Prof. G.G. Agarwal, Department of Statistics, University of
Lucknow, during the course of my research work.
Laboratory facilities provided by Head Department of
Zoology,University of Lucknow are also acknowledged. Further,
help received from Dr. Monisha Banerjee of the Department of
Zoology, her students and Dr. A.M. Saxena is also acknowledged.
I also undertake the opportunity to express my thanks to
Dr. M.K. Upadhyay, Scientist 'C', Biotech Park, Lucknow, for
helping in 1analysis part of my research and to Mr. L.K. Gupta, a
friend of mine, working at Zydus Cadila Healthcare, for lending his
scientific and technical support.
Thanks are also due to my labmates Saroj Rajvanshi, Amrin
Ali and Shailendra Ray and seniors Dr. Amita Devak, Dr. Priyanka
Tripathi, Dr. B.K.Gupta and Dr. K.K. Singh.
Words will fail to show my gratitude for overwhelming
response, utmost care, moral support and pains, taken by my family
members including my mother, father, brother, uncles, aunts, my
relatives, my in-laws, my friends, hostel inmates who stood beside me
all these years.
My husband Mr. Abhishek Sinha, Judge, U.P. Judicial
Services gave me much needed space and time and stood steadfastly
with me through thick and thin during the last phase of my work.
For this, I will remain obliged to him ever.
INTRODUCTION
The Cypriniformes is an order of teleosts and includes carps,
minnows and loaches (about 4000 species). Furthermore, thousands of
species of freshwater fishes still remain to be described, most of them in
tropics (Lundberg et al., 2000). They are distributed throughout Africa
(except Madagascar), Asia, Europe and North America. They are the
most diverse in Asia, but are entirely absent from Australia and South
America. India is one of the mega biodiversity countries in the world and
occupies ninth position in terms of freshwater mega biodiversity. In India
itself there are 2,500 species of fishes of which 930 are freshwater.
Cypriniformes is of considerable economic significance in India.
Fish represents a major group of organisms serving as hosts for
many adult platyhelminth parasites (Sukontason et al., 1999; Wongsawad
et al., 2004; Kue–A-Pai and Wiwanikit., 2005). The Monogenoidea is one
of the largest groups of parasitic flatworms (Platyhelminthes), possessing
simplest life cycle among parasitic Platyhelminthes. They have no
intermediate hosts and are mainly found on skin or gills of fishes
(Bychowsky, 1957; Malmberg and Fernholm, 1989). They are obligate
parasites of aquatic and semi-aquatic organisms (Bychowsky, 1957).
They attach to hosts using hooks, anchors, clamps and a variety of other
specialized structures. Most species are oviparous but a few are
viviparous.
In recent times, molecular techniques (that utilize genetic markers
in nuclear and mitochondria DNA) are used in taxonomy and
phylogenetics of species and have emerged as valuable supplementary
tools in providing authentic and unambiguous identification of taxa. The
gene segment of eukaryotic rDNA contains highly conserved, 18S, 5.8S,
1
and 28S tracts and forms a tandem repetitive cluster to highly variable,
transcribed and non-transcribed or intergenic spacer regions. rDNA is
used to resolve taxonomic issues of helminth parasites (Powers et al.,
1997; Gasser and Newton, 2000; Winchell et al., 1999, 2001, 2002, 2004;
Lockyer et al., 2003 and Andrea et al., 2007). Advantage of using this
region is that the tandem repeated copies provide a large number of target
sequences for PCR amplification (Jousson et al., 1998). Since it is easy to
amplify them even from small quantities of DNA, and because of having
a high degree of variation between closely related species, these markers
are extensively used for taxonomic studies (Hancock et al., 2001). There
is increasing evidence that the 28S rDNA gene sequences add useful and
often significant resolution to molecular systematic estimates of
phylogeny, particularly for older, deep branching lineages. 28S rDNA
markers have been used to detect species boundaries (Kaukas and
Rollinson, 1997).
The present work investigates the morphology of monogenoidean
parasites found on few commonly available cypriniformes fishes and also
the molecular phylogeny of these parasites using 28S rDNA. Species
belonging to two genera were examined for the monogenoidean infection
i.e. Puntius Hamilton, 1882 and Esomus Swainson, 1839, which are
popular larvivorous fish to the consumers of North-India, Eastern India
and North-East as well. They are small fishes, can easily be maintained
the laboratory aquaria for a long period. Their habitat is rivers, streams,
and ponds in plains and submontane regions. They are used in fish
aquarium trade. They are highly valuable source of macro and
micronutrients that play an important role to provide essential nutrients
for the people. Vitamins and minerals are found to be much more in small
fishes than in large fish. Esomus are rich in iron and calcium respectively.
2
The thesis has been divided in four chapters. Chapter I includes
record of seven species of Dactylogyrus and its molecular phylogenetics.
Chaptor II has record of four species of Dactylogyroides and molecular
phylogenetic study whereas Chaptor III consists of study of three species
of the newly proposed genus Esomocleidus n.g. and their molecular
phylogeny. Finally in Chapter IV combined molecular analysis of all the
14 species included in Chapter I, II, and III is summarized. Five species
of the genus Dactylogyrus included in Chapter I have been given
numbers 1-5 because they are part of an unpublished data of one of my
senior lab mates. Sequence of Dactylogyroides longicirrus is from
GENBANK. The Molecular analysis of these monogenoideans was a
daunting task, as very closely related species occur on the same hosts. To
further strengthen the identification with this useful tool, my Ph.D.
supervisor Prof. Nirupama Agrawal of the Department of Zoology,
therefore, suggested me to undertake the work “Molecular Analysis of
Monogenoidean Parasites in some Cyprinids”.
3
A BRIEF HISTORY OF WORK DONE
Our knowledge of molecular biology of monogenoideans is
currently lagging behind to that of other groups of helminthes. This group
has number of interesting characteristics making them suitable models for
molecular study. In early nineties, with innovation of molecular markers,
the genetic characterization of monogenoideans started. Partial small
subunit (SSU) ribosomal RNA (rRNA) genes of Dictyocotyle coeliaca,
Diclidophora merlangi and Anaplodiscus cirrusspiralis were first
determined by Baverstock et al. (1991) and their sequences were
subsequently used in phylogenetic analysis of numerous platyhelminths.
This work now encompasses a wide range of techniques and target
regions of the genome. Molecular investigation of monogenoideans
began with the PCR amplification of 18S rRNA gene of Gyrodactylus
salaris. Its sequence was used to examine the phylogenetic relationship of
this species with other platyhelminths (Cunningham et al., 1995).
The valuable contributions in this field from abroad are those of
Cunningham (1997), Littlewood et al. (1998), Jousson et al. (1998),
Cable et al. (1999), Zardoya and Doadrio (1999), Zardoya et al. (1999a &
b), Winchell et al. (1999, 2001, 2002, 2004), Gasser and Newton (2000),
Mollaret et al. (2000), Bentz et al. (2001), Bruno et al. (2001), Desdevises
(2001, 2002), Sicard et al. (2001), Chisholm et al. (2001), Matejusova et
al. (2001a & b, 2004), Verneau et al. (2002), Lockyer et al. (2003),
Simkova et al. (2002, 2003, 2004, 2006), Zietara and Lumme (2003),
Matejusova and Cunningham (2004), Whittington et al. (2004), ,
Waeschenbach et al. (2007), Andrea et al. (2007), Wu et al. (2007),
Mendlova et al. (2010, 2012), Poisot et al. (2011), Hahn et al. (2011).
Indian workers are Tandon (2007), Singh and Chaudhary (2010, 2011),
Chaudhary and Singh (2012a, b, c & d),Verma et al. (2012), Chaudhary
4
et al. (2013), Chiary et al. (2013), Rajvanshi and Agrawal (2013) and
Agrawal and Ali (2013).
Identification of monogenoideans was done using “An
Encyclopaedia of Indian Monogenoidea” (Pandey & Agrawal, 2008),
which includes taxonomical work done till 2008. Later contributions are
those of Agrawal et al. (2008), Tripathi et al. (2009a & b, 2012), Agrawal
et al. (2010a & b), Saxena et al. (2010), Upadhyay et al. (2011), Ali et al.
(2012), Rajvanshi and Agrawal (2011, 2012) and Porwal et al. (2012).
5
MATERIALS AND METHODS
Collection of parasites
Fish were collected from different sites of U.P., India (Table 1) with
the help of local fishermen, transferred to laboratory and maintained in
small glass aquaria. They were dissected as per requirement. Parasites
were gently scrapped from the gills and then transferred to small droplets
of water onto slides, flattened under clean coverslips and examined under
a phase contrast microscope (Olympus CX 41 U-DA 4E 03365 Japan).
Some specimens were mounted unstained in glycerin by sealing the
margins with a nail enamel (semi-permanent slides). Permanent
preparations were also made, stained with Aceto-alum carmine and
Gomori’s Trichrome, dehydrated in ascending grades of alcohol of 70%,
90% and 100%, and mounted in Canada Balsam.
Drawings were made from live, semi permanent as well as
permanent preparations, using a drawing tube attached to phase contrast
microscope. Measurements (in micrometers) were made using a
calibrated micrometer.
The taxonomy of monogenoideans follows “An Encyclopaedia of
Indian Monogenoidea” (Pandey & Agrawal, 2008) and those of fish
follow “Fishbase” (Froese and Pauly, 2007). A list of parasites recovered
from their respective hosts and accession numbers of sequences (analyzed
in present study and retrieved from NCBI) is included in Table 2.
DNA Isolation and Amplification
Identified specimens of monogenoidean were fixed in either 95 or 100%
ethanol for extraction of genomic DNA. The genomic DNA was isolated
6
by using DNeasy Tissue Kit, Qiagen, Hilden, Germany (procedure given
in flow chart).
Flow Chart for DNA Extraction Tissue + 200µl ATL Buffer
↓ Add 20 µl Proteinase K,
Mix thoroughly by vortexing (Incubate 20 min at 56º C)
↓ Add 200 µl AL Buffer,
Mix thoroughly by vortexing ↓
Add 200 µl Ethanol, Mix thoroughly by vortexing
↓ Pipet the mixture into spin coloum placed in a 2ml collection tube,
Centrifuge at 8000 rpm for 1 min ↓
Discard flow-through and collection tube ↓
Place the column in a new 2ml collection tube ↓
Add 500µl AW1 Buffer, Centrifuge at 8000rpm for 1 min
↓ Discard flow-through and collection tube
↓ Place the column in a new 2ml collection tub
↓ Add 500µl AW2 Buffer,
Centrifuge at 14000rpm for 1 min ↓
Discard flow-through and collection tube ↓
Place the column in a new 2ml microcentrifuge tube ↓
Pipet 80 µl AE Buffer directly onto the membrane ↓
Incubate at room temperature for 1 min, Centrifuge for 1 min at 8000 rpm to elute (store at -20°C)
7
DNA amplification
The 28S rDNA region was amplified with the universal primers.
Sequences of the primer both reverse and forward are as follows:-
Forward- 5’ ACCCGCTGAATTTAAGCAT 3’
Reverse- 5’ CTCTTCAGAGTACTTTTCAAC 3’
Fig. 1 :- rDNA showing regions of 18S, ITS and 28S
(Source: Current Biology)
8
Composition of reaction mixture for PCR
Each amplification reaction was performed in a final volume of 25 ml
containing:
Components Quantity
1 Taq buffer 2.5 µl
2 DNTPs 1 µl
3 Forward 1 µl
4 Reverse 1 µl
5 Taq polymerase 0.5 µl
6 MgCl2 1 µl
7 Genomic DNA 5 µl
8 MilliQ 13 µl
Steps and conditions for PCR
S.No. Step Temperature Time
1 Initial Denaturation 95º 5min X 1 Cycle
2 Denaturation 95 º 30sec 35
X cycle 3 Annealing 55 º 40 sec
4 Extension 72 º 30 sec
5 Final Extension 72 º 7min X 1 Cycle
6 Hold 4 º
PCR products were examined on 1.5% Agarose–TAE gels, stained
with Ethidium Bromide and visualized in Gel Doc. Gel pictures are given
in the following pages (Fig. A-F).
