Sequence and Haplotype Analyses of Ligula intestinalis in Acanthobrama Marmid (Cyprinidae) in Turkey

doi:10.21203/rs.3.rs-1629631/v1

Ligulosis caused by Ligula intestinalis adversely affects the fisheries carried out in the lakes and ponds of in Turkey. This parasite causes economic losses in the fish industry. In this study, it was aimed to reveal the molecular characterization of L. intestinalis isolates obtained from woodfish (Acanthobrama marmid) in Keban Dam Lake in Elazig province of Turkey by using mt-CO1 gene sequences and to determine the genetic differences and haplotypes between the isolates. In the examination made in terms of L. intestinalis, the intestine of the fish was opened with the help of fine-tipped scissors, the contents were allowed to come out, and the parasites were taken into a petri dish containing physiological water. Then, L. intestinalis plerocercoids were taken into 15 ml falcon tubes containing 70% ethanol and stored at -20ºC until further analysis. From each isolate, total gDNA was extracted from the plerocercoids. A partial (480 bp) mt-CO1 gene was amplified by PCR and sequenced unidirectionally. The final size of the trimmed sequences was 392 bp for 43 sequences. Sequence and haplotype analyses were performed, followed by phylogenetic analyses. All isolates were confirmed as L. intestinalis by BLAST analysis. In addition, 87 nucleotide mutation positions were determined among 43 CO1 gene sequences. As a result of the haplotype network performed for the mt-CO1 gene region of L. intestinalis isolates; arranged in a star-like configuration with the main haplotype (Hap05), separated from other haplotypes by 1–6 mutation steps, and 29 haplotypes were identified, covering 13.9% (6/43) of the total isolates. Also, 75 variable (polymorphic) sites were determined, 52 of which were parsimony informative sites.

Ligulosis

Ligula intestinalis

fish

haplotype

Turkey

Seafood is a cheaper and richer source of protein to meet animal protein needs, and it is an industry branch that has a significant proportion of the world's nutritional needs (Yuksel et al. 2011). Infection of fish, especially with parasitic diseases, causes significant problems in aquatic ecosystems. Sometimes even a parasite species can threaten the existing reserves (Dorucu 2008) Ligula intestinalis takes the first place among the cestodes among the parasitic agents in the fish. It has a significant prevalence in Turkey and creates a problem especially in dam lakes (Erer 2002). Ligulosis caused by Ligula intestinalis adversely affects the fisheries carried out in the lakes and ponds of in Turkey (Arslan et al. 2015; Doosti and Yılmaz 2020).

Ligula intestinalis, classified in the Diphyllobothrium family, is a cestode reaching 28 cm in adults and 40 cm in plerocercoids (Ciloglu et al. 2013; Hajirostamloo 2008). It is common in fresh waters in the Northern Hemisphere, including Turkey. This parasite causes economic losses in the fish industry (Ciloglu et al. 2013h; Hajirostamloo 2008). Ligula intestinalis; It has been reported in many fish families such as Cyprinidae, Catostomidae, Salmonidae or Galaxiidae. However, it shows a remarkable heterogeneity in host preference according to the geographical area studied (Bouzid et al. 2008a). Nowadays, the species in the Ligula taxon are classified as two genus (Ligula and Digramma) and four species (L. intestinalis, L. colymbi and D. interrupta, D. nemachili) according to their morphological and anatomical features (Dubinina 1980). Ligula intestinalis is a pseudophyllidean cestode species and has the general characteristics of cestodes. Scolex typically has dorsal and ventral bothriums. Sometimes bothriums are absent, and instead they have slits in the central part of the dorsal and ventral sides. The neck is quite short, almost absent. There are procercoid and plerocercoid stages in their development (Guralp 1974; Hoffman 1967; Tolgay 1973).

Ligula intestinalis is a pseudophyllidean cestode with a three-host life cycle. There are two larval stages, procercoid and pleroscoid stages in their life cycle. The dominant phase of the life cycle of the parasite is the pleroscoid phase, which lives in the body cavity of the fish (Guralp 1974; Hoffman 1967; Tolgay 1973). The first intermediate hosts are copepod species belonging to the Cyclops and Diaptomus genus. The larval form in these intermediate hosts are procercoids. The second intermediate hosts are freshwater fish, and their larval form is plerocercoids. Waterfowl such as seagulls, ducks, water divers and sea divers are also the last hosts (Olson et al. 2002). Freshwater fish are second intermediate hosts of L. intestinalis and become infected by eating parasitic copepods (Erer 2002; Olson et al. 2002; Islam 2019).

Plerocercoides of L. intestinalis most commonly develop in tench, red-eye, perch and pike perch. These plerocercoides are found in the abdominal cavity of fish. It causes the belly of the fish to be swollen and the swimming slows down. In severe infections, the abdomen bursts and causes the death of the fish. Plerocercoids exert mechanical pressure on the reproductive organs and destroy the gonads. However, it is known that it adversely affects reproductive physiology by secreting antigonadotropic hormone. This effect causes infertility and pernicious anemia in fish (Ozcel et al. 2016). In the fight against ligulosis, it is not practical and almost impossible to fight with copepod species belonging to the Cyclops and Diaptomus genus, which are the first intermediate host. In order to reduce the number of adult parasite-carrying and fish-eating waterfowl, measures should be taken periodically around the lake. Infected fish that move passively in the water should be collected and destroyed as much as possible (Akkaya et al. 1998; Arda et al. 2005; Ozcan and Kartal 2020)

Molecular techniques are used to identify species that cannot be distinguished morphologically according to their genetic differences (Aksakal and Erdogan 2007). Nuclear 28S rDNA and mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) gene sequencing of Ligula intestinalis are used for molecular identification of the parasite (Li et al. 2000). In the event that both the existing fish species in aquatic ecosystems and the species that are grown intensively in net cages being exposed to this parasitic effect, molecular identification is important in order to prevent economic depreciation and to take the necessary measures (Ozcan and Kartal 2020)

In this study, it was aimed to reveal the molecular characterization of L. intestinalis isolates obtained from woodfish (Acanthobrama marmid) in Keban Dam Lake in Elazig province of Turkey by using mt-CO1 gene sequences and to determine the genetic differences and haplotypes between the isolates.

