Molecular Study On Lernaeosis In Arwana Fish (Scleropages Jardinii).
MOLECULAR STUDY ON LERNAEOSIS IN ARWANA FISH (Scleropages
jardinii)
SHATRIE, D. N. 1, KURNIASIH2, NURCAHYO, W., TRIYANTO
1.)
Centre for Fish Quarantine, Ministry of Marine Affair and Fisheries, Jakarta, Indonesia
Faculty of Veterinary Medicine Gadjah Mada University, Jogjakarta, Indonesia
2.)
ABSTRACT
The Arwana Irian fish is one of the endangered species. Lernaeosis is one of
the diseases that cause the high mortality in juvenile fish. The study was to find out
the species of Lernaea (Copepoda) often attacked Arwana Irian fish. Lernaea sp was
collected from Papua and Jakarta (Java). They were fixed in the glutaraldehyde for
SEM and some of them in the ethanol absolute solution for molecular study. It was
found five different species of Lernaea and two of them were thought as a new
species, based on the morphology and SEM. Lernaea species had a slightly different
based on RFLP of DNA in the 18S region. However, they showed three different
variation based on the DNA sequencing result.
Keyword : Scleropages jardinii, Lernaeos
INTRODUCTION
Lernaeid copepods cause serious deleterious effects on their freshwater fish
hosts (Kabata, 1985). One factor which may have contributed to the increased
infestation level during the drying phase was the reduction in water volume. The
economic importance of the lernaeid ectoparasites has increased due to numerous
epizootics occuring among the most important farmed fish in various parts of the
world (Tasawar et al., 2007). A lernaeid population found only on jaws of Tilapia
(Oreochromis) spp. Lernaea victoria Fryer, 1961 has been identified as Lernaea
cyprinacea. Other lernaeids of African fish were endemic, Lernaea barnimiana may
be found on both cyprinid and cichlid hosts (Paperna, 1996). The embedded anchors
were surrounded by fibrous granulation tissue, and there was considerable leucocytic
response below the dermis. Myofibril degeneration and hemorrhage were noted in
most sections (Berry et al., 1991). Puntius gonionotus occurred in Selangor, West
Malaysia as Lernaea minuta. Lernaea minuta is a parasite of Javanese carp, (Puntius
International Seminar Biotechnology
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gonionotus), Lernaea minuta followed by secondary bacterial invasions (Kularatne et
al, 1993). Lernaea cyprinacea was reported from several fish such as goldfish,
Carassius auratus (L), Helostoma temmincki, Cyprinus carpio, Carassius auratus (L)
(Shariff et al., 1986). Infestations of Lernaea cyprinacea on four native fish species
(Galaxias occidentalis Ogilby ; Edelia vittata Castelnau; Tandanus bostocki Whitley)
and three introduced fish species (Carassius auratus L.; Gambusia holbrooki
(Girard); Phalloceros caudimaculatus (Hensel) (Marina et al., 2008).
MATERIALS AND METHODS
A total of 20 of parasitic copepodes belonging to Lernaeidae were collected
from Arwana fish in Papua, Indonesia. Copepode was examined under stereo
microscope to identify species. Specimens were fixed in glutaraldehyde for SEM and
ethanol absolute for molecular study. Primers were used, 18SF (5’ – AAG GTG
TGM CCT ATC AAC T – 3’), 18SR (5’ – TTA CTT CCT CTA AAC GCT C – 3’),
28SF (5’ – ACA ACT GTG ATG CCC TTA G – 3’), and 28SR(5’ – TGG TCC GTG
TTT CAA GAC G – 3’). Following conditions of cycles were 94 0C for 5 minutes,
followed by 30 cycles of 94 0C for 30 second, 54 0C for 30 second and 72 0C for 1
minute with a final extension at 72 0C for 5 minutes (Song et al., 2008). Products of
PCR were then digested with HaeIII and Alu enzyme in 18S and 28S regions, then
were visualized and stained wit h silver of 8% gel polyacrilamide method. Six
specimens of Lernaea were then purified and sequenced in the 28S region.
RESULTS AND DISCUSSIONS
There were 6 different species of Lernaea collected from Arwana fish in
Papua and Jakarta, based on morphology. They were Lernaea cyprinacea, L.
lophiara, Harding 1950, L.papuensis (Boxshall, 1981), L.devastatrix (Boxshall et al.,
1997) and 2 new species of Lernaea . The fine structure of body surface was smooth.
