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Mitochondrial DNA, Early Online: 1–5

! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.987269

SHORT COMMUNICATION

Linking eggs and adults of Argulus spp. using mitochondrial DNA

barcodes
K. Feroz Khan, G. Sanker, and C. Prasanna Kumar

Faculty of Marine Sciences, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai, Tamil Nadu, India

Abstract Keywords
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We have created barcode library for common Argulus spp. infecting Carassius auratus, which Argulus spp., COI gene, DNA barcode,
could also be used to identify premature forms of Argulus spp. even by non-professionals. ornamental fishes, parasite eggs
Infected C. auratus was examined and purchased from ornamental fish-trading centers and the
adult life stage of Argulus spp. was identified and DNA barcoded. The eggs of Argulus spp. were History
collected using bottle implants. The collected eggs are barcoded and precisely identified by
matching with the adult sequences. Four species of adult Argulus spp. were identified, namely Received 20 April 2014
Argulus japonicus, Argulus indicus, Argulus siamensis, and Argulus foliaceus. Precise identification Revised 9 November 2014
of egg samples was done by two different analyses, namely (i) BLAST analysis and (ii) Accepted 10 November 2014
phylogenetic clustering of adults and eggs. All egg samples including the control were Published online 10 December 2014
precisely identified by BLAST analysis and the results are consistent with phylogenetic
clustering of adult and egg’s DNA barcodes. In order to establish the DNA barcode technology
for the identification of all Argulus spp and its premature forms, the development of full-
For personal use only.

fledged barcode library that includes all species of this genus is very important for the benefit
of ornamental fish industries.

