First description study on molecular characterization of Hemiclepsis khankiana (Hirudinea: Glossiphoniidae) in China

Abstract

Background

Leeches are an integral component of aquatic biocenosis and can be found in a wide range of ecosystems such as freshwater, saltwater, flowing, and still-water ecosystems. It especially plays an important role in the freshwater benthic community and is an important part of the food web. In this study, a leech species was found in the mantle cavity of wild freshwater mussels in Zigong City, Sichuan Province, China, and its identity was determined through morphological analysis and molecular biological analysis.

Results

The leech is Hemiclepsis khankiana, a new species of Hemiclepsis that has been discovered in Russia in recent years. Through morphological analysis, the current survey observed that the morphological characteristics of Hemiclepsis khankiana eyespots were significantly different from the first reported description. The first pair of eyespots on the leech were separated and clear, while it had been reduced to unclear shadows in the previous report. The phylogenetic tree based on the COI gene showed that the COI gene sequence obtained in this study was in the same evolutionary branch as Hemiclepsis khankiana (MN295420, MN295421). Genetically, it was most closely related to Hemiclepsis kasmiana (mean COI p-distance = 3.98%).

Conclusions

The current study reported on the new distribution range of Hemiclepsis khankiana, which was initially discovered in China. This study indicates that the distribution range of the leech species has expanded, laying a foundation for further studies in China.

Background

Leeches are an integral component of aquatic biocenosis and can be found in a wide range of ecosystems such as freshwater, saltwater, flowing, and still-water ecosystems [1, 2]. It has the largest distribution in coastal areas, especially plays an important role in the freshwater benthic community in coastal areas, and is an important part of the food web [3]. In addition to the above-mentioned roles, leeches are accumulators of toxic substances and biological indicators of water pollution and are also directly related to the spread of bacterial and viral infections [2].

The freshwater leech genus Hemiclepsis Vejdovsky, 1884 (Hirudinea: Glossiphoniidae) currently contains at least 16 species, five of which have been recently discovered. Among them, four species were found in Russia in 2019, namely Hemiclepsis khankiana, Hemiclepsis myanmariana, Hemiclepsis schrencki and Hemiclepsis tumniniana, and one species Hemiclepsis yangtzenensis was found in China in 2021 [4, 5]. Bolotov et al. [4] found that Hemiclepsis originated from East Asia, and the Asian Hemiclepsis mussel-associated leeches have had a wide range of ancestors in both East Asia and Southeast Asia. Most of the reported species belonging to the genus Hemiclepsis are mainly distributed in some countries on the Asian continent, such as China, India, Japan, South Korea and Russia, and the hosts are mainly freshwater leeches and freshwater fish [4, 6]. In China, six species of Hemiclepsis have been reported: H. erhaiensis, H. guangdongensis, H. hubeiensis, H. kasmiana, H. marginata and H. yangtzenensis. Among this species, H. erhaiensis and H. hubeiensis were found on the gills of freshwater fish, H. guangdongensis was collected from the skin of Cuora amboinensis, H. kasmiana and H. yangtzenensis were collected from the body surface of Monopterus albus, and H. marginata also has been found on the body surface of M. albus [5,6,7,8,9].

According to Bolotov’s previous report, there were seven stages in the life cycle of Hemiclepsis mussel-associated leeches [4]. Firstly, the mature leech leaves the mantle cavity and fixes the egg cluster on the back edge of the host shell near the umbo, and covers the brood with its body to develop the eggs. Then, the eggs hatch into larvae and attach to the ventral surface of the parent, entering the mantle cavity of the host mussel with the parent. Subsequently, the larvae leave the parent body and probably start to feed on the host mussel. The leech larvae grow into adults. Proposed feeding on the host mussel (still to be confirmed). Finally, adult leeches grow into maturation leeches feeding on freshwater fish as their primary host. From the above description of life history, it can be found that most stages in the life cycle of Hemiclepsis mussel-associated leeches mainly occur in the mantle cavity of the mussel.

