genetic characterization of dactylogyroides longicirrus

Sci Parasitol 11(3):119-127, September 2010 ISSN 1582-1366 ORIGINAL RESEARCH ARTICLE

119

Genetic characterization of Dactylogyroides longicirrus (Tripathi,

1959) Gussev, 1976 by nuclear 28S segment of ribosomal DNA with a

morphological redescription

Hridaya S. Singh�, Anshu Chaudhary

Department of Zoology, Chaudhary Charan Singh University, Meerut (U.P.), 250004, India.

Correspondence: Tel. 09412204430, Fax 91-121-2760577, E-mail [email protected]

Abstract. Present communication deals with the redescription of Dactylogyroides longicirrus (Tripathi, 1959)

Gussev, 1976 from Puntius sophore at Hastinapur, Meerut U.P., India. The redescription is based on the fresh

materials collected by the author. Besides describing detailed morphological features, nuclear ribosomal DNA

(rDNA) has also been used to establish the phylogenetic position and genetic diversity of the parasite. Phylogenetic

analysis using the neighbor joining (NJ) and maximum parsimony (MP) methods was performed. This study

suggests that morphological characters along with molecular identification are essential for validating and

identifying species. This is the first published sequence of a species from the genus Dactylogyroides Gussev, 1963.

Keywords: Monogenea, Dactylogyroides, 28S rDNA gene, Puntius sophore, Meerut, India.

Received 13/06/2010. Accepted 29/08/2010.

Introduction

Genus Dactylogyroides was proposed by

Gussev (1963) for the worms previously

described under the genus Dactylogyrus

Diesing, 1850 by Tripathi viz., Dactylogyrus

tripathii (Tripathi, 1959; Yamaguti, 1961)

Gussev, 1963 from Puntius ticto and P. stigma

at Lucknow, India. These are parasites of

freshwater cyprinids. Gussev (1963)

differentiated the genus from Dactylogyrus by

having anchors with their points directed

towards each other, dorsal bar usually double

or single with different degree of separation

into two parts. Dactylogyrus longicirrus was

described from P. ticto and P. stigma by

Tripathi (1959) from local fish ponds of

Lucknow, India. Gussev (1976) transferred

Dactylogyrus longicirrus into the genus

Dactylogyroides based on the fore mentioned

morphological features.

During a general survey of freshwater fishes of

Western U.P. for parasitic monogeneans, we

came across specimens of Puntius sophore at

Hastinapur, Meerut, India, infected with

monogeneans belonging to the genus

Dactylogyroides Gussev, 1963. After detailed

study we found that the original description

suffers from certain lacunae and it was

described on the basis of only morphology of

hard parts of the body. In view of the above, we

re-described Dactylogyroides longicirrus

(Tripathi, 1959) Gussev, 1976. The aim of the

present study was to establish, using the

genetic marker, the molecular identification


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features of the genus Dactylogyroides and the

differentiation of D. longicirrus from other

congeneric species.

Materials and methods

Parasites were collected carefully from the gills

of Puntius sophore (Hamilton, 1822), captured

in Hastinapur, U.P., India. Morphological

studies were made as suggested by Malmberg

(1970). Parasites were identified to species

morphologically, according to existing keys and

descriptions.

For genomic DNA extraction, an individual

specimen was fixed in 100% ethanol. DNA was

extracted from one parasite using the Qiagen

DNeasy Tissue Kit according to the

manufacturer’s instructions. A region

comprising partial 28S rDNA was amplified

using the universal primers, forward (5’-

ACCCGCTGAATTTAAGCAT-3’) and the reverse

primer (5’-CTCTTCAGAGTACTTTTCAAC-3’).

