Morphological and Molecular Identiﬁcation of Dactylogyrus gobiocypris (Monogenea: Dactylogyridae) on Gills of a Model Fish, Gobiocypris rarus (Cypriniformes: Gobionidae)

: The rare minnow Gobiocypris rarus is an ideal model organism for toxicological research. Dactylogyrus species are usually found on the gills of this rare minnow in laboratory farming systems. Dactylogyrid infection may change the sensibility of ﬁsh to toxicants and affect toxicological evaluations. In the present study, dactylogyrid infection was investigated, and species of Dactylogyrus collected from rare minnows were determined. Based on the observed ‘ D. wunderi ’ type anchors, with a shorter outer root and elongated inner root, and accessory piece consisting of two parts, the dactyl-ogyrids were identiﬁed as D. gobiocypris . A partial 18S-ITS1 rDNA sequence was ﬁrstly sequenced, and the highest sequence identity (86.7%) was to D. cryptomeres . Phylogenetic analysis revealed that D. gobiocypris formed a clade with D. squameus , D. ﬁnitimus , and D. cryptomeres , all of which have been recorded in the family Gobionidae. Histopathology analysis indicated that a heavy burden of D. gobiocypris caused necrosis of gill ﬁlaments. Inﬂammatory responses, such as tumefaction and hyperaemia, were also observed on gills with severe dactylogyrid infection. Supplementary morphological characteristics and 18S-ITS1 rDNA sequence provided basic data for identiﬁcation of this parasite species.


Introduction
The rare minnow Gobiocypris rarus Ye et Fu, 1983 (Gobionidae) is a small gobionid fish endemic to China, mainly distributed at the edge of the west and northwest area of the Sichuan Basin [1,2]. This rare minnow possesses particular biological characteristics, such as high sensitivity to chemicals, small body size, short life cycle, and being easy to rear in laboratory, which make it an excellent model organism for ecotoxicology studies [3][4][5]. Since 1995, rare minnows have been widely used in acute and subchronic toxicity experiments on heavy metals [6,7], organics [8][9][10], and endocrine-disrupting chemicals [11][12][13].
The monogenean family Dactylogyridae Bychowsky, 1933 is one of the most speciesrich groups of helminths, with more than 1000 species recognised worldwide [14]. Fortyone species of Dactylogyrus have been recorded from fishes in the family Gobionidae in China [15], and twenty-six species of Dactylogyrus are found on fishes in the family Gobionidae in Europe [16]. Species of Dactylogyrus were found on gills of rare minnows in a laboratory in China, and D. gobiocypris Yao, 1995 was described based on sclerotized parts of the anchor and copulatory complex [17]. No studies have reported D. gobiocypris since.
The present study provides supplementary morphological characteristics, novel sequences of the 18S ribosomal RNA subunit and the first internal transcribed spacer region of rDNA (ITS1), and histopathological analysis of D. gobiocypris parasitizing G. rarus specimens reared in the laboratory in the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan City, Hubei province, China.

Parasite Collection
Samples of rare minnow were obtained from the laboratory in the Institute of Hydrobiology, Chinese Academy of Sciences in April 2022. Thirty fish (with total body length of 4.2 ± 0.9 cm) were randomly selected and anesthetized with 0.02% MS-222 (tricaine methanesulfonate) (Sigma, St Louis, MO, USA). Specimens of species of Dactylogyrus were then examined and collected using micro surgical needles under a stereoscopic microscope. Worms were rinsed several times with distilled water for further analyses.

Morphological Identification
A random subsample of dactylogyrids were mounted on a microscope slide and fixed in ammonium picrate glycerine (GAP) as whole mount following the procedure described by Ergens [24] and Malmberg [25] for morphological identification. GAP and Canada balsam were performed according to Ergens [24]. Additional specimens, with opisthaptors excised using a scalpel, were then individually subjected to proteolytic digestion according to the method described by Paladini et al. [26] and Tu et al. [22]. The tissue-free opisthaptoral sclerotized parts were mounted in GAP, and the excised body of each specimen was preserved in 95% ethanol for subsequent molecular analysis. Specimens were photographed using an optical microscope (Axioplan 2 imaging and Axiophot 2, Zeiss, Oberkochen, Germany). Measurements were taken according to Šimková et al. [27], and are given in micrometers (µm) unless otherwise stated. Identification of individual specimens was performed by comparing the morphology and measurements of anchors and the copulatory complex to the literature [17]. Five full worms, embedded in GAP and mounted on Canada balsam, were deposited as voucher specimens (accession nos. CJW-DG 202201-05) in the Museum of the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan City, Hubei province, China.