Hemiclepsis khankiana is a new species that belonging to the genus Hemiclepsis, discovered in the Russian Far East in 2019 [4]. This species was considered a possible obligate inhabitant of the mantle cavity of freshwater mussels [4]. Bolotov et al. [4] discovered that the secondary host and shelter of this species were freshwater mussels Nodularia (Unionidae: Unioninae), and the primary host were freshwater fishes Rhodeus (Cyprinidae), the distribution ranges were in the Khanka Lake Basin and Amur Basin system, Russia Far East and probably China. However, there have been no recent reports of this species in China [4].

Freshwater mussels have become one of the most important fauna in freshwater ecosystems due to their potential to enhance biodiversity and ecosystem functions [10,11,12,13,14]. China is one of the countries with the highest species diversity of freshwater mussels, especially the Yangtze River is the center of freshwater mussel diversity in East Asia [15]. As a province rich in water resources in the upper reaches of the Yangtze River, Sichuan has developed freshwater aquaculture industries such as fish, crustaceans and shellfish. Recently, a freshwater leech species was found in the mantle cavity of freshwater mussels in Zigong, Sichuan Province. In the current study, this experiment observed the morphology of the freshwater leech by stereomicroscope and scanning electron microscope and identified the characterization by molecular biology methods. Finally, the distribution range, morphological differences, and phylogenetic analysis of this species were discussed.

Results

Morphological analysis

In this study, all of the 48 freshwater mussel (Unionidae: Unioninae) specimens were examined, with 19 freshwater mussels being infested by 84 leeches. The leech observed in this study was a medium-sized rice-shaped leech with dorsoventrally flatted and dorsum smoothly. The body length of the representative specimen from the anterior tip to the rear posterior sucker was 7.85 mm, the body widest point was 2.21 mm, and the posterior sucker diameter was 1.69 mm, broader than the oral sucker width (1.12 mm). The characteristics of leech samples could be summarized as follows: the dorsal of the leech samples was pale yellow, with six vertical, widely brown stripes; the posterior sucker had no strips but with dense and diffuse brown spots. Two pairs of obvious eyespots, the first pair of eyespots was small but visible and was located ahead of the second pair of eyes, both pairs were drop-like and separated (Fig. 1). Scanning electron microscopy provided H. khankiana surface morphology structures (Fig. 2). The photographic evidence showed a small oral sucker (Fig. 2B and C) and a large deeply cupped posterior sucker ((Fig. 2D and E)). In the center of the oral sucker was a stellate, radial pit (Fig. 2C). The surface of the whole oral sucker was full of small pore structures (Fig. 2C).

Fig. 1

External morphology of Hemiclepsis khankiana’s head region (dorsal view and ventral view) and posterior sucker (dorsal view and ventral view). The part circled by the dotted line represents the two pairs of eyespots of the leech. The part circled by the solid line represents the oral sucker (ventral view) of the leech. Black arrows indicate the posterior sucker (dorsal view and ventral view) of leech

Fig. 2

Scanning electron photomicrographs of Hemiclepsis khankiana. (A) Side view of the Hemiclepsis khankiana. (B) and (C) Side view of the oral sucker. (D) and (E) Side view of the caudal sucker. The part circled by the dotted line represents the stellate, radial pit of the leech oral sucker. The white arrows indicate small pore structures of the leech oral sucker

Molecular and phylogenetic analyses

The BLASTn analysis showed that the COI partial sequence (ON778569) obtained in the current study had 98.03% identity with the H. khankiana COI sequence (MN295420) in Russia. Phylogenetic analysis revealed that COI partial sequence (ON778569) was in the same clade as H. khankiana (MN295420 and MN295421) isolated from Russia (Fig. 3). Genetic distance among various Hemiclepsis clades was 3.98–12.83% and H. khankiana with 3.98–11.41% genetic distance with other Hemiclepsis species. Genetically, this Hemiclepsis species was most closely related to H. kasmiana (mean COI p-distance = 3.98%) (Fig. 4).