Each amplification reaction was performed in a

final volume of 25 µl containing 3 µl of genomic

DNA extract, 10X PCR buffer, 0.4 mM dNTP, 10

pM of each primer pair, 1 U Taq polymerase

(Biotools) and Milli-Q water in a thermocycler

(Eppendorf Mastercycler Personal) under the

following conditions: 3 minutes at 94°C (initial

denaturation), followed by 35 cycles of 30

seconds at 94°C (denaturation), 45 seconds at

56°C (annealing) and 1 minute at 72°C

(extension) and a final extension at 72°C for 10

minutes. PCR products were examined on 1.5%

agarose-TBE gels, stained with ethidium

bromide and visualized under ultraviolet light.

Cycle sequencing of 28S amplicon was

performed on products purified from gels by

Chromous PCR clean up Kit (#PCR 10)

according to manufacturer’s instructions. Both

DNA strands were sequenced using a Big Dye

Terminator version 3.1 cycle sequencing kit in

an ABI 3130 Genetic Analyzer. The primers,

forward primer (5’-ACCCGCTGAATTTAAGCAT-

3’) and the reverse primer (5’-

CTCTTCAGAGTACTTTTCAAC-3’) were used for

sequencing.

Sequence similarity searching for D. longicirrus

was performed using the NCBI BLAST online

application. Analyses of multiple sequence

alignments were done using the Clustal W

software (Thompson et al., 1994). DNA

sequences of closely related species were also

downloaded and used in the phylogenetic

analysis. The phylogenetic trees were

reconstructed using MEGA version 4.0

(Tamura et al., 2007). Phylogenetic analyses

were performed based on neighbor-joining

(NJ) approach and maximum parsimony (MP)

methods. In constructing the NJ tree, the

Kimura-2-parmeter model (Kimura, 1980) was

used to estimate the distances. The robustness

of the inferred phylogeny was assessed using a

bootstrap procedure with 1,000 replications.

The long subunit of rDNA gene sequence of D.

longicirrus generated in this study was 301 bp

and deposited in GenBank under the accession

no. GU903482.

Results

Dactylogyroides longicirrus (Tripathi, 1959)

Gussev, 1976.

Type host – Puntius sophore (Hamilton, 1822).

Type locality – Hastinapur (29001’N and

77045’E), Meerut, U.P., India.

Additional localities – Tungabhadra Dam, River

Gomti, Lucknow, U.P., India.

Site of infection – Gills.

Type material – The holotype and paratype

slides have been deposited in the museum of

Department of Zoology (Voucher numbers.

HS/Monogenea/2009/01), Ch. C.S. University,

Meerut, U.P., India.

Redescription: The worm body is moderate

sized, elongated, 0.56-0.60 mm in length and

0.10-0.12 mm in width (figure 1). Two pair of

eye spots are present. Eyespots are very well

developed, located slightly anterior to pharynx.

The posterior pair of eyespot is considerably

larger than the anterior. Pharynx is spherical,

has muscular structure, measuring 0.003-0.005

mm. Intestinal caeca bifurcated and crura

confluent posterior to testis.


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Figure 1. Dactylogyroides longicirrus (Tripathi, 1959) Gussev, 1976:

1 – whole mount; 2 – egg; 3 – male copulatory complex; 4 – haptor;

5 – dorsal anchor; 6 – dorsal bar; 7 – ventral bar; 8 – hooks


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Ovary is round to oval, in post-equatorial

position, intercaecal, median and measures

0.030-0.032 mm in length and 0.032-0.034 mm

in width. Eggs oval in shape with a stumpy spur,

0.040-0.042 mm in length and 0.030-0.032 mm

in width (figure 1.2). The testis is single,

elongated, intercaecal, with post-equatorial

position, dorsal to ovary, 0.060-0.062 mm in

length and 0.020-0.024 mm in width. Copulatory

complex is composed of a copulatory tube and an

accessory piece. Copulatory tube, 0.022-0.026

mm in length, is comma shaped with a swollen

base tapering towards its ends, The sclerotised

accessory piece measures 0.020-0.024 mm, is

articulated at base of the copulatory tube, initially

thin, medially broad, leaf like, and terminally

claw like with an aperture through which

copulatory tube glides (figure 1.3).