2.1. Study location and samples collection

The fish to be examined for the study were obtained from Keban Dam Lake in Elazig province of Turkey between June and August, 2021. The fish were caught by fishing with gill nets number 22 in the lake. The fish, either alive or dead, were procured from the fishermen and brought to the Bingol University Faculty of Veterinary Fisheries Diseases Laboratory with ice in styrofoam boxes. After the external examination of the fish, the internal examination was carried out in accordance with the necropsy technique and examined for L. intestinalis plerocercoids. In the examination made in terms of L. intestinalis, the intestine of the fish was opened with the help of fine-tipped scissors, the contents were allowed to come out, and the parasites were taken into a petri dish containing physiological water. Then, L. intestinalis plerocercoids were taken into 15 ml falcon tubes containing 70% ethanol and stored at -20ºC until further analysis. The samples terminal segments were stained with aceto-carmine and mounted with the Canada balsam. The specimens were identified as L. intestinalis by using characters that are suitable for species identification according to taxonomic keys (Chubb et al. 1987). The morphological characterization of plerocercoid was completed by observing them under a light microscope.

2.2. Genomik DNA Isolation

Total genomic DNA (gDNA) was extracted using a Hibrigen Genomic DNA Isolation Kit (Hibrigen, Kocaeli- Turkey) according to the manufacturer’s instructions, with some slight modifications. The samples were washed five times using phosphate buffered saline (PBS) to remove the alcohol and then the plerocercoids was dissected into small segments using a scalpel, and transferred into Eppendorf tubes (1.5 ml). Two hundred microliters of lysis solution and 20 µl of Proteinase-K (20 mg/ml) were added to the tubes, mixed, and incubated overnight at 65°C. To obtain support the lysis, the samples were vortexed every 1 h for at least two to three times. The other day kit procedures were followed and total gDNA was isolated. The quantity of gDNA was determined using a device (Nanodrop Technologies, Wilmington, DE, USA) and those samples containing insufficient gDNA were re-extracted to obtain a sufficient amount of gDNA. The isolated gDNA’s were stored at -20°C until further use.

2.3. PCR Amplification of mt-CO1 gene fragment

To determine the genetic diversity of L. intestinalis isolates, the mt-CO1 gene region (480 bp) was amplified using specific primers COIA2 (5’CATATGTTTTGATTTTTTGG3’) and COIB2 (5’AKAACATAATGAAAATGAGC3’) as previously described (Bouzid et al. 2008b). The PCR reaction comprised: 10 X PCR buffer (5 µl), MgCl₂ (25 mM), dNTP's (400 uM), primers (each 20 pmol), TaqDNA Polimeraz (5 U/µl) ve PCR grade water (28.8 µl) and 5 µl of gDNA. The cycle parameters of the PCR reaction were: initial denaturation for 5 minutes at 95°C; 35 cycles of 45 seconds at 95°C, 45 seconds at 57°C, and 1 minute at 72°C; and a final extension step for 5 minutes at 72°C. A thermal cycler from SensoQuest GmbH (Göttingen, Germany) was used to perform PCR, and the PCR products were separated using 1.4% agarose gel electrophoresis.

2.4. DNA Sequences

PCR products of the mt-CO1 gene belonging to L. intestinalis isolates (n = 50) were sent to a commercial company (DNA Laboratory Systems, Istanbul, Turkey) for unidirectional Sanger DNA sequences.

2.5. Alignment and Phylogenetic Analysis of the sequences

FinchTV 1.4.0 was utilized to analyze the sequence data. BLAST search (www.ncbi.nlm.nih.gov/BLAST/) was used to compare and trim the sequences. The MEGA X tool was used to evaluate the trimmed sequences (Kumar et al. 2018). Using NCBI Pubmed published reference sequences as outgroups, alignment of the sequences was performed. The evolutionary tree was generated using the Maximum Likelihood method in MEGA X, which was after finding the most accurate and efficient model the find best DNA model, with MEGA X program (Kumar et al. 2018).

2.6. Haplotype networks, Nucleotide Polymorphism, Diversity, and Neutrality indices

Following a published protocol (Rozas et al. 2017), all sequence data were uploaded into the DnaSP6 software. The software was used to determine the diversity of population indices and neutrality indices. Population diversity indices included nucleotide diversity (πd), haplotype diversity (hd) and haplotype number (hn), while neutrality indices consisted of Tajima’s D (Tajima 1989), Fu’s statistics (Fu 1997), Fu’s Fs (Fu 1997), Fu and Li's D test (FLD), Fu and Li's F (FLF) statistics test and parsimony informative analysis were performed using DnaSP6. Data in the NEXUS output format were generated using DnaSP6 software. To create the haplotype network, the Minimum Spanning Networks (MSN) method was used (Bandelt et al. 1999) in the PopART-1.7 tool (Leigh and Bryant 2015).

A total of 50 Ligula intestinalis plerocercoids were obtained from the abdominal cavity of the fishes (Acanthobrama marmid) (Fig 1). To identify the exact species of the isolates, the mt-CO1 gene fragment (480 bp) was amplified using PCR. After sequence analysis, nucleotide sequences were compared with published reference sequences. The final size of the trimmed sequences was 392 bp and 7 sequences were excluded because of the low quality sequence reads although twice attempts. Totally 43 sequences of mt-CO1 samples were submitted to GenBank.

After the alignment, point mutations in the sequences were identified. Variated nucleotide positions based on the reference sequence are shown in the Table 1. Ligula intestinalis (MZ359928, NC039445) was used as the reference sequence for phylogenetic tree construction while Taenia saginata (AY684274) and Echinococcus granulosus (NC044548) sequences were used as outgroup. The most suitable phylogenetic tree model for the sequences was determined as Tamura-Nei+Gamma distribution (+G) by the MEGAX program. A phylogenetic tree was created with the bootstrap test (1000 replicates) by applying the Maximum Likehood statistical method and is shown in Fig 2.

