CC BY
62
DOI 10.18551/rjoas.2019-08.35
IDENTIFICATION OF ANISAKID NEMATODE L3 LARVAE INFECTION ON SKIPJACK TUNA (KATSUWONUS PELAMIS L.) FROM KUPANG WATERS, EAST NUSA TENGGARA OF INDONESIA
Haryadi Linda1,4, Suprayitno Eddy2, Aulanni'am Aulanni'am3, Amin Mohamad5,
Hariati Anik Martinah2*
1Doctoral Program, Faculty of Fisheries and Marine Sciences, University of Brawijaya,
Malang, Indonesia
2Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang, Indonesia 3Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Malang, Indonesia 4Fish Quarantine and Inspection Agency Kupang, Ministry of Marine Affairs and Fisheries
of Republic of Indonesia, Kupang, Indonesia 5Department of Biology, Faculty of Mathematics and Sciences, University of Negeri Malang,
Malang, Indonesia *E-mail: a [email protected]
ABSTRACT
The larvae of Anisakis were living parasites and made marine mammals, birds and reptiles the definitive host. Identification of Anisakis larvae using morphological observation methods will be difficult, especially if there are only a few samples that can be identified. PCR is an identification method using DNA from a small sample quantity and can provide DNA sequence samples. This study aimed to determine the type and infection level of Anisakis sp. at skipjack tuna (Katsuwonus pelamis) from the Oeba Fish Auction (TPI) Kupang City morphologically and molecularly. Morphological analysis results of 30 Anisakis larvae showed the body parts of Anisakis larvae, namely the head, digestive tract, and tail. The infection of Anisakis nematodes in skipjack tuna found five individual nematodes in muscle tissue, 59 individuals in stomach tissue, and 1991 individuals in internal organs. Alignment results between isolates At1 and At2 against isolates A. typica comparing (outgroup), isolate At1 and At2 have high homologs. Based on the results of the study concluded that the type I Anisakis isolated from skipjack tuna (Savu Sea) was Anisakis typica.
KEY WORDS
Cetacean, dolphin, molecular, savu, whale.
Nematodes from the Anisakidae family are living parasites and make marine mammals, birds and reptiles a definitive host. Although the life cycle of this family is unclear, it is known that marine fish can act as intermediaries, paratenic hosts or definitive. Nematode Anisakis Djuardin, 1845 (Mladineo, Simat, Miletic, Beck, & Poljak, 2012), generally inhabits the digestive tract of aquatic vertebrates, where euphausiid crustaceans act as intermediate hosts, fish and cephalopods as paratenic, and cetaceans as final hosts. Some Anisakis species, such as Anisakis simplex and Anisakis pegriffii, are known to cause clinically significant disease in humans (Zhang et al., 2007).
Anisakis spp uses fish or aquatic invertebrates such as squid and shrimp as intermediary hosts. Anisakis larval stages in intermediate hosts are called L3 larvae (Sakanari & Mckerrow, 1989) and (Nagasawa & Moravec, 1995). Anisakis spp is commonly found living on the intestinal wall, liver and muscle of fish flesh and can cause pathological effects on fish (Yoshinaga, Kinami, Hall, & Ogawa, 2006); (Hassan, Mohamed, & Osman,
2013) (Koinari, Karl, Elliot, Ryan, & Lymbery, 2013); (Anshary, Sriwulan, Freeman, & Ogawa,
2014); (Palm et al., 2017); and (Setyobudi et al., 2019).
Specific identification of nematode larvae using morphological observation methods will be difficult, especially if there is only a small amount to identify. One way of identification, to overcome this, is the Polymerase Chain Reaction (PCR). The PCR method enables the
identification process using DNA from a small quantity of material (nanograms to picograms) and provides a target DNA sequence (X. Zhu, Gasser, Podolska, & Chilton, 1998). The results have shown that Internal Transcribe Spacers (ITS-1 and ITS-2) from ribosomal nuclear DNA (rDNA) provide genetic markers for identification of adult Anisacidae, including A. simplex, Hystemthylacium aduncum and Contracaecum rudolphii (XQ Zhu et al., 2002 ). Identification of Anisakis nematodes requires accuracy at the life cycle stage and each host center. It aims to understand Anisakis ecology and epidemiology, diagnosis, and key components of disease control and control (Cheng, 1982).