The opisthohaptor is distinctly set off from the

body, and measures 0.10-0.12 mm in length and

0.13-0.15 mm in width (figure 1.4). The

armature of the haptor consists of large dorsal

anchors with inner length 0.034-0.036 mm,

outer length 0.033-0.035 mm, inner root 0.011-

0.013 mm, outer root 0.008-0.010 mm and

recurved point 0.010-0.012 mm (figure 1.5).

Dorsal connecting bar ‘V’ shaped, divided into

two parts, each 0.03 mm (figure 1.6). Ventral

bar wide ‘U’ shaped, measuring 0.040-0.042 mm

(figure 1.7). The marginal hooklets are 7 pairs,

with a dilated sickle and small thin handle. The

handle is divided into a fairly long and a thin

pivot; the distal end is swollen and with the

shape of a small bulb, known as the heel of the

handle. The sickle filament loop is short and

extends to the middle part of pivot of handle.

Hooks pairs 1, 2, 3 and 5 are 0.010-0.012 mm,

pair 4 is 0.013-0.015 and pairs 6 and 7 measure

0.06-0.08 mm (figure 1.8).

Phylogenetic analysis

28S rDNA sequence was aligned using the Clustal

W software to perform the phylogenetic analysis.

The reference sequences used in this study are

listed in table 1. Nucleotide sequence data from

the 28S rDNA region of D. longicirrus generated

in this study produced an alignment of 301

nucleotide sites. Pairwise comparisons were

made by Kimura-2 parameter model in the 28S

sequences between different species are shown

in table 2. The phylogenetic reconstructions

inferred from analyses of the 28S rDNA

sequences showed great resolution for the

species of the monogeneans. This species shows

nucleotide identity of 90-91% with the different

species of genus Dactylogyrus from which it was

differentiated by Gussev in 1963. The

Dactylogyrus species appeared to be the most

closely related and is much more divergent with

a well supported clade by neighbor joining (NJ)

and maximum parsimony (MP) with a high

degree of confidence (figures 2 and 3) with

MEGA 4.0 and the species of the genus

Dactylogyroides shows a different clade.

Bootstrap values, indicating the robustness of the

internal nodes, were set at 1000 replications.

Both the methods gave trees with similar

topology and approximate relatively

bootstrapped values. These sequences were

aligned with the 28S rDNA genes and revealed

clear differences in nucleotide sequences among

different species (figure 4). Therefore, we

consider the congruence of the both methods as

an indication of the strength of present analysis.

Discussion

Gussev (1973) recorded four related

monogeneans from different piscine hosts

Puntius stigma, Barbus mahecola and B. dorsalis.

The original description of these specimens was

far from complete and the author (Gussev, 1973)

was not sure about the taxonomic status of these

worms namely Dactylogyroides tripathii f.

dorsalis, D. tripathii f. filamentosi, D. tripathii f.

mahecoli and D. tripathii f. stigmi. Singh and Jain

(1988) reported Dactylogyroides wallagonius

from Wallago attu at Meerut. The description of

this later species also suffers from serious lapses

besides its presence on a silurid fish, W. attu as

also pointed out by Dubey et al. (1997). Dubey et

al. (1997) redescribed Dactylogyroides tripathii

(Tripathi, 1959) Gussev, 1973 and D. longicirrus

(Tripathi, 1959) Gussev, 1973 from Puntius

sophore at Raipur. Agrawal et al. (2002) made a

comprehensive review of Indian species of

Dactylogyroides Gussev, 1963 and described a

new species D. osteobramii from Osteobrama

cotio at Lucknow. This description of Agrawal et

al. (2002) also suffers from several incongruities

and also they have not consulted papers

published from Indian subcontinent by Singh and

Jain (1988) and Dubey et al. (1997). As far as

genus Dactylogyroides is concerned, various


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species of this genus reported from Indian,

Chinese and African region are listed in table 3.