9
Fig A-F: Gel Pictures
PCR Products of Monogenoidean Parasites
M= DNA Marker (100 bp ladder)
M 1 2 3 1 M 2 3 4 5
Fig. A- Lane 1 Dactylogyrus longiacus Fig. B- Lane 1 Dactylogyrus sp. 1
Lane 3 Dactylogyrus subtilis Lane 5 Dactylogyrus sp. 2
M 1 2 3 4 1 M 2 3
Fig C- Lane 4 Dactylogyrus sp. 4 Fig D- Lane 1 Dactylogyrus sp.5
Lane 3 Dactylogyrus sp.3
10
1 2 3 4 M 5 6 1 2 M 3 4 5
Fig. E- Lane 1 Dactylogyroides tripathii Fig. F- Lane 1 Esomocleiodus esomi
Lane 4 Dactylogyroides dorsali Lane 3 Esomocleiodus chakrabarthi
Lane 6 Dactylogyroides mahecoli Lane 5 Esomocleiodus lucknowensis
11
Sequencing of Purified DNA
Amplified products were purified by a PCR clean up kit and
sequenced by Xcelris Labs Limited, Ahemadabad. The concentration of
the purified DNA was determined and was subjected to automated DNA
sequencing on ABI3730xl Genetic Analyzer (Applied Biosystems, USA).
Sequencing was carried out using BigDye® Terminator v3.1 Cycle
sequencing kit.
Cycle Sequencing
Cycle sequencing was performed following the instructions
supplied along with BigDye® Terminator v3.1 Cycle Sequencing Kit.
The reaction was carried out in a final reaction volume of 20µl having
10ng of PCR product with ABI ready reaction mix and sequencing buffer
and carried out in thin wall PCR tube. The cycling protocol was designed
for 25 cycles with the thermal ramp rate of 1ºC per second. After the
cycling, the extension products were purified and mixed well in 10 µl of
Hi-Di formamide. The contents were mixed on shaker for 30 minutes at
300 g. Eluted PCR products were placed in a sample plate and covered
with the septa. Sample plate was heated at 95ºC for 5 min, snap chilled
and loaded into autosampler of the instrument.
Sequence Analysis For sequence analysis various bioinformatics tools detailed below
were used. All obtained 13 sequences were submitted in GENBANK.
BLAST (Basic Local Alignment SearchTool)
BLAST is one of the most widely used bioinformatics programs
because it addresses a fundamental problem and the heuristic algorithm it
12
uses is much faster than calculating an optimal alignment. BLAST, is
an algorithm for comparing primary biological sequence information. A
BLAST search enables to compare a query sequence with database of
sequences. Different types of BLASTs are available according to the
query sequences. BLAST program was designed by Altschul et al., 1990.
CLUSTAL W
Clustal is a widely used multiple sequence alignment computer
program. Clustal W (Chenna, 2003) is a general purpose multiple
sequence alignment program for DNA or proteins.It produces
biologically meaningful multiple sequence alignments of divergent
sequences. It calculates the best match for the selected sequences, and
lines them up so that the identities, similarities and differences can be
seen.
MEGA 5
MEGA, Molecular Evolutionary Genetics Analysis, is used for
making dendrograms, or phylogenetic trees using nucleotide or protein
sequences. It is developed by Tamura et al., 2011. MEGA is an integrated
tool for conducting sequence alignment, inferring phylogenetic trees,
mining web-based databases, estimating rates of molecular evolution,
inferring ancestral sequences, and testing evolutionary hypotheses.
13
Table 1:- Collection sites of different fish hosts
S.N. Collection Site Geographical Coordinates
1. Lucknow 26.85° N, 80.92° E
2. Sitapur 27.57° N, 80.68° E
3. Sultanpur 26.27° N, 82.07° E
4. Barabanki 27°.92° N, 80.20° E
5. Pilibhit 28.55° N, 80.10° E
6. Tanakpur 29.08° N, 80.11° E
7. Unnao 26.53° N, 80.50° E
14
Table 2:- List of hosts, parasites, sequence length (bp) and their
accession number
S.N. Name of fish Name of parasites Sequence
Length(bp)
GenBank
Number
1. Puntius
sophore
Dactylogyroides tripathii
(Tripathi, 1959, Yamaguti,1963)
Gusev 1963
368 JX960645
Dactylogyroides longicirrus
(Tripathi, 1959) Gusev, 1976
(Retrieved from GenBank)
301 GU903482
Dactylogyrus longiacus
Gusev,1976
377 JX947854
Dactylogyrus subtilis Gusev,1976 371 JX947851
Dactylogyrus sp.1 359 JX947853
2. Puntius chola Dactylogyroides mahecoli
(Gussev,1976) Agrawal et al.,
2002
381 JX960644
Dactylogyroides dorsali
(Gussev,1976) Agrawal et al.,
2002
364 JX960643
Dactylogyrus sp.2 368 JX960642
3. Puntius ticto Dactylogyrus sp.3 357 JX947855
Dactylogyrus sp.4 361 JX947856
Dactylogyrus sp.5 336 JX947857
4. Esomus
danricus
Esomocleidus chakrabartii
(Gusev, 1976)
337 KC962224
Esomocleidus esomi (Gusev,
1963)
367 KC962226
Esomocleidus lucknowensis 358 KC962225
15
Dactylogyrus longiacus Gusev, 1976
(Plate. 1)
Host- Puntius sophore Hamilton, 1882
Type Locality- River Gomti
Infection site- Gills
No. of host examined-12
Body elongate 275 (250-320; n=12); maximum width 80 (75-95;
n=12). Cephalic region well developed and divided into four lobes. Two
pairs of eye spots, posterior pair larger. Intestinal caeca confluent
posterior to testis. Pharynx spherical 16(14-22; n=12) in diameter.
Copulatory complex consists of copulatory tube and accessory piece.
Copulatory tube 72 (65-90; n=12) long, comprising a coil with a swollen
base. Accessory piece complex, consist of three distal pincers and a broad
basal flap. Vaginal apparatus sclerotised, opening into seminal receptacle
through a vaginal tube. Ovary oval, 19 (18-22; n=12) long, 21 (15-26;
n=12) wide. Vitelline follicles dense, throughout body, except gonadal
region. Testis single, 28 (24-30; n=12) long, 16 (14-22; n=12) wide.
Single prostatic reservoir opens at the base of copulatory complex. Vas
deferens arises from the anterior end of testis, runs anteriorly to loop left
intestinal caecum, dilating to form spindle- shaped seminal vesicle which
in turn opens at the base of copulatory tube. Haptor 35 (32-45; n=12)
long, 50 (45- 65; n=12) wide. Dorsal anchor inner length 36 (32-38;
n=12), outer length 27 (25-30; n=12), recurved point 13 (12-15; n=12).
Ventral anchors absent. Dorsal connecting bar 14 (12-16; n=12). Hooks
seven pairs, similar, hook pair 4, 5and 6, 12-18 (n=12), hook pairs 1, 2, 3
and 7, 18- 21 (n=12).
16
Remarks:- Gusev (1976) described D. longiacus from P.stigma (now
known as P.sophore) from the water bodies near Lucknow. The
description was based on hard parts only. Agrawal et al. (2003)
redescribed the species in detail and have added structure of soft parts and
variations in sclerotised structures. It is therefore briefly recorded here.
17
Dactylogyrus longiacus Whole- mount (ventral view)
(Plate. 1)
18
Dactylogyrus subtilis Gusev, 1976
(Plate. 2)
Host- Puntius sophore Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-10
Body elongate 250 (210-320; n=10); maximum width 85 (70-95;
n=10). Cephalic region well developed and divided into four lobes. Two
pairs of eye spots, posterior pair larger. Intestinal caeca confluent
posterior to testis. Pharynx spherical 23 (18-26; n=10) in diameter.
Copulatory complex consist of copulatory tube and accessory piece,
copulatory tube 29 (27-34; n=10) with swollen base; accessory piece
forms two pincers. Prostatic reservoir not observed. Ovary oval 43 (42-
48; n=10) long, 32 (30-36; n=10) wide.Vitelline follicles dense,
throughout body except gonadal region. Testis single, 31 (30-37; n=10)
long,14(12-22; n=10) wide, vas deferense arises from the anterior end of
testis and dilating to form a seminal vesicle, opens at the base of
copulatory complex. Haptor 55 (45-58; n=10) long, 72 (70-95; n=10)
wide. Dorsal anchor inner length 25 (23-28; n=10), outer length 20 (18-
25; n=10), recurved point 10 (9-12; n=10). Dorsal connecting bar, 19 (18-
22; n=10) long. Ventral bar stick shaped, 13(10-15) long. Hooks seven
pairs, similar, hook pairs 1, 2, 4, 5 and 7, 14- 20 (n=10), hook pair 3, 23-
28 (n=10), hook pairs 6, 14-19 (n=10).
Remarks:- This species was described by Gusev (1976) from P. stigma
(now known as P. sophore), from a water bodies near Lucknow. Its
19
description was only based on hard parts. Agrawal et al. (2003)
redescribed the species, adding information on soft parts and some
differences in sclerotised structures. It is briefly recorded in the present
work.
20
Dactylogyrus subtilis Whole-mount (dorsal view)
(Plate. 2)
21
Dactylogyrus sp.1
(Plate. 3)
Host- Puntius sophore Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-12
Body elongate 430 (350-550; n=12); maximum width 130 (110-
150; n=12). Cephalic region well developed and divided into four lobes.
Two pairs of eye spots, posterior pair larger. Intestinal caeca confluent
posterior to testis. Pharynx spherical 22 (20-26; n=12) in diameter.
Copulatory complex consist of copulatory tube and accessory piece,
copulatory tube 62 (50-70; n=12) with swollen base; accessory piece 25
(24-28; n=12) long. Ovary oval 55 (54-60; n=12) long, 35 (29-49; n=12)
wide. Vitelline follicles dense, throughout body except gonadal region.
Testis single, 48 (45- 80; n=12) long, 16 (16-22; n=12) wide, vas
deferense arises from the anterior end of testis, dilates to form seminal
vesicle which in turn opens at the base of copulatory complex. Single
prostatic reservoir opens at the base of copulatory Haptor 85 (75-95;
n=12) long, 100 (95-125; n=12) wide. Dorsal anchor inner length 45 (40-
58; n=12), outer length 42 (35-50; n=12), recurved point 9 (8-9; n=12).
Dorsal bar 20 (18-22; n=12). Hooks seven pairs, similar, hook pairs 1, 5
and 7, 18- 22 (n=12), hook pair 2, 3, 23-24 (n=12), hook pairs 4, 6, 12-17
(n=12) long.
Remarks:-The present species was earlier described in detail by Tripathi
(unpublished work) from P. sophore from river Gomti, Lucknow. It is
therefore briefly recorded here. 22
Dactylogyrus sp.1 Whole-mount (dorsal view).
(Plate. 3)
23
Dactylogyrus sp.2
(Plate. 4)
Host- Puntius chola Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-8
Body elongate 355 (280-435; n=8); maximum width 95 (85-135;
n=8). Cephalic region well developed and divided into four lobes. Two
pairs of eye spots. Intestinal caeca confluent posterior to testis. Pharynx
spherical, 15 (12-26; n=8) in diameter. Copulatory complex consist of
copulatory tube and accessory piece, copulatory tube 40 (32-42; n=8)
long. Vaginal apparatus sclerotised, opening into seminal receptacle
through a vaginal tube. Ovary oval, 28 (25-35; n=8) long, 22 (20-22;
n=10) wide. Vitelline follicles dense, throughtout body, except gonadal
region. Testis single, 31 (29-37;n=10) long,17 (16-22;n=10) wide, vas
deferense arises from the anterior end of testis, dilates to form seminal
vesicle which in turn opens at the base of copulatory complex. Single
prostatic reservoir opens at the base of copulatory complex. Haptor 85
(72-90; n=8) long, 98 (95-115; n=8) wide. Dorsal anchor inner length 39
(36-50; n=10), outer length 28 (25-32; n=8), recurved point 10 (8-12;
n=8), dorsal bar 19 (17-20). Hooks seven pairs, similar, hook pairs 1, 2, 3
and 5, 18- 25 (n=10), hook pair 4, 29-34 (n=10), hook pairs 6 and 7, 16-
19 (n=10).
Remarks: This species was earlier described by Tripathi (unpublished
work) from P. chola from river Gomti, Lucknow, in detail. It is briefly
recorded in the present work.
24
Dactylogyrus sp.2 Whole-mount (ventral view) (Plate. 4)
25
Dactylogyrus sp. 3
(Plate. 5)
Host- Puntius ticto Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-15
Body elongate 335 (280-385; n=15); maximum width 65 (90-130;
n=15). Cephalic region well developed and divided into four lobes. Two
pairs of eye spots. Intestinal caeca confluent posterior to testis. Pharynx
spherical 22 (17-26; n=15) in diameter. Copulatory complex consist of
copulatory tube and accessory piece, copulatory tube 29 (27-37; n=15)
with swollen base; accessory peice 25 (24-28; n=15) long. Vaginal
apparatus sclerotised, opening into seminal receptacle through a vaginal
tube. Ovary oval, 40 (38-45; n=15) long, 24 (20-28; n=15) wide. Vitelline
follicles dense, throughtout body, except gonadal region. Testis single, 31
(29-37; n=15) long, 16 (16-22; n=15) wide, vas deferens arises from the
anterior end of testis, single seminal vesicle opens at the base of
copulatory complex. Prostatic reservoirs two, opens at the base of
copulatory complex. Haptor 48 (40-55; n=15) long, 98 (95-135; n=15)
wide. Dorsal anchor inner length 42 (36-52; n=15), outer length 28 (25-
32; n=15), recurved point 9 (8-9; n=15). Dorsal bar 22 (20-25; n=15)
long. Hooks seven pairs, similar, hook pairs 1, 2, 3, 18- 25 (n=10), hook
pairs 6 and 7, 10-12 (n=10), hook pair 4, 5, 6, (14-18; n=10).