The hold last, cephalothorax, swim leg of specimen number 3 was the same as L.
devastatrix. Two specimens were proposed as new species based on morphology and
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SEM. The type of Lernaea n.sp.1 had a large dorsal branched of holdfast which was
clearly chitineous, like a finger nail (fig.1).
The extraction and PCR product had a good result (Figure 2). There were
slightly differences of restriction sites of RFLP using HaeIII in the 18S region (Figure
4) compared with Alu enzyme (Figure 4). Specimens no.3, 4 and 5 were clearly
different,. specimen no.1 was different from no.2 and three others. Lernaea
cyprinacea showed variation strain and paraphyletic with L. orientalis and L.
chinensis from freshwater fish in China (Song et al., 2008).
A new species of Lernaea,closely related to Lernaea cyprinacea, is desribed
from both native and introduced fish species at a Brazil. Lernaea devastatrix, can be
distinguished from Lernaea cyprinacea by the reflexed claw on its antenna (Boxshall
et al., 1997). Lernaceae papuensis, Lernaea devastatrix, Lernaea lophiara and
Lernaea cyprinacea were never identify based on the molecular study.
Figure 1. Lernaea cyprinacea, with simple holdfast C1, Lernaea sp.n.kedua pasang
cabang C3.; Lernaea lophiara with holdfast like butterfly,C4.
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1 2 3 4 5
6 7 8 9 10 11 12 13 14
Figure 2. DNA Extraction of Lernaea species lanes 2-7, amplification product in 18S
region of Lernaea species lanes 8-14, Lane 1 = marker
M
1
2
3
4
5
6
7
8
Figure 3. Restriction endonuclease sites of Lernaea species using Aluenzyme in 18S
region. M = marker, 1 = Lernaea lophiara, 2 = Lernaea cyprinacea, 3 =
Lernaea devastatrix, 4 = Lernaea sp.Lanes 5-8 using HaeIII enzyme
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M 1 2 3 4 5
6 7 8 9 10
Figure 4. Restriction endonuclease sites of Lernaea species using HaeIII enzyme in
18S region. M = marker, 1 = Lernaea lophiara, 2 = Lernaea cyprinacea, 3
= Lernaea devastatrix, 4 = Lernaea sp1, 5 = Lernaea sp2.Lanes 6-10 was
restriction endonuclease sites of Lernaea species in 28S region.
The sequense results showed that there were several gene variation between
Lernaea 1, 10, 11 (one group), Lernaea 2,3 (one group), and Lernaea 4 (Figure 5).
The sequences result of Lernaea in Indonesia has not been reported.
L 1 : AGCACTGA –CCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
50
L 2 : AGCACTGAAC- GCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L 3 : AGCACTGAAC- GCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L 4 : AGCACTGAACCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L10: AGCACTGA - CCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L11: AGCCTGGA - CCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L 1 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
100
L 2 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L 3 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
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L 4 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L10: GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L11 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L 1 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
150
L 2 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 3 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 4 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 10: ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 11: ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 1 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
200
L 2 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L 3 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L 4 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L10: GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L11: GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L 1 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
250
L 2 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L 3 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L 4 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L10: GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L11: GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L 1 : GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
300
L 2 : GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L 4 : GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L10: GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L11: GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L 1 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
350
L 2 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L 3 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L 4 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
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L10: ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L11: ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L 1 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
400
L 2 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L 3 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L 4 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L10 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L11 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L 1 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
450
L 2 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L 3 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L 4 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L10 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L11 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L 1 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
500
L 2 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L 3 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L 4 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAAAGCGAATCAAGTG
L10 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L11 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L 1 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
550
L 2 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L 3 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L 4 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGGGTTGGGAGCCCCC
L10 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L11 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L 1 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
600
L 2 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L 3 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L 4 : CGTTTTCTGGCTTCAATTTCGGGGGTCCTTAAAAAATGAGATAGGGACAC
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L10 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L11 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L 1 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
650
L 2 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L 3 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L 4 : CACTCCAAAATAGCGGAGTGCCGGCCTTTGTTGGCCCTGTTCTTTTGTGC
L10 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L11 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L 1 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
L 2 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
L 3 : TCGCGAGTACGTCGGTGGCCTCTCTGACCCGTCTTG
L 4 : GTTGGCCCCTGGTTCTTTGTGGCGGGACATACGGG
L10 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
L11 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
Figure 5. Sequencing result of Lernaea species from Arwana Irian fish
CONCLUSION
There were 6 species of Lernaea from Arwana fish from Papua based on
morphology. Lernaea n.sp.1 had a chitineous tip on the dorsal large branch of
holdfast. Lernaea n.sp.2 had a clearly different type of holdfast from other.