The evaluation of generic-level monophyly and

morphological criteria for genus erection seems

to be the most controversial area in phylogenetic

and taxonomic studies of the Monogenea

(Desdevises, 2001; Wu et al., 2005; 2006; 2007;

Pouyaud et al., 2006). Morphological characters

for the differentiation of closely related genera or

species group are usually selected based on

practical purposes, taking no account of the

evolutionary values of the characters. It is really

difficult to establish a criterion for genus erection

for the diversified monogeneans. Analyses of 28S

region in this study revealed that this gene is a

very good molecular marker for inferring

relationship between closely related species.

Table 1. GenBank reference sequences used in this study, their geographical origins as well as accession numbers

Species Origin Genbank accession no.

Dactylogyroides longicirrus

Dactylogyrus parabramis

Dactylogyrus petruschewskyi

Dactylogyrus nanus

Dactylogyrus pekinensis

Dactylogyrus hemiamphibothrium

Dactylogyrus sp.YY

Dactylogyrus sp.LY1

Dactylogyrus hypophalmichthys

Dactylogyrus cryptomeres

Dactylogyrus sphyrna

Dactylogyrus inexpectatus

Dactylogyrus quanfami

Dactylogyrus extensus*

Dactylogyrus extensus

India

China

China

Czech Republic

China

Czech Republic

China

China

China

Czech Republic

Czech Republic

Czech Republic

China

Czech Republic

China

GU903482*

EF100534

AY548927

AJ969942

EF100535

AJ969946

EF100538

EF100537

EF100532

AJ969947

AJ969943

AJ969945

EF100536

AJ969944

AY553629 *Species sequenced during present study

Table 2. Pairwise genetic distances (estimated using the Kimura 2- parameter model; in % differences)

for the long subunit rDNA sequence of the Dactylogyroides sp. and Dactylogyrus sp.

Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

D. longicirrus

D. parabramis

D. petruschewskyi

D. nanus

D. pekinensis

D. hemiamphibothrium

D. sp.YY

D. sp.LY

D. hypophalmichthys

D. cryptomeres

D. sphyrna

D. inexpectatus

D. quanfami

D. extensus*

D. extensus

0.1187

0.1268

0.1266

0.1348

0.1270

0.1350

0.1429

0.1429

0.1354

0.1394

0.1437

0.1359

0.1476

0.1476

0.0208

0.0385

0.0279

0.0459

0.0315

0.0314

0.0314

0.0537

0.0496

0.0720

0.0878

0.0992

0.0992

0.0243

0.0069

0.0386

0.0103

0.0314

0.0314

0.0389

0.0350

0.0757

0.0720

0.0911

0.0911

0.0314

0.0458

0.0314

0.0421

0.0421

0.0423

0.0244

0.0716

0.0793

0.0986

0.0986

0.0458

0.0173

0.0385

0.0385

0.0460

0.0422

0.0832

0.0795

0.0987

0.0987

0.0458

0.0642

0.0642

0.0497

0.0459

0.0718

0.0796

0.0793

0.0793

0.0351

0.0351

0.0499

0.0422

0.0833

0.0796

0.0989

0.0989

0.0000

0.0646

0.0532

0.0869

0.1029

0.1187

0.1187

0.0646

0.0532

0.0869

0.1029

0.1187

0.1187

0.0498

0.0642

0.0757

0.0870

0.0870

0.0832

0.0873

0.0987

0.0987

0.0720

0.0607

0.0607

0.0756

0.0756

0.0000

In the present study, D. longicirrus shows the

close similarity with the genus Dactylogyrus

from which it was distinguished. The tree

topologies derived from the phylogenetic

analysis inferred from 28S rDNA data depicted

that Dactylogyroides and Dactylogyrus as

genetically closely related sister taxa. They are

also in agreement in showing that they are

formed different clade. Therefore, based on our

molecular results and morphological

examinations we propose that the species D.

longicirrus was correctly accommodating in the

genus Dactylogyroides. These studies further

indicate that molecular markers such as those

provided by rDNA are useful markers

(barcodes) for distinguishing sister genera or


124

species. DNA barcodes may be helpful in

discriminating species especially when

morphological differences are often difficult to

determine. We believe that such taxonomic

revisions based on molecular biology will

continue with the increasing number of species

being used for morphological and molecular

phylogenetic investigations in the future. To

the best of our knowledge, there have been no

such previous studies of this species based on

both morphological and DNA evidence. In the

absence of molecular data for many species of

Dactylogyroides, a thorough analysis of the

taxonomic affinities of group must await a

more thorough molecular coverage.