Introduction The low host specificity of Argulus spp. makes them a more
potent ecto-parasite. There are many methods of control, preven-
Members of the family Argulidae are highly modified crustaceans
tion, and treatment of argulosis (Gault et al., 2002; Hakalahti
which have become specialized as external parasites of fishes.
et al., 2004; Kabata, 1985; Poulin & FitzGeral 1987; Puffer &
The varied morphological adaptations by argulids for a parasitic
Beal 1981; Singhal et al., 1986). Earlier detection could lessen the
existence are of considerable taxonomic significance. The
economic loss. Usually Argulus spp. in water is unnoticed by
argulids mostly parasitize the fresh water fish but few have
the consignment dispatchers. Furthermore, inspection of fishes
been reported from marine fish (Cressey, 1978; Devraj & Ameer
for transport to other places does not normally include conscien-
Hamza, 1978). Earlier, 10 species of fish lice have been reported
tious efforts to locate fish lice. The adult fish lice may be
from India (Natrajan, 1982), while only two species, Argulus
obvious on fish being inspected, but intermediate stages may be
indicus and Argulus japonicus have been reported from Pakistan
passed over. Even, the basic health management practices
(Jafri & Ahmed, submitted for publication). Recently,
might be easily over looked due to dearth of trained personal,
A. japonicus has been reported (Kazmi, 2003) from a gold fish
which may cause the shifting of pathogens along with their hosts
in an aquarium. Most branchiurans occur in fresh water, but a few
(Iqbal et al., 2013).
species of the genus Argulus are ectoparasites on the skin of
DNA barcoding is technique which uses the organism’s
marine fish. The damage and stress caused by Argulus in cultured
mitochondrial DNA to identify it as particular species.
fishery is the beginning of the infection and hence the economic
Cytochrome C oxidase subunit I gene sequence was proposed as
loss to aquaculture farmers. Usually argulosis is associated
a potent barcode for species of animal kingdom. DNA barcoding
with secondary bacterial infestations. Several studies have
works in two simple steps. First, DNA barcode of known species
examined the role of parasites as vectors for other diseases such
is identified, sequenced, and deposited in barcode library. A bit of
as Aeromoniasis or Pseudomoniasis (Bauer, 1991; Richards,
tissue or adult specimens or immature forms can be sequenced
1977) associated with argulosis.
and compared with the library for the species identification. In the
India looses US$ 1428 per hectare per year in carp culture due
present study, we have created barcode library for common
to argulosis (Sahoo et al., 2013). Argulus inducus, A. japonicus,
Argulus spp. infecting Carassius auratus, which could also be
and A. siamenses have been reported to cause mortality in major
used to identify premature forms of Argulus spp. even by non-
and Chinese carps (Anev, 2006; Bauer et al., 1973; Jaffri &
professionals.
Ahmad, 1994; Nandp & Das, 1991). Argulus foliaceus and
Infected C. auratus was examined and purchased from
Argulus bengalensis have been reported to infect 11 freshwater
ornamental fish trading centers and the adult life stage of
fishes in Bangladesh (Chandra, 2004; Faruk et al., 2004).
Argulus spp. was identified and DNA barcoded. The eggs of
Argulus spp. were collected using bottle implants. The collected
Correspondence: C. Prasanna Kumar, Centre of Advanced Study in eggs are barcoded and precisely identified by matching with the
Marine Biology, Faculty of Marine Sciences, Annamalai University, adult sequences. The study proved the efficacy of DNA barcodes
Parangipettai 608502, Tamil Nadu, India. E-mail: micropras@gmail.com in precise identification of eggs and adults of Argulus spp.
 2 K. F. Khan et al.
Materials and methods
Sample collection, identification, and storage
The study was carried out between the months of May 2012 and
August 2013. Twenty ornamental fish trading centers were visited
and 150 live specimens of C. auratus were examined for Argulus
spp. infections. Fifty infected C. auratus from various trading
centers were selected and acclimatized in aquarium tanks in
laboratory condition. Two to three Argulus spp. were removed
manually from the fishes. Adult Arguls spp. were identified to
species level using the key morphological characters described
elsewhere (Alas et al., 2010; Hoffman, 1977; Wadeh et al., 2008).
The identified specimens are stored in 95% ethanol until
molecular analysis. A cube of lateral tissue of C. auratus was
exercised and preserved in 95% ethanol from molecular taxo-
nomic analysis.
Experimental setup for Argulus egg collection
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We employed bottle implant technique to harvest the egg loads of
Argulus spp. Dark brown bottles (500 ml) are used as a substratum
for the egg mass attachments. Four to six brown bottles
(depending on the size of the tank) are implanted in an inverted
position inside the aquarium tanks where the Argulus spp.
infected with C. auratus are maintained. The bottles are taken out
once in every 24 h interval and examined for eggs on the surface.
The egg capsules are stored in 95% ethanol until further analysis.
To enable effective DNA barcode comparison analysis, C. auratus
infected with single species of Argulus sp. was quarantined in
separate tank with bottle implants.
For personal use only.
Host DNA isolation
About 5 mm  5 mm of fish lateral tissue was taken for molecular
analysis. The preserved tissue is made ethanol free by repeated