DNA extraction, Amplification and Sequencing
Genomic DNA was extracted from the excised bodies of 12 specimens using a Tissue Cell Genome Kit (TaKaRa, Beijing, China) according to the manufacturer's instructions. The region of rDNA spanning the 3 end of the 18S ribosomal RNA subunit, the entire ITS1 gene, and the 5 end of the 5.8S ribosomal RNA subunit were targeted using primers S1 (5 -ATTCCGATAACGAACGAGACT-3 ) and H7 (5 -GCTGCGTTCTTCATCGATACTCG-3 ) [28,29]. PCR amplification was conducted using LA Taq polymerase (TaKaRa, Beijing, China) with the following profile: 5 min at 95 • C, 35 cycles of 1 min at 94 • C, 1 min at 55 • C, 1 min 30 s at 72 • C, and a final extension of 10 min at 72 • C. After purification, PCR products were cloned into the pGEM-T vector (Promega, Madison, USA), sequenced with the primers described above, produced by Sangon Biotech (Shanghai, China), and assembled manually with DNAStar's SeqMan software (DNAStar, Madison, WI, USA).

Molecular Analyses
The obtained sequences of partial 18S rDNA, ITS1, and the flanking sequence of 5.8S rDNA were compared using BLAST in GenBank to assess similarity with other Dactylogyrus species. From the 12 specimens, 12 sequences of 18S-ITS1 rDNA were obtained to evaluate the intraspecific variation using BLAST. Sequences (Table 1) (Siwak, 1932), in the family Ancylodiscoididae, was used as the outgroup. Sequences were imported into PhyloSuite [30] and aligned with available 18S-ITS1 rDNA sequences of other Dactylogyrus spp. in GenBank using MAFFT 7.149 [31]. Ambiguously aligned fragments were trimmed using Gblocks [32] with the following parameter settings: minimum number of sequences for a conserved/flank position (6/6), maximum number of contiguous nonconserved positions (8), minimum length of a block (10), allowed gap positions (with half). Phylogenetic analyses based on the 18S-ITS1 sequences were performed using maximum likelihood (ML) and inference (BI) methods. TNe+G4 and K2P+G4 were chosen as the best-fit partition model for nucleotide evolution for ML and BI analyses, respectively using ModelFinder [33]. ML phylogenies were inferred using IQ-TREE [34], for 1000 standard bootstraps, as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test. BI phylogenies were inferred using MrBayes 3.2.6 [35], with two parallel runs (2,000,000 generations) in which the initial 25% of sampled data were discarded as burn-in.

Morphological Characterization
Dactylogyrus gobiocypris Yao, 1995 was the only Dactylogyrus species described on the gills of this rare minnow in China [17]. More detailed morphometric measurements are provided herein, since new data on the morphology and phylogeny of D. gobiocypris were obtained in the present study. The measurements and shape of the sclerotized parts of the anchors of the specimens collected in the present study were almost identical to the original descriptions of D. gobiocypris by Yao [17]. However, the ventral bar was flatter and straighter, and the copulatory complex was shorter, than that of the D. gobiocypris described by Yao [17] (penis length 10-14 μm vs. 15-20 μm, and accessory piece 13-17 μm vs. 18-21, respectively). The morphometrical parameters of the sclerotized parts of dactylogyrids sometimes vary over seasons, temperature, and fixation and measurement procedure [29,40,41]. We used a substantial sample size in the present study, while the original descriptions by Yao were based on seven specimens. Thus, these discrepancies are judged to demonstrate intraspecific variation.
According to the studied morphological characteristics, D. gobiocypris most closely resembles D. trullaeformis in the shape of the anchors, with 'D. wunderi' type anchors, having a shorter outer root and elongated inner root. However, D. gobiocypris differs from D. trullaeformis [39] in: (1) the longer length (15-21 μm vs. 14-16 μm, respectively) and shape of the middle portion of ventral bar (slightly convex posteriorly), which is flatter and straighter in D. trullaeformis; (2) the accessory piece of D. gobiocypris consists of two parts, one of which is horseshoe-shape and the other semicapsular, while the accessory piece of