Figure 2. A phylogenetic tree constructed by neighbor-joining method (1,000 bootstraps) for 28S region of different species.

Bootstrap values (as percentages) are shown at internal nodes. The scale bar indicates the proportion of sites changing along

each branch. The asterisk is used due to same species name.

Figure 3. Phylogenetic relationship of the D. longicirrus inferred from 28S region using the Maximum parismony (MP)

method (1,000 bootstraps). The scale bar indicates the proportion of sites changing along each branch.

The asterisk is used due to same species name

Dactylogyrus Dactylogyrus Dactylogyrus Dactylogyrus sp. LY1sp. LY1sp. LY1sp. LY1

Dactylogyrus hypophalmichthysDactylogyrus hypophalmichthysDactylogyrus hypophalmichthysDactylogyrus hypophalmichthys

Dactylogyrus parabramis Dactylogyrus parabramis Dactylogyrus parabramis Dactylogyrus parabramis

Dactylogyrus Dactylogyrus Dactylogyrus Dactylogyrus sp. YYsp. YYsp. YYsp. YY

Dactylogyrus petruschewskyiDactylogyrus petruschewskyiDactylogyrus petruschewskyiDactylogyrus petruschewskyi

Dactylogyrus pekinensisDactylogyrus pekinensisDactylogyrus pekinensisDactylogyrus pekinensis

DactylogyrDactylogyrDactylogyrDactylogyrus nanusus nanusus nanusus nanus

Dactylogyrus sphyrnaDactylogyrus sphyrnaDactylogyrus sphyrnaDactylogyrus sphyrna

Dactylogyrus cryptomeres Dactylogyrus cryptomeres Dactylogyrus cryptomeres Dactylogyrus cryptomeres

Dactylogyrus hemiamphibothrium Dactylogyrus hemiamphibothrium Dactylogyrus hemiamphibothrium Dactylogyrus hemiamphibothrium

Dactylogyrus quanfamiDactylogyrus quanfamiDactylogyrus quanfamiDactylogyrus quanfami

Dactylogyrus inexpectatus Dactylogyrus inexpectatus Dactylogyrus inexpectatus Dactylogyrus inexpectatus

Dactylogyrus extensusDactylogyrus extensusDactylogyrus extensusDactylogyrus extensus**** Dactylogyrus extensusDactylogyrus extensusDactylogyrus extensusDactylogyrus extensus

Dactylogyroides longicirrusDactylogyroides longicirrusDactylogyroides longicirrusDactylogyroides longicirrus

99

69

56

52

61

67

63

99

5

Dactylogyrus Dactylogyrus Dactylogyrus Dactylogyrus sp. LY1sp. LY1sp. LY1sp. LY1

Dactylogyrus hypophalmichthysDactylogyrus hypophalmichthysDactylogyrus hypophalmichthysDactylogyrus hypophalmichthys

Dactylogyrus parabramisDactylogyrus parabramisDactylogyrus parabramisDactylogyrus parabramis

Dactylogyrus Dactylogyrus Dactylogyrus Dactylogyrus sp.sp.sp.sp. YY YY YY YY

Dactylogyrus petruschewskyiDactylogyrus petruschewskyiDactylogyrus petruschewskyiDactylogyrus petruschewskyi

Dactylogyrus pekinensisDactylogyrus pekinensisDactylogyrus pekinensisDactylogyrus pekinensis

