Molecular Identification of Parasitic Copepods Using Cytochrome c Oxidase Subunit 1 from Chinese Marine Waters

It has been reviewed that less attention has been given to parasitic copepods of mollusc species found in Chinese coastal waters and meager information is available about their morphological and molecular based taxonomy. Therefore, this study is designed to understand the taxonomic status of parasitic co-pepods of molluscs inhabiting Chinese coastal waters using cytochrome c oxidase subunit 1 (COX1) molecular marker. The results revealed seven parasitic copepods i.e. Conchyliurus quintus, Lichomolgus similis, Modiolicola bifida, Mytilicola orientalis, Trochicola japonica, Ostrincola koe and Pseudomyicola spinosus . All the sequences were submitted to NCBI, under accession numbers OQ725274-OQ725305. Evolutionary distance within families and between families ranged from 0.007 to 0.113 and 0.299 to 0.350 respectively. Pairwise distance ranged from 0.186 to 0.360. The neighbor joining phylogenetic tree described three main clades of these species. The identification of seven parasitic species of copepods found in Chinese marine waters is confirmed by the current investigation. The present study will be useful for future taxonomic investigations.


INTRODUCTION
T o date approximately 280 parasitic copepods of mollusc species have been reported as parasites or associates of molluscs (Boxshall and O'Reilly, 2015), and more than 430 species of molluscs were known to be hosts of copepods, with most of them being bivalves (Humes, 1994;Kim, 2004).The study of the marine mollusc parasitic copepod fauna from China seas is relatively limited and short of systematic taxonomy.
So far, 14 species of copepod parasitic species have been found with mollusc hosts (Humes and Lee, 1985;Humes and Boxshall, 1988;Ho and Zheng, 1994;Lin and Ho, 1999;Ho et al., 2012;Du et al., 2018;Du and Sun, 2022), from the Yellow Sea, including 9 species (Ostrincola The aim of the present study was to identify parasitic copepod of molluscs applying molecular approaches supported by morphological features.To our knowledge, it is a first attempt to identify the parasitic copepods using molecular based approach in China.

Sample collection
A total of 32 species and 8628 individuals of bivalves were sampled from Shandong, Jiangsu and Liaoning provinces along the coasts of Yellow Sea and Bohai Sea (Du and Sun, 2022).After being washed with filtered water, each mollusc was opened in a petri dish containing a small amount of filtered sea water.The parasitic copepods were isolated under a dissecting microscope (Zeiss Stemi,508).Some copepod specimens were isolated from the hosts that were sampled from seven localities along the coasts of Yellow Sea and Bohai Sea and then fixed and stored in 75% ethanol until study (Fig. 1).The parasitic species were first identified using major morphological taxonomic characters (Huys and Boxshall, 1991;Boxshall and Halsey, 2004).

Molecular procedures
Genomic DNA from each of 32 samples was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer's instructions.The COX1 region was amplified by polymerase chain reaction (PCR) using the forward primer LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and the reverse primer HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al., 1994).The total volume of 20 µl of PCR reaction mixture comprised 5.8 µl of distilled water, 2 µl of DNA template, 1 µl each of the forward and reverse primers (Diluted 20 times for using), 2× reaction mix 10 µl and golden DNA polymerase (Beijing, Tiangen, China) 0.2 µl.The thermo-cycling profile was 5 min at 94 °C for initial denaturation, 30 sec at 94 °C for denaturation, 30 sec at 50°C for annealing, 1 min at 72 °C for extension, and 7 min at 72 °C for final extension.The thermal cycle from denaturation to extension was repeated 35 times.
PCR products were checked on Gold View-stained 1.5% agarose gels.Sanger dideoxy sequencing of each sample was carried out on both strands.The DNA sequences obtained herein were deposited in the National Center for Biotechnology Information (NCBI) database (http:// www.ncbi.nlm.nih.gov) and compared (using the Clustal W) with those available in the GenBank database.The accession number of each sequence is submitted under accession numbers OQ725274-OQ725305 in Table I.

RESULTS
Seven bivalves including Ruditapes philippinarum, Meretrix petechialis, Mactra chinensis, Mytilus galloprovincialis, Mactra quadrangularis, Leukoma jedoensis, and Crassostrea gigas hosts were found to harbor seven parasitic copepods of the order Cyclopoida (Table I).The parasitic species were first identified using taxonomic characters.The molecular identification using 621 bp of COX1 molecular marker sequence revealed

Myicolidae
Clausidiidae The average evolutionary distances within families were ranging from 0.007 to 0.113 (Table II), whereas those between families ranged from 0.299 to 0.350 (Table III).
The pairwise distance ranged from 0.186 to 0.360 (Table IV).The neighbor joining results revealed three major clades and certain sub-clades belongs to four families (Fig. 2).Only the individuals of family Lichomolgidae showed differentiated clades whereas the rest of the families showed sub-clades within a single major clade.