Remarks:- The present species was earlier described in detail by Tripathi
(unpublished work) from P. ticto from river Gomti, Lucknow. It is
therefore briefly recorded here. It is therefore briefly recorded here.
26
Dactylogyrus sp.3 Whole-mount (ventral view)
(Plate. 5)
27
Dactylogyrus sp. 4
(Plate. 6)
Host- Puntius ticto Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-8
Body elongate 310 (280-410; n=8); maximum width 95 (75-120;
n=8). Cephalic region well developed and divided into four lobes. Two
pairs of eye spots. Intestinal caeca confluent posterior to testis. Pharynx
spherical 20 (17-26; n=8) in diameter. Copulatory complex consist of
copulatory tube and accessory piece, copulatory tube 29 (27-34; n=8)
with swollen base; accessory peice 25 (24-28; n=8) long. Vaginal
apparatus sclerotised, opening into seminal receptacle through a vaginal
tube. Ovary round to oval, 35 (32-42; n=8) long, 30 (20-38; n=8) wide.
Vitelline follicles dense, throughtout body, except gonadal region. Testis
single, 33 (29-38;n=8) long,17 (16-22; n=8) wide, vas deferens arises
from the anterior end of testis, single seminal vesicle opens at the base of
copulatory complex. Prostatic reservoirs two, opens at the base of
copulatory complex. Haptor 65 (50-80; n=8) long, 110 (95-135; n=8)
wide. Dorsal anchor inner length 45 (36-52; n=8), outer length 34 (30-
38; n=8), recurved point 9 (8-9; n=8). Dorsal bar 12 (11-15; n=8) long,
Similar hooks; hook pairs 1, 2, 3 and 7, 11-14 (n=8), hook pair 2 and bar
Hooks seven 6, 15-16 (n=8), hook pairs 4, 17-19 (n=8).
Remarks:-This species was earlier described in detail by Tripathi
(unpublished work) from P. ticto from river Gomti, Lucknow. It is briefly
recorded in the present work.
28
Dactylogyrus sp.4 Whole-mount (dorsal view)
(Plate. 6)
29
Dactylogyrus sp.5
(Plate. 7)
Host- Puntius ticto Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-12
Body elongate 395 (300-415; n=12); maximum width 95 (90-130;
n=12). Cephalic region well developed and divided into four lobes. Two
pairs of eye spots, posterior pair larger. Intestinal caeca confluent
posterior to testis. Pharynx spherical, 25 (17-30; n=10) in diameter.
Copulatory complex consist of copulatory tube and accessory piece,
copulatory tube 52 (45-64; n=10) with swollen base; accessory piece 24
(24-28; n=10) long. Vaginal apparatus sclerotized, opening into seminal
receptacle through a vaginal tube. Ovary oval, 56 (42-62; n=10) long, 32
(28-40; n=10) wide. Vitelline follicles dense, upto haptoral region. Testis
single, 31 (25-37; n=10) long, 18 (16-22; n=10) wide, vas deferens arises
from the anterior end of testis, single seminal vesicle opens at the base of
copulatory complex. Prostatic reservoirs two, opens at the base of
copulatory complex. Haptor 45 (40-52; n=10) long, 98 (85-135; n=10)
wide. Dorsal anchor inner length 38 (36-52; n=10), outer length 26 (23-
35; n=10), recurved point 10 (8-12; n=10), dorsal bar 18 (16-20) long.
Hooks seven pairs, similar, hook pairs 1, 2, 3, 4 and 7, 18- 20 (n=10),
hook pair 5 and 6, 14-16 (n=10).
Remarks:- The present species was earlier described in detail by Tripathi
(unpublished work) from P. ticto from river Gomti, Lucknow. It is
therefore briefly recorded here. It is therefore briefly recorded here.
30
Dactylogyrus sp.5 Whole –mount (dorsal view).
(Plate. 7)
31
Molecular Phylogenetics
Nucleotide Sequence Analysis
Average of all the seven Dactylogyrus species nucleotide
sequences had total of 338 positions, in the final data set (Detailed
species wise compositions included in Table 1). It revealed the fewest
Cytosine (22.7%). The degree of bias depends upon the codon
composition i.e. 25.4% Cytosine in the first position, 20.7% in second
position and 21.8% in third position. The first position rich in Guanines
was 27.1%, the second position rich in Thymine was 29.0% and third
position rich in Adenines was 30.2%.
Average Evolutionary Divergence over all Sequence Pairs is 2.98.
Estimated results showed minimum 0.17 distance between Dactylogyrus
sp.1 and Dactylogyrus sp.3 where maximum distance 4.90 was shown
between Dactylogyrus subtilis and Dactylogyrus sp.5 (Table 2).
Substitution pattern rates were estimated under Kimura 2-
parameter model (Kimura 1980). Substitution Matrix showed estimated
values between 11.13 (minimum) - 14.33 (maximum) and each entry
showed the probability of substitution (r) from one base to another base
(Table 3). The transition/transversion rate ratios are k1 = 1.806 (purines)
and k2 = 2.288 (pyrimidines). The overall transition/transversion bias is R
= 1.018, where R = [A*G*k1 + T*C*k2]/[(A+G)*(T+C)].
In Codon Based Z- Test of Selection, the values of P less than
0.05 were considered significant at 5% level (Table 4). The variance of
the difference was computed using bootstrap method (1000 replicates). In
Tajima Test of Neutrality, number of segregating sites (ps) was 1.000000,
32
nucleotide diversity 0.677656 and the Tajima Test statistic calculated (D)
3.884346 (Table 5).
Phylogeny
The phylogenetic reconstructions (Figs. 1&2), inferred from
analysis of 28S rDNA sequences showed great resolution for the species
of the monogenoideans. 28S rDNA sequences were aligned using Clustal
W (Thompson et al. 1994). All seven Dactylogyrus species under study
and two Onchobdella bopeleti Paperna, 1968 (Accession no. HQ010033)
and Onchobdella aframae Bilong Bilong Euzet, 1995 (Accessesion no.
HQ010034) (Ancyrocephaline monogenoideans), recovered from
GENBANK for phylogenetic reconstruction, revealed clear differences in
nucleotide sequences among different species. BLAST analysis showed
unique feature of the sequences. They had 92% (minimum) - 93%
(maximum) similarity for D. subtilis, 91%- 93% for D. longiacus, 90%-
91% for Dactylogyrus sp.1, 89%- 91% for Dactylogyrussp.2, 90- 92%
for Dactylogyrus sp.3, 90- 92% for Dactylogyrussp.4, 88- 92% for
Dactylogyrus sp. 5, with the sequences of species of genus Dactylogyrus
available at NCBI.
For phylogenetic analysis, the phylogenetic trees were computed
with Neighbor- Joining (NJ) method and Minimum Evolution (ME)
method. The evolutionary distances were computed using p-distance
method and are in the units of the number of base differences per site.
Codon position included were 1st+2nd+3rd+Noncoding. Gaps and missing
data were eliminated. The ME tree was constructed using close-neighbor-
interchange (CNI) algorithm. Branch lengths are generally not obtained
for each topology; the sequences at each node are inferred to be those that
require the least number of changes to give each of two immediately
33
descendant sequences (Dopazo, 1994). Bootstrap values were included to
test the reliability of inferred trees (Felsentein, 1985) and the estimation
of evolutionary divergence between sequences was computed. The
phylogenies were tested with 1000 bootstrap replicates. Bootstrap values,
indicating the robustness of the internal nodes were set at 1000
replications. Above mentioned two methods- Neighbor- Joining (NJ) and
Minimum Evolution (ME) gave trees with similar topology and
approximately similar bootstrapped values. These trees showed three
groups of parasites, first group included three species Dactylogyrus sp 1,
Dactylogyrus sp 3 and Dactylogyrus sp.4 having bootstrap value 89%,
second group included two species Dactylogyrus sp.2, Dactylogyrus sp.5
having bootstrap value between 51% to 55% , third group included two
species Dactylogyrus subtilis, Dactylogyrus longiacus having bootstrap
value 67%-68% .
Discussion
Family Dactylogyridae (Dactylogyrinae: Dactylogyroidae)
represented a highly diversified group (Gibson et al. 1996; Timofeeva et
al. 1997). Phylogenetic analysis using morphological characters had been
performed previously by Kritsky and Boeger (1989) to resolve the
phylogenetic relationships among families and subfamilies of
Dactylogyridae. Traditional study was based, to a large extent, on the
morphology of the sclerotized components of the haptoral parts.
Within the Monogenoidea, sequences of ribosomal subunits were
widely used to infer phylogenetic relationships at the level of families and
subfamilies (Simkova et al. 2003; Plaisance et al. 2005) or also to
investigate evolutionary associations between parasites and their hosts
(Desdevises et al. 2002; Simkova et al. 2004).
34
The present study provided the first insight data on 28S genes of
seven species of Dactylogyrus occurring on fish Puntius in India, an
important genus of freshwater food and ornamental fish belonging to the
family Cyprinidae. All parasites of Puntius are strictly specialists, none
being generalist. The main process of Dactylogyrus diversification
corresponded to sympatric speciation, correlated to strict host specificity.
Although distribution of some species might overlap, their reproductive
organs kept them apart (Shrivastava et al. 2012).
PCR technology and DNA sequencing techniques permitted
identification of species easier, as 28S gene was highly conserved. In
total, 338 bp sequences of 28S rDNA gene were taken for 7 species of
Dactylogyrus from family Dactylogyridae (also see Simkova et al. 2002,
Chowdhary and Singh, 2012). On the basis of nucleotide sequence
variation analysis, it was aptly clear that values of A T G C contents
(Table 1) of parasites having similarity formed distinct groups.
Phylogenetic analysis further highlighted that Dactylogyrus species under
study are similar and clustered together. Within these seven Dactylogyrus
species, three lineages were observed. The tree obtained from the p-
distance with bootstrap proportions (BP) supported the monophyly of
Dactylogyrus. The first lineage (Fig.1&2) was composed of two sister
subgroups. The first associated two species Dactylogyrus sp.1 and
Dactylogyrus sp.3. The second one associated only one species
Dactylogyrus sp.4. The second lineage (Fig.1&2) is composed of two
species, Dactylogyrus sp 2 and Dactylogyrus sp.5. The third lineage
(Fig.1&2) is also composed of two species, Dactylogyrus subtilis and
Dactylogyrus longiacus. It therefore proved the concept of monophyly in
Dactylogyrus (also supported by Simkova et al. 2004). Dactylogyrus
sp.1, Dactylogyrus sp.3, Dactylogyrus sp.4 belonging to first lineage did
35
not occur on the same species of fish but one on P. sophore and two on P.
ticto. Dactylogyrus sp.2 and Dactylogyrus sp.5 belonging to second
lineage occurred on P. chola and P. ticto respectively and Dactylogyrus
subtilis and Dactylogyrus longiacus belonged to third lineage on P.
sophore. The results showed significant relationship in phylogenetic
attribute of Dactylogyrus species under study. On the basis of NJ and ME
trees we can say that the first group in which D. subtilis and D. longiacus
evolved first (Also supported by evolution of host species P. sophore, as
observed by Pallavi et al. 2012). Group second composed of
Dactylogyrus sp.1. Dactylogyrus sp.3, Dactylogyrus sp.4 came next. Of
these, Dactylogyrus sp.1 was on P. sophore and two other infected P.
ticto. Third group consisting of Dactylogyrus sp.2 and Dactylogyrus sp.5
were found on P. chola and P.ticto respectively. According to Pallavi et
al. 2012, P. ticto evolved after P. chola. In our opinion probably the third
group evolved from second group as a result speciation of parasite
species. Moreover, the present analysis suggested the independent
secondary colonization of Dactylogyrus sp. 2 and Dactylogyrus sp. 5 on
their respective hosts. In conclusion, the phylogeny inferred from the
partial 28S rDNA supported the hypothesis that colonization of
Dactylogyrus parasites followed the pattern of diversification of their host
species.