washing with saline and subjected to DNA isolation procedures as
per Prasanna kumar et al. (2011). Briefly, the tissue was placed in
1.5 ml Eppendorf tube and 500 ml of solution I (50 mM Tris-HCl
pH 8, 20 mM EDTA pH 8, and 2% SDS) was added. The tissue
was homogenized with sterile micro-pestle and 10 ml of proteinase
K (20 mg/ml) was added and quickly vortexed. The sample was
incubated at 55  C in water bath for 2 h with occasional mixing.
Following incubation, the sample was chilled over ice for 10 min
and 250 ml of solution II (6 M NaCl) was added and inverted
several times for thorough mixing. The tube was chilled on ice for
5 min and centrifuged at 8000 rpm for 15 min. About 500 ml of
supernatant was carefully collected into newly labeled 1.5 ml tube
and twice the volume (i.e., 1 ml) of 100% AR grade ethanol was
added to precipitate the DNA. The precipitate was pellet down at
8000 rpm for 5 min and the supernatant was removed without
touching the pellet. The DNA pellet was rinsed with 500 ml of
cold ethanol and centrifuged at 11,000 rpm for 5 min. The
supernatant was carefully removed and the excess liquid was
drained using pipette.
The pellet was partially dried (devoid of ethanol) with lid off
at 55  C on a heating block. The pellet was re-suspended with
50–200 ml of fresh sterile H2O depending on the size of pellet
(100 ml average) by gently pipetting sample with wide-bore filter
tip until dissolved. This dissolved DNA acted as a template for
polymerase chain reaction (PCR).
DNA isolation from Argulus spp.
The same protocol was followed for isolating DNA from adult and
egg specimens of Argulus spp. with volume of reagent has been
reduced to one-fifth quantity with the same gravity for centrifu-
gation. In case of adult specimen, entire animal was used for DNA
Mitochondrial DNA, Early Online: 1–5
extraction whereas in case of egg samples, 1–2 cm of egg capsule
was used. About 10 ml of proteinase K (20 mg/ml) was used for
digesting eggs whereas 5 ml of proteinase K (20 mg/ml) was
sufficient for digesting adult specimens of Argulus spp.
Polymerase chain reaction and DNA sequencing
In the present study, for all PCRs, the fragment of COI gene was
amplified by GeneAmp PCR system 9700 (QIAGEN Inc.,
Germantown, MD). PCR was carried out in 25 ml volumes
[2.5 ml of 10  PCR buffer, 1.5 ml of MgCl2 (2 mM/ml), 1 ml of
DNA template, 1 ml of each primer (10 pmoles/ml), 2 dNTPs
(1 mM/ml), 10 U of 1 ml of Taq polymerase (Bioserve
Biotechnologies Pvt, Ltd, Hyderabad, India), and 15 ml of sterile
Mill Q water].
COI gene of C. auratus was amplified using the primer
pair Fish F1: 50 -TCAACCAACCACAAAGACATTGGCAC-30
and Fish F2: 50 -TAGACTTCTGGGTGGCCAAAGAATCA-30
(Ivanova et al., 2007) and following PCR conditions were
followed. Heating starts at 94  C for 1 min, 30 five cycles of
94  C for 30 s, annealing at 45  C for 40 s, and extension at 72  C
for 1 min, with a final extension at 72  C for 10 min, followed by
indefinite held at 4  C. For amplifying COI gene fragment of adult
and egg specimens of Argulus spp. the primer pair LCO1490: 50 -
GGTCAACAAATCATAAAGATATTGG-30 and HCO2198: 50 -
TAAACTTCAGGGTGACCAAAAAATCA-30 (Folmer et al.,
1994) were used. The PCR condition for amplifying Argulus’s
COI gene included, heating starts with 94  C for 1 min, five cycles
of 94  C for 30 s, annealing at 49  C for 40 s, extension at 72  C
for 1 min, and final extension at 72  C for 10 min.
Following PCR, about 10 ml of PCR product with 2 ml of
bromo thymol blue were added to 2% agarose gel, prepared with
2.5 ml of 1% ethidium bromide and electrophorized at 90 V till the
dye moved 6 cm from the well in the gel. The reaction was
stopped and the gel was examined under UV trans-illuminator gel
doc system (ABI, Abilene, TX). Following confirmation of PCR
amplicon, sequencing PCR was carried out using dye terminator
mix v3.1 (ABI, Abilene, TX) and quantified in Euro bio-agarose
gel (ABI, Abilene, TX). The samples were loaded onto MegaBace
sequencer (Bioserve Biotechnologies, Pvt. Ltd., Hyderabad,
India) for DNA sequencing.
DNA sequence analysis
The electrophenerogram generated by automated DNA sequencer
was read by Chromas Pro v1.42 (USB, Cleveland, OH) and the
sequences were manually double checked for mis-calls and base
spacing. ClustalX 2.0.6 was used to align the COI gene sequences
(Thomson, 1997). MEGA 4.1 (Tamura et al., 2007) was used to
construct phylogenetic trees via the neighborhood joining method
using the Kimura 2-parameter distance model and the pair-wise
distances between the sequences were calculated using the same
model. Closest matches Argulus spp. sequences in the Genbank
were compared with Basic Local Alignment Search Tool
(BLASTN) ver. 2.2.30+. BLAST works by aligning the query
sequence with that of closest match of reference sequence in
Genbank database (Morgulis et al., 2008; Zhang et al., 2000).
The quarantined tank contained C. auratus infected with
A. japonicus only. Two adult specimen and egg capsules of
A. japonicus were sampled from quarantined tank for DNA
sequencing as a confirmation that COI gene sequences of adult
could be linked with its eggs. All 30 DNA barcodes (18 from eggs
and 12 from adults) were subjected to pair-wise distant analysis.
The pair-wise distances between the barcode sequences were
calculated using Kimura 2-parameter distance model (Kimura,
1980). The average of distances between and within sequences
was calculated and expressed in percentage of variations.
 DOI: 10.3109/19401736.2014.987269 Linking eggs and adults of Argulus spp. using mitochondrial DNA barcodes 3

