Limnotrachelobdella hypophthalmichthysa n. sp. (Hirudinida: Piscicolidae) on Gills of Bighead Carp Hypophthalmichthys nobilis in China

We describe the characterization of a novel fish leech species found on the gills of bighead carp (Hypophthalmichthys nobilis) from lakes and reservoirs in China. This leech is morphologically similar to Limnotrachelobdella sinensis recorded on goldfish and common carp. However, there are 0–2 pairs of symmetrical or asymmetrical eyes and 10 pairs of pulsatile vesicles in the newly discovered leech, in remarkable contrast to L. sinensis. Except for bighead carp, where it demonstrated a higher than 90% prevalence, and silver carp (H. molitrix), where there was low infection, this leech was not detected on any other fish from the Qiandao reservoir in China that were examined during this investigation. Molecular analyses indicated 87.8% ITS sequence identity with L. sinensis and 85.0 and 86.1% COX1 sequence identity with L. sinensis and L. okae, respectively. The uncorrected p-distance based on the COX1 sequence was found to be 15.1 and 14.0% for L. sinensis and L. okae, respectively, suggesting interspecific variation. Phylogenetic analyses based on the combination of 18S and COX1 sequences showed that the newly discovered leech groups with Limnotrachelobdella species. Histopathological observation indicated that attachment of the leech on the gill rakers and gill arches causes a loss of connective tissue, hemorrhage, and ulceration. Based on the morphology, molecular analyses, and host specificity, we conclude that this leech is a new species of Limnotrachelobdella and named it Limnotrachelobdella hypophthalmichthysa n. sp.

In recent years, a kind of leech was only found on the bighead carp, Hypophthalmichthys nobilis (Richardson, 1845), in reservoirs and lakes in China. A preliminary investigation showed that this leech is morphologically similar to L. sinensis, which has been recorded on goldfish (Carassius auratus) and common carp (Cyprinus carpio) in China [15]. However, the leech found on bighead carp is distinct from L. sinensis and has not been found on goldfish or common carp during investigations. Therefore, the leech on bighead carp was identified by its morphological characteristics and further characterized based on molecular sequence analyses as well histopathological observation.

Sample Collection and Preservation
Fish were collected for the examination of leeches in the Qiandao reservoir (29 • 33 44.32 N, 119 • 01 51.98 E), Zhejiang province, China, during 2021-2022. Leeches were detached from fish gills using forceps. Living leeches were stocked in fresh water for morphological analyses. Some leeches were preserved in 100% ethanol and stored at −20 • C for DNA extraction, including one specimen each from Hubei and Jiangxi provinces. In addition, gills infected with leeches were preserved in 4% paraformaldehyde for histopathological observation.

Morphological Identification
First, living leeches were examined for the number of eyes within 48 h after detachment from gills. Then, specimens were anesthetized by adding ethanol in a dropwise manner for a subsequent observation of the digestive and reproductive system under a stereomicroscope. The epidermis of the slack leech was separated using a scalpel applied along the longitudinal centerline dorsally or ventrally. The ventral nerve cord was then separated from the epidermis using ophthalmic forceps to determine its positional relationship to the reproductive and digestive systems. The digestive system was directly observed in small leeches collected from December to February when it has thinner and more transparent muscle layers. In addition, specimens fixed in 4% paraformaldehyde were photographed using a Panasonic DMC-GX85CGK digital camera. The materials were deposited in the collection of the Institute of Hydrobiology, Chinese Academy of Science, Hubei, China [Holotype (accession no. LH-Z3) and four paratypes (accession nos. LH-Z2, Z4, H1, and J1)].

Histopathological Observation
Gills fixed in 4% paraformaldehyde were trimmed into 1 cm × 1 cm tissue blocks and dehydrated using a series of graded ethanol solutions and cleared in xylene. After the paraffin wax solidified, 4 µm paraffin sections were taken and mounted on slides for hematoxylin and eosin (H and E) staining. The slides were subsequently examined under a stereomicroscope.

DNA Extraction, Amplification, and Sequencing
Muscles of the lateral body and caudal sucker were removed for DNA extraction to avoid the presence of host blood in the leech gut. Genomic DNA was isolated using an Aidlab Genomic DNA extraction kit (Aidlab Co., Beijing, China) according to the manufacturer's instructions. Ribosomal encoded genes, including the 18S DNA gene, internal transcribed spacer 1-5.8S rDNA-internal transcribed spacer 2 (ITS) and fragments of the mitochondrial gene, cytochrome c oxidase subunit I (COX1), were amplified from the genomic DNA. All the genes used, following polymerase chain reaction (PCR) condition, were according to the manufacturer's instructions (Takara Bio, China): 98 • C for 2 min, 40 cycles of 98 • C for 10 s, 50 • C for 15 s, and 68 • C for 1 min, and final extension at 68 • C for 10 min. Primer pairs were designed according to the sequence in similar species (Table 1). PCR products were sequenced using an ABI 3730 automatic sequencer (Sanger Sequencing). The sequences were assembled manually using DNAstar software v7.1 (Madison, Wisconsin, USA).