Restriction endonuclease using HaeIII enzyme in 18S and using Alu in 28S showed
slightly different sites among them. There were 3 group variation of Lernaea from
Arwana Irian fish in Indonesia based on the sequencing result.
REFERENCES
BERRY,C.R., BABEY,G.J., SHRADER,T. Effect of Lernaea cyprinacea (Crustacea
: Copepoda) on stocked rainbow trout (Onchorhynchus mykiss).
J. of
Wildlife Dis., 27(2):.206-213. 1991.
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BOXSHALL,G.A. A new species of Lernaea (Copepoda : Cyclopoida) from PapuaNew Guinea. J.Bull. Br. Mus. nat. Hist.(Zool.).40(4) :117-120.1981.
BOXSHALL,G.A., MONTU,M.A and SCHWARZBOLD,A. A new species of
Lernaea L. (Copepoda : Cyclopoida) from Brazil, with notes on its ontogeny.
J.Systemic Parasitology, 37 :195-205.1997.
KABATA,Z. Parasites and diseases of fish cultured in the tropics. London &
Philadelphia, Taylor & Francis, 1985.
KULARATNE,M., SUBASINGHE, R.P and SHARIFF, M. Investigations on the
lack of aqcuired immunity by ht e Javanese carp, Punctius gonionotus
(Bleeker), against the crustacean parasite, Lernaea minuta (Kuang). J.Fish &
Shellfish Imun., 4: 107-114.1998.
MARINA, H., BEATTY, S.J., MORGAN D.L., DOUPE, R.G., LYMBERY, A.J. An
introduced parasite, Lernaea cyprinacea L., found on native freshwater fishes
in the South West of Western Australia. J. of the Royal Soc.of Westen
Australia, 91:149-153. 2008.
PAPERNA,I. Parasites, infections and diseases of fishes in Africa : an update.
Israel.EIFAA Technical Paper – CIFA/T31. 1996.
SHARIFF,M, KABATA,Z., SOMMERVILLE,C. Host susceptibility to Lernaea
cyprinacea L. and its treatment in a large aquarium system. J. of Fish
Dis.,9:393-401, 1986.
SONG,Y., WANG,G.T., YAO,W.J., GAO,G and NIE,P. Phylogeny of freshwater
parasitic copepods in the Ergasilidae (Copepoda : Poecilostomatoida) based
on 18S and 28S rDNA sequences. J.Parasitol, 102:299-306. 2008
TASAWAR,Z., HANIF,M., LASHARI,M.H and HAYAT,C.S. The prevalence of
lernaeid ectoparasites in Mori (Cirrhinus migala) fish. J.Pakistan Vet, 27(4)
:176-178. 2007
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jardinii)
SHATRIE, D. N. 1, KURNIASIH2, NURCAHYO, W., TRIYANTO
1.)
Centre for Fish Quarantine, Ministry of Marine Affair and Fisheries, Jakarta, Indonesia
Faculty of Veterinary Medicine Gadjah Mada University, Jogjakarta, Indonesia
2.)
ABSTRACT
The Arwana Irian fish is one of the endangered species. Lernaeosis is one of
the diseases that cause the high mortality in juvenile fish. The study was to find out
the species of Lernaea (Copepoda) often attacked Arwana Irian fish. Lernaea sp was
collected from Papua and Jakarta (Java). They were fixed in the glutaraldehyde for
SEM and some of them in the ethanol absolute solution for molecular study. It was
found five different species of Lernaea and two of them were thought as a new
species, based on the morphology and SEM. Lernaea species had a slightly different
based on RFLP of DNA in the 18S region. However, they showed three different
variation based on the DNA sequencing result.