Haplotype analysis of the 43 sequences with a size of 392 bp belonging to the mt-CO1 gene fragment of L. intestinalis isolates was performed. As a result of the haplotype network performed for the mt-CO1 gene region of L. intestinalis isolates; arranged in a star-like configuration with the main haplotype (Hap05), separated from other haplotypes by 1-6 mutation steps, and 29 haplotypes were identified, covering 13.9% (6/43) of the total isolates. (Fig 3; Table 2). As a result of haplotype analysis, 75 variable (polymorphic) sites were determined, 52 of which were parsimony informative sites. High haplotype variation and low nucleotide diversity were observed for the mt-CO1 gene fragment (Table 3). Population build-up and/or refinement of selection was indicated by Tajima’s D value. Uncommon haplotypes from recent population expansion or hitchhiking were expected because of the significant negative Fu’s Fs value. Hence, the fact that 79.3% (23/29) of the haplotype groups were single haplotypes supported these results. The sequences identity among the published reference sequence (NC_039445) and the current sequences (OM760000 - OM760042) for L. intestinalis are presented in Table 2. After a BLAST search several novel L. intestinalis haplotypes were arised. The Hap02 and Hap29 were matched 100% with the published sequences in the Genbank. Although, Hap06 and Hap21 had 99.74% similarity rate, Hap13 had the lowest similarity with 90.67%. Finally, 27 haplotypes were determined as unique haplotypes in the current study.

One of the health problems encountered in fish farming is parasitic diseases. In recent years, it has been reported that parasitic diseases are quite common in freshwater fish farming in our country. The diseases not only have harmful effects such as growth retardation and reproductive problems in fish, but also lead to death when the parasite population is high (Ozcan et al. 2019). The recognition of parasitic fish diseases and the investigation of their treatment are of great importance for the developing fishery sector today. Parasites are encountered intensively when the fish density is high, malnutrition and environmental conditions change. Weakness and intense parasitic invasions developing with increasing stress factors can be fatal for the fish (Arda et al. 2005; Cetin et al. 1983; Ekingen 1983).

In recent studies in Turkey, parasitic diseases are frequently encountered in fish species belonging to the cyprinidae family found in natural waters (Dorucu and Barata 2014; Gul et al. 2014; Ozbek and Ozturk 2010; Ozturk 2012; Vilizzi et al. 2015). Turkey has a rich location in terms of both its geographical location and water resources (lake, river, sea, etc.) and fish population. Thus, parasitic infections encountered in fish facilities and fisheries are very important for Turkey's economy. In our study, the presence of L. intestinalis infection in the Acanthobrama marmid species was investigated and the internal examinations of the fish were examined by necropsy technique and 50 L. intestinalis plerocercoids were obtained from the abdominal cavities of the fishes. In this study, detection of L. intestinalis parasite in A. marmid was in agreement with the studies in terms of detection of the parasite in freshwater fish. In some studies, the changing in L. intestinalis prevalance depend on the seasons, age groups and sex characteristics of the fish were investigated. (Kır et al. 2004, Kurupınar and Ozturk 2009, Ozbek and Ozturk 2010, Demirtas and Altındag 2011). Moreover, the effect of the parasite on the fish condition factor and physiological-anatomical structures was also investigated (Harris and Wheeler 1974; Uzbilek and Yıldız 2002, Ergonul and Altındag 2005, Museth 2001, Innal and Keskin 2006, Akmirza 2007, Tekin-Ozan and Barlas 2008).

Today, PCR-based DNA sequence analysis and other molecular methods are frequently preferred for the identification of parasite agents and their phylogenetic characterization. (Bouzid et al. 2008a). Studies on L. intestinalis species are mostly anatomical-morphologically based in Turkey (Innal et al. 2007), and the molecular studies are limited (Logan et al. 2004). However, genetic markers such as ITS and CO1 have been frequently used in recent years to accurately determine the taxonomic positions of organisms (Li et al. 2000). The traditional classification of ligulid cestodes Ligula (Bloch, 1782) and Digramma (Cholodkovsky, 1914) is a controversial issue. Especially, the low nucleotide diversity between the genus Ligula and Digramma indicates that Digramma is probably not an independent genus. For this reason, it showed that Ligula and Digramma should be considered as two species within the genus Ligula. As a result of the studies, it has been reported that ITS1, ITS2 and ND1 sequences are useful genetic markers to distinguish between Ligula and Digramma (Ahmadiara et al. 2018; Li et al. 2018; Li and Liao 2003; Luo et al. 2003). Lagrue et al. (2018) reported a case of Ligula sp. in fish species Gobiomorphus cotidianus and Oncorhynchus tshawytscha from Lake Hawea, South Island of New Zealand. Parasites have been quite recorded in large sizes (60–300 mm). In addition, low prevalences in fish populations suggested that the infection was rare or local. The ITS1 and ITS2 sequences confirmed that these isolates belong to the Ligula genus (Islam 2019). In present study, sequence of the mt-CO1 gene fragment was preferred to determine the genetic diversity and phylogenetic characterization of L. intestinalis isolates collected from Acanthobrama marmid fish species belonging to the Cyprinidae family. As a result all sequences were match with L. intestinalis in the current study.