Morphological Characterization
Dactylogyrus gobiocypris Yao, 1995 was the only Dactylogyrus species described on the gills of this rare minnow in China [17]. More detailed morphometric measurements are provided herein, since new data on the morphology and phylogeny of D. gobiocypris were obtained in the present study. The measurements and shape of the sclerotized parts of the anchors of the specimens collected in the present study were almost identical to the original descriptions of D. gobiocypris by Yao [17]. However, the ventral bar was flatter and straighter, and the copulatory complex was shorter, than that of the D. gobiocypris described by Yao [17] (penis length 10-14 µm vs. 15-20 µm, and accessory piece 13-17 µm vs. 18-21, respectively). The morphometrical parameters of the sclerotized parts of dactylogyrids sometimes vary over seasons, temperature, and fixation and measurement procedure [29,40,41]. We used a substantial sample size in the present study, while the original descriptions by Yao were based on seven specimens. Thus, these discrepancies are judged to demonstrate intraspecific variation.
According to the studied morphological characteristics, D. gobiocypris most closely resembles D. trullaeformis in the shape of the anchors, with 'D. wunderi' type anchors, having a shorter outer root and elongated inner root. However, D. gobiocypris differs from D. trullaeformis [39] in: (1) the longer length (15-21 µm vs. 14-16 µm, respectively) and shape of the middle portion of ventral bar (slightly convex posteriorly), which is flatter and straighter in D. trullaeformis; (2) the accessory piece of D. gobiocypris consists of two parts, one of which is horseshoe-shape and the other semicapsular, while the accessory piece of D. trullaeformis consists of only one part and is shaped as a heterogeneous groove (Figure 4).

Molecular Analyses
Sequences (18S-ITS1) collected from the 12 specimens were identical; the length was 1042 bp. The BLAST search showed that Dactylogyrus gobiocypris displayed the highest sequence identity, 86.7%, to D. cryptomeres, which was collected from Gobio gobio (Linnaeus, 1758) (Cypriniformes: Gobionidae). The sequence of D. gobiocypris was then submitted in GenBank for the first time.
Phylogenetic analyses, based on the BI and ML criteria of the 18S rDNA-ITS1 sequence, showed identical topology and only minor differences in statistical support values for some nodes ( Figure 5). Dactylogyrus gobiocypris formed a clade with D. squameus, D. finitimus, and D. cryptomeres, all of which parasitize on the family Gobionidae. D. lamellatus, parasitic on Ctenopharyngodon Idella (Valenciennes, 1844) (Cypriniformes: Xenocyprididae), then formed a clade with those Dactylogyrus species above.

Histopathology Analysis
The histopathological responses of the host to D. gobiocypris were investigated by serially sectioning the gills of naturally infected fishes. The gill lamellae of uninfected G. rarus were structurally intact, with consistent thickness at the base and end, uniform morphology, and visible gaps between gill lamellae (Figure 6 A, C). Histological examination showed that the infected gills were damaged, to some extent, by necrosis. Additionally, the infection caused hyperplasia of the respiratory epithelium between gill lamellae, with a tendency for adjacent gill filaments to fuse (Figure 6 B). Gill lamellae were affected by the anchors of D. gobiocypris, with a breakdown of cell integrity. Cell proliferation was also observed on the base of gill lamellae, which resulted in adhesion of adjacent gill lamellae (Figure 6 D).

Molecular Analyses
Sequences (18S-ITS1) collected from the 12 specimens were identical; the length was 1042 bp. The BLAST search showed that Dactylogyrus gobiocypris displayed the highest sequence identity, 86.7%, to D. cryptomeres, which was collected from Gobio gobio (Linnaeus, 1758) (Cypriniformes: Gobionidae). The sequence of D. gobiocypris was then submitted in GenBank for the first time.
Phylogenetic analyses, based on the BI and ML criteria of the 18S rDNA-ITS1 sequence, showed identical topology and only minor differences in statistical support values for some nodes ( Figure 5). Dactylogyrus gobiocypris formed a clade with D. squameus, D. finitimus, and D. cryptomeres, all of which parasitize on the family Gobionidae. D. lamellatus, parasitic on Ctenopharyngodon Idella (Valenciennes, 1844) (Cypriniformes: Xenocyprididae), then formed a clade with those Dactylogyrus species above.

Histopathology Analysis
The histopathological responses of the host to D. gobiocypris were investigated by serially sectioning the gills of naturally infected fishes. The gill lamellae of uninfected G. rarus were structurally intact, with consistent thickness at the base and end, uniform morphology, and visible gaps between gill lamellae (Figure 6 A,C). Histological examination showed that the infected gills were damaged, to some extent, by necrosis. Additionally, the infection caused hyperplasia of the respiratory epithelium between gill lamellae, with a tendency for adjacent gill filaments to fuse (Figure 6 B). Gill lamellae were affected by the anchors of D. gobiocypris, with a breakdown of cell integrity. Cell proliferation was also observed on the base of gill lamellae, which resulted in adhesion of adjacent gill lamellae ( Figure 6D).