Therefore, the results of adult Anisakis sequencing function as a reference to identify the larval stage. The PCR mutation scanning process is combined with a selective sequence of ITS-1 and/or ITS-2. This process provides a powerful approach to identifying and differentiating Anisakis nematodes (at any stage of development). This identification process aims to diagnostic or taxonomy, explore the genetic composition of Anisakis larvae populations, and to investigate their ecology (X. Q. Zhu et al., 2007).
Molecular identification results by Palm, Damriyasa, Linda, & Oka (2008) and Anshary et al. (2014) found A. typica species as the dominant species in the waters of Bali and the Makassar Strait. Both of these sea waters are close to the waters of East Nusa Tenggara. The study of A. typica in these two waters are inseparable from the life pattern of Anisakis nematodes, the distribution of skipjack tuna and mackerel tuna, and the migration patterns of several marine mammals as parentic hosts.
In this study, we want to develop and apply diagnoses based on molecular DNA bonds using ITS (Internal Transcribed Spacer) analysis. This study wants to prove the most dominant species found in fish samples, assuming A. typica is the species most often found in Indonesian waters. Fish sampling locations are Kupang waters, East Nusa Tenggara Province, Indonesia. This research is also based on the existence of reports of human cases infected with Anisakis nematodes. It needs to be investigated about the protein profile that is thought to be an allergen in humans. This study aimed to determine the type and level of Anisakis sp. infection on skipjack tuna (Katsuwonus pelamis) from the Oeba Fish Auction Place (TPI) of Kupang City morphologically and molecularly.
MATERIALS AND METHODS OF RESEARCH
Anisakis nematodes collected from skipjack tuna (Katsuwonus pelamis) which purchased from a fish auction place in the city of Kupang. Nematode larvae collected from the surface of the internal cavity and organs (liver, intestine, stomach, and gonads). The fresh nematodes washed several times using sterile water then with 0.9% NaCl solution, then with pure water and finally with a 0.9% NaCl solution. Furthermore, nematode larvae stored in 0.9% NaCl solution at -20 °C for protein extraction and partly in 70% ethanol at 4 °C for DNA extraction.
The morphological identification process refers to J. Grabda (1991). Anisakis L3 larvae purified using glycerin-phenol-lactic acid distilled water solution (2: 1: 1: 1). Morphological characteristics measured were body width, esophageal length, ventricular length, tail length, body length/body width, body length/esophageal length, body length/ventricular length, and body length/tail length (Setyobudi, Jeon, Lee, Seong, & Kim, 2011). Nematodes observed with microscope (Axio Lab.A1 Zeiss).