Fig. 3

Phylogenetic tree of the leech based on the COI gene sequences. The tree was constructed with the neighbor-joining method using MEGA 7.0. Numbers at nodes represent the percentage occurrence of clades in 1000 bootstrap replications of data. The solid circle indicates the sequence from this study. All sequence from this study was bolded

Fig. 4

Genetic distances between Hemiclepsis khankiana and other same genus species based on the COI gene. The bars indicate standard error estimates of the distance values based on the bootstrap approach (1000 replications)

Discussion

The current study described a freshwater leech species parasitic in the freshwater mussel mantle cavity. This species was identified as H. khankiana by morphological analysis and molecular biology. This Hemiclepsis species also has been reported for the first time in China.

Hemiclepsis khankiana, a new Hemiclepsis species that was discovered in recent years, had only one record. The name of this species was derived from the Khanka Lake basin in the Russian Far East [4]. As shown in Fig. 5, Khanka Lake is the boundary lake between China and Russia, which lies on the border of Primorsky Krai (Russia) and the Heilongjiang province of China. Its only outflow is the Songacha (Song’acha) River, a tributary of the Ussuri (Wusuli) River. Then, the Ussuri (Wusuli) River heads north to join the Amur River near Khabarovsk. Lake Khanka belongs to the Ussuri River System, which is part of the Amur River System. The Amur Basin is also called the Heilongjiang River Basin in China. This suggested that H. khankiana may also exist in the Heilongjiang River Basin, China. In addition, the primary host of H. khankiana is the freshwater fish Rhodeus (Cyprinidae), and Rhodeus is widely distributed in China, as well as in the Yangtze River Basin and Heilongjiang River Basin [16]. Although in this study, this species was collected in the Yangtze River Basin and has not been reported in the Heilongjiang River Basin, this investigation confirmed Bolotov’s guess that H. khankiana may also be distributed in China, and updated and expanded the distribution range of H. khankiana. At present, it is unclear whether the distribution of this species in China was caused by natural factors or factors such as the transportation of aquatic products, and whether the expansion of the species would have an impact on freshwater mussels and freshwater fish in the distribution area.

Fig. 5

The geographical location of the Heilongjiang (Amur) River Basin (the blue filling)

The dorsal view of the leech sample head region (Fig. 2), showed that the sample collected in this study was different in the description of the eyespots of H. khankiana. Bolotov et al. [4] described eyespots of H. khankiana as “the first pair strongly reduced up to an unclear shading, the second pair was cup-like”, but from observed in this study, the first pair of eyes was very obvious, and the second pair of eyes closer to the shape of water drops (Fig. 2). The same species had stable morphological characteristics, This result suggested the phenotype of a species was the result of the interaction between its genotype and the environment. Whether the difference between the morphological characteristics of H. khankiana described in this study and the Bolotov et al. [4] reported was caused by environmental factors was unknown, and further information mining may be required for this species.

The phylogenetic tree and genetic distance analysis of the COI gene showed the evolutionary relationship clearly between H. khankiana and other Hemiclepsis species. Among them, H. khankiana and H. kasmiana were closely related in evolution, and they were in a large evolutionary branch, with a bootstrap value of up to 98. Moreover, the mean COI p-distance between H. khankiana and H. kasmiana was the closest, with a value of 3.8%. In addition, we found that H. yangtzenensis, a new species of Hemiclepsis species discovered in 2021, was significantly different from other Hemiclepsis species, and had a relatively far genetic distance (mean COI p-distance = 10.41–12.83%) from other species in the analysis of genetic distance [5].