DISCUSSION
According to Ho and Kim (1991), the edible bivalve species are commercially important, and their populations experience mass mortalities as a result of various problems including parasitic copepods as causative agent of such mass mortality.Consequently, there are instability in the bivalve species' culture.Parasitic copepods are one of the many factors that contribute to mass mortality, like in the case of the Meretrix meretrix mass mortality in Jiangsu's southern shore (Ho and Zheng, 1994).Meretrix lusoria had mass mortality in the early 1970s, which contributed to the abrupt fall in annual productivity (Ho and Kim, 1991).The parasitic species like C. quintus, M. bifida, O. koe, and P. spinosus depends on variety of bivalve species, and these have negative impact on numerous host species which are significant from an economic perspective (Tanaka, 1961;Ko et al., 1962;Humes, 1968;Ko and Yoshikoshi, 1974;Do and Kajihara, 1986;Yoosukh, 1991;Ho and Zheng, 1994;Ho and Kim, 1991).According to Ho (1992), there are 51 different species of bivalves worldwide that contain P. spinosus.Fundamentally, copepod identification is based on anatomical and morphological characters (Huys and Boxshall, 1991;Boxshall and Halsey, 2004), yet, molecular identification of these significant microscopic organisms has received relatively less attention.Therefore, in order to understand the relationships between the parasitic copepod molecular-based identification of copepods and their phylogenetic studies are of utmost relevance.The traditional method of identification and associated investigations are being quickly replaced by molecular techniques (Khodami et al., 2019).Parasitic copepods can find a wide variety of hosts in marine environments, practically in every phylum (Boxshall and Halsey, 2004).

O n l i n e F i r s t A r t i c l e
In China, the molecular based identification of parasitic copepods associated with marine bivalve species have not been reported yet, however, the parasites of fish have, such as the work by Song et al. (2008).The traditional based identification can cause confrontations because of complex structure and way of understanding of morphofeatures, usually the taxonomic used certain character keys, and these keys some time being misinterpreted and misunderstood, therefore, the present study designed to identify the parasitic copepods of bivalve species using COX1.
The outcome of the present study consists of seven species which are re-confirmed using molecular marker such as, C. quintus, L. similis, M. bifida, M. orientalis, O. koe, P. spinosus and T. japonica.Earlier investigations on parasitic copepods of mollusc focused the identifications based on the morphological taxonomic characters (Humes and Lee, 1985;Humes and Boxshall, 1988;Ho and Zheng, 1994;Lin and Ho, 1999;Ho et al., 2012;Du et al., 2018;Du and Sun, 2022).To complement the study of the phylogenetic relationships among parasitic copepods of molluscs based on the morphological characters, a different approach employing molecular data is necessary (Elsner et al., 2010;Feis et al., 2019).
It has been said that partial COX1 region in 600 bp in length is valuable to identify out a cryptic species but might not be possible to conclude the phylogenetic relationship in certain copepods (Machida and Tsuda, 2010;Blanco-Bercial et al., 2011, 2014;Aarbakke et al., 2014).Nevertheless, several publications on different copepods provides profound importance of COX1 in taxonomy of species.Bucklin et al. (2010) detailed as the sequence for determining the difference between species, Laakmann et al. (2013) used proteome finger printing and DNA sequencing for comparison of calanoid copepods and the authors used several markers like 18S rDNA, COX1 etc.As mentioned above, COX1 molecular marker also has profound importance for phylogenetic studies for

O n l i n e F i r s t A r t i c l e
Molecular Identification of Parasitic Copepods Using COX1 5 diversified taxa, such as Aarbakke et al. (2014) investigated the five sibling species of Pseudocalanus demography and phylogeny.Kozol et al. (2012), used multigene approach to find the divergence between two Calanus species.The phylogenetic study of present investigation denoted three main groups along with sub-clades.
In the current investigation, these seven parasitic copepod species were reported on seven different bivalve hosts (Table I).The present research confirms the identification of seven copepod parasite species using molecular marker.

CONCLUSION
Rare are the molecular-based investigations of copepods from Chinese waters.The identification of seven parasite species of copepods found in Chinese marine waters is confirmed by the current investigation.This exploratory research will be useful for taxonomists as they plan future investigations of parasitic copepods in many scientific areas.Additionally, molecular studies based on DNA sequences are of immense power in the analysis of phylogenetic relationships of parasitic copepods in molluscs, when new parasitic copepods or two similar copepods are appeared, molecular data may provide useful information to determine the phylogenetic affiliation of the species, and a period of profound change is to be expected.

O n l i n e F i r s t A r t i c l e O n l i n e F i r s t A r t i c l e
koe, Octopicola huanghaiensis, Conchyliurus quintus, Herrmannella soleni, Lichomolgus similis, Modiolicola bifida, Pseudomyicola spinosus, Mytilicola orentalis and Trochicola japonica), of which 5 species (O.koe, C. quintus, L. similis, M. bifida and T. japonica) were reported from the Bohai Sea, 4 species (Anthessius mytilicolus, O. koe, L. similis and Myicola formosanus) were known from the East China Sea and 3 species (A.mytilicolus, A. pinnae and Panjakus platygyrae) were recorded from the South China Sea.Two methods of taxonomic identification have been used up till now.The morphological based characteristic and molecular marker based identification.The DNA barcode-based identifications are valued around the world, especially for all kinds of morphologically complicated species, because traditional morphological identifications are significant but raise accuracy concerns.The complex morphological characteristics confuses the identification of species, therefore, it becomes intuitive A species accurately to understand the diversity, determine the reference basis of resources and assess potential diversity resources of the region using modern techniques like the DNA barcode-based identifications are appreciated worldwide especially for the animals of similar morphological characteristics.The mitochondrial DNA (mtDNA) are potential markers because of maternal inheritance, fast evolutionary rate.Therefore, cytochrome c oxidase subunit 1 has been used in present study.

Fig. 1 .
Fig. 1.The map showing the collection areas mainly samples were collected from Bohai Sea and Yellow Sea area of Shandong, Jiangsu and Liaoning provinces, respectively.

Fig. 2 .
Fig. 2. Neighbor joining tree (NJ) based on thirty-two COX1 sequences of seven parasitic copepods of order Cyclopoida belongs to four families.The colors are representing the families, respectively.