36
Table 1:- Computed Nucleotide Composition
Domain: Data T(U) C A G Total T-1 C-1 A-1 G-1 Pos #1 T-2 C-2 A-2 G-2 Pos #2 T-3 C-3 A-3 G-3 Pos #3
Dactylogyrus
subtilis 21.6 21.6 25.9 31.0 371.0 19 24.2 27.4 29.8 124.0 23 23.4 21.8 32.3 124.0 24 17.1 28.5 30.9 123.0
Dactylogyrus
longiacus 24.8 21.3 26.1 27.8 371.0 26 26.6 21.8 25.8 124.0 28 18.5 25.0 28.2 124.0 20 18.7 31.7 29.3 123.0
Dactylogyrus
sp.1 24.2 21.7 26.7 27.3 359.0 22 25.8 26.7 25.8 120.0 33 18.3 20.8 28.3 120.0 18 21.0 32.8 27.7 119.0
Dactylogyrus
sp. 2 27.2 26.9 22.8 23.1 368.0 25 27.6 22.0 25.2 123.0 33 26.8 18.7 22.0 123.0 24 26.2 27.9 22.1 122.0
Dactylogyrus
sp.3 24.9 20.4 26.6 28.0 357.0 24 23.5 26.1 26.9 119.0 29 16.8 22.7 31.9 119.0 23 21.0 31.1 25.2 119.0
Dactylogyrus
sp.4 24.1 21.1 25.8 29.1 361.0 24 24.8 22.3 28.9 121.0 26 17.5 22.5 34.2 120.0 23 20.8 32.5 24.2 120.0
Dactylogyrus
sp.5 27.1 25.9 24.1 22.9 340.0 25 25.4 21.9 27.2 114.0 32 23.7 23.7 21.1 114.0 24 28.6 26.8 20.5 112.0
Avg. 24.8 22.7 25.4 27.1 361.0 23 25.4 24.0 27.1 120.7 29 20.7 22.2 28.3 120.6 22 21.8 30.2 25.8 119.7
37
Table 2:- Pairwise Distance (Maximum Composite Likelihood)
Dactylogyrus subtilis
Dactylogyrus longiacus 2.66
Dactylogyrus sp.1 3.61 3.25
Dactylogyrus sp.2 3.54 3.15 3.61
Dactylogyrus sp.3 3.29 2.56 0.17 4.58
Dactylogyrus sp.4 3.23 2.79 0.28 3.59 0.17 Dactylogyrus sp.5 4.90 2.89 3.49 2.79 4.48 3.50
Overall distance- 2.98
Table 3:- Maximum Composite Likelihood Subtitution Matrix
(Tamura Nei Model) Maximum Composite Likelihood Estimate of the Pattern of Nucleotide
Substitution
A T C G
A - 6.26 5.8 11.77
T 6.16 - 13.28 6.52
C 6.16 14.33 - 6.52
G 11.13 6.26 5.8 -
38
Table 4:- Codon Based Z- Test of Selection
Dactylogyrus subtilis 1.03 -0.78 -2.99 -1.28 -0.40 1.21
Dactylogyrus longiacus 0.30 0.35 0.68 0.23 0.65 0.62
Dactylogyrus sp.1 0.44 0.73 -1.45 0.34 0.96 -0.37
Dactylogyrus sp.2 0.00 0.50 0.15 -0.89 0.60 0.74
Dactylogyrus sp.3 0.20 0.82 0.74 0.38 -0.33 -0.31
Dactylogyrus sp.4 0.69 0.52 0.34 0.55 0.74 -0.12
Dactylogyrus sp.5 0.23 0.54 0.71 0.46 0.76 0.90
Table 5:- Tajima Test of Neutrality
M S ps Θ Π D
7 338 1.000000 0.408163 0.677656 3.884346
Abbreviations: m = number of sequences, S = Number of segregating
sites, ps = S/m, Θ = ps/a1, π = nucleotide diversity, and D is the Tajima
test statistic.
39
Fig:- 1 Phylogenetic analysis by Neighbor-Joining Method
Neighbor Joining Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
40
Fig:-2 Phylogenetic analysis by Minimum Evolution Method
Minimum Evolution Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
41
Dactylogyroides tripathii (Tripathi, 1959) Gusev, 1963
(Plate. 8)
Host- Puntius sophore Hamilton, 1882
Type Locality- River Gomti
Infection site- Gills
No. of host examined-10
Body elongate 310 (240-430; n=10) long; maximum width 80 (75-
115; n=10). Cephalic region well developed and divided into four lobes.
Two pairs of eye spots. Intestinal caeca confluent posterior to testis.
Pharynx spherical 21 (17-27; n=10) in diameter. Copulatory complex
consist of copulatory tube and accessory piece. Copulatory tube 29 (27-
31; n=10) long, comma shaped, with a swollen base; accessory piece 26
(26-28; n=10) long. Vaginal apparatus sclerotised, opening into seminal
receptacle through a vaginal tube. Ovary oval, 38 (36-42; n=10) long, 32
(30-35; n=10) wide. Vitelline follicles dense, throughtout body, except
gonadal region. Testis single, 43 (40-48; n=10) long 18 (16-20; n=10)
wide. Vas deferens arises from the anterior end of testis and forms two
seminal vesicles, which in turn opens at the base of copulatory complex.
Single prostatic reservoir opens at the base of copulatory complex.
Haptor 44 (40-52; n=10) long, 84 (75-95; n=10) wide. Dorsal anchor
inner length 41(38-45; n=10), outer length 40 (38-45), inner root 9 (8-10;
n=10), outer root 5 (5-7; n=10) recurved point 9 (8-9; n=10). Ventral
anchors absent. 'V' shaped dorsal connecting bar present. It is divided into
two parts, each 30 (29-32; n=10) long, ventral bar wide U- shaped 35 (32-
42
38; n=10) and a median groove present. Hooks seven pairs; hook pairs 1,
2 and 3, 17-22 (n=10), hook pairs 5, 6 and 7, 16-19 (n=10), hook pair 4,
27-33 (n=10).
Remarks:- This species was described by Gusev (1963) from a water
bodies near Lucknow. Agrawal et al. (2002) redescribed the species,
adding information on soft parts and some differences in sclerotised
structures. It is briefly recorded in the present work.
43
Dactylogyroides tripathii Whole- mount (dorsal view)
(Plate. 8)
44
Dactylogyroides longicirrus (Tripathi, 1959) Gusev, 1976
(Plate. 9)
Host- Puntius sophore Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-10
Body elongate 340 (280-430; n=10) long; maximum width 125
(90-185; n=10). Cephalic region well developed and divided into four
lobes. Two pairs of eye spots. Intestinal caeca confluent posterior to
testis. Pharynx spherical 20 (17-27; n=10) in diameter. Copulatory
complex consist of copulatory tube and accessory piece, copulatory tube
29 (27-32; n=10) long with swollen base; accessory piece 26 (24-29;
n=10) long. Vaginal apparatus sclerotised, opening into seminal
receptacle through a vaginal tube. Ovary oval, 45 (36-48; n=10) long, 32
(30-35; n=10) wide. Vitelline follicles dense, throughtout body, except
gonadal region. Testis single, 41 (39-45; n=10) long, 20 (17-22; n=10)
wide. Vas deferens arises from the anterior end of testis and forms two
seminal vesicles, which in turn opens at the base of copulatory complex.
Single prostatic reservoir opens at the base of copulatory complex.
Haptor 45 (40-52; n=10) long, 150 (140-165; n=10) wide. Dorsal anchor
inner length 75 (72-78; n=10), outer length 75 (70-80; n=10), recurved
point 9 (8-9; n=10). Ventral anchors absent. 'V' shaped dorsal connecting
bar present. It is divided into two parts, each 30 (30-34; n=10) long,
ventral bar wide U-shaped 32 (32-38; n=10) and a median groove present.
Hooks seven pairs; hook pairs 1, 2, 3 and 5, 22- 25 (n=10), hook pairs 6
and 7, 16-19 (n=10), hook pair 4, 29-34 (n=10) long.
45
Remarks:- Gusev (1976) described D. longicirrus from the water bodies
near Lucknow. Agrawal et al. (2002) redescribed the species in detail. It
is therefore briefly recorded here.
46
Dactylogyroides longicirrus Whole-mount (ventral view) (Plate. 9)
47
Dactylogyroides mahecoli Gusev, 1976
(Plate.10)
Host- Puntius chola Hamilton, 1882
Type Locality- River Gomti
Infection site- Gills
No. of host examined-10
Body elongate 380 (280-445; n=10) long; maximum width 110
(90-125; n=10). Cephalic region well developed and divided into four
lobes. Two pairs of eye spots. Intestinal caeca confluent posterior to
testis. Pharynx spherical 22 (17-30; n=10) in diameter. Copulatory
complex consist of copulatory tube and accessory piece, copulatory tube
27(24-32; n=10) long with swollen base; accessory piece 23 (26-29;
n=10) long. Vaginal apparatus sclerotised, opening into seminal
receptacle through a vaginal tube. Ovary oval, 55 (36-65; n=10) long, 38
(30-42; n=10) wide. Vitelline follicles dense, throughout body except
gonadal region. Testis single, 42 (39-58; n=10) long, 18 (17-22; n=10)
wide. Vas deferens arises from the anterior end of testis and forms two
seminal vesicles, which in turn opens at the base of copulatory complex.
Single prostatic reservoir opens at the base of copulatory complex.
Haptor 48 (40-55; n=10) long, 160 (140-175; n=10) wide. Dorsal anchor
inner length 42 (35-46; n=10), outer length 38 (34-42; n=10), recurved
point 8 (8-9; n=10). Ventral anchors absent. 'V' shaped dorsal connecting
bar present. It is divided into two parts, each 32 (30-34; n=10) long,
ventral bar wide U-shaped 29 (26-38; n=10) long and a median groove
present. Hooks seven pairs; hook pairs 1, 2, 3, 20- 24 (n=10), hook pairs
6 and 7, 16-20 (n=10), hook pair 4, 26-32 (n=10) long.
48
Remarks:- Gusev (1976) described from the water bodies near
Lucknow. Agrawal et al. (2002) redescribed the species in detail. It is
therefore briefly recorded here.
49
Dactylogyroides mahecoli Whole-mount (dorsal view)
(Plate. 10)
50
Dactylogyroides dorsali Gusev, 1976
(Plate.11)
Host- Puntius chola Hamilton, 1882
Type Locality- River Gomti
Infection site - Gills
No. of host examined-10
Body elongate 320 (280-425; n=10) long; maximum width 95 (80-
130; n=10). Cephalic region well developed and divided into four lobes.
Two pairs of eye spots. Intestinal caeca confluent posterior to testis.
Pharynx spherical 19 (17-26; n=10) in diameter. Copulatory complex
consist of copulatory tube and accessory piece, copulatory tube 29 (27-
34; n=10) long with swollen base; accessory piece 26 (24-28; n=10) long.
Vaginal apparatus sclerotised, opening into seminal receptacle through a
vaginal tube. Ovary oval, 44 (42-48; n=10) long, 21 (20-22; n=10) wide.
Vitelline follicles dense, throughtout body, except gonadal region. Testis
single, 34 (29-37; n=10) long, 18 (16-22; n=10) wide. Vas deferens arises
from the anterior end of testis and forms two seminal vesicles, which in
turn opens at the base of copulatory complex. Single prostatic reservoir
opens at the base of copulatory complex. Haptor 42 (40-52; n=10) long,
115 (95-135; n=10) wide. Dorsal anchor inner length 43 (36-52; n=10),
outer length 36 (32-40; n=10), recurved point 9 (8-9; n=10). Ventral
anchors absent. 'V' shaped dorsal connecting bar present. It is divided into
two parts, each 29 (28-32; n=10) each, ventral bar wide U-shaped 35 (32-
40; n=10) and a median groove present. Hooks seven pairs; hook pairs 1,
2, 3 and 5, 18- 25 (n=10), hook pair 4, 29-34 (n=10), hook pairs 6 and 7,
16-19 (n=10) long.
51
Remarks:- Gusev (1976) described from the water bodies near
Lucknow. Agrawal et al. (2002) redescribed the species in detail. It is
therefore briefly recorded here.
52
Dactylogyroides dorsali Whole-mount (ventral view)
(Plate. 11)
53
Molecular Phylogenetics
Nucleotide Sequence Analysis
Average of all the four Dactylogyroides species nucleotide
sequences had total of 254 positions, in the final data set (Detailed
species wise compositions included in Table 1). It revealed the fewest
Cytosine (22.8%). The degree of bias depends upon the codon
composition i.e. 23.7% Cytosine in the first position, 23.1% in second
position and 21.6 % in third position. The first position rich in Adenine
was 26.6 %, the second position rich in Thymine was 28.0 % and third
position was also rich in Thymine 27 %.
Average Evolutionary Divergence over all Sequence Pairs is 9.51.
Estimated results showed minimum 5.54 distance between
Dactylogyroides dorsali and Dactylogyroides longicirrus where
maximum distance 11.65 was shown between Dactylogyroides mahecoli
and Dactylogyroides longicirrus (Table 2).