Several studies have been carried out on the molecular identification of Ligula intestinalis infection, which causes serious yield and economic losses in fish facilities. Li et al. (2000), certain nucleotides of 393 bp for the CO1 gene of seven L. intestinalis samples were directly sequenced from each sample. Interestingly, no nucleotide variation was detected in the sequences of the CO1 gene among the seven Ligula samples studied. The authors reported that this homology in sequences may be due to the mutation patterns of the mitochondrial genome and the sequence similarity of CO1 genes of closely related taxon. In another study, a single pattern and reliable band were recorded in all gene sequences of 6 identified L. intestinalis isolates. The size of the bands were determined as 480 bp. No nucleotide variation was detected in any of the CO1 gene products of the samples. Only one mutation was detected at nucleotide 225 of one sample (Islam 2019). In support of the data of the above researchers, in this study similar Ligula intestinalis specific primers were used. A single pattern and a reliable band were recorded in all of the gene sequences of the 43 L. intestinalis isolates. The bands were 480 bp in size. All isolates were confirmed as L. intestinalis by BLAST analysis. In addition, 87 nucleotide mutation positions were determined among 43 CO1 gene sequences. This suggests that L. intestinalis plerocercoids in Acanthobrama marmid fish show higher nucleotide diversity compared to other fish species.

Bouzid et al. (2008b), a study was conducted to determine the phylogeny and genetic structure analysis of L. intestinalis according to geography and fish host preference. The analyzed data consisted of 109 parasites from 13 host fish species in 18 different locations on a macrogeographic scale. The two mitochondrial genes CO1 and cytb have also been used to study the population genetic structure of L. intestinalis on a local and global scale. Besides, the nuclear sequence of intergenic transcribed spacer 2 (ITS2) was used for genetic reconstruction. Different evolutionary diversity were found at local and global geographic scales. Although the ITS2 sequences were found to show significant intragenomic variability, their associations were generally stated to be in good agreement with the topology derived from mitochondrial genes. In another study, the genetic diversity of L. intestinalis was analyzed by inter-simple sequence repeat markers (ISSR). In order to investigate the genetic diversity of L. intestinalis populations, nine ISSR markers were applied to populations from nine geographical areas around the world and 10 host species. The 110 loci selected from the ISSR patterns produced revealed high variability among the analysed samples, with a polymorphism of 100% and a global coefficient of gene differentiation estimated by Nei's index (GST) of 0.776. Major genetic differentiation was associated with five broad geographic regions (Europe, China, Canada, Australia and Algeria). Nevertheless, no significant genetic variation was found, even though isolates in Europe were obtained from different geographic regions and different hosts. In conclusion, the ISSR approach has been shown to be rapid and inexpensive and provides reliable indicators to assess the genetic diversity of L. intestinalis (Bouzid et al. 2008a). In present study, we determined 29 haplotypes among the 43 L. intestinalis isolates and observed multiple nucleotide mutation positions (87 point mutations) within these 29 haplotypes. Among the analyzed sequences, the highly prevalent haplotypes were Hap05 and Hap12. The identification of 29 haplotypes in 43 isolates proved the significant genetic variability of L. intestinalis. According to the haplotype analysis for the mt-CO1 gene, high haplotype diversity and low nucleotide variation were noted. Tajima’s D value indicated expansion of the population and the influence of purifying selection. The negative value of Fu’s Fs indicated that the presence of the rare haplotypes occurred because of hitchhiking or recent population enlargement. Therefore, it was not surprising that there were 29 haplotypes out of 43 sequences. Besides, 79.3% (23/29) of the haplotype comprised a single sequence.

This study was conducted by taking into account that it is necessary the molecular characterization of L. intestinalis, which causes serious economic losses in fishing. Molecular characterization studies of L. intestinalis are limited in Turkey. As a result of this study, the haplotype analysis of L. intestinalis was revealed for the first time. Hence, the molecular characterization of L. intestinalis in woodfish (Acanthobrama marmid) was determined for the first time. The data of this study will also contribute to the epidemiology, control and treatment options of L. intestinalis infection, which is one of the most serious economic problems of the country's fishing. With this study, will open the way for more original and comprehensive research on molecular identification of parasitic disease agents in all regions of our country in the future.

Funding

This study has been supported by a grant from the Bingol University Scientific Research Projects Unit (Project No: BAP-VF.2021.00.002).

Conflicts of interest

All authors have read and agreed to the published version of the manuscript. The authors declare that they have no conflicts of interest.

Availability of data and material:

Not applicable

Author contributions

HKK, FC, SGK and SS contributed to the conception and design of the study. HKK, FC, CT, SGK and SS collected the samples, performed the laboratory activities and organized the database. HKK and SS wrote the manuscript. HKK, SS and AG critically oversaw the substantial revisions of the manuscript.

Ethics approval:

Not applicable

Consent to participate:

Not applicable

Consent for publication:

