First Report of Cyamus Boopis from A Humpback Whale (Megaptera novaeangliae) in the Coastal East China Sea

Background: Cyamus boopis is an amphipod crustacean that obligately parasitizes the body surface of the humpback whale, Megaptera novaeangliae. The life cycle of C. boopis does not include a swimming stage, and the crustacean spends its entire life on the body of M. novaeangliae. Methods: On November 15, 2017, a male humpback whale was found stranded on the coast of the cape of Yuan Tuo, Qidong, Nantong, JiangSu province, China. Parasites were collected from the carcass of this whale and identied by morphological techniques and molecular analysis. Results: A total of 15 C. boopis specimens were collected, and eight females and seven males were morphologically identied. A phylogenetic tree of cytochrome c oxidase subunit 1 revealed that the collected specimens clustered together with previously reported C. boopis sequences from the Northern Hemisphere. Conclusion: This is the rst report of C. boopis in a humpback whale from the East China Sea and supplements data from humpback whales found off the coast of China. In addition, our data provide supplementary data on the migration paths of humpback whales.

Acute ventral processes formulas and maxilliped palp characters are usually used to identify cyamids [2,[7][8][9][10]. However, a lack of available descriptions and variations in morphological characteristics have led to the incorrect identi cation of some Cyamus juveniles [2,7]. In particular, identi cation of C. boopis over the decades has been controversial. C. boopis was rst described by Lütken, who collected specimens from M. novaeangliae in 1870 [11]. In the following few years, C. boopis was transferred to the new genus Paracyamus based on the uni-articulate maxilliped palp present in adults [12]. Later C. boopis was returned to the Cyamus genus [2]. At the genus level, C. boopis and Cyamus catodontis are similar, as both lack ventral processes on pereonites 3 and 4 and have a smooth grinding surface on the right lacinia mobilis [7,10]. This has led previous authors to confuse these two species. Therefore, in addition to morphological identi cation, molecular methods are required for proper identi cation.
The life cycle of whale lice does not include a swimming stage, and the parasite spends its entire life on the body of its whale host [4,13]. Therefore, whale lice are a potential tool for studying humpback whale migratory routes around the world [6,14]. Although there have been persistent reports of whale lice in the Northern Hemisphere, molecular data on these parasites are still relatively scant [5,14,15]. M. novaeangliae are one of the most cosmopolitan whales in the world [16]. The migration patterns of humpback whales have been extensively studied over the last few decades. However, there is still a gap in data on M. novaeangliae in the Northern Hemisphere, especially near China.
In this study, we describe the rst known case of whale lice collected from a humpback whale stranded on the coast of the East China Sea. Based on morphological identi cation and cytochrome c oxidase subunit 1 (COI) gene sequence analysis, we con rmed that the lice belonged to C. boopis. This report supplements data on C. boopis and its humpback whale hosts off the coast of China and provides information on the possible migration paths of humpback whales.

Methods
Whale lice (JS isolate) were collected from the carcass of a male humpback whale (body length, 6.62 m) on the coast of the East China Sea on the cape of Yuan Tuo, Qidong, Nantong, Jiangsu province, China (N 31°44′28″, E 121°56′10″) on November 15, 2017 (Graphical Abstract).
Parasites were xed and preserved in 70% ethanol. Microscopic examinations were performed using an Olympus BX53 (Olympus, Tokyo, Japan) with an adapted Canon EOS R camera (Canon, Tokyo, Japan).
Morphological characteristics and descriptions were used to identify parasites according to previous reports [2,7,10]. Drawings were digitally prepared using Adobe Illustrator CS6 (Adobe Inc., San Jose, CA, USA).
To examine phylogenetic relationships, homologous sequences were selected and multiple alignments were performed using ClustalX2. A maximum likelihood tree was constructed using the online program PhyML 3.0 (http://www.atgc-montpellier.fr/phyml/) with Smart Model Selection [17]. The Akaike Information Criterion model was selected with Subtree Pruning and Regrafting as the type of tree improvement. The number of bootstrap replicates was 100. All sequences included in the phylogenetic tree were retrieved from the GenBank database (Table 1).

Results
All whale lice exhibited speci c features that allowed for sex and species identi cation. In total, eight female and seven male C. boopis specimens were identi ed. C. boopis adults differ from other Cyamus species by the number of ventral processes on pereonites 5-7. Sex was assigned based on several characteristics: pereonites 3 and 4 are broader than those of males; subequal in width to pereonites 5 and 6; and the pleons of females lack pleopods (Fig. 1).
The phylogenetic tree showed that the sequence of the collected specimens (JS isolate) clustered with all other reported C. boopis COI sequences but was separate from other Cyamus species (Fig. 2). The sequence was deposited in the GenBank database (accession number, MT551876).

Discussion
To the best of our knowledge, this is the rst report of C. Using morphological identi cation, the specimens collected from the humpback whale stranded on the coast of the East China were found to belong to C. boopis. Combined with mitochondrial DNA analysis of the COI sequence, we found that the C. boopis specimens clustered together with all other specimens from the Northern Hemisphere. A previous study compared C. boopis from three M. novaeangliae breeding stocks from the Southern Hemisphere with C. boopis from the Northern Hemisphere using the COI gene sequences and found that C. boopis from the two locations differed greatly in genetic structure [6]. However, data from the Northern Hemisphere was based on only three samples of C. boopis. Therefore, data on C. boopis and humpback whales in the Northern Hemisphere are lacking compared with those from the Southern Hemisphere.
Some studies have suggested that, in addition to C. boopis, C. elongatus may also parasitize humpback whales [13]. However, molecular studies of Brazilian C. boopis con rmed that all specimens of whale lice collected from M. novaeangliae were from the same species [2,7]. Our study con rmed that the specimen collected from the humpback whale in China was also C. boopis. Therefore, more data are needed to support the presence of C. elongatus on humpback whales.
Whale lice can provide valuable information about whale population histories. Because whale lice have no alternative hosts or free-living stages and have a long history of living on their whale hosts [4,19]. In addition, because of their short generation time [19] and large population size [20], synonymous sequence divergence of homologous markers of Cyamidae can be ten times faster than that of their whale hosts [14]. The genetic structure of cyamids could also reveal encounters between whales of different stocks [6,13]. Although records have been collected of whale lice from Cetacea in coastal waters all over the world, molecular information on whale lice is still limited. Therefore, the collection of whale louse samples and their genetic data could provide a scienti c basis for the classi cation and identi cation of whale lice and provide information for the further study of whale populations and migration.

Conclusions
To the best of our knowledge, this is the rst report of C. boopis in humpback whale from China. This study extends the track of humpback whale over the world.