Keyword : Scleropages jardinii, Lernaeos
INTRODUCTION
Lernaeid copepods cause serious deleterious effects on their freshwater fish
hosts (Kabata, 1985). One factor which may have contributed to the increased
infestation level during the drying phase was the reduction in water volume. The
economic importance of the lernaeid ectoparasites has increased due to numerous
epizootics occuring among the most important farmed fish in various parts of the
world (Tasawar et al., 2007). A lernaeid population found only on jaws of Tilapia
(Oreochromis) spp. Lernaea victoria Fryer, 1961 has been identified as Lernaea
cyprinacea. Other lernaeids of African fish were endemic, Lernaea barnimiana may
be found on both cyprinid and cichlid hosts (Paperna, 1996). The embedded anchors
were surrounded by fibrous granulation tissue, and there was considerable leucocytic
response below the dermis. Myofibril degeneration and hemorrhage were noted in
most sections (Berry et al., 1991). Puntius gonionotus occurred in Selangor, West
Malaysia as Lernaea minuta. Lernaea minuta is a parasite of Javanese carp, (Puntius
International Seminar Biotechnology
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gonionotus), Lernaea minuta followed by secondary bacterial invasions (Kularatne et
al, 1993). Lernaea cyprinacea was reported from several fish such as goldfish,
Carassius auratus (L), Helostoma temmincki, Cyprinus carpio, Carassius auratus (L)
(Shariff et al., 1986). Infestations of Lernaea cyprinacea on four native fish species
(Galaxias occidentalis Ogilby ; Edelia vittata Castelnau; Tandanus bostocki Whitley)
and three introduced fish species (Carassius auratus L.; Gambusia holbrooki
(Girard); Phalloceros caudimaculatus (Hensel) (Marina et al., 2008).
MATERIALS AND METHODS
A total of 20 of parasitic copepodes belonging to Lernaeidae were collected
from Arwana fish in Papua, Indonesia. Copepode was examined under stereo
microscope to identify species. Specimens were fixed in glutaraldehyde for SEM and
ethanol absolute for molecular study. Primers were used, 18SF (5’ – AAG GTG
TGM CCT ATC AAC T – 3’), 18SR (5’ – TTA CTT CCT CTA AAC GCT C – 3’),
28SF (5’ – ACA ACT GTG ATG CCC TTA G – 3’), and 28SR(5’ – TGG TCC GTG
TTT CAA GAC G – 3’). Following conditions of cycles were 94 0C for 5 minutes,
followed by 30 cycles of 94 0C for 30 second, 54 0C for 30 second and 72 0C for 1
minute with a final extension at 72 0C for 5 minutes (Song et al., 2008). Products of
PCR were then digested with HaeIII and Alu enzyme in 18S and 28S regions, then
were visualized and stained wit h silver of 8% gel polyacrilamide method. Six
specimens of Lernaea were then purified and sequenced in the 28S region.
RESULTS AND DISCUSSIONS
There were 6 different species of Lernaea collected from Arwana fish in
Papua and Jakarta, based on morphology. They were Lernaea cyprinacea, L.
lophiara, Harding 1950, L.papuensis (Boxshall, 1981), L.devastatrix (Boxshall et al.,
1997) and 2 new species of Lernaea . The fine structure of body surface was smooth.
The hold last, cephalothorax, swim leg of specimen number 3 was the same as L.
devastatrix. Two specimens were proposed as new species based on morphology and
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SEM. The type of Lernaea n.sp.1 had a large dorsal branched of holdfast which was
clearly chitineous, like a finger nail (fig.1).
The extraction and PCR product had a good result (Figure 2). There were
slightly differences of restriction sites of RFLP using HaeIII in the 18S region (Figure
4) compared with Alu enzyme (Figure 4). Specimens no.3, 4 and 5 were clearly
different,. specimen no.1 was different from no.2 and three others. Lernaea
cyprinacea showed variation strain and paraphyletic with L. orientalis and L.
chinensis from freshwater fish in China (Song et al., 2008).
A new species of Lernaea,closely related to Lernaea cyprinacea, is desribed
from both native and introduced fish species at a Brazil. Lernaea devastatrix, can be
distinguished from Lernaea cyprinacea by the reflexed claw on its antenna (Boxshall
et al., 1997). Lernaceae papuensis, Lernaea devastatrix, Lernaea lophiara and
Lernaea cyprinacea were never identify based on the molecular study.
Figure 1. Lernaea cyprinacea, with simple holdfast C1, Lernaea sp.n.kedua pasang
cabang C3.; Lernaea lophiara with holdfast like butterfly,C4.
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1 2 3 4 5
6 7 8 9 10 11 12 13 14
Figure 2. DNA Extraction of Lernaea species lanes 2-7, amplification product in 18S
region of Lernaea species lanes 8-14, Lane 1 = marker
M
1
2
3
4
5
6
7
8
Figure 3. Restriction endonuclease sites of Lernaea species using Aluenzyme in 18S
region. M = marker, 1 = Lernaea lophiara, 2 = Lernaea cyprinacea, 3 =
Lernaea devastatrix, 4 = Lernaea sp.Lanes 5-8 using HaeIII enzyme
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M 1 2 3 4 5
6 7 8 9 10
Figure 4. Restriction endonuclease sites of Lernaea species using HaeIII enzyme in
18S region. M = marker, 1 = Lernaea lophiara, 2 = Lernaea cyprinacea, 3
= Lernaea devastatrix, 4 = Lernaea sp1, 5 = Lernaea sp2.Lanes 6-10 was
restriction endonuclease sites of Lernaea species in 28S region.