Uploaded

Ahmadiara E, Hosseini SH, Jalousian F, Mousavi HAE (2018) The taxonomic position and phylogenetic relationship between digramma interrupta and Ligula intestinalis based on morphological and molecular diagnosis. Iranian Journal of Parasitology 13(4):648
Akkaya H, Turkmen H, Vuruşaner C (1998) Balık parazitleri. Teknik yayınları, İstanbul
Akmirza A (2007) Ligula intestinalis L. plerocercoidlerinin acı balığın (Rhodeus amarus Bloch, 1782) büyümesi üzerine etkisi. J Black Sea/Mediterranean Environment 13:155-160
Aksakal E, Erdoğan O (2007) PCR-RFLP Uygulamalarının Su Ürünlerinde Kullanım Alanları. Türk Sucul Yaşam Dergisi 5:747-753
Arda M, Seçer S, Sarıeyyupoglu M (2005) Balık Hastalıkları. Medisan Yayın Serisi, Ankara
Arslan MO, Yilmaz M, Tasci GT (2015) Infections of Ligula intestinalis on freshwater fish in Kars plateau of north-eastern Anatolia, Turkey. Türkiye Parazitolojii Dergisi 39(3):218
Bandelt H-J, Forster P, Röhl A (1999) Median-joining networks for inferring intraspecific phylogenies. Molecular biology and evolution 16(1):37-48
Bouzid W, et al. (2008a) Genetic diversity of Ligula intestinalis (Cestoda: Diphyllobothriidea) based on analysis of inter‐simple sequence repeat markers. Journal of Zoological Systematics and Evolutionary Research 46(4):289-296
Bouzid W, et al. (2008b) Geography and host specificity: two forces behind the genetic structure of the freshwater fish parasite Ligula intestinalis (Cestoda: Diphyllobothriidae). International Journal for Parasitology 38(12):1465-1479
Cetin ET, Ang O, Toreci K (1983) Tıbbi Parazitoloji. İstanbul Tıp Fakültesi Yayınları, İstanbul
Chubb J, Pool D, Veltkamp C (1987) A key to the species of cestodes (tapeworms) parasitic in British and Irish freshwater fishes. Journal of Fish Biology 31(4):517-543
Ciloglu A and Biskin Z (2013) Balıklarda Görülen Parazit Hastalıkları: Respirator Sistem, Deri ve Bağ Dokularda Cestod ve Nematod Hastalıkları. In: Ozcel MA(ed) Veteriner Hekimliğinde Parazit Hastalıkları, Turkiye Parazitol Dern Yay, Izmir, pp 1426s
Demirtas M, Altındag A (2011) Terkos Gölü'ndeki Bazı Balıklarda (Cyprinidae) Ligula intestinalis Plerocercoid L., 1758 Enfeksiyonunun Mevsimsel Dağılımı. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi 22(3):147-151
Doosti S, Yılmaz F (2020) Occurrence of Ligula sp. plerocercoids in Ladigesocypris irideus (Ladiges, 1960) from South-Western Turkey: new host and new locality records. Journal of the Institute of Science and Technology 10(4):2416-2423
Dorucu M (2008) Paraziter balik hastaliklari ve ilaçla tedavileri. Ecological Life Sciences 3(2):372-380
Dorucu M, Barata S (2014) Karakaya Baraj Gölü Kömürhan Bölgesinden yakalanan bazı balıklarda endohelmintlerin araştırılması.
Dubinina MN (1980) Tapeworms (Cestoda, Ligulidae) of the fauna of the USSR. Tapeworms (Cestoda, Ligulidae) of the fauna of the USSR
Ekingen G (1983) Tatlı Su Balık Parazitleri, Fırat Üniversitesi Su Ürünleri Yüksek Okulu FÜ Basımevi, Elâzığ.
Erer H (2002) Balık hastalıkları. Selçuk Üniversitesi Basımevi, Konya
Ergonul M, Altındag A (2005) Ligula intestinalis pleurocercoidlerinin kadife balığının büyüme özelliklerine etkisi. Turkish J Vet Anim Sci 29:1337-1341
Fu Y-X (1997) Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147(2):915-925
Gul A, Ispir U, Turk C, Kırıcı M, Taysı M, Yonar M (2014) The Investigation of Diplostomum sp. Metacercariae in Some Cyprinids from Murat River (Genç Area), Bingöl, Turkey. Türk Tarım ve Doğa Bilimleri Dergisi 1(4):547-551
Guralp N (1974) Helmintoloji. Ankara Universitesi Veteriner Fakultesi Yayınları, Ankara
Hajirostamloo M (2008) The occurrence and parasite-host of Ligula intestinalis in Sattarkhan Lake (East Azerbaijan-Iran).
Harris MT, Wheeler A (1974) Ligula infestation of bleak Alburnus alburnus (L.) in the tidal Thames. Journal of Fish Biology 6(2):181-188
Hoffman GI (1967) Parasites of North American Freshwater Fishes. University of California Pres, Berkeley and Los Angeles
Innal D, Keskin N (2006) The Infection of European Chub Leuciscus cephalus L 1758 with Ligula intestinalis Plecercoids in Çamkoru Lake Turkey. Journal of Animal and Veterinary Advances, 5 (2): 108-110.
Innal D, Keskin N, Erk'akan F (2007) Distribution of Ligula intestinalis (L.) in Turkey. Turkish Journal of Fisheries and Aquatic Sciences 7: 19-22
Islam E (2019) Dna dizisi tabanlı olarak Ligula intestinalis L.'in moleküler tanımlaması. Dissertation, Afyon Kocatepe Üniversitesi
Kır I, Ayvaz Y, Barlas M, Ozan ST (2004) Karacaören I Baraj Gölü’nde yaşayan Sazan (Cyprinus carpio L., 1758)’lardaki parazitlerin mevsimsel dağılımları ve etkileri. Türkiye Parazitoloji Dergisi 28(1):45-49
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular biology and evolution 35(6):1547
Kurupınar E, Ozturk MO (2009) Mevsimsel değişime ve boy büyüklüğüne bağlı olarak Leuciscus cephalus l.’un (Örenler Baraj Gölü, Afyonkarahisar) helmint faunası üzerine bir araştırma. Türkiye Parazitoloji Dergisi 33(3):248-253
Lagrue C, Presswell B, Dunckley N, Poulin R (2018) The invasive cestode parasite Ligula from salmonids and bullies on the South Island, New Zealand. Parasitology research 117(1):151-156
Leigh JW, Bryant D (2015) POPART: full-feature software for haplotype network construction. Methods in Ecology and Evolution 6(9):1110-1116
Li J, Liao X (2003) The taxonomic status of Digramma (Pseudophyllidea: Ligulidae) inferred from DNA sequences. Journal of Parasitology 89(4):792-799
Li J, Liao X, Yang H (2000) Molecular characterization of a parasitic tapeworm (Ligula) based on DNA sequences from formalin-fixed specimens. Biochemical Genetics 38(9):309-322
Li WX, Fu PP, Zhang D, Boyce K, Xi BW, Zou H, Li M, Wu SG, Wang GT (2018) Comparative mitogenomics supports synonymy of the genera Ligula and Digramma (Cestoda: Diphyllobothriidae). Parasit Vectors 11(1):324.
Logan FJ, Horák A, Štefka J, Aydogdu A, Scholz T (2004) The phylogeny of diphyllobothriid tapeworms (Cestoda: Pseudophyllidea) based on ITS-2 rDNA sequences. Parasitology Research 94(1):10-15
Luo H, Nie P, Yao W, Wang G, Gao Q (2003) Is the genus Digramma synonymous to the genus Ligula (Cestoda: Pseudophyllidea)? Parasitology research 89(5):419-421
Museth J (2001) Effects of Ligula intestinalis on habitat use, predation risk and catchability in European minnows. Journal of Fish Biology 59(4):1070-1080
Olson P, Littlewood D, Griffiths D, Kennedy C, Arme C (2002) Evidence for the co-existence of separate strains or species of Ligula in Lough Neagh, Northern Ireland. Journal of Helminthology 76(2):171-174
Ozbek M, Ozturk MO (2010) Kunduzlar Baraj Gölü (Kırka, Eskişehir)’nde yaşayan bazı balıkların Ligula intestinalis plerocercoid L., 1758 enfeksiyonu üzerine araştırmalar. Türkiye Parazitoloji Dergisi 34(2):112-117
Ozcan M, Kartal B (2020) Molecular Identification of the Parasite Ligula intestinalis Identified in Alburnus adanensis. Asian Journal of Science and Technology 11(1): 10700-10707.
Ozcan M, Yılmaz Y, Donat E, Kılavuz D, Tuncel M (2019) A Research On Endoparasıtıc Fauna In Fısh Specıes Caught In Menzelet Dam Lake Kahramanmaraş (Turkey). Middle East Journal of Science 5(1):33-40
Ozcel MA, Inci A, Koroglu E, Karaer Z, Eren H (2016) Veteriner hekimliğinde parazit hastalıkları. Türkiye Parazitoloji Derneği
Ozturk M (2012) Emre Baraj Gölü (Afyonkarahisar)'ndeki Üç Cyprinid Balık Türünün Argulus foliaceus Enfeksiyonu Üzerine Araştırma. İstanbul Üniversitesi Veteriner Fakültesi Dergisi 38(2):183-190
Rozas J, et al. (2017) DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular biology and evolution 34(12):3299-3302
Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123(3):585-595
Tekin-Ozan S, Barlas M (2008) Concentrations of selected heavy metals in Ligula intestinalis L., 1758 plerocercoids (Cestoda) compared to it host’s (Tinca tinca L., 1758) organs from Beyşehir Lake (Turkey). Helminthologia 45(2):76-80
Tolgay N (1973) Evcil ve yabani kanatlıların önemli parazitleri. A.U. Vet. Fak. Yayın no: 249, Ankara
Uzbilek MK, Yıldız HY (2002) A report on spontaneous diseases in the culture of grass carp (Ctenopharyngodon idella Val. 1844), Turkey. Turkish Journal of Veterinary and Animal Sciences 26(2):407-410
Vilizzi L, Tarkan AS, Ekmekci FG (2015) Parasites of the common carp Cyprinus carpio L., 1758 (Teleostei: Cyprinidae) from water bodies of Turkey: updated checklist and review for the 1964-2014 period. Turkish Journal of Zoology 39(4):545-554
Yuksel F, Kuzgun NK, Ozer EI (2011) Tunceli ili balık tüketim alışkanlığının belirlenmesi. Karadeniz Fen Bilimleri Dergisi 2(3):28-36