Substitution matrix estimated between minimum7.08 to maximum
38.25 and each entry shows the probability of substitution (r) from one
base (row) to another base (column) (Table 3). The transition/transversion
rate ratios are k1 = 3.351 (purines) and k2 = 17.047 (pyrimidines). The
overall transition/transversion bias is R = 4.924, where R = [A*G*k1 +
T*C*k2]/[(A+G)*(T+C)]. In estimation of substitution pattern disparity,
the probability of rejecting the null hypothesis that sequences have
evolved with the same pattern of substitution, as judged from the extent
of differences in base composition biases between sequences (Disparity
Index test). A Monte Carlo test (1000 replicates) was used to estimate the
P-values, which are shown below the diagonal. P-values smaller than
0.05 are considered significant.
54
In Codon Based Z- Test of Selection, the values of P less than 0.05
were considered significant at 5% level (Table 4). The variance of the
difference was computed using bootstrap method (1000 replicates). In
Tajima Test of Neutrality, number of segregating sites (ps) was 0.984252,
nucleotide diversity 0.677656 and the Tajima Test statistic calculated (D)
4.269047 (Table 5).
Phylogeny
The phylogenetic reconstructions (Figs. 1&2), inferred from
analysis of 28S rDNA sequences showed great resolution for the species
of the monogenoideans. 28S rDNA sequences were aligned using Clustal
W (Thompson et al., 1994). All four Dactylogyroides species under study
and two Ligophorus chabaudi Euzet and Suriano, 1977 (Accession no.
JN996834) and Ligophorus vanbenedenii (Parona & Perugia, 1890)
Johnston & Teigs, 1922 (Accession no. JN996802) (Ancyrocephaline
monogenoideans), recovered from GENBANK for phylogenetic
reconstruction, revealed clear differences in nucleotide sequences among
different species. Sequence analysis was conducted using the nucleotide
BLAST program in the NCBI database (National Center for
Biotechnology Information, NIH, Bethesda, Maryland, USA) for
similarity and nucleotide length (Tatusova and Madden, 1999). We also
calculated the fractional GC contents of the nucleic acid sequences. The
length of 28S partial rDNA sequence of D.tripathii, D.longicirrus,
D.mahecoli and D.dorsali were 368, 301, 381 and 364 bases respectively.
They had 92% (minimum) - 93% (maximum) similarity for D.tripathii,
91%- 93% for D.longicirrus, 90%- 91% for D.mahecoli, 89%- 91% for
D.dorsali, with the sequences of other available at NCBI in BLAST
search. GC content of these four sequences were between 49.2% - 50.8%.
55
For phylogenetic analysis, the phylogenetic trees were computed
with Neighbor- Joining (NJ) method and Minimum Evolution (ME)
method. The evolutionary distances were computed using p-distance
method and are in the units of the number of base differences per site.
Codon position included were 1st+2nd+3rd+Noncoding. Gaps and missing
data were eliminated. The ME tree was constructed using close-neighbor-
interchange (CNI) algorithm. Branch lengths are generally not obtained
for each topology; the sequences at each node are inferred to be those that
require the least number of changes to give each of two immediately
descendant sequences (Dopazo, 1994). Bootstrap values were included to
test the reliability of inferred trees (Felsentein, 1985) and the estimation
of evolutionary divergence between sequences was computed. The
phylogenies were tested with 1000 bootstrap replicates. Bootstrap values,
indicating the robustness of the internal nodes were set at 1000
replications. Above mentioned two methods- Neighbor- Joining (NJ) and
Minimum Evolution (ME) gave trees with similar topology and
approximately similar bootstrapped values. These trees showed two
monohyletic groups of parasites and forming two subclade, first subclade
included two species Dactylogyroides tripathii and Dactylogyroides
mahecoli and having bootstrap value 51-53%, second also included two
species Dactylogyroides longicirrus and Dactylogyroides dorsali having
bootstrap value between 53% to 57% .
Discussion
Traditional study was based, to a large extent, on the morphology
of the sclerotized components of the haptoral parts. In recent times,
molecular techniques are being increasingly used in taxonomy and
phylogenetic species and have emerged as valuable supplementary tools
in providing authentic and unambiguous identification of species. 28S
56
rDNA markers have been used to detect species boundaries (Kaukas and
Rollinson, 1997).
Phylogenetic relationships based on morphological characters and
molecules are mostly concordant (Bernardi and Crane 2005: Ward et al.
2005). From our data, we observed genetic variation among species in
parasitic Platyhelminthes. The each variation is very variable based on
each gene among species. We think that these differences in the
nucleotide length, GC percentage, nucleotide differences, and number of
gaps of gene can be attributed largely to varying numbers of repeat, copy
numbers, deletions, alignment gaps, and base substitutions and additions.
Base substitutions and additions are characterized by very high C content
which can, at time represent pure poly C structures. In addition this may
be a consequence of mutations in the lineage.
Genus Dactylogyroides showed close similarity with the genus
Dactylogyrus of the same subfamily Dactylogyrinae. Only difference lies
with the structure of bar. Dactylogyroides possess ‘V’ shaped bar in two
parts. Only five species of this genus are so far reported in India four on
two species of fish Puntius and one on fish Osteobrama cotio (not
included in the present study).
Two subclade were formed in this study. Those Dactylogyroides
species which were found in same subclade do not parasitize the same
fish species. The result from the phylogenetic analyses did not indicate
that Dactylogyroides species coexisting on the same host evolve by intra-
host speciation, which was inferred to be an important process of parasite
diversification in cyprinid fish species (Simkova et al., 2004). We
compared gene sequences by NJ and ME analysis for phylogenetic
analysis, and then we acquired the same tree pattern.
57
The molecular phylogenetic analyses showed that Dactylogyroides
from different Puntius species clustered together. The two species D.
tripathii and D.mahecoli, in the first cluster showed that on the basis of
nucleotide composition shows similarity at an average of T position and
T-1 and G-1 position. Molecular phylogeny shows similarity between D.
tripathii and D. mahecoli having bootstrap value 51-53% in ME and NJ
methods. These observations are further supported by similarity in the
structure of taxonomically important sclerotised structures like haptoral
armature and copulatory complex, although they infect two different
species of the host (Agrawal et al., 2002). It seems most likely that the
two species co-speciated, infecting two different host species of the same
host genus. Similarly, the second cluster of D. longicirrus and D. dorsali,
have different nucleotide composition, as supported by ME tree
(bootstrap value 57). It is worthwhile to mention here that these species
are morphologically quite distinct as well (Gusev, 1976, Agrawal et al.,
2002).
58
Table 1- Compute Nucleotide composition
Domain: Data
T(U) C A G Total T-1 C-1 A-1 G-1 Pos #1 T-2 C-2 A-2 G-2 Pos #2 T-3 C-3 A-3 G-3 Pos #3 Dactylogyroides
tripathii 24.7 22.6 26.1 26.6 368.0 22 19.5 30.1 28.5 123.0 25 26.0 21.1 27.6 123.0 27 22.1 27.0
23.8 122.0 Dactylogyroides
longicirrus 30.7 20.2 22.6 26.5 257.0 31 24.1 21.8 23.0 87.0 34 17.4 22.1 26.7 86.0 27 19.0 23.8
29.8 84.0 Dactylogyroides
mahecoli 24.7 19.6 27.4 28.3 368.0 22 20.3 29.3 28.5 123.0 27 20.3 23.6 29.3 123.0 25 18.0 29.5
27.0 122.0 Dactylogyroides
dorsali 27.1 28.2 22.2 22.5 365.0 25 31.1 23.8 19.7 122.0 26 27.0 20.5 26.2 122.0 30 26.4 22.3
21.5 121.0
Avg. 26.5 22.8 24.7 25.9 339.5 25 23.7 26.6 25.1 113.8 28 23.1 21.8 27.5 113.5 27 21.6 25.8
25.2 112.3
59
Table 2:- Pairwise Distance (Maximum Composite Likelihood)
Dactylogyroides tripathii
Dactylogyroides longicirrus 8.91
Dactylogyroides mahecoli 9.08 11.65 Dactylogyroides dorsali 11.35 5.54 10.50
Overall Distance- 9.51
Table 3:- Maximum Composite Likelihood Substitution Matrix
(Tamura Nei Model)
Maximum Composite Likelihood Estimate of the Pattern of Nucleotide
Substitution
A T C G
A - 2.24 1.82 7.08
T 2.11 - 31.01 2.11
C 2.11 38.25 - 2.11
G 7.08 2.24 1.82 -
60
Table 4:- Codon Based Z- Test of Selection
Dactylogyroides tripathii -0.60 0.99 -1.25
Dactylogyroides longicirrus 0.55 0.57 -0.21
Dactylogyroides mahecoli 0.33 0.57 -1.82
Dactylogyroides dorsali 0.21 0.83 0.07
Table 5:- Tajima Test of Neutrality
Table. Results from Tajima's Neutrality Test [1]
m S ps Θ Π D
4 250 0.984252 0.536865 0.755249 4.269047
Abbreviations: m = number of sequences, S = Number of segregating
sites, ps = S/m, Θ = ps/a1, π = nucleotide diversity, and D is the Tajima
test statistic.
61
Fig:- 1 Phylogenetic analysis by Neighbor-Joining Method
Neighbor Joining Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
62
Fig:- 2 Phylogenetic analysis by Minimum Evolution Method
Minimum Evolution Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
63
Esomocleidus esomi (Gusev, 1963) n. comb.
(Plate. 12 & 13)
Syn - Ancyrocephalus esomi Gusev, 1963
Type host- Esomus danricus Hamilton, 1822
Type locality- Nugegoda, Sri Lanka
Infection site- Gills
No. of host examined- 15
Redescription
Body elongate 170 (165-172; n= 15) long; greatest width 37 (32-
40; n= 15). Tegument annulated, a membranous covering is thrown out
all around the body margin, when parasite is relaxed or in dead condition.
Cephalic lobes well developed; head organs conspicuous, three pairs.
Eyes four, anterior pair larger, lens present at posterior pair. Pharynx oval
13 (12-14; n= 15) long, 14 (13-15; n= 15) wide. Testis oval 20 (19-22; n=
15) long, 15 (14-18; n= 15) wide; seminal vesicle two, dilation of vas
deferens; prostatic reservoir not seen. Copulatory complex consists of
copulatory tube and accessory piece, copulatory tube 10 (9-11; n= 15),
with broad base 6 (7-9; n= 15), forked-shaped, having thickened wall;
accessory piece 8 (7-9; n= 15) long, tri-radiate shaped. Ovary oval, 31
(29-33; n= 15) long, 17 (16-18; n= 15) wide. Vagina 3 (2-3; n= 15)
lightly sclerolized, thin tube-like, anterior or parallel to copulatory
complex, opening into balloon-shaped seminal receptacle. Vitelline
follicles dense, throughot body, except gonadal region. Haptor
subtrapezoidal 50 (49-52; n= 15) long, 22 (21-24; n= 15) wide. Hooks 14;
64
dissimilar, hook pair 1, 8, 8-9 (n= 15); hook pair 2, 9, 9-10 (n= 15);
hook pair 3, 11, 10-11 (n= 15); hook pair 4, 12, 12-13 (n= 15); hook pair
5, 11, 11-12 (n= 15); hook pair 6, 11, 11-12 (n= 15) and hook pair 7, 7-8
(n= 15) long. Dorsal anchor 17 (16-18; n= 15) long, outer root well
developed, 4 (4-5; n= 15) long, inner root well developed, 7 (7-8; n= 15)
long, evenly curved shaft. Ventral anchor 14 (13-15; n= 15) long, roots
feebly developed, inner root 3 (3-4; n= 15) long, shaft slightly curved,
point 3 (3-4; n= 15) long. Dorsal bar 16 (15-18; n= 15) long, narrow, rod-
shaped, usually arched posteriorly. Ventral bar 19 (19-20; n= 15) long,
bent downwards, with elongate antero-medial projection, 7 (7-8; n= 15).
Egg round to oval, filament at one end, large 52 (50-54; n= 15) long,
filament small 3 (3-4; n=15).
Remarks:- The original description of Ancyrocephalus esomi is brief and
lacks whole mount drawing. Further, Gusev (1963) overlooked certain
important morphological features; Gusev (1963) stated the intestinal
caeca to be confluent in Ancyrocephalus esomi. However, in the genus
Ancyrocephalus, the intestinal caeca are separate. In specimens, under
study, however, the intestinal caeca are confluent. Gusev (1963) also
overlooked vaginal armature, annulations on body margin, projection in
ventral bar, seminal vesicle, (two in present worm) and gonads, as well as
the whole mount drawings were also not given. Esomocleidus gen. n.