Discussion
Species of the genus Dactylogyrus are a group of monogenean gill parasites that are highly specific to freshwater fishes of the family Cyprinidae [28]. Basing on the measurements and shape of sclerotized parts of opisthaptor and copulatory complex, the dactylogyrids collected from gills of a rare minnow were identified as D. gobiocypris. To date, D. gobiocypris represents the only monogenean species reported infecting this rare minnow in China [17]. The present study provides additional measurements of sclerotized structures of the opisthaptor of this species, along with its molecular characterization and histopathological responses.
In general, the taxonomy of dactylogyrid monogeneans depends on accurate descriptions of the size and shape of the sclerotized parts of the opisthaptor and reproductive organs [29]. The measurements and morphology of the sclerotized parts of the specimens collected in the present study were almost identical to those of D. gobiocypris provided by Yao [17]. Of the other species infecting closely related hosts in the Gobionidae family, D. gobiocypris most closely resembles D. trullaeformis in the shape of the anchors. However, there are distinct differences in the structure of the copulatory complex between the two species. The accessory piece of D. gobiocypris consists of two parts, one of which is horseshoe-shaped and the other semicapsular, whereas in D. trullaeformis it consists of only one part and is shaped as a heterogeneous groove. The 18S ribosomal RNA subunit and the internal transcribed spacer region (ITS1) are common molecular markers for identification of Dactylogyrus species [29,42]. The results of the BLAST search suggested the sequence of D. gobiocypris displayed the highest overall identity (86.7%) to D. cryptomeres, collected from Gobio gobio. The sequence of D. gobiocypris was obtained and submitted in GenBank for the first time.
Phylogenetic trees (BI / ML) of Dactylogyrus species, constructed based on partial 18S-ITS1 rDNA sequences, are divided into two clades: (1) one clade includes dactylogyrids from Cyprinus carpio (Linnaeus, 1758) and Carassius auratus Linnaeus, 1758, both representatives of Cyprinidae; (2) the other clade includes parasite species of C. idella (Xenocyprididae) and Gobionidae. Dactylogyrus gobiocypris exhibited a relatively close phylogenetic relationship with D. squameus, D. finitimus, and D. cryptomeres, all of which parasitize fishes of the Gobionidae family. The molecular phylogeny shows a consistent pattern of relationships among Dactylogyrus species. This suggests that there is a high degree of host specificity among the Dactylogyrus species that parasitize Gobionidae fishes, which has been displayed in previous molecular phylogenetic studies [28,42,43].
Dactylogyrus gobiocypris was found on all individuals of G. rarus investigated, with a high abundance which reached 60.8±84.5 parasites per fish. Prevalence and mean abundance of Dactylogyrus infection in cultured rare minnow under laboratory conditions is higher than Dactylogyrus spp. in wild and farmed goldfish Carassius auratus [42,44]. The IHB rare minnow is a closed laboratory animal colony, the offspring of 50 wild G. rarus specimens collected in Hanyuan County of Sichuan Province in 2006 and bred using methods that prevent inbreeding [45]. The higher prevalence and mean abundance of Dactylogyrus gobiocypris infection may be related to declining genetic diversity and regular supplementation of the number of susceptible hosts. Dactylogyrus gobiocypris can be achieved by in vivo culture under laboratory conditions, and the host is singly infected with D. gobiocypris. The rare minnow (Gobiocypris rarus)-D. gobiocypris artificial infection system can be used as a new host-parasite laboratory model, which will provide support for further investigation.
Observation of histopathological sections of gills of G. rarus infected with D. gobiocypris indicated that D. gobiocypris infection could lead to damage of gill lamellae, causing serious hyperplasia and fusion of the gill filament epithelium. These lesions may reduce the area of gas exchange, affect the respiratory function of gills, and even cause potential secondary infections leading to serious disease with adverse consequences [20,21,46]. In the present study, G. rarus infected with a high abundance of D. gobiocypris did not have obvious typical clinical symptoms or high mortalities. This lack of symptoms is perhaps caused by decreased parasite virulence or increased host tolerance with a long coevolutionary history.
Parasitic infection may be capable of modifying the resistance of the host to other stressors [46][47][48]. The susceptibility to toxicants of G. rarus may be affected under the stress of high abundance of D. gobiocypris, thus interfering with the outcome of toxicological evaluation [49]. Fish hosts infected with parasites have been proven to be more sensitive to toxicants than uninfected conspecifics [50][51][52][53]. Most research to date on tolerance to chemicals and environmental pollutants appears to have overlooked the effects of parasites.
Therefore, parasite infection in model organisms should be considered during aquatic toxicity testing and chemical safety assessment.