Results Table 1. Result of BLAST analysis of 18 egg barcodes.

DNA barcoding with infected C. auratus BLAST similarity

The COI gene sequence of the infected host shared 98% similarity Specimen % & reference Accession
name accession number Identified as number
with the DNA sequence obtained from C. auratus (KF558297) of
San Francisco estuary (Brandl et al., 2013) published in GenBank. AgE1 99 & KF723409 Argulus japonicus KF713304
The COI gene sequence of C. auratus used in the present study AgE2 100 & KF723410 Argulus japonicus KF713305
could be accessed through the accession number KC292220. AgE3 100 & KF723413 Argulus indicus KF713306
AgE4 100 & KF723411 Argulus japonicus KF713307
AgE5 100 & KF723415 Argulus siamensis KF713308
Bottle implant experiment AgE6 100 & KF723412 Argulus japonicus KF713309
The bottle implant experiment was conduced for a span for AgE7 99 & KF723409 Argulus japonicus KF713310
AgE8 100 & KF723415 Argulus siamensis KF713311
3 months. Egg masses on the bottle surface were observed after AgE9 99 & KF723412 Argulus japonicus KF713312
24 h of infected fish introduction. However, laying of eggs AgE10 100 & KF723411 Argulus japonicus KF713313
depends on the maturity of the infested Argulus spp. Average of AgE11 99 & KF723408 Argulus japonicus KF713314
three egg capsules at the length of 3–5 cm was observed in each AgE12 100 & KF723415 Argulus siamensis KF713315
bottle. The location of the bottles inside the tank does not have AgE13 100 & KF723408 Argulus japonicus KF713316
any influence on the density of egg capsules found. Each capsule AgE14 99 & KF723411 Argulus japonicus KF713317
AgE15 100 & KF723415 Argulus siamensis KF713318
contained minimum of 100 to maximum of 300 eggs. Totally 18
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AgE16 99 & KF723415 Argulus foliaceus KF713319

egg capsules are randomly scrapped and preserved in ethanol for AgE17 100 & KF723408 Argulus japonicus KF713320
DNA barcoding. From the quarantined tank, two egg capsules AgE18 100 & KF723409 Argulus japonicus KF713321
were taken for barcoding.

Construction of Argulus spp. barcode library

Four species of adult Argulus spp. were identified, namely
A. japonicus, A. indicus, A. siamensis, and A. foliaceus. Five
specimens of A. japonicus, three specimens of A. indicus, two
specimens of A. foliaceus and A. siamensis were sequenced for
the COI gene. Totally 12 DNA sequences of adult Argulus spp.
were submitted in GenBank. The DNA barcodes of the adult
For personal use only.
of egg samples of A. japonicus could be accessed through
accession numbers JX025023 and JX025024. For phylogenetic
clustering, all adult and egg’s DNA barcodes were pooled
(including the COI gene sequences from the quarantined tank)
and subjected to phylogenetic analysis. The constructed phylo-
gram clearly clustered eggs of same species with its adult

sequences (Figure 1).

specimens could be accessed through the accession numbers
KF723408–KF723419.
The DNA barcodes of eggs (18 numbers) were obtained and
deposited in GenBank which could be accessed through accession
numbers KF713304–KF713321. The DNA barcodes of all four
species were produced for first time as Genbank did not contain
any reference sequences for the species described in the present
study. We used these deposited sequences in Genbank as the
reference sequences for identifying eggs of Argulus spp.
Identification of Argulus eggs using DNA barcodes
Precise identification of egg samples was done by two different
analyses, namely (i) BLAST analysis and (ii) phylogenetic
clustering of adults and eggs.
BLAST analysis
All 18 egg barcodes were subjected to BLAST analysis and their
closest matches were tabulated (Table 1). The abbreviation
‘‘AgE’’ in the table denotes Argulus egg.
All 18 eggs (AgE1–AgE20) showed closest matches with their
corresponding species in the reference database and are precisely
identified. The DNA barcodes of eggs do not show more than 1%
variation compared with adult barcodes of the respective species.
The similarity percentage of all 18 egg barcodes with that of adult
barcode library ranged from 99% to 100%.
Phylogenetic clustering of adults with eggs and its
pair-wise distance analysis
Phylogenetic clustering was done for the identification of eggs
with its corresponding adult species. The tank maintained
separately with A. japonicus acted as a proof that DNA barcodes
of eggs and adults could be linked. The DNA barcodes of two
adult specimens of A. japonicus could be accessed through
accession numbers JX025025 and JX025026. The DNA barcodes
The results were consistent with BLAST analysis (Table 1).
Phylogram cleared contained four major clades clustering each
species in single clade. Argulus japonicus forms the top most
major clade in phylogram clustering 14 egg barcodes with that its
respective adult barcode sequences (n ¼ 5). Second clade from the
top was occupied by A. foliaceus consisting of two adult barcodes
with single egg barcode sequence. However, second major clade
belongs to A. siamensis which contained four egg barcodes
clustered with two adult barcode sequences. In this phylogenetic
analysis, A. indus formed the separate clade which contained
single egg barcode sequence clustered with three adult barcode
sequences. Over all, all egg barcodes of the phylogram clustered
with the adult barcodes indicating the genetic relatedness of
premature forms with its adult counter parts. Between the species
of Argulus, a maximum distance of 8.5% was observed between
A. indicus and A. foliaceus and least of 0.2% was observed
between A. siamensis and A. foliaceus (Table 2). Within Argulus
spp., a maximum variation in DNA barcodes was observed for
A. japonicus (0.7%), which might indicate high genetic variations
within this species. The overall pair-wise distance observed for all
barcodes produced in this study is 2.3%.
Discussion
The present study created a DNA barcode library for four
species of adult Argulus spp. namely A. japonicus, A. indicus,
A. siamensis, A. foliaceus, and precisely identified randomly
collected Argulus egg masses with that of created barcode library.
The designed experimental set-up using bottle implants for egg
mass collection is fruitful as sampling egg mass for molecular
analysis was easy. It is very evident that eggs of individual
Argulus sp are impossible to be named. Even though conventional
morphological keys exist for Ichthyoplanktons, it is very
complicated to define such morphological keys for smaller
crustaceans like Argulus spp. The present study, for the first
time, proved that fish parasite eggs can be precisely identified
 4 K. F. Khan et al. Mitochondrial DNA, Early Online: 1–5