(Monogenoidea: Dactylogyridae) is, therefore, proposed to include three
species, collected from the gills of Indian pencil fish, Esomus danricus
(Hamilton, 1822) (Cyprindae); Esomocleidus esomi (Gusev, 1963) n.
comb. (syn. Ancyrocephalus esomi Gusev, 1963), Esomocleidus
chakrabarti (Gusev, 1976) n. comb. (syn. Ancyrocephalus chakrabarti
Gusev, 1976) and Esomocleidus lucknowensis n. sp. The new genus is
characterized by body annulations/serrations, a shield-like or tube-like
65
vagina located parallel or anterior to the copulatory complex and an
elongate antero-medial projection of the ventral bar. The present species
differs from E. chakrabarti (Gusev, 1963) n. comb. and E. lucknowensis
n.sp., described in the following pages, by tube-like vagina, tri-radiate
accessory piece, larger eggs and structure and shape of hooks.
Generic Diagnosis of Esomocleidus n.g.
Body abrasive or textured, comprising body proper (cephalic region,
trunk and peduncle) and haptor. Tegument annulated. Head organs 2 to 3
pairs. Eye spots 2 pairs, anterior larger than posterior. Eye lens present at
posterior pair. Mouth subterminal. Pharynx comprising muscular and
glandular bulb. Oesophagus short to non-existent. Intestinal caeca two,
confluent, posterior to gonads, lacking diverticula. Gonads inter-caecal,
overlapping, lying near posterior region of body. Vas deferns looping left
intestinal caecum. Two seminal vesicle, both dilation of vas deferens.
Prostatic reservoirs generally present. Copulatory complex comprising
copulatory tube & accessory piece. Copulatory tube sickle-shaped,
tubular; accessory piece tetra-radiate or tri-radiate. Vagina sclerotised
shield-like or tube-like, parallel or anterior to copulatory complex.
Seminal receptacle balloon-shaped, pre-ovarian. Vaginal pore sub-
marginal, ventral. Vitelline follicles in trunk, absent from regions of
reproductive organs. Haptor expands to form two lateral wings,
sometimes appearing wedge-like with dorsal, ventral anchor/bar
complexes. Seven pairs of dissimilar hooks with ancyrocephaline
distribution, hook thumb erect, proximal region slightly inflated. Eggs
round to oval, with filament at one end. Parasites of gills of Indian
pencilfish (Cyprinidae: Rasborinae).
Etymology: The generic name reflects the genus of fishes that serve as
hosts for species of the genus + the Greek word Kleidos (= key).
66
Esomocleidus esomi Whole-mount (ventral view). (Plate. 12)
67
1.Ventral anchor; 2. Dorsal anchor ; 3.Dorsal bar ; 4.Ventral bar; 5. Copulatory complex; 6.Vagina and seminal receptacle ; 7.Hook pairs 1-7 ; 8.Egg.
(Plate. 13)
68
Esomocleidus chakrabartii (Gusev, 1976) n.comb.
(Plate. 14 &15)
Syn.- Ancyrocephalus chakrabartii Gusev, 1976
Type host- Esomus danricus (Hamilton, 1822)
Type locality- Water bodies of Lucknow
Infection site- Gills
No. of host examined- 15
Redescription
Body elongate 150 (140-165; n= 15) long; greatest width 41
(39-44; n= 15). Tegument annulated, margin serrated. Cephalic lobes well
developed; head organs conspicuous, two pairs. Eyes four, anterior pair
larger, lens present at posterior pair. A distinct neck present. Pharynx oval
14 (12-14; n= 15) long, 13 (13-15; n= 15) wide. Testis oval 16 µm (15-
17; n= 15) long, 12 (11-13; n= 15) wide; seminal vesicle two, dilation of
vas deferens; prostatic reservoir three, balloon-shaped, opening at base of
copulatory tube. Copulatory complex consists of copulatory tube and
accessory piece, copulatory tube 25 (23-28; n= 15) sickle-shaped, with
broad base 11 (9-14; n= 15) having thickened wall; accessory piece 28
(27-30; n= 15) long, tetra-radiate. Ovary ovate, 28 (27-29; n= 15) long,
19 (18-20; n= 15) wide. Vaginal apparatus 11 (10-12; n= 15) highly
sclerotised, perforated, shield-like, anterior or parallel to copulatory
complex, opening into balloon-shaped seminal receptacle. Vitelline
follicles dense, throughout body, except gonadal region. Haptor sub-
trapezoidal 55 (51-60; n= 15) long, 27 (25-28; n= 15) wide. Hooks 14;
dissimilar, hook pairs 1, 2, 3 & 5, 18-20 (n= 15), hook pair 4, 21-22 (n=
15), hook pair 6, 12-13 (n= 15), hook pair 7, 14-15 (n= 15) long. Dorsal
69
anchor 19 (18-20; n= 15) long, outer root well developed, 5 (5-6; n= 15)
long, inner root well developed, 8 (7-9; n= 15) long, evenly curved shaft.
Ventral anchor 26 (26-27; n= 15) long, outer roots feebly developed,
inner root well developed, 9 (8-9; n= 15) long, shaft slightly curved, point
8 (7-9; n= 15) long. Dorsal bar 21 (20-22; n= 15) long, narrow, rod-
shaped, usually arched posteriorly. Ventral bar 18 (18-19; n= 15) long,
bent downwards, with elongate antero-medial projection, 8 (8-10; n= 15).
Egg round to oval, filament at one end, large 40 (38-42; n= 15) long,
filament small 3 (2-4; n=15).
Remarks:- The original description of Ancyrocephalus chakrabarti
includes diagrammatic figures and misinterpreted structures. Gusev
(1976) stated the intestinal caeca to be confluent in Ancyrocephalus
chakrabarti but in the generic diagnosis of Ancyrocephalus indicates the
intestinal caeca to be separate. In the present specimens, the intestinal
caeca are confluent. He overlooked vaginal armature but we have
observed perforated shield-like vagina in the present specimens. He also
missed the prostatic reservoirs, (three in present specimen), seminal
vesicle, (two in present worm) and gonads. Ancyrocephalus chakrabarti
is transferred under the new genus Esomocleidus as E. chakrabarti
n.comb. The present species significantly differs from E. esomi (Gusev,
1963) n. comb. and E. lucknowensis n.sp. by having perforated shield-like
vaginal apparatus, tetra-radiate accessory piece and sickle-shaped
copulatory tube.
70
Esomocleidus chakrabartii Whole-mount (ventral view).
(Plate. 14)
71
1. Copulatory complex; 2. Dorsal bar; 3. Ventral bar; 4. Dorsal anchor; 5. Ventral anchor; 6. Vagina; 7. Hook pairs; 8. Egg.
(Plate. 15)
72
Esomocleidus lucknowensis n.sp.
(Plate. 16 & 17)
Type host- Esomus danricus (Hamilton, 1822)
Type locality- River Sai, Lucknow
No. of host examined- 12
Infection site- Gills
Body elongate 120 (112-125; n= 12) long; greatest width 22 (20-
25; n= 12).Tegument annulated. Cephalic lobes well developed; head
organs conspicuous, two pairs. Eyes four, anterior pair larger, lens present
at posterior pair. Pharynx oval 10 (10-11; n= 12) long, 8 (7-9; n= 12)
wide. Testis oval 14 (12-15; n= 12) long, 10 (10-12; n= 12) wide; seminal
vesicle two, dilation of vas deferens. Copulatory complex consists of
copulatory tube and accessory piece, copulatory tube 11 (10-11; n= 12)
having thickened wall; accessory piece funnel-shaped, 6 (5-6; n= 12)
long, bi-radiate. Ovary ovate, 22 (20-22; n= 12) long, 15 (14-16; n= 12)
wide. Vaginal apparatus 4 (4-5; n= 12) non sclerotised, muscular, tubular,
anterior or parallel to copulatory complex, opening into balloon-shaped
seminal receptacle. Vitelline follicles dense, throughtout body, except
gonadal region. Haptor wedge shaped, 47 (46-48; n= 12) long, 22 (21-23;
n= 12) wide. Hooks 14; dissimilar, hook pairs 1, 3, 20-21 (n= 12), hook
pairs 2, 5, 22-23 (n= 12); hook pair 4, 24-25 (n= 12), hook pair 6, 17-18
(n= 12) and hook pair 7, 19-20 (n= 12) long. Dorsal anchor 16 (15-16; n=
12) long, outer root well developed, 4 (4-5; n= 12) long, inner root well
developed, 7 (7-8; n= 12) long, evenly curved shaft. Ventral anchor 25
(24-25; n= 12) long, outer root feebly developed, inner root 9 (8-9; n= 12)
long, shaft slightly curved, point 8 (7-9; n= 12) long. Dorsal bar 19 (18-
19; n= 12) long, thick, rod-shaped, slightly bent downwards. Ventral bar
73
18 (18-19; n= 12) long, bent downwards, with elongate antero-medial
projection, 1 µm (1-2; n= 12). Egg round to oval, filament at one end,
large 42 (42-44; n= 12) long, filament small 2 (2-3; n=12).
Remarks:- This species differs from E. esomi (Gusev, 1963) n. comb.
and E. chakrabarti (Gusev, 1963) n. comb. by having as projection in
ventral bar, shape of dorsal bar, muscular vaginal apparatus and shape
and size of hooks.
74
Esomocleidus lucknowensis n. sp. Whole-mount (ventral view). (Plate. 16)
75
1.Ventral anchor; 2. Dorsal anchor ; 3. Copulatory complex ; 4.Vagina and seminal receptacle; 5. Ventral bar; 6. Dorsal bar ; 7.
Hook pairs 1-7; 8. Egg. (Plate. 17)
76
Molecular Phylogenetics
Nucleotide Sequence Analysis
Average of all the four nucleotide sequences in which three
sequences of genus Esomocleidus and one for Ancyrocephalus paradox
had total of 337 positions, in the final data set (Detailed species wise
compositions included in Table 1). It revealed the fewest Adenine
(23.3%). The degree of bias depends upon the codon composition i.e.
24.3% Adenine in the first position , 24.1% in second position and 21.5%
in the third position. The first position rich in Thymine was 26%, the
second position rich in Cytosine was 26.2% and third position rich in
Guanine was 26.9%. Average of nucleotide sequences of three species of
Esomocleidus (E.chakrabartii, E.esomi and E.lucknowensis) at first,
second and third position are similar with each other but differ with
A.paradoxus.
Average Evolutionary Divergence over all sequence pairs is 0.77.
Estimated results showed minimum 0.72 distance between E.esomi and
E.lucknowensis where maximum distance 0.82 was shown between
E.esomi and Ancyrocephalus paradox (Table 2). Substitution pattern and
rates were estimated under Kimura (1980) 2-parameter model.
Substitution Matrix showed estimated values between 20.14 (minimum) –
22.8 (maximum) and each entry showed the probability of substitution (r)
from one base to another base (Table 3). The transition/transversion rate
ratios are k1 = 12.101 (purines) and k2 = 11.16 (pyrimidines). The overall
transition/transversion bias is R = 5.799, where R = [A*G*k1 +
T*C*k2]/[(A+G)*(T+C)].
77
In Codon Based Z- Test of Selection, the values of P less than 0.05
were considered significant at 5% level (Table 4). The variance of the
difference was computed using bootstrap method (1000 replicates). In
Tajima Test of Neutrality, number of segregating sites (ps) was 0.997033,
nucleotide diversity 0.845697 and the Tajima Test statistic calculated (D)
5.828348 (Table 5).
Phylogeny
The phylogenetic reconstructions (Figs. 1&2) inferred from
analysis of 28S rDNA sequences showed great resolution for the species
of the monogenoideans. 28S rDNA sequences were aligned using Clustal
W. All three Esomocleidus species and one Ancyrocephalus species
under study and one Dactylogyrus sp.1, recovered from GENBANK for
phylogenetic reconstruction, revealed clear differences in nucleotide
sequences among different species. For phylogenetic analysis, the
phylogenetic trees were computed with Neighbor- Joining method and
Minimum Evolution method. The evolutionary distances were computed
using p-distance method and are in the units of the number of base
differences per site. Codon position included were
1st+2nd+3rd+Noncoding. Gaps and missing data were eliminated.
Minimum Evolution was also based on p-distance method. The ME tree
was searched using close-neighbor- interchange (CNI) algorithm. Branch
lengths are generally not obtained for each topology; the sequences at
each node are inferred to be those that require the least number of
changes to give each of two immediately descendant sequences (Dopazo,
1994). Bootstrap values were included to test the reliability of inferred
trees (Felsentein, 1985) and the estimation of evolutionary divergence
between sequences was computed. The phylogenies were tested with
78
1000 bootstrap replicates. Bootstrap values, indicating the robustness of
the internal nodes were set at 1000 replications. Above mentioned two
methods- Neighbor- Joining (NJ) and Minimum Evolution (ME) gave
trees with similar topology and approximately similar bootstrapped
values. These trees showed two groups of parasites, first group included
two species Esomocleidus chakrabarti and Ancyrocephalus paradoxus
having bootstrap value 53% -60% and the second group included two
species Esomocleidus esomi and Esomocleidus lucknowensis having
bootstrap value between 93% to 94% .