The DNA extraction was modified from D 'Amelio et al. (2000). The nematodes rinsed with PBS and put in a 1.5 mL microtube. Then added with 200 mL extract buffer (50 mM TrisCl pH 8, 100 mMNaCL, 5 mM EDTA, 10% SDS, 10 mg/mL Proteinase K). After that, it is homogenized and incubated in a water bath at 56 ° C for 2 hours. Then, added with 125 mL of 5 M NaCl and stirred with vortex for 10 sec and centrifuged at 13,000 rpm for 5 min. A total of 200 mL PCI (25: 24: 1) added in the supernatant, then centrifuged at 13,000 rpm for 10 min. This centrifugation repeated with the addition of 200 mL CI (24: 1). The supernatant added with 500 mL absolute ethanol, then incubated for 1 hour at -20 ° C. The sample centrifuged for 10 min at 13,000 rpm, 4 ° C. After that, Pellets added with 500 mL of 70% ethanol then centrifuged for 5 min at 13,000 rpm 4 ° C. The pellet dried at 55 °C. Then 50 mL
of TE buffer pH 7.6 added to the tube. The isolated DNA PCR amplified using ITS (ITS 1.58S rDNA and ITS 2), primers NC5 (forward; 5'-GTAGGTGAACCTGCGGAAGATCATT-3') and NC2 (reverse: 5' TTAGTTTCTTTTCCTCCGCT-3'). PCR program as many as 30 cycles at a temperature of 95°C for 15 min (predenaturation), 95°C for 1 min (denaturation), 55°C for 1 min (annealing), 72°C for 1 min (extension) and 72°C for 5 min (post extension). The reaction mixture for PCR includes PCR Mix 12.5 |jL, ddH2O 8.5 |jL, Primary Forward 1 |jL, Primary Reverse 1 jL, DNA sample 2 jL.
RESULTS OF STUDY
Morphological Characters and Infection Rates of A. typica in Skipjack Tuna (K. pelamis). Anisacid larvae collected from 30 skipjack tuna (K. pelamis from NTT waters). The larvae were white, attached to the infected part, membranes wrapped with different distribution and intensity of infection. Internal organs (especially the stomach) are the organs with the highest infection rates. Morphological analysis was carried out on 30 individual larvae taken randomly, showing that the body parts of Anisacid larvae, namely the head, digestive tract, and tail. Part A is the anterior end or head to describe the nematodes having three lips around the mouth and tooth on the top of the head (larvae). Part B is part of the ventricles or digestive tract, which consists of the esophagus, ventricles, and intestines. Part B is an essential part because it used as a basis for identification of nematodes at the genus level. Part C is the posterior end or tail consisting of the anal glands, anus, and mucus (Figure 1).
(A). The Anterior Kn<l / (B). The Ventricals
/
Mucron
(C). The Posterior End
Figure 1 - Morphology of Anisakis Nematode. A). Anterior; B). Ventriculus; and C). Posterior
A total of 30 skipjack tuna, found five individuals nematodes in muscle tissue, 59 individuals nematodes in stomach tissue, and 1991 individuals nematodes in internal organs. Morphologically, the total body length is between 7.27 -14.42 mm, ventricular length 0.020.08 mm, mucron length 0.001-0.012 mm. Morphological characters were presented in Table 1.
The parasitic A. typica found on the inner surface of the body cavity and also found in muscles. However, the highest infection intensity of the parasite A. typica found in internal organs (stomach, liver, and intestine) (Palm et al., 2008). Morphologically the total body length is between 9-15mm, ventricular length 0.02-0.07mm, mucron length 0.02-0.03mm
(Quiazon, Yoshinaga, Santos, & Ogawa, 2009). The size of the ventricular length is one of the important parameters in identifying Anisakis spp. morphologically. The use of ventricular display in identifying Anisakis species has been applied to Anisakis simplex (ss) and A. Pegreffii. Anshary (2011) also confirmed that Anisakis type I is characterized by the presence of boring tooth at the anterior end and the mucron at the posterior end.
Table 1 - Morphological characters of A. typica isolation from skipjack tuna (K. pelamis)
from NTT waters*
Morphological Characters Size Range (mm) Average Size, (Mean ± SD) (mm)
Maximum Length 7.27 - 14.42 11.53 ± 1.83
Maximum Width 0.20 - 0.48 0,33 ± 0.07
Esophagus Length 0.01 - 0.80 0.09 ± 0.15
Ventriculus Length 0.02 - 0.08 0.04 ± 0.01
Ventricular width 0.01 - 0.02 0.01 ± 0.003
Mucron Length 0.001 - 0.012 0.003 ± 0.002
*The number of larval samples: 40.