Table 1

Nucleotide variation positions of the mt-CO1 (392 bp) gene among 29 haplotypes analyzed.

Nucleotid positions

14

18

21

22

25

27

31

34

35

36

39

40

42

45

46

48

54

59

60

63

69

72

78

92

NC_039445 (Reference sequence)

G

T

C

T

A

G

C

G

T

G

C

T

C

A

T

Hap01

C

T

C

T

G

Hap02

T

C

T

G

Hap03

C

T

C

T

G

Hap04

C

T

C

T

G

Hap05

C

T

C

T

G

Hap06

T

C

T

G

Hap07

C

T

C

T

G

Hap08

C

T

C

T

G

Hap09

C

T

C

T

G

Hap10

C

T

C

T

G

Hap11

C

T

C

T

G

Hap12

C

T

C

T

G

Hap13

C

T

C

T

G

Hap14

T

C

T

C

T

G

Hap15

C

T

C

T

G

Hap16

C

T

C

T

G

Hap17

C

T

C

T

A

T

G

Hap18

C

T

C

G

C

T

G

Hap19

C

T

C

T

A

T

G

Hap20

C

T

C

T

A

T

G

Hap21

T

C

T

G

Hap22

C

T

C

T

G

Hap23

T

C

T

G

Hap24

T

C

T

C

T

G

C

Hap25

T

C

T

C

T

G

Hap26

C

T

C

T

G

Hap27

C

T

C

T

G

Hap28

C

T

C

T

G

Hap29

T

C

T

G

Nucleotid positions

93

96

99

102

105

108

117

120

123

132

136

138

142

145

150

157

159

162

165

167

171

172

174

180

NC_039445

(Reference sequence)

C

T

A

T

A

C

T

C

A

G

T

G

T

C

G

A

G

C

G

Hap01

G

T

A

T

G

T

Hap02

T

G

T

C

T

Hap03

G

T

A

T

G

T

Hap04

G

A

T

Hap05

G

A

T

Hap06

T

G

T

C

T

Hap07

G

A

T

Hap08

G

T

A

T

Hap09

G

T

A

T

G

T

Hap10

G

A

T

G

T

Hap11

G

T

A

T

Hap12

G

A

T

G

T

Hap13

G

C

G

T

A

T

A

T

C

T

Hap14

G

A

T

A

T

C

T

Hap15

G

C

G

A

T

Hap16

G

A

T

Hap17

G

A

T

Hap18

G

A

T

A

T

Hap19

G

A

T

Hap20

G

T

A

T

G

A

T

Hap21

T

G

T

C

T

Hap22

G

T

A

T

G

T

Hap23

T

G

T

A

C

T

Hap24

T

C

G

T

C

T

G

C

T

Hap25

G

A

T

Hap26

G

T

A

T

G

T

Hap27

G

T

A

C

T

Hap28

G

A

T

Hap29

T

G

T

C

T

Nucleotid positions

182

186

188

189

193

195

196

198

199

201

202

204

213

222

225

231

240

243

253

255

273

282

285

288

NC_039445

(Reference sequence)