Previous studies have shown that global mussel-associated leech assemblage includes at least 12 species belonging to the family Glossiphoniidae [4]. Mussel-associated leech taxa have been reported in East and Southeast Asia, India, Nepal, and Africa, but have not been reported in Europe, the Middle East, North and Central Asia, Australia, the Indonesian archipelago, New Guinea, and the Philippines [4]. Research on the genera Batracobdelloides and Hemiclepsis were largely overlooked by researchers, and the number of Hemiclepsis species in China was still very small, but with the discovery of the new species, we are still largely underestimating the number of Hemiclepsis species [5]. The freshwater leech genus Hemiclepsis has great value in respect of aquaculture matters. M. albus infection with a large amount of H. marginata will cause massive blood loss of M. albus, leading to M. albus dying in large numbers [17]. In addition, H. marginata is also an important vector for the spread of fish diseases, such as trypanosomes and pancreatic necrosis viruses [18,19,20]. H. khankiana also is parasitic on freshwater fish, but whether the species can have the ability to spread the pathogenic to the host like other freshwater leeches needs further exploration. Whether a large number of H. khankiana parasites can be harmful to fish has not been reported. In short, there is currently only one relevant report about H. khankiana, and more information about it needs further research.

So far, only a few species have been described in the world, and the actual number of invasive species may exceed the current record and will further increase. For newly discovered species or alien species that have not been understood by science, their functions have not been fully observed or understood [21, 22]. There are six broad mechanisms for alien species to be introduced into a region, among which ‘contaminant’ and ‘stowaway’ are common for invertebrates, algae, fungi and microorganisms, because these organisms are widespread and inconspicuous, and are often introduced through transportation. For example, pathogens and parasites are frequently introduced as contingents with their hosts [23,24,25,26]. In China, the invasion of Pomacea canaliculata has not only caused serious economic losses to local agricultural production, wetland ecosystems and freshwater resources but also served as an intermediate host for many human and livestock parasites and pathogens, the most famous of which is Angiostrongylus cantonensis, which can cause diseases such as eosinophilic meningitis or meningoencephalitis when infecting humans [27,28,29].

The impact of exotic parasites and pathogens is usually hidden [30]. Even if a species was detected in its alien range, even though its ecological function and changes are not mysterious, it may still not be found if it is affected by seasonal, annual or regional variations, temporal and spatial variations and human beings. Pathogens can also evolve rapidly, so many exotic pathogens with serious impacts are not considered threats before they are established in new hosts [30]. Moreover, the transmission of parasites needs to reach a certain population density and the specific space-time conditions required for transmission [31, 32]. The freshwater leech parasitic on freshwater mussels found in this study was reported for the first time in China. It cannot be ruled out that the species invaded China through natural factors or freshwater aquatic product trades between Russia and China, and other factors.

With the rapid growth of world maritime trade, the world is connected by shipping corridors. Two of these, the Panama and Suez canals, are major shipping corridors that are considered hotspots for biological invasions [33]. Some species of halobios can freely shuttle between the two ends of the canal and become exotic organisms in another sea area. In recent years, a variety of fish has entered the Mediterranean through the Suez Canal, increasing the number of species in the Mediterranean by 8%. In addition, a variety of previously unreported marine fish has also been found in Gatun Lake in Panama. The number of some species in Gatun Lake has increased significantly, almost completely replacing the previously existing freshwater fish in the lake [34]. Although the primary host of freshwater leech parasitized on freshwater mussels found in this study is widely distributed in China, this does not exclude that this species may invade China through natural factors such as “corridor”. After all, the Amur River basin and the Heilongjiang River basin belong to the same water system. Of course, this problem needs further study.

Conclusions

In conclusion, firstly, the current study reported the new distribution range of H. khankiana, this species was found in China. This study also gave a simple description of H. khankiana in morphology. It was necessary to further survey the host range of H. khankiana and whether H. khankiana transmits pathogens and can harm its host.

Methods

Sample collection

The leech specimens were collected in the mantle cavity of freshwater mussels of the Zigong (29°21′49.36″N, 104°46′26.04″E), Sichuan Province, China (Fig. 6). The collected samples were partially stored at − 80 °C for the following experiments, and partly stored in absolute ethanol for observation under the microscope. The remaining samples were stored in the electron microscopy fixative for scanning electron microscope.

