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
2.)
Faculty of Veterinary Medicine Gadjah Mada University, Jogjakarta, Indonesia
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
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 with 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.
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 etal., 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 2. DNA Extraction of Lernaea species lanes 2-7, amplification product in 18S region of Lernaea species lanes 8-14, Lane 1 = marker
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
1 2 3 4 5
6
7
8 9 10 11 12 13 14
1
2
3
4
5
6
7
8
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
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
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
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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