T

G

C

A

T

G

C

A

T

G

C

T

C

T

A

C

T

C

Hap01

A

T

C

G

T

C

T

Hap02

T

C

T

Hap03

A

T

C

G

T

C

T

Hap04

A

T

C

G

T

C

T

Hap05

A

T

C

G

T

C

T

Hap06

T

C

T

Hap07

A

T

C

G

T

C

T

Hap08

A

T

C

G

T

C

T

Hap09

T

A

T

C

G

T

C

T

Hap10

A

T

C

G

T

C

T

Hap11

A

T

C

G

T

C

T

Hap12

A

T

C

G

T

C

T

Hap13

A

C

A

T

C

T

A

T

A

T

C

G

T

C

T

Hap14

A

T

C

G

T

C

T

Hap15

A

T

C

G

T

C

T

Hap16

A

T

C

G

T

C

T

Hap17

A

T

C

G

T

C

T

Hap18

T

A

C

A

T

C

G

T

C

T

Hap19

A

T

C

G

T

C

T

Hap20

A

T

A

T

C

G

T

C

T

Hap21

T

C

T

Hap22

A

T

C

G

T

C

T

Hap23

T

C

T

Hap24

T

C

T

Hap25

A

T

C

G

T

C

T

Hap26

C

A

T

C

G

T

C

T

Hap27

A

C

T

Hap28

A

T

C

G

T

C

T

Hap29

T

C

T

C

T

Nucleotid positions

294

297

301

306

318

321

399

342

345

350

351

366

369

373

381

NC_039445 (Reference sequence)

T

A

G

C

T

G

A

T

A

T

G

C

Hap01

C

A

T

C

G

A

G

T

Hap02

A

C

T

A

G

C

T

Hap03

C

A

T

C

G

A

G

T

Hap04

C

T

A

C

G

A

G

T

Hap05

C

T

A

C

G

A

G

T

Hap06

A

C

T

A

G

C

T

Hap07

C

T

A

C

G

A

G

T

Hap08

C

T

A

C

G

A

G

T

Hap09

C

A

T

C

G

A

G

T

Hap10

C

A

T

C

G

A

G

T

Hap11

C

T

A

C

G

A

G

T

Hap12

C

A

T

C

G

A

G

T

Hap13

C

T

A

C

G

A

G

T

Hap14

C

A

T

C

G

A

G

T

Hap15

C

T

A

C

G

A

G

T

Hap16

C

T

A

C

G

A

G

T

Hap17

C

T

A

C

G

A

G

T

Hap18

C

T

A

C

G

A

G

A

T

Hap19

C

T

A

C

G

A

G

T

Hap20

C

A

T

C

G

A

G

T

Hap21

A

C

T

A

G

C

T

Hap22

C

A

T

C

G

A

G

T

Hap23

A

C

T

A

G

C

A

C

T

Hap24

A

C

T

A

G

C

T

Hap25

C

T

A

C

G

A

G

T

Hap26

C

A

C

G

A

G

T

Hap27

A

C

T

A

G

C

T

Hap28

C

T

A

C

G

A

G

T

Hap29

A

C

T

A

G

C

T

Table 2

Haplotypes of mt-CO1 sequences of Ligula sp. and accession numbers of isolates forming groups.

No	Haplotype name	Number of isolates	Isolate codes (Accession numbers)
1	Hap01	1	TRLig01(OM760000)
2	Hap02	2	TRLig02(OM760001) TRLig31(OM760030)
3	Hap03	1	TRLig03(OM760002)
4	Hap04	1	TRLig04(OM760003)
5	Hap05	6	TRLig05(OM760004) TRLig06(OM760005) TRLig12(OM760011) TRLig15(OM760014) TRLig27(OM760026) TRLig39(OM760038)
6	Hap06	1	TRLig07(OM760006)
7	Hap07	1	TRLig08 (OM760007)
8	Hap08	3	TRLig09(OM760008) TRLig16(OM760015) TRLig21(OM760020)
9	Hap09	1	TRLig10(OM760009)
10	Hap10	1	TRLig11(OM760010)
11	Hap11	2	TRLig13(OM760012) TRLig42(OM760041)
12	Hap12	5	TRLig14(OM760013) TRLig19(OM760018) TRLig35(OM760034) TRLig38(OM760037) TRLig43(OM760042)
13	Hap13	1	TRLig17(OM760016)
14	Hap14	1	TRLig18(OM760017)
15	Hap15	1	TRLig20(OM760019)
16	Hap16	2	TRLig22(OM760021) TRLig37(OM760036)
17	Hap17	1	TRLig23(OM760022)
18	Hap18	1	TRLig24(OM760023)
19	Hap19	1	TRLig25(OM760024)
20	Hap20	1	TRLig26(OM760025)
21	Hap21	1	TRLig28(OM760027)
22	Hap22	1	TRLig29(OM760028)
23	Hap23	1	TRLig30(OM760029)
24	Hap24	1	TRLig32(OM760031)
25	Hap25	1	TRLig33(OM760032)
26	Hap26	1	TRLig34(OM760033)
27	Hap27	1	TRLig36(OM760035)
28	Hap28	1	TRLig40(OM760039)
29	Hap29	1	TRLig41(OM760040)

Table 3

Diversity and neutrality indices obtained using nucleotide data of the Ligula sp. mt-CO1 gene (392 bp).

Table 4

Sequence identity (%) between mt-CO1 gene lineages of Ligula intestinalis.

Sequence identity (%) between the reference genome of Ligula intestinalis and the mt-CO1 gene strains of the haplotypes detected in this study.