A. typica was commonly found in tropical fish species. A. typica was reportedly identified from the waters of the Southwest Atlantic, West and East Atlantic, the Mediterranean Sea, the Central Pacific. Latest data from IndoPacific waters, and free-living on bottlenose dolphins (Tursiops aduncus) from the Hurghada coastline in the northern Red Sea, Egypt (Palm et al., 2017).
Previous studies also reported that most Anisakis larvae that infect fish in Indonesian waters were identified as A. typica (Anshary et al., 2014 and Palm et al., 2017). Anisakis typica populations have been genetically detected over a wide geographical range, extending from 30 southern latitudes to 35 northern latitudes in warm and tropical climates (Simonetta Mattiucci & Nascetti, 2008). Whales (Kogia breviceps and Peponochephala electra) and dolphins (Sotalia guieanensis, Sotalia fluviatilis, and Stenella cyclimene) were identified as parentic hosts of A. typica found in Brazilian waters (Iniguez, Carvalho, Motta, Pinheiro, & Vicente, 2011 and S Mattiucci et al., 2002). According to KKP (2014) K. breviceps and P. electra as well as several species identified as Anisakis parentic hosts reportedly also migrated through the waters of East Nusa Tenggara. A. typica has been reported from marine fish around the world such as in Korea, Japan, China, Portugal, Taiwan, Brazil, Maroco, Papua New Guinea and the Mediterranean Sea (X. Q. Zhu et al., 2007; Farjallah et al., 2008; Umehara et al., 2010; and Koinari et al., 2013).
Genetic Characterization of A. typical. Isolate A. typica after observing its clinical symptoms; a PCR assay was performed to determine the genetic character of A. typica from skipjack tuna (Katsuwonus pelamis). The positive amplicon was identified as A. typica, carried out DNA purification. After that, sequencing and phylogenetic analysis were carried out. The results of PCR amplification using ITS primers (ITS 1.58S rDNA and ITS 2), showed that the individuals identified were A. typica in the 975 bp band (Figure 2).
Figure 2 - PCR assay results for A. typica species (At1 and At2 = samples; M = Marker)
Based on the alignment results in Figure 3, between At1 and At2 isolates against A. typica (Outgroup), showed that At1 and At2 isolates had high homologs. The results of the nucleotide/sequence sequences of At1 and At2 isolates with their isolates showed that there were differences in nucleotide base pairs or mutations in the isolates of A.typica isolates of At1 and At2, which indirectly changed the composition of their amino acids.
At_1
At_2
KC928262.1 KC9282 61.1 AB432909.1 AB432908.1
At_1
At_2
KC928262.1 KC928261.1 AB432909.1 AS432 90 8.1
At_1
At_2
KC928262.1 KC 92 82 61.1 AB432909.1 AB432908.1
At_1
At_2
KC928262.1 KC928261.1 AB432909.1 AB432 908.1
At_1
At_2
KC928262.1 KC928261.1 AB432909.1 AB432 90 8.1