Dactylogyrus nanusDactylogyrus nanusDactylogyrus nanusDactylogyrus nanus

Dactylogyrus sphyrnaDactylogyrus sphyrnaDactylogyrus sphyrnaDactylogyrus sphyrna

Dactylogyrus cryptomeres Dactylogyrus cryptomeres Dactylogyrus cryptomeres Dactylogyrus cryptomeres

Dactylogyrus hemiamphibothriumDactylogyrus hemiamphibothriumDactylogyrus hemiamphibothriumDactylogyrus hemiamphibothrium

Dactylogyrus quanfami Dactylogyrus quanfami Dactylogyrus quanfami Dactylogyrus quanfami

Dactylogyrus inexpectatusDactylogyrus inexpectatusDactylogyrus inexpectatusDactylogyrus inexpectatus

Dactylogyrus extensus*Dactylogyrus extensus*Dactylogyrus extensus*Dactylogyrus extensus*

Dactylogyrus extensusDactylogyrus extensusDactylogyrus extensusDactylogyrus extensus

Dactylogyroides longicirrusDactylogyroides longicirrusDactylogyroides longicirrusDactylogyroides longicirrus

99

67

61

63

71

75 68

100

0.01


125

D.longicirrus -------CTG TATATCTCCC TGTTTTTGTT TTTAGGACAA TGTGGTGTTC [50] D.parabramis CCCATCA.C. -.AG...AT. CACG.....G GAC....... .........T [50] D.petruschewskyi CCCATCA.C. -.AG...ATT CACG.....G GAC....... .......... [50] D.nanus CCCATCA.C. -.AG...ATT CAC......G GAC....... .......... [50] D.pekinensis CCCATCA.C. -.AG...ATT CACG.....G GAC....... .......... [50] D.hemiamphibothrium CCCATCA.C. -.AG...ATT CAC.G....G GAC....... .......... [50] D.sp.YY CCCATCA.C. -.AG...ATT CACG.....G GAC....... .......... [50] D.sp.LY1 CCCATCA.C. -.AG...ATT CACG.....G AAC....... .........T [50] D.hypophalmichthys CCCATCA.C. -.AG...ATT CACG.....G AAC....... .........T [50] D.cryptomeres CCCATCA.C. -.AG...ATT CAC......G GAC....... .......... [50] D.sphyrna CCCATCA.C. -.AG...ATT CAC......G GAC....... ........CT [50] D.inexpectatus CCCATCA.C. -.AG...ATT CACG.....G GAC....... .........A [50] D.quanfami CCCATCA.C. -.AG...ATT CACG.....G GAC.A..... .......... [50] D.extensus* CCCATCA.C. -.AG...ATT CACAC....G GAC....... .......... [50] D.extensus CCCATCA.C. -.AG...ATT CACAC....G GAC....... .......... [50]

D.longicirrus AGGTGTGTAC CTGGGAGCGC TTGCCTACCC GAAGTCCAAT TCCGATTCTG [100] D.parabramis .......... .......... ........T. .........C .......A.. [100] D.petruschewskyi .......... .......T.. ........T. .........C .......A.. [100] D.nanus ....T..... .......T.. ........T. .........C .......A.. [100] D.pekinensis ........T. .......T.. ........T. .........C .......A.. [100] D.hemiamphibothrium .......... .......... ........T. .........C .......A.. [100] D.sp.YY .......... .......T.. ........T. .........C .......A.. [100] D.sp.LY1 ....T..... .......... .G......T. .........C .......A.. [100] D.hypophalmichthys ....T..... .......... .G......T. .........C .......A.. [100] D.cryptomeres G......C.. .......... ........T. .........C .......A.. [100] D.sphyrna .A..T..... .......T.. ........T. .........C .......A.. [100] D.inexpectatus G..CA..C.. .......... ........T. ........TC ..T....A.. [100] D.quanfami G..C...C.. ......AT.. .......... ........GC .......A.. [100] D.extensus* .A.C...C.. ......A..A .......... ........TC .......A.. [100] D.extensus .A.C...C.. ......A..A .......... ........TC .......A.. [100]