	NC_039445	Hap01	Hap02	Hap03	Hap04	Hap05	Hap06	Hap07	Hap08	Hap09	Hap10	Hap11	Hap12	Hap13	Hap14	Hap15	Hap16	Hap17	Hap18	Hap19	Hap20	Hap21	Hap22	Hap23	Hap24	Hap25	Hap26	Hap27	Hap28	Hap29
NC_039445	100.00	89.29	92.60	90.82	90.82	91.07	92.35	90.56	90.82	89.54	90.31	90.82	90.56	85.97	88.78	90.82	90.82	90.31	88.78	90.82	90.05	92.35	90.31	92.35	91.07	90.82	90.56	92.60	90.82	92.35
Hap01		100.00	89.80	98.47	97.70	97.70	89.54	97.96	97.45	99.74	98.98	97.96	98.72	94.90	97.70	97.45	97.45	97.45	96.68	97.45	97.70	90.05	98.47	90.31	89.54	96.94	97.70	92.86	97.45	89.80
Hap02			100.00	91.33	90.31	90.56	99.74	90.05	90.82	90.05	90.82	90.31	91.07	85.46	89.29	90.31	90.31	89.80	88.27	90.31	90.56	99.74	90.82	99.49	97.96	90.31	90.56	96.94	90.31	99.74
Hap03				100.00	98.72	98.72	91.07	98.47	98.47	98.72	99.49	98.47	99.74	93.37	96.68	98.47	98.47	97.96	96.68	98.47	98.72	91.58	99.49	91.84	91.07	97.96	98.72	93.88	98.47	91.33
Hap04					100.00	99.74	90.05	99.74	99.49	97.96	98.72	99.49	98.98	94.64	96.43	99.49	99.49	98.98	97.96	99.49	97.96	90.56	98.72	91.84	89.80	98.98	98.47	95.15	99.49	90.31
Hap05						100.00	90.31	99.49	99.74	97.96	98.72	99.74	98.98	94.39	96.17	99.74	99.74	99.23	97.70	99.74	97.96	90.82	98.72	91.07	89.80	99.23	98.47	93.62	99.74	90.56
Hap06							100.00	89.80	90.56	89.80	90.56	90.05	90.82	85.20	89.03	90.05	90.05	89.54	88.01	90.05	90.31	99.49	90.56	99.23	97.70	90.05	90.31	96.68	90.05	99.49
Hap07								100.00	99.23	98.21	98.98	99.74	98.72	94.90	96.68	99.23	99.23	99.23	98.21	99.23	97.70	90.31	98.47	99.23	89.54	98.72	98.21	93.11	99.23	90.05
Hap08									100.00	97.70	98.47	99.49	98.72	94.13	95.92	99.49	99.49	98.98	97.45	99.49	97.70	91.07	98.47	91.33	90.05	98.98	98.21	93.37	99.49	90.82
Hap09										100.00	99.23	98.21	98.98	94.64	97.45	97.70	97.70	97.70	96.94	97.70	97.96	90.31	98.72	90.56	89.80	97.19	97.96	93.11	97.70	90.05
Hap10											100.00	98.98	99.74	93.88	97.19	98.47	98.47	98.47	97.19	98.47	98.72	91.07	99.49	91.33	90.56	97.96	98.72	93.88	98.47	90.82
Hap11												100.00	98.72	94.64	96.43	99.49	99.49	99.49	97.96	99.49	97.70	90.56	98.47	90.82	89.54	98.98	98.21	93.37	99.49	90.31
Hap12													100.00	93.62	96.94	98.72	98.72	98.21	96.94	98.72	98.98	91.33	99.74	91.58	90.82	98.21	98.98	94.13	98.72	91.07
Hap13														100.00	94.13	94.64	94.13	94.39	94.13	94.13	93.62	85.71	93.37	85.97	85.46	93.62	93.11	88.52	94.64	85.20
Hap14															100.00	95.92	95.92	95.92	94.90	95.92	96.94	89.54	96.68	89.80	88.78	96.94	96.43	91.84	95.92	89.29
Hap15																100.00	99.49	98.98	97.45	99.49	97.70	90.56	98.47	90.82	90.05	98.98	98.21	93.37	99.49	90.31
Hap16																	100.00	98.98	97.45	99.49	97.70	90.56	98.47	90.82	89.54	98.98	98.21	93.37	99.49	90.31
Hap17																		100.00	97.70	99.49	97.70	90.05	97.96	90.31	89.03	98.47	97.70	92.86	99.23	89.80
Hap18																			100.00	97.45	95.92	88.52	96.68	88.78	87.76	96.94	96.43	91.33	97.96	88.27
Hap19																				100.00	98.21	90.56	98.47	90.82	89.54	98.98	98.21	93.37	99.49	90.31
Hap20																					100.00	90.82	98.72	91.07	90.31	97.19	98.47	93.62	97.70	90.56
Hap21																						100.00	91.07	99.23	97.70	90.56	90.82	97.19	90.56	99.49
Hap22																							100.00	91.33	90.56	97.96	98.72	93.88	98.47	90.82
Hap23																								100.00	97.45	90.82	91.07	96.94	90.82	99.23
Hap24																									100.00	89.54	90.31	93.62	89.54	97.70
Hap25																										100.00	97.70	92.86	98.98	90.31
Hap26																											100.00	93.62	98.21	90.56
Hap27																												100.00	93.37	96.68
Hap28																													100.00	90.31
Hap29																														100.00

No competing interests reported.

Sequence and Haplotype Analyses of Ligula intestinalis in Acanthobrama Marmid (Cyprinidae) in Turkey

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Material And Methods

2.1. Study location and samples collection

2.2. Genomik DNA Isolation

2.3. PCR Amplification of mt-CO1 gene fragment

2.4. DNA Sequences

2.5. Alignment and Phylogenetic Analysis of the sequences

2.6. Haplotype networks, Nucleotide Polymorphism, Diversity, and Neutrality indices

3. Results

4. Discussion

5. Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Journal Publication

Version 1