Molecular Identification and Phylogenetic Analyses
Fish were collected for leech examination in the Qiandao reservoir (29 • 33 44.32 N, 119 • 01 51.98 E), Zhejiang province, China, during 2021-2022. For molecular analyses, ITS and COX1 were chosen to compare specimens collected from three places: Zhejiang, Hubei, and Jiangxi provinces. In addition, the new species' similarity to L. sinensis and L. okae was verified in the NCBI GenBank database. The uncorrected p-distance was used to examine COX1 genetic variation among Limnotrachelobdella species with 1000 replicates in MEGA11 [18].
Phylogenetic analyses were performed, combining 18S and COX1 gene datasets using 29 sequences of Piscicolidae leech species from GenBank ( Table 2). All of the sequences were imported into PhyloSuite [19] and aligned using MAFFT 7.149 [20] with auto strategy and normal alignment mode; further adjustments to sequence alignments were manually applied. Then, the 18S and COX1 sequences were concatenated using the "concatenate sequence" function in PhyloSuite. The best partitioning scheme and evolutionary models for 2 predefined partitions were selected using PartitionFinder2 [21] with a greedy algorithm. Bayesian inference (BI) phylogenies were inferred using MrBayes 3.2.6 [22] under the partition model (2 parallel runs, 100,000 generations), with the initial 25% of sampled data discarded as burn-in and sampling frequency of 3000.

Infection by Fish Leeches
In total, 16 species of fish from the Qiandao reservoir were examined, including Carassius auratus, Megalobrama amblycephala, and Opsariichthys bidens. Fish leeches were found on bighead carp at higher than 90% prevalence and silver carp (Hypophthalmichthys molitrix) at extremely low prevalence (only one silver carp infected with one leech).

Description
External morphology: The body distinctly divides into trachelosome and urosome, with a total length of 4.6-70.3 mm and width of 0.9-16.2 mm ( Figure 1A). Body skin is smooth, but with numerous brown pigments on the surface of the trachelosome region ( Figure 2B). Body color of the living leech is reddish or reddish brown ( Figure 1B). Eyes, 0-2 pairs, symmetrical or asymmetrical or degenerated eyes on oral sucker ( Figure 2B-F). Clitellum is ring-like, sharply distinct from pre-clitellum and urosome. In the urosome region, the body musculature is well developed. Complete somite is composed of six annuli, but the external appearance suggests it is divided into fourteen annuli, which is consistent with L. sinensis. Grape-like tissue is present in the area before somite XXII in the mature individuals. Pulsatile vesicles are of 10 pairs, present separately on each somite of somites XIV-XXIII and matched against sequential numbers of the ventral nerve cord ganglion, respectively. Caudal sucker is deep cup-like, thick, and ventrally directed, and its diameter is smaller than the maximum body width. Anal is separated by the last two annuli of somite XXVII and is difficult to be observed.  Internal morphology: Esophagus passes posteriorly from the proboscis to somite XI. Crop extends from somite XIII, and with seven chambers, each with three pairs of pouches (the first two bigger than the last). Posterior crop caeca fused with five fenestraes, forming   Internal morphology: Esophagus passes posteriorly from the proboscis to somite XI. Crop extends from somite XIII, and with seven chambers, each with three pairs of pouches (the first two bigger than the last). Posterior crop caeca fused with five fenestraes, forming Internal morphology: Esophagus passes posteriorly from the proboscis to somite XI. Crop extends from somite XIII, and with seven chambers, each with three pairs of pouches (the first two bigger than the last). Posterior crop caeca fused with five fenestraes, forming five well-developed chambers similar to crop chambers. Intestine consists of four chambers without lateral pouches (Figure 2A). Male gonopore is large, round and crinkled. Female gonopore is below the male gonopore, particularly smaller, and elliptical ( Figure 3C). Because of the smooth skin of the clitellum, it was difficult to determine the number of annuli between the two gonopores. Testisacs are five pairs, elliptical in shape, located on somites XIV-XVIII and alternating with crop caeca, slightly posterior to the ganglion of the ventral nerve cord ( Figure 3A). Ejaculatory duct and atrial cornua are both in kidney-like shape. Accessory gland cells are absent. Common atrium is near-spherical, connecting a thick and large bursa that leads to the male gonopore. The ejaculatory duct, atrial cornua, and common atrium are all semitranslucent and threaded before fixation and are often wrapped by the brusa, then evert out of the male gonopore ( Figure 3B). Conducting tissue and vector tissue are absent. Paired ovisacs are sac-like, extending to somite XV. Each ovisac consists of three lobes with the third lobe the largest.
Pathogens 2023, 12, x FOR PEER REVIEW 6 of 11 five well-developed chambers similar to crop chambers. Intestine consists of four chambers without lateral pouches (Figure 2A). Male gonopore is large, round and crinkled. Female gonopore is below the male gonopore, particularly smaller, and elliptical ( Figure  3C). Because of the smooth skin of the clitellum, it was difficult to determine the number of annuli between the two gonopores. Testisacs are five pairs, elliptical in shape, located on somites XIV-XVIII and alternating with crop caeca, slightly posterior to the ganglion of the ventral nerve cord ( Figure 3A). Ejaculatory duct and atrial cornua are both in kidney-like shape. Accessory gland cells are absent. Common atrium is near-spherical, connecting a thick and large bursa that leads to the male gonopore. The ejaculatory duct, atrial cornua, and common atrium are all semitranslucent and threaded before fixation and are often wrapped by the brusa, then evert out of the male gonopore ( Figure 3B). Conducting tissue and vector tissue are absent. Paired ovisacs are sac-like, extending to somite XV. Each ovisac consists of three lobes with the third lobe the largest.