D.longicirrus GCTTGGATAC TACCCACAGC GGGTGAAAGG CCCGTACGGG TAGGCC-CCT [150] D.parabramis ...A...CCA .G....G... .......... .....T.... ....TT-GT. [150] D.petruschewskyi ...A...CCA .G....G... .......... .....T.... ....TT-GT. [150] D.nanus .G....G... .......... .....T.... ....TT-TTG .-.GTTTTG. [150] D.pekinensis .G....G... .......... .....T.... ....TT-GT. ..CAATTTG. [150] D.hemiamphibothrium .G....G... .......... .....T.... .....T-TA. ..C-ATTT.. [150] D.sp.YY .G....G... .........T .....T.... ....TT-AT. ..CAATTTG. [150] D.sp.LY1 .G....G... .........T .....T.... ....TT-GA. ..CAAT.TGG [150] D.hypophalmichthys .G....G... .........T .....T.... ....TT-GA. ..CAAT.TGG [150] D.cryptomeres .G....G... .......... .....T.... ....TT-G-C ....ATTTG. [150] D.sphyrna .G....G... .......... .....T.... ....TT-TTG .-.GTTTTG. [150] D.inexpectatus .G....G... .......... .....G.... .....T-TG. GA..----.. [150] D.quanfami .G........ .......... .......... ....ATGTG. ...--TTT.T [150] D.extensus* .G....G... .......... .....G.... .....T-TG. G.A.ATT... [150] D.extensus .G....G... .......... .....G.... .....T-TG. G.A.ATT... [150]

D.longicirrus TGTTGCAATA AGCGTTCCTC AGATGTGCAT ACCGTCGAGT CGGATTGCTT [200] D.parabramis ..CAATG... ....C.T... ......A... ...T...... .......... [200] D.petruschewskyi ..CAATTTG. ....C..... ......A... ..TT...... .......... [200] D.nanus ...A...CCA ....C..... ......A... T..T...... .......... [200] D.pekinensis ...A...CCA ....C..... .....AA... ..TT...... .......... [200] D.hemiamphibothrium ...A.T.CCA ....C..... .......... G.TT...... .......... [200] D.sp.YY ...A...CCA ....C..... ......A... ..TT...... .......... [200] D.sp.LY1 ...A...CCA ....C..... ......A.T. ...T...... .......... [200] D.hypophalmichthys ...A...CCA ....C..... ......A.T. ...T...... .......... [200] D.cryptomeres ...A...CCA ....C....T .......T.. G.TC...... .......... [200] D.sphyrna ...A...CCA ....C..... .......... ..TT...... .......... [200] D.inexpectatus .......CTA ....C..... ..C....T.. G..T...... .......... [200] D.quanfami .......CCA G..A...... .......... G.TT...... .......... [200] D.extensus* ....C..CTG .T........ .......T.. G.TT...... .......... [200] D.extensus ....C..CTG .T........ .......T.. G.TT...... .......... [200]

Figure 4. Alignment of 28S sequences for comparative purposes of different species from different geographical locations

showed nucleotide identical to D. longicirrus. Dots indicate identity with the first sequence and dashes are inferred insertion-

deletion events. The asterisk is used due to same species name


126

D.longicirrus GAGAATGCAG TCCAAAGTGG GTGG-TAAAC TCCATCCAAG GCTAAATACT [250] D.parabramis .......... .......... ....-..... .......... .......... [250] D.petruschewskyi .......... .......... ....-..... .......... .......... [250] D.nanus .......... .......... ....-..... .......... .......... [250] D.pekinensis .......... .......... ....-..... .......... .......... [250] D.hemiamphibothrium .......... .......C.. ....G..... .......... .......... [250] D.sp.YY .......... .......... ....-..... .......... .......... [250] D.sp.LY1 .......... .......... ....-..... .......... .......... [250] D.hypophalmichthys .......... .......... ....-..... .......... .......... [250] D.cryptomeres .......... .......... ....-..... .......... .......... [250] D.sphyrna .......... .......C.. ....-..... .......... .......... [250] D.inexpectatus .......... .......... ....-..... .......... .......... [250] D.quanfami .......... .......... ....-..... .......... .......... [250] D.extensus* .......... .......... ....-..... .......... .......... [250] D.extensus .......... .......... ....-..... .......... .......... [250]