Discussion
Twelve species of the genus Esomus are found only in the Asian
countries. Distribution of these fish in Asian countries include India,
Pakistan, Nepal, Bangladesh, Afghanistan, Srilanka and Myanmar. Gusev
(1963) have reported two species from the gills of pencil fish, i.e.
Ancyrocephalus esomi Gusev, 1963 at Nugegoda, Sri Lanka and A.
chakrabartii Gusev, 1976 at Lucknow, Uttar Pradesh. For the present
study, pencil fish from aquarium stores at Lucknow and river Sai, near
Lucknow district were examined and three species of dactylogyrids were
found from the gills of Esomus danricus (Hamilton, 1822), these are
Esomocleidus esomi (Gusev, 1976) n.comb. Syn. Ancyrocephalus esomi
Gusev, 1976; Esomocleidus chakrabartii (Gusev, 1976) n.comb. Syn.
Ancyrocephalus chakrabarti Gusev, 1976; Esomocleidus lucknowensis
n.sp. The original description of Ancyrocephalus esomi includes
diagrammatic figures and misinterpreted structures. Gusev (1963) stated
the intestinal caeca to be confluent in Ancyrocephalus esomi but in
generic diagnosis of Ancyrocephalus, the intestinal caeca are stated to be
separate. In the E. esomi (Gusev, 1976) n.comb., the intestinal caeca are
confluent. Gusev (1976) also overlooked vaginal armature, annulations
79
on body margin, projection in ventral bar, seminal vesicle, (two in present
worm) and gonads, as well as the whole mount drawings were also not
given, but in the present study whole mount drawing and all the above
characteristic features are observed. Similarly in Esomocleidus
chakrabartii (Gusev, 1976) n.comb., Gusev (1976) overlooked vaginal
armature, which is a perforated shield-like structure. He could not
observe three prostatic reservoirs, two seminal vesicles and the gonads.
The new species Esomocleidus lucknowensis n.sp. is characterized by a
projection in ventral bar, shape of dorsal bar, muscular vaginal apparatus
and shape and size of hooks.
Describing new species solely on the basis of their morphology is
often not straightforward, and especially so for small-bodied organisms
that display few morphological features on which to rely. A good
illustration is highlighted in monogenoidean parasitic flatworms, where
the main morphological structures used for species identification, namely
the hard parts of the host attachment apparatus (haptor) and male
copulatory organ, often require expert advice to discriminate closely
related species.
Phylogenetic analysis highlighted that Esomocleidus species under
study are similar and clustered together. Within these three Esomocleidus
species, two lineages were observed. The tree obtained from the p-
distance with bootstrap proportions (BP) supported the monophyly of
Esomocleidus. The first lineage composed of two species E.chakrabartii
and A.paradoxus. The second lineage is also composed of two species
E.esomi and E. lucknowensis.
80
According to nucleotide variation analysis Ancyrocephalus
paradoxus differs from three species of Esomocleidus, proving its distinct
identity (Table 2). Esomus sp.have a widespread distribution in India
(Talwar & Jhingran, 1991; Jayaram, 1999) and in Srilankan streams.
Occurrence of similar host specific monogenoideans on their fish also
supports common origin of host and parasites in these land masses.
81
Table 1: Computed Nucleotide composition
Domain: Data
T(U) C A G Total T-1 C-1 A-1 G-1 Pos #1 T-2 C-2 A-2 G-2 Pos #2 T-3
C-3
A-3 G-3 Pos #3
Esomocleidus
chakrabarthii 23.7 21.1 26.1 29.1 337.0 27 18.6 29.2 25.7 113.0 23 20.5 28.6 27.7 112.0 21
24.1
20.5 33.9 112.0
Esomocleidus
esomi 24.0 29.7 22.6 23.7 337.0 23 27.4 24.8 24.8 113.0 25 33.0 18.8 23.2 112.0 24
28.6
24.1 23.2 112.0
Esomocleidus
lucknowensis 27.0 26.7 22.3 24.0 337.0 25 26.5 21.2 27.4 113.0 29 25.9 24.1 20.5 112.0 27
27.7
21.4 24.1 112.0
Ancyrocephalus
paradoxus 33.3 21.0 20.0 25.7 105.0 38 20.5 17.9 23.1 39.0 21 23.5 26.5 29.4 34.0 41
18.8
15.6 25.0 32.0
Avg. 25.7 25.4 23.3 25.6 279.0 26 23.8 24.3 25.7 94.5 25 26.2 24.1 24.3 92.5 26
26.1
21.6 26.9 92.0
82
Table 2:- Pairwise Distance (Maximum Composite Likelihood)
Esomocleidus chakrabarthii
Esomocleidus esomi 0.77
Esomocleidus lucknowensis 0.78 0.72
Ancyrocephalus paradoxus 0.74 0.82 0.76
Overall Distance- 0.77
Table 3:- Maximum Composite Likelihood Subtitution Matrix
(Tamura Nei Model) Maximum Composite Likelihood Estimate of the Pattern of Nucleotide
Substitution
A T C G
A - 1.99 1.81 22.8
T 1.67 - 20.14 1.89
C 1.67 22.13 - 1.89
G 20.21 1.99 1.81 -
83
Table 4:- Codon Based Z- Test of Selection
Esomocleidus chakrabarthii 0.43 0.53 0.36
Esomocleidus esomi 0.67 0.86 0.10
Esomocleidus lucknowensis 0.60 0.39 1.14
Ancyrocephalus paradoxus 0.72 0.92 0.26
Table 5:- Tajima's Neutrality Test Table. Results from Tajima's Neutrality Test [1]
m S ps Θ π D
4 336 0.997033 0.543836 0.845697 5.828348
Abbreviations: m = number of sequences, S = Number of segregating
sites, ps = S/m, Θ = ps/a1, π = nucleotide diversity, and D is the Tajima
test statistic.
84
Fig: 1:- Phylogenetic analysis by Neighbor-Joining Method
Neighbor Joining Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
85
Fig:- 2 Phylogenetic analysis by Minimum Evolution Method
Minimum Evolution Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
86
Molecular Phylogenetics
The phylogenic relationships among 14 species of monogenoidea (7
species belonging to the genus Dactylogyrus, 4 species of
Dactylogyroides and 3 species of genus Esomocleidus) were investigated
via use of partial 28S ribosomal DNA (rDNA) sequences. All 28S rDNA
sequences were aligned using Clustal W software. Length of sequences
ranged between 301- 381 bp. Sequences of Tetraonchus monenteron
Diesing, 1858 (Accession no. AJ969953) and Anoplodiscus
cirrusspiralis Roubal, Armitage & Rohde, 1983 (Accession no.
AF382060) recoverd from GENBANK (placed in an outgroup) for
phylogenetic reconstruction, revealed clear differences in nucleotide
sequences among different species. Outgroup choice is especially critical
in resolving basal relationships from molecular data because of long-
branch attraction problems (Felsentein, 1978) and uncertainty in rooting.
Nucleotide Sequence Analysis
Average Evolutionary Divergence overall Sequence Pairs is 0.72.
Estimated results showed minimum 0.15 distance between Dactylogyrus
sp.3 & Dactylogyrus sp.2 where maximum distance 0.82 was shown
between Dactylogyroides longicirrus and Dactylogyroides tripathii
(Table 1).
Substitution pattern rates were estimated under Kimura 2-
parameter model (Kimura 1980). Substitution Matrix showed estimated
values between 9.58 (minimum) - 14.33 (maximum) and each entry
showed the probability of substitution (r) from one base to another base
.The transition/transversion rate ratios are k1 = 2.2 (purines) and k2 =
87
1.589 (pyrimidines). The overall transition/transversion bias is R =0.941,
where R = [A*G*k1 + T*C*k2]/[(A+G)*(T+C)] (Table 2).
In Codon Based Z- Test of Selection, the values of P less than 0.05
were considered significant at 5% level (Table 3). The variance of the
difference was computed using bootstrap method (1000 replicates). In
Tajima Test of Neutrality, number of segregating sites (ps) was 1.000000,
nucleotide diversity 0.760452 and the Tajima Test statistic calculated (D)
6.409283 (Table 4).
The nucleotide sequences were analyzed by length and G + C % in
order to determine the analyzed phylogenic relationships. For
phylogenetic analysis, the phylogenetic trees were computed with
Neighbor Joining (NJ) method and Minimum Evolution (ME) method.
The evolutionary distances were computed using p-distance method and
are in the units of the number of base differences per site. Codon position
included were 1st+2nd+3rd+Noncoding. Gaps and missing data were
eliminated. Minimum Evolution was also based on p-distance method.
The ME tree was constructed using close-neighbor- interchange (CNI)
algorithm. Branch lengths are generally not obtained for each topology;
the sequences at each node are inferred to be those that require the least
number of changes to give each of two immediately descendant
sequences (Dopazo, 1994). Bootstrap values were included to test the
reliability of inferred trees (Felsentein, 1985) and the estimation of
evolutionary divergence between sequences was computed. The
phylogenies were tested with 1000 bootstrap replicates. Bootstrap values,
indicating the robustness of the internal nodes were set at 1000
replications. Above mentioned two methods- Neighbor- Joining (NJ) and
Minimum Evolution (ME) gave trees with similar topology and
88
approximately similar bootstrapped values. These trees showed two
clearly divided clades. In first clade, genus Dactylogyrus and
Dactylogyroides were included and in the second clade species of the
genus Esomocleidus were found. In first clade five subclades were found.
In first subclade, three species Dactylogyrus longiacus, Dactylogyrus
subtilis and Dactylogyroides longicirrus were found, having bootstrap
value 52-53%. In the second subclade two species Dactylogyrus sp. 2 and
Dactylogyrus.sp 5 species were included, having bootstrap value 58-60%
and in the third subclade only one species Dactylogyroides dorsali was
found. In fourth subclade Dactylogyroides mahecoli and Dactylogyroides
tripathii were found, having bootstrap value 55-60% and in fifth subclade
Dactylogyrus sp.4, Dactylogyrus sp.3, and Dactylogyrus.sp.1 lied, having
bootstrap value 100%. However, in the second clade three species of the
genus Esomocleidus were found, having bootstrap value 62-69%.
Disscussion
Molecular phylogenetic approaches has advantages over the
morphology based approach. Molecular phylogenetic studies can use
powerful methods which accommodate various well characterized
features of molecular evolution, including different rates for different
types of nucleotide substitution. These model approaches are not
available for morphological analysis.
Resolving the interrelationship of these genera is the main aim of
present study. For this comparision, 28S rDNA sequences of all the
species were analysed. We conducted conventional bootstrap analyses,
under Neighbor Joining and Minimum Evolution criteria for phylogenetic
89
analysis, and then we acquired the same tree pattern. 28S rDNA genes are
conserved to a high degree.
Dactylogyrus, Dactylogyroides and Esomocleidus are under family
Dactylogyridae. However, the genera Dactylogyrus and Dactylogyroides
belong to the dactylogyrinae subfamily and Esomocleidus to subfamily
Ancyrocephalinae. All these analyses provide moderate to very strong
support for the monophyly of these genera.
Dactylogyrus and Dactylogyroides are somewhat similar genera.
The only difference is in the shape of the bar. All Dactylogyroides have V
shaped bar. In both NJ and MP trees Dactylogyrus and Dactylogyroides
were found in the first clade.