----I----| ----I----I
510
GATCCACGAA CCGAGTGATC GATCCACGAA CCGAGTGATC
GATCCACGAA CCGAGTGATC GATCCACGAA CCGAGTGATC GATCCACGAA CCGAGTGATC
GATCCACGAA CCGAGTGATC
.1----| ----I----I
530 S4\
CACCGCCAAG ATTTGTACAT CACCGCCAAG ATTTGTACAT CACCGCCAAG ATTTGTACAT CACCGCCAAG ATTTGTACAT CACCGCCAAG ATTTGTACAT CACCGCCAAG ATTTGTACAT
----I----| ----I----I ----I----| ----I----I ----I----I----I----I
550 :c1 570 580 590 600
AATCATCATC AACAATGTCA AATGACAACA ATTGGTGTGT TTTGGGTTCT GGCGGCCACC AATCATCATC AACAATGTCA AATGACAACA ATTGGTGTGT TTTGGGTTCT GGCGGCCACC AATCATCATC AACAATGTCA AATGACAACA ATTGGTGTGT TTTGGGTTCT GGCGGCCACC AATCATCATC AACAATGTCA AATGACAACA ATTGGTGTGT TTTGGGTTCT GGCGGCCACC AATCATCATC AACAATGTCA AATGACAACA ATTGGTGTGT TTTGGGTTCT GGCGGCCACC AATCATCATC AACAATGTCA AATGACAACA ATTGGTGTGT TTTGGGTTCT GGCGGCCACC
----I----| ----|----I ----|----| ----I----I ----|----I ----|----| ----|----| ----|----I ----|----|----|----|
€10 620 630 640 650 660 670 6e0 690 700
TAGAGCGTGG CTCATTAAGT CTGCTCAACT CTGACGAGGA ACTTCCTCCG CATCTTTCAT CGCTACCGAC GGTCCAGGCG GAGGCTAGAT ACGGCGTAGA
TAGAGCGTGG CTCATTAAGT CTGCTCAACT CTGACGAGGA ACTTCCTCCG CATCTTTCAT CGCTACCGAC GGTCCAGGCG GAGGCTAGAT ACGGCGTAGA TAGAGCGTGG CTCATTAAGT CTGCTCAACT CTGACGAGGA ACTTCCTCCG CATCTTTCAT CGCTACCGAC GGTCCAGGCG GAGGCTAGAT ACGGCGTAGA TAGAGCGTGG CTCATTAAGT CTGCTCAACT CTGACGAGGA ACTTCCTCCG CATCTTTCAT CGCTACCGAC GGTCCAGGCG GAGGCTAGAT ACGGCGTAGA TAGAGCGTGG CTCATTAAGT CTGCTCAACT CTGACGAGGA ACTTCCTCCG CATCTTTCAT CGCTACCGAC GGTCCAGGCG GAGGCTAGAT ACGGCGTAGA TAGAGCGTGG CTCATTAAGT CTGCTCAACT CTGACGAGGA ACTTCCTCCG CATCTTTCAT CGCTACCGAC GGTCCAGGCG GAGGCTAGAT ACGGCGTAGA
----I----| ----|----I ----|----| ----I----| ----|----| ----|----| ----|----| ----|----| ----|----|----|----|
710 720 730 "4: 750 760 770 7B0 790 8C0
CGCCAAATTG GTCACCGTTG TTCAACAAGC GCACAGCCAC TCGTAAGCCA TTGTCTGCCC AACGAACGCA CAGCAGACGG CTATCACCAC CACAAACGTG CGCCAAATTG GTCACCGTTG TTCAACAAGC GCACAGCCAC TCGTAAGCCA TTGTCTGCCC AACGAACGCA CAGCAGACGG CTATCACCAC CACAAACGTG CGCCAAATTG GTCACCGTTG TTCAACAAGC GCACAGCCAC TCGTAAGCCA TTGTCTGCCC AACGAACGCA CAGCAGACGG CTATCACCAC CACAAACGTG CGCCAAATTG GTCACCGTTG TTCAACAAGC GCACAGCCAC TCGTAAGCCA TTGTCTGCCC AACGAACGCA CAGCAGACGG CTATCACCAC CACAAACGTG CGCCAAATTG GTCACCGTTG TTCAACAAGC GCACAGCCAC TCGTAAGCCA TTGTCTGCCC AACGAACGCA CAGCAGACGG CTATCACCAC CACAAACGTG CGCCAAATTG GTCACCGTTG TTCAACAAGC GCACAGCCAC TCGTAAGCCA TTGTCTGCCC AACGAACGCA CAGCAGACGG CTATCACCAC CACAAACGTG
- I -
■ I
- I -
- I -
.1 ----I----I ----|----I ----I----I ----I----I ----|----I ----|----I ----|----|
810 S20 630 840 e50 860 670 8B0 890 900
TACGAGTTTC CGTGCGGCTC ACAACAACAT GTAAATGGGC GGTATGCACG TTGGGAGACT TTTTCGTTTT GGATTCGCTC GATAATGATC CTTCCGCAGT
TACGAGTTTC CGTGCGGCTC ACAACAACAT GTAAATGGGC GGTAT GCACG TTGGGAGACT TTTTCGTTTT GGATTCGCTC GATAATGATC CTTCCGCAGT