Molecular Identification and Phylogenetic Analyses
From the three specimens (Z1, H1 and J1), the 18S rDNA (1712-1713 bp), ITS (1489-1495 bp), and COX1 (1537 bp) genes sequence were obtained. Molecular analyses indicate that the ITS and COX1 sequence identity of the three specimens (collected from Zhejiang, Hubei, and Jiangxi) ranges from 98.6 to 99.7%. The new leech shares 87.8% of ITS sequence identity with L. sinensis and 85.0 and 86.1% of COX1 sequence identity with L. sinensis and L. okae, respectively. The uncorrected p-distance of COX1 sequence was found to be 0.3% for the three specimens in pairwise comparison, and 15.1 and 14.1% with L. sinensis and L. okae, respectively.
The phylogenetic tree produced by BI analyses of a combination of 18S and COX1 sequences indicates that L. hypophthalmichthysa n. sp. groups with L. sinensis and L. okae (Figure 4). for the three specimens in pairwise comparison, and 15.1 and 14.1% with L. sinensis and L. okae, respectively. The phylogenetic tree produced by BI analyses of a combination of 18S and COX1 sequences indicates that L. hypophthalmichthysa n. sp. groups with L. sinensis and L. okae (Figure 4).    Type material: Holotype (accession no. LH-Z3) and four paratypes (accession nos. LH-Z2, Z4, H1, and J1) were deposited in the Museum of the Institute of Hydrobiology, Chinese Academy of Sciences, China.

Histopathological Analyses
Attachment of the leeches on gill rakers and gill arches with the caudal sucker causes loss of connective tissue ( Figure 5A). Hemorrhage and ulceration were also observed on gills infected by leeches ( Figure 5B).

Histopathological Analyses
Attachment of the leeches on gill rakers and gill arches with the caudal sucker causes loss of connective tissue ( Figure 5A). Hemorrhage and ulceration were also observed on gills infected by leeches ( Figure 5B).

Discussion
Yang [17] presented identification keys for the genus Limnotrachelobdella: the distinction between trachelosome and urosome, deeply cup-shaped caudal sucker with a smaller diameter than the maximum width of the body, six annuli in each complete somite, ten to thirteen pairs of pulsatile vesicles on the urosome, a fused posterior crop caeca, and five to six pairs of testisacs. The morphological identification of the fish leeches we collected revealed conformance to these features. So far, five nominal species of Limnotrachelobdella have been recorded: L. sinensis (Blanchard, 1896), L. okae (Moore, 1924), L. taimeni (Epshtein, 1957), L. fujianensis (Yang, 1987), and L. turkestanica (Stschegolew, 1912). Owing to its similar appearance, fish leech L. hypophthalmichthysa n. sp. collected from bighead carp can be easily mistaken for L. sinensis. However, it has 10 pairs of pulsatile vesicles, while the latter has 11 pairs. Meanwhile, the following characteristics are helpful in distinguishing it from five known species: no eyes or one to two pairs of symmetrical, asymmetrical, or degenerated eyes, seven pairs of crop caeca, and five pairs of testisacs (Table  3). In addition, there are numerous brown pigments on the surface of the trachelosome region, and the appearance of the atrial cornua, which extends dorsally, is typical.