AgE18
QE2
AgE17
57
AgE13
QE1
62
QA2 Argulus japonicus
39 QA1 Argulus japonicus
AgE7
28
AgE1
AgE11
28
99 A1 Argulus japonicus A. japonicus
AgE2
AgE9
43
AgE14
37 AgE10
62
55 A2 Argulus japonicus
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AgE6
73
A3 Argulus japonicus
29 AgE4
A5 Argulus japonicus
94 A4 Argulus japonicus
AgE16
40 33 A11 Argulus foliaceus A. foliaceus
100 A12 Argulus foliaceus
AgE12
AgE15
For personal use only.

A8 Argulus siamensis
A. siamensis
89 AgE8
A7 Argulus siamensis
AgE5
98 A6 Argulus indicus
AgE3
A. indicus
A9 Argulus indicus
100 A10 Argulus indicus

0.005

Figure 1. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The
evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. Codon
positions included were 1st+2nd+3rd+non-coding. All positions containing gaps and missing data were eliminated from the dataset (complete deletion
option). There were a total of 623 positions in the final dataset. The abbreviations QA and AE with the triangle marks in the top most clade of the
phylogram represent the adult and egg samples sampled from quarantined tank. The abbreviation A and AgE represent adult and egg samples,
respectively.

Table 2. Estimates of pair-wise divergence between the sequences of four different species of Argulus. Defining such precise species
different Argulus spp. boundaries could aid in further invention and discovery of new
species of Argulus.
A. foliaceus A. siamensis A. japonicus A. indicus
In order to establish the DNA barcode technology for the
A. foliaceus 0 identification of all Argulus spp and its premature forms,
A. siamensis 0.2 ± 0.05 0.1 ± 0.05 development of full-fledged barcode library that includes all
A. japonicus 5.5 ± 0.5 2.0 ± 0.6 0.7 ± 0.3 species of this genus is very important. The present study does not
A. indicus 8.5 ± 1.2 4.5 ± 0.9 6.0 ± 0.8 0.5 ± 0.01
attempt to define barcode gaps within Argulus spp. due to limited
number of species we sampled. Sampling multiple specimens
per species and attaining maximum species coverage for DNA
barcoding for establishing DNA barcode library will facilitate the
using DNA barcodes. This barcoding technology can be used as a utility of DNA barcodes in the identification and discovery of
diagnostic tool. Further, earlier detection of preliminary life forms Argulus spp.
of Argulus spp could facilitate in preventing the spread as most of
the Argulus spp infections becomes epidemic through the spread
of gelatinous egg masses. The pair-wise distance analysis Acknowledgements
indicated the necessity for defining species boundary among The authors are thankful to the head of the institute for support and
Argulus spp. with increased number of multiple samples from encouragement and university authorities for the facility provided.
 DOI: 10.3109/19401736.2014.987269 Linking eggs and adults of Argulus spp. using mitochondrial DNA barcodes
Authors are thankful to the reviewer for improving the quality of the
manuscript.
Declaration of interest
The authors report that they have no conflicts of interest. First
author thanks University Grant Commission, Government of
India, for financial support through UGC-CPEPA project.
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