TACGAGTTTC CGTGCGGCTC ACAACAACAT GTAAATGGGC GGTAT GCACG TTGGGAGACT TTTTCGTTTT GGATTCGCTC GATAATGATC CTTCCGCAG—
TACGAGTTTC CGTGCGGCTC ACAACAACAT GTAAATGGGC GGTAT GCACG TTGGGAGACT TTTTCGTTTT GGATTCGCTC GATAATGATC CTTCCGCAG—
TACGAGTTTC CGTGCGGCTC ACAACAACAT GTAAATGGGC GGTATGCACG TTGGGAGACT TTTTCGTTTT GGATTCGCTC GATAATGATC CTTCCGCAG—
TACGAGTTTC CGTGCGGCTC ACAACAACAT GTAAATGGGC GGTAT GCACG TTGGGAGACT TTTTCGTTTT GGATTCGCTC GATAATGATC CTTCCGCAG—
----I----| ----
910
GTTCACCCTA CAAG GTTCACCCTA CAAG GTTCACC-TA CAAG GTTCACC—TA CAAG
GTTCACC-------
GTTCACC-------
Figure 3 - Alignment Results in between At1 and At2 isolates against A. typica (Outgroup)
58
KM273048.1 Anisakis simplex KM273046.1 Anisakis simplex MF959775.1 Anisakis simplex JX535521.1 Anisakis simplex - MH197040.1 Anisakis pegreffii JQ900763.1 Anisakis pegreffii KF214797.1 Anisakis ziphidarum KF214799.1 Anisakis ziphidarum 791 KF214798.1 Anisakis ziphidarum KXD98558.1 Anisakis brevispiculata KXD98559.1 Anisakis brevispiculata k0<D98557.1 Anisakis brevispiculata GU295976.1 Anisakis paggiae GU295975.1 Anisakis paggiae GU295974.1 Anisakis paggiae GU295973.1 Anisakis paggiae At 1 At 2
KC9282G2.1 Anisakis typica KC928261.1 Anisakis typica AB432909.1 Anisakis typica
Figure 4 - Phylogenetic trees of At1 and At2 isolates with 19 comparative isolates
Molecular identification results by Palm et al. (2008) and Anshary et al. (2014) found A. typica as the dominant species in the waters of Bali and the Makassar Strait. Both of these waters were adjacent to the waters of East Nusa Tenggara. The results of A. typica in these two waters are inseparable from the life pattern of Anisakis nematodes, the distribution of skipjack tuna and the migration patterns of some marine mammals as parent hosts.
The waters of East Nusa Tenggara were the migration area of 30 species of Cetaceans, especially whales and dolphins. Whales and dolphins migrate from the Pacific Ocean to the Indian Ocean through Indonesian waters, especially through the waters of the Savu Sea, East Nusa Tenggara. The Savu Sea is a deep-sea bounded by the islands of Timor, Rote, Sumba, Flores, Solor, Alor, and Lembata and was reported to have a high diversity of cetaceans (there are 19 species of cetaceans in these waters) (Kahn, JamesKahn, & Pet, 2000).
CONCLUSION
The conclusions of this study were the results of morphological and PCR-sequencing of 2 samples observed from Savu sea , showed that the type I Anisakis species isolated from skipjack tuna was A. typica. The results of this study can be used to identify Anisakis spp parasites in Indonesian waters.
ACKNOWLEDGMENTS
We thank the Ministry of Marine Affairs and Fisheries, Republic of Indonesian and Fish Quarantine and Inspection agency Kupang for research funding (grant No.113/SJ/KP.532/III/2016).
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