D.longicirrus GGCACGAGTC CGATAGTAGA CAAGTACCGT GAGGGAAAGT TGAAAAGTAC [300] D.parabramis .......... .......... .......... .......... .......... [300] D.petruschewskyi .......... .......... .......... .......... .......... [300] D.nanus .......... .......... .......... .......... .......... [300] D.pekinensis .......... .......... .......... .......... .......... [300] D.hemiamphibothrium ...G...... .......... .......... .......... .......... [300] D.sp.YY ....T..... .......... .......... .......... .......... [300] D.sp.LY1 .......... .......... .......... .......... .......... [300] D.hypophalmichthys .......... .......... .......... .......... .......... [300] D.cryptomeres .......... .......... .......... .......... .......... [300] D.sphyrna .......... .......... .......... .......... .......... [300] D.inexpectatus .......... .......... .......... .......... .......... [300] D.quanfami .......... .......... .......... .......... .......... [300] D.extensus* .......... .......... .......... .......... .......... [300] D.extensus .......... .......... .......... .......... .......... [300]

D.longicirrus TCTGAAGAGA [310] D.parabramis .......... [310] D.petruschewskyi .......... [310] D.nanus .......... [310] D.pekinensis .......... [310] D.hemiamphibothrium .......... [310] D.sp.YY .......... [310] D.sp.LY1 .......... [310] D.hypophalmichthys .......... [310] D.cryptomeres .......... [310] D.sphyrna .......... [310] D.inexpectatus .......... [310] D.quanfami .......... [310] D.extensus* .......... [310] D.extensus .......... [310]

Figure 4. (cont.)

Table 3. List of various species of the genus Dactylogyroides Gussev, 1963

Species Host Region D. bimaculati Gussev, 1963

D. dorsalis (Gussev, 1963) Lim and Furtado, 1984

D. fernandoi (Gussev, 1963) Lim and Furtado, 1984

D. gussevi Tripathi, 1975 [macracanthus (Tripathi, 1959) Gussev, 1963 renamed]

D. longicirrus (Tripathi, 1959) Gussev, 1976

D. tripathii (Tripathi, 1959, Yamaguti, 1963) Gussev, 1963

D. tripathii f. dorsalis of Gussev 1976

D. tripathii f. filamentosi of Gussev 1976

D. tripathii f. mahecoli of Gussev 1976

D. tripathii f. stigmi of Gussev 1976

D. vittati Gussev, 1963

D. wallagonius Singh and Jain, 1988

D. osteobramii Agrawal et al., 2002

D. gussevia Singh et al., 2002

D. biradius Birgi and Lambert, 1987

D. malayensis Lim and Furtado, 1984

D. tubiformis Lang, 1981

Puntius (Barbus)

Puntius (Barbus)

Puntius (Barbus)

Puntius (Barbus)

Puntius (Barbus)

Puntius (Barbus)

Barbus

Puntius (Barbus)

Barbus

Puntius (Barbus)

Puntius (Barbus)

Wallago attu

Osteobrama cotio

Tor putitora

Barbus

Osteochilus

Osteochilus

Indian

Indian

Indian

Indian

Indian

Indian

Indian

Indian

Indian

Indian

Indian

Indian

Indian

Indian

African

Chinese

Chinese


127

Acknowledgments

The authors are thankful to Department of

Zoology, C.C.S. University, Meerut, for

providing laboratory facilities. This work was

funded by the Department of Science and

Technology (DST), through grant

(SR/SO/A543/2005) awarded to HSS.

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genetic characterization of dactylogyroides longicirrus

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