Discussion
Yang [17] presented identification keys for the genus Limnotrachelobdella: the distinction between trachelosome and urosome, deeply cup-shaped caudal sucker with a smaller diameter than the maximum width of the body, six annuli in each complete somite, ten to thirteen pairs of pulsatile vesicles on the urosome, a fused posterior crop caeca, and five to six pairs of testisacs. The morphological identification of the fish leeches we collected revealed conformance to these features. So far, five nominal species of Limnotrachelobdella have been recorded: L. sinensis (Blanchard, 1896), L. okae (Moore, 1924), L. taimeni (Epshtein, 1957), L. fujianensis (Yang, 1987), and L. turkestanica (Stschegolew, 1912). Owing to its similar appearance, fish leech L. hypophthalmichthysa n. sp. collected from bighead carp can be easily mistaken for L. sinensis. However, it has 10 pairs of pulsatile vesicles, while the latter has 11 pairs. Meanwhile, the following characteristics are helpful in distinguishing it from five known species: no eyes or one to two pairs of symmetrical, asymmetrical, or degenerated eyes, seven pairs of crop caeca, and five pairs of testisacs (Table 3). In addition, there are numerous brown pigments on the surface of the trachelosome region, and the appearance of the atrial cornua, which extends dorsally, is typical. L. hypophthalmichthysa n. sp. is also different from other Limnotrachelobdella species in terms of its host range. L. sinensis has been recorded on goldfish/silver crucian carp such as Carassius auratus and C. cuvieri in Korea [23][24][25], C. cuvieri and C. auratus langsdorfii in Japan [7,26], and C. gibelio in Russia [17,27]. L. sinensis has also been found on common carp such as Cyprinus carpio in China [15] and Japan [28] and C. carpio haematopterus in Russia [20,27]. Meanwhile, L. taimeni was found on Hucho taimen (Pallas, 1773) [14] and L. fujianensis was found on Epinephelus akaara (Temminck and Schlegel, 1842) and Planiliza affinis (Gunther, 1861) [15,17], respectively. However, L. okae was recorded on fish in the Acipenseridae, Cyprinidae, Salmonidae, Lateolabracidae, Carangidae, Paralichthyidae, and Tetraodontidae families [29,30], exhibiting a wide host range. L. turkestanica was also considered to have a wide range of fish hosts [31,32]. Therefore, unlike L. okae and L. turkestanica, L. sinensis, L. taimeni, and L. fujianensis are fish leeches with high host specificity. L. hypophthalmichthysa n. sp. has only been found on Hypophthalmichthys nobilis and H. molitrix, which suggests that it is a highly host-specific species. In addition, L. sinensis was found on the operculums of goldfish and common carp [7,26,28], whereas the leech representing the focus of this study has been found on gill arches and gill rakers of bighead carp. Therefore, its different host species and infection sites compared with L. sinensis suggests that this leech is a new species.
The ITS sequences of the species in genus Whitmania (Hirudinida: Haemopidae) (W. acranulata, JX885692; W. laevis, JX885693; W. plgra, EU652726) was obtained on Genbank database and the 92.7 to 98.4% of sequence identity could be calculated easily. In this study, the high ITS sequence identity of 98.6 to 99.5% of the specimens from Zhejiang, Hubei, and Jiangxi indicates that they are of the same species. However, only 87.8% ITS sequence identity was found between the new leech and L. sinensis, which was smaller than the range of interspecific divergence in genus Whitmania. Based on the COX1 sequence, the uncorrected p-distance is 15.1% with L. sinensis and 14.0% with L. okae, which are higher than the intraspecific divergence of 0.3% for specimens of the new leech from Zhejiang, Hubei, and Jiangxi. For COX1, a 2.0% genetic distance was found to be indicative of intraspecific divergence [33][34][35], with interspecific divergence of 13.3 to 23.6% between Helobdella blinni (Hirudinidea: Glossiphoniidae) and other species in the genus Helobdella [36]. In the present study, the p-distance of 14.0 to 15.1% is within this range of interspecific divergence. Therefore, the low ITS sequence identity and high genetic distance between the new leech and other species in Limnotrachelobdella provides molecular evidence that the leech found on bighead carp is a novel species of the genus Limnotrachelobdella.
Williams and Burreson [11] attempted to sequence 18S rDNA, COX1, and ND1 gene data of a large number of species in Piscicolidae and illuminated the phylogeny of the family by considering the sequences above in combination with morphological data. In this study, the BI tree is consistent with our expectations, implying that L. hypophthalmichthysa n. sp. groups together with L. sinensis and L. okae.
In summary, based on the morphology, molecular analyses, host specificity, and infection site, the leech infecting bighead carp is a novel species of Limnotrachelobdella, which we named Limnotrachelobdella hypophthalmichthysa n. sp.  Institutional Review Board Statement: The animal study protocol was approved by the Animal Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences (project identification code: IHB/LL/2020025; date of approval: 27 July 2022).

Informed Consent Statement:
Written informed consent has been obtained from the patient(s) to publish this paper. Data Availability Statement: Data are available from the authors upon reasonable request.