The sequense results showed that there were several gene variation between
Lernaea 1, 10, 11 (one group), Lernaea 2,3 (one group), and Lernaea 4 (Figure 5).
The sequences result of Lernaea in Indonesia has not been reported.
L 1 : AGCACTGA –CCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
50
L 2 : AGCACTGAAC- GCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L 3 : AGCACTGAAC- GCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L 4 : AGCACTGAACCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L10: AGCACTGA - CCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L11: AGCCTGGA - CCGCCAGCTTTTGAAAGGGTTGCGCGGAATGTAGTGTTTG
L 1 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
100
L 2 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L 3 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
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L 4 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L10: GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L11 : GGAGAGCCTTCTCATGATGCGCGGTGCAAAATCTGTCTAAGTCCACCTTG
L 1 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
150
L 2 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 3 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 4 : ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 10: ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 11: ACTGGGGCCACTACCCATAGAGGGTGATAGGCCCGTAAGACAGTCTGCGT
L 1 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
200
L 2 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L 3 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L 4 : GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L10: GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L11: GTTGTGCTGGCTTTTTCCCTAGAGTCGAGTTGCTTGGGAGTGCAGCTCAAA
L 1 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
250
L 2 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L 3 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L 4 : GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L10: GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L11: GTGCGTGGTAAACTCCACGTAAGGCTAAATATCACCCCGAGACCGATAGC
L 1 : GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
300
L 2 : GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L 4 : GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L10: GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L11: GAACAAGTACCGTGAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCA
L 1 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
350
L 2 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L 3 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L 4 : ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
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L10: ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L11: ATAGTACGTGAAACTGTGTAGCGGTAAACAGAGGGGCTCTCGAAGTCCAG
L 1 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
400
L 2 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L 3 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L 4 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L10 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L11 : GCTGGAGATTCAGGTTGCCAGATGGCTAGTTTGGCTGGTGCGAAGATCTG
L 1 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
450
L 2 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L 3 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L 4 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L10 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L11 : CATAGGACTTTGTGCCTGGTCAAATGTTGTTGGCTGGATGGCGATTGCAC
L 1 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
500
L 2 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L 3 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L 4 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAAAGCGAATCAAGTG
L10 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L11 : TTCTCTGGCCTAGCAATGGGCGCGACGAGCCACTGAGAGCGAATCAAGTG
L 1 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
550
L 2 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L 3 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L 4 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGGGTTGGGAGCCCCC
L10 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L11 : CGTGGGTGAAGTTTGCTTCAACAGTCTTATGGCTGGTGTTGTGAGCCCCG
L 1 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
600
L 2 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L 3 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L 4 : CGTTTTCTGGCTTCAATTTCGGGGGTCCTTAAAAAATGAGATAGGGACAC
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L10 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L11 : CGTTTTCTGGCTTCGATTTCGGTGGTCTTATGTATGGAGATAGGACAGAC
L 1 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
650
L 2 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L 3 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L 4 : CACTCCAAAATAGCGGAGTGCCGGCCTTTGTTGGCCCTGTTCTTTTGTGC
L10 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L11 : TCGTTTATAGCGAGTGCCGCTTTTGTGGCACTGTCTTTGTCCGACATCTG
L 1 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
L 2 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
L 3 : TCGCGAGTACGTCGGTGGCCTCTCTGACCCGTCTTG
L 4 : GTTGGCCCCTGGTTCTTTGTGGCGGGACATACGGG
L10 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
L11 : TCGCGAGTAGGTCGGTGGCCTCTCTGACCCGTCTTG
Figure 5. Sequencing result of Lernaea species from Arwana Irian fish
CONCLUSION
There were 6 species of Lernaea from Arwana fish from Papua based on
morphology. Lernaea n.sp.1 had a chitineous tip on the dorsal large branch of
holdfast. Lernaea n.sp.2 had a clearly different type of holdfast from other.
Restriction endonuclease using HaeIII enzyme in 18S and using Alu in 28S showed
slightly different sites among them. There were 3 group variation of Lernaea from
Arwana Irian fish in Indonesia based on the sequencing result.
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