90
Table 1 - Pairwise Distance (Maximum Composite Likelihood)
Dactylogyrus subtilis
Dactylogyrus longiacus 0.73
Dactylogyrus sp.1 0.77 0.75
Dactylogyrus sp.2 0.77 0.77 0.77
Dactylogyrus sp.3 0.75 0.71 0.15 0.79
Dactylogyrus sp.4 0.74 0.72 0.23 0.77 0.15
Dactylogyrus sp.5 0.77 0.74 0.78 0.73 0.80 0.78
Dactylogyroides tripathii 0.78 0.47 0.77 0.74 0.73 0.72 0.78
Dactylogyroides longicirrus 0.71 0.77 0.81 0.73 0.78 0.79 0.77 0.82
Dactylogyroides dorsali 0.73 0.75 0.79 0.34 0.81 0.80 0.77 0.78 0.72
Dactylogyroides mahecoli 0.23 0.79 0.75 0.75 0.75 0.73 0.75 0.76 0.69 0.73
Esomocleidus chakrabarthii 0.75 0.73 0.77 0.78 0.76 0.79 0.76 0.73 0.74 0.79 0.73
Esomocleidus lucknowensis 0.74 0.71 0.79 0.78 0.79 0.76 0.75 0.73 0.76 0.77 0.73 0.74
Esomocleidus esomi 0.74 0.75 0.77 0.77 0.76 0.74 0.76 0.75 0.77 0.78 0.77 0.70 0.70
91
Table 2 -Maximum Composite Likelihood Subtitution Matrix (Tamura Nei Model)
Maximum Composite Likelihood Estimate of the Pattern of Nucleotide Substitution
A T C G
A - 6.99 6.03 14.33
T 6.17 - 9.58 6.51
C 6.17 11.11 - 6.51
G 13.58 6.99 6.03 -
92
Table 3:- Codon Based Z- Test of Selection
Dactylogyrus subtilis 0.89 -1.78 -2.29 -1.48 -1.68 1.30 -0.59 1.05 -0.73 1.17
Dactylogyrus longiacus 0.37 -0.08 0.75 0.07 0.23 0.69 -0.01 1.28 -0.53 2.02
Dactylogyrus sp.1 0.08 0.94 -1.47 0.72 0.72 -0.26 0.31 0.04 -0.29 -1.53
Dactylogyrus sp.2 0.02 0.46 0.15 -1.07 0.45 0.68 -0.43 -1.64 0.03 -2.37
Dactylogyrus sp.3 0.14 0.95 0.47 0.29 -0.45 0.09 -0.31 -0.44 -0.53 -0.62
Dactylogyrus sp.4 0.10 0.82 0.48 0.66 0.65 -0.27 -0.72 0.61 0.50 -0.39
Dactylogyrus sp.5 0.20 0.49 0.80 0.50 0.93 0.79 -0.05 -1.02 -0.20 -0.44
Dactylogyroides tripathii 0.56 0.99 0.75 0.67 0.76 0.48 0.96 -0.25 -1.59 0.66
Dactylogyroides longicirrus 0.30 0.20 0.97 0.10 0.66 0.55 0.31 0.80 -1.68 1.27
Dactylogyroides dorsali 0.47 0.60 0.77 0.98 0.59 0.62 0.85 0.11 0.10 -0.96
Dactylogyroides mahecoli 0.24 0.05 0.13 0.02 0.54 0.70 0.66 0.51 0.21 0.34
Esomocleidus chakrabarthii 0.04 0.94 1.00 0.72 0.54 0.57 0.45 0.24 0.65 0.25 0.03
Esomocleidus lucknowensis 0.20 0.75 0.15 0.24 0.34 0.57 0.05 0.15 0.59 0.39 0.03
Esomocleidus esomi 0.06 0.20 0.25 0.88 0.37 0.79 0.38 0.46 0.09 0.57 0.20
93
Table 4:- Tajima Test of Neutrality
Results from Tajima's Neutrality Test [1]
M S ps Θ π D 14 368 1.000000 0.314452 0.760452 6.409283
Abbreviations: m = number of sequences, S = Number of segregating sites, ps = S/m, Θ = ps/a1, π = nucleotide diversity,
and D is the Tajima test statistic.
94
Fig:- 1 Phylogenetic analysis by Neighbor-Joining Method
Neighbor Joining Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
95
Fig:-2 Phylogenetic analysis by Minimum Evolution Method
Minimum Evolution Distance Tree with distance estimated by p-distance
method. Score derived from interior branch test analysis with 1000
replications respectively are shown above the branches.
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Summary
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The thesis entitled “Molecular Analysis of Monogenoidean
Parasites in some Cyprinids” comprises work done during 2009-2013
on characterisation of monogenoideans infecting some commonly
available cypriniformes fishes of two genera Puntius Hamilton, 1882
(Puntius sophore Hamilton, 1882, Puntius chola Hamilton, 1882, Puntius
ticto Hamilton, 1882) and Esomus Swainson, 1839 (Esomus danricus
Hamilton, 1822). During this period, 14 monogenoidean species were
collected and identified. They were finally subjected to molecular
phylogenetics, using 28S rDNA.
An introduction has been given in the beginning of the thesis. It is
followed by a brief history of work done on molecular phylogenetics of
monogenoids and taxonomy of Indian worms is included. In the
following pages materials and methods is also appended. In the end of the
thesis, literature consulted for the present work is given. The thesis has
been divided into four chapters: I, II, III & IV.
Chapter I It includes records of seven species of the genus Dactylogyrus
Deising, 1850 of which five species are unknown and two are known and
their molecular phylogenetic studies.
1. Dactylogyrus longiacus Gusev, 1976
Gusev (1976) described D. longiacus from P.stigma (now
known as P.sophore) from the water bodies near Lucknow. The
description was based on hard parts only. Agrawal et al. (2003)
redescribed the species in detail and have added structure of soft
parts and variations in sclerotised structures. It is therefore briefly
recorded here.
2. D. subtilis Gusev, 1976
This species was described by Gusev (1976)
from P. stigma (now known as P. sophore), from a water bodies near
Lucknow. Its description was only based on hard parts. Agrawal et al.
(2003) redescribed the species, adding information on soft parts and some
differences in sclerotised structures. It is briefly recorded in the present
work.
3. Dactylogyrus sp.1
The present species was earlier described in detail by Tripathi
(unpublished work) from P. sophore from river Gomti, Lucknow. It is
therefore briefly recorded here.
4. Dactylogyrus sp.2
This species was earlier described by Tripathi (unpublished
work) from P. chola from river Gomti, Lucknow, in detail. It is briefly
recorded in the present work.
5. Dactylogyrus sp.3
The present species was earlier described in detail by Tripathi
(unpublished work) from P. ticto from river Gomti, Lucknow. It is
therefore briefly recorded here. It is therefore briefly recorded here.
6. Dactylogyrus sp.4
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This species was earlier described in detail by Tripathi
(unpublished work) from P. ticto from river Gomti, Lucknow. It is
briefly recorded in the present work.
7. Dactylogyrus sp.5
The present species was earlier described in detail
by Tripathi (unpublished work) from P. ticto from river Gomti, Lucknow.
It is therefore briefly recorded here. It is therefore briefly recorded here.
Molecular Phylogenetics
Phylogenetic analysis is largely based on partial 28S
ribosomal DNA (rDNA) sequences. The present study provided the
first insight data on 28S genes of seven species of Dactylogyrus,
occurring on Puntius in India. Sequences of Onchobdella bopeleti
Paperna, 1968 (Accession no. HQ010033) and Onchobdella
aframae Bilong Bilong Euzet, 1995 (Accessesion no. HQ010034)
(Ancyrocephaline monogenoideans), recovered from GENBANK
are used as outgroup for phylogenetic reconstruction. The
phylogenetic trees were computed with Neighbor- Joining (NJ)
method and Minimum Evolution (ME) method. The evolutionary
distances were computed using p-distance method and are in the
units of number of base differences per site. The phylogenies were
tested with 1000 bootstrap replicates. The tree constructed by the
two methods- Neighbor- Joining (NJ) and Minimum Evolution
(ME) have similar topology and approximately similar
bootstrapped values.
Chapter II
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It includes records of four species of the genus Dactylogyroides Gusev,
1976 and their molecular phylogenetic studies.
Dactylogyroides tripathii (Tripathi, 1959) Gusev, 1963
This species was described by Gusev (1963) from a water bodies near
Lucknow. Agrawal et al. (2002) redescribed the species, adding
information on soft parts and some differences in sclerotised
structures. It is briefly recorded in the present work.
2. D. longicirrus (Tripathi, 1959) Gusev, 1976
Gusev (1976) described D. longicirrus from the water bodies near
Lucknow. Agrawal et al. (2002) redescribed the species in detail. It is
therefore briefly recorded here.
3. D. mahecoli Gusev, 1976
Gusev (1976) described D.mahecoli from the water bodies near
Lucknow. Agrawal et al. (2002) redescribed the species in detail. It is
therefore briefly recorded here.
4. D. dorsali Gusev, 1976
Gusev (1976) described D.dorsali from the water bodies near
Lucknow. Agrawal et al. (2002) redescribed the species in detail. It is
therefore briefly recorded here.
Molecular Phylogenetics
This chapter includes the phylogenetic relationships among four
monogenoideans belonging to genus Dactylogyroides found on two
species of freshwater cypriniformes fish Puntius as Puntius sophore and
Puntius chola in the River Gomti, India. These relationships were
investigated using partial 28S ribosomal DNA (rDNA). Phylogenetic
trees were computed with Neighbor- Joining (NJ) method and Minimum
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Evolution (ME) method. The evolutionary distances were computed
using p-distance method and are in the units of number of base
differences per site. Bootstrap values were included to test the reliability
of inferred trees. The phylogenies were tested with 1000 bootstrap
replicates. Ligophorus chabaudi Euzet and Suriano, 1977 (Accession no.
JN996834) and Ligophorus vanbenedenii (Parona & Perugia, 1890)
Johnston & Teigs, 1922 (Accession no. JN996802) (Ancyrocephaline
monogenoideans), recovered from GENBANK are used as outgroup.
Chapter III
It consists of study of three species of the newly proposed genus
Esomocleidus n.g. and their molecular phylogeny.
1. Esomocleidus esomi (Gusev, 1963) n. comb.
The original description of Ancyrocephalus esomi is brief and
lacks whole mount drawing. Gusev (1963) overlooked certain important
morphological features. He stated the intestinal caeca to be confluent in
Ancyrocephalus esomi. However, in the genus Ancyrocephalus, the
intestinal caeca are separate. In specimens, under study, however, the
intestinal caeca are confluent. Gusev (1963) also overlooked vaginal
armature, annulations on body margin, projection in ventral bar, seminal
vesicle, (two in present worm) and gonads, as well as the whole mount
drawings were also not given. The new genus is characterized by body
annulations/serrations, a shield-like or tube-like vagina located parallel or
anterior to the copulatory complex and an elongate antero-medial
projection of the ventral bar. The present species differs from E.
chakrabartii (Gusev, 1963) n. comb. and E. lucknowensis n.sp. by tube-
like vagina, tri-radiate accessory piece, larger eggs and structure and
shape of hooks.
2. Esomocleidus chakrabartii (Gusev, 1976) n.comb.
The original description of Ancyrocephalus chakrabartii includes
diagrammatic figures and misinterpreted structures. Gusev (1976) stated
the intestinal caeca to be confluent in Ancyrocephalus chakrabartii but in
the generic diagnosis of Ancyrocephalus indicates the
intestinal caeca to be separate. In the present specimens, the
intestinal caeca are confluent. He overlooked vaginal armature but we
have observed perforated shield-like vagina in the present specimens. He
also missed the prostatic reservoirs, (three in present specimen), seminal
vesicle, (two in present worm) and gonads. Ancyrocephalus chakrabartii
is transferred under the new genus Esomocleidus as E. chakrabarti
n.comb. The present species significantly differs from E. esomi (Gusev,
1963) n. comb. and E. lucknowensis n.sp. by having perforated shield-like
vaginal apparatus, tetra-radiate accessory piece and sickle-shaped
copulatory tube.
3. Esomocleidus lucknowensis n.sp.
This species differs from E. esomi (Gusev, 1963) n. comb. and E.
chakrabarti (Gusev, 1963) n. comb. by having as projection in ventral
bar, shape of dorsal bar, muscular vaginal apparatus and shape and size of
hooks.
Molecular Phylogenetics
The phylogenetic analysis is based on partial 28S ribosomal DNA
(rDNA) sequences. Sequences of Dactylogyrus sp.1, recovered from
GENBANK is used as outgroup for phylogenetic reconstruction. The
phylogenetic trees were computed with Neighbor- Joining method and
Minimum Evolution method. The evolutionary distances were computed
using p-distance method and are in the units of the number of base
differences per site. The phylogenies were tested with 1000 bootstrap
replicates.
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Chapter IV
This chapter deals with the phylogenic relationships
among 14 species of monogenoidea (7 species
belonging to the genus Dactylogyrus, 4 species of Dactylogyroides
and 3 species of genus Esomocleidus). These relationships were
investigated via use of partial 28S ribosomal DNA (rDNA)
sequences. Resolving the interrelationship of these genera is the
main aim of present study. Phylogenetic trees were computed with
Neighbor- Joining (NJ) method and Minimum Evolution (ME)
method. The evolutionary distances were computed using p-
distance method and are in the units of the number of base
differences per site. Bootstrap values were included to test the
reliability of inferred trees. The phylogenies were tested with 1000
bootstrap replicates. Sequences of Tetraonchus monenteron
Diesing, 1858 (Accession no. AJ969953) and Anoplodiscus
cirrusspiralis Roubal, Armitage & Rohde, 1983 (Accession no.
AF382060) recoverd from GENBANK (placed in an outgroup) are
used for phylogenetic reconstruction.
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