Fig. 6

The geographical location of the sample sampling (red solid diamond). The green filling indicates the Yangtze River basin. (The figure was modified based on the map in the article by XU et al., 2021 [5])

Morphological analyses

In this study, the leech samples were observed by the stereoscopic microscope. According to the key of mussel-associated leech species (Glossiphoniidae: Batracobdelloides and Hemiclepsis) written by Bolotov et al. [4], we mainly observed the color and pattern of the dorsum of the leeches, the size and color of the suckers, the shape and number of the eyespots, and preliminarily determined the species of leeches samples according to the external morphological characteristics through the above observation results [4].

To better observe the external characteristics of the leech samples, the current study also observed them by scanning electron microscopy, which was briefly described as follows: the samples fixed with electron microscopy fixative were washed three times with 0.1 M phosphate buffer (pH 7.4) for 15 minutes each. Then, the samples were transferred to a 1% OsO₄ solution prepared with a 0.1 M phosphate buffer (pH 7.4) and incubated at room temperature for 1-2 hours. After that, the samples were washed in 0.1 M phosphate buffer (pH 7.4) three times for 15 minutes each. Then, the samples were sequentially placed in 30%, 50%, 70%, 80%, 90%, 95%, 100% and 100% alcohol for dehydration, with each step lasting 15 minutes. Then, isoamyl acetate was used to exchange the ethanol for 15 minutes. Dried samples with a Critical Point Dryer, then sputter-coated with gold for 30 seconds, and finally digital images were captured using a scanning electron microscope.

Molecular analyses

The mitochondrial cytochrome c oxidase subunit I gene (COI) sequence, known as an available DNA barcoding marker for animals, is frequently used for phylogenetic estimation of leeches [35, 36]. Therefore, we selected COI as the target gene to be amplified by polymerase chain reaction (PCR). The COI partial sequence for the leeches was obtained using the following primers: LoboF1 (5′-KBT CHA CAA AYC AYA ARG AYA THG G-3′) and LoboR1 (5′-TAA ACY TCW GGR TGW CCR AAR AAY CA-3′) [37]. Total genomic DNA was extracted from samples that were stored -80 °C using the TIANamp Genomic DNA Kit (TIANGEN, China), following the manufacturer’s protocol. PCR reaction volume of 25 μL containing 3 μL of DNA template, 0.5 μL each of forward and reverse primers (10 μM), 0.25 μL of Taq polymerase (0.5 U; New England BioLab, USA), 1.25 μL of deoxyribonucleotide triphosphate (200 μM; New England BioLab, USA), 2.5 μL of 10 × ThermoPol Reaction Buffer (New England BioLab, USA), and double-distilled water to a total volume of 25 μL. PCR reaction programs for COI are as follows: 95 °C (5 min), followed by 35 cycles at 95 °C (50 s), 48 °C (50 s), 68 °C (1 min), and a final extension at 68 °C (5 min). PCR product was purified using EasyPure® Quick Gel Extraction Kit (TransGen, China) and cloned into E. coli DH5α using the PMD™ 19-T Vector Cloning Kit (TaKaRa, Japan). At least two positive clones were selected for sequencing at Sangon Biotech (Shanghai) Co., Ltd.

Sequence and phylogenetic analyses

Nucleotide sequence identities were determined by performing GenBank BLASTn analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence was then submitted to GenBank to obtain the accession number. For phylogenetic analyses, the newly obtained sequence of H. khankiana (ON778569) was added. Further more, we obtained several published Hemiclepsis COI sequences as follows: Hemiclepsis kasmiana (MN295423, MN295430, MN295432, MN295437); H. khankiana (MN295420, MN295421); H. myanmariana (MN295403, MN295456, MN295397); Hemiclepsis marginata (MN295390, MN295388); H. tumniniana (MN295410, MN295417); H. schrencki (MN295415, MN295416); H. yangtzenensis (MW699107), and other Glossiphoniidae leeches species.

Based on the above sequences, the Neighbor-Joining method was used to construct a phylogenetic tree, and calculated genetic distance between H. khankiana and other Hemiclepsis species by using the p-distance model in MEGA7.0 [38].