Identification and Characterization of Myxobolus pronini (Cnidaria: Myxozoa) from Gibel Carp Carassius auratus gibelio and Goldfish C. auratus: New Fish Host, Infection Site, and Geographic Distribution in China

Abstract

Myxozoans represent a significant group of parasitic pathogens affecting both wild and farmed fish. For accurate and comprehensive early detection, the composition of myxozoan pathogens in fish has consistently been a primary focus for parasitologists. In China, we undertook an investigation into myxozoan infection in fish from Shandong Province, China, successfully isolating a myxozoan species from gibel carp Carassius auratus gibelio Bloch, 1782 and goldfish C. auratus Linnaeus, 1758. In the present study, this myxozoan species was identified by detailed morphological and molecular analysis. This parasite is characterized by the presence of visible plasmodia in various organs (intestine, liver, gallbladder, and abdominal cavity) of gibel carp as well as the abdominal cavities of goldfish. In comparison to all documented myxozoan species, this myxozoan species exhibits morphological identity with Myxobolus pronini Liu, Batueva, Zhao, Zhang, Zhang, Li, Li, 2016, in terms of spore morphology. Molecular sequence analysis, including sequence similarity, variable site, and the secondary structure of SSU rRNA, indicates that the present myxozoan species closely resembles M. pronini. Furthermore, phylogenetic analyses revealed that all isolates collected from different organs and hosts in this study clustered with M. pronini, forming a distinct branch within the Carassius-infecting clade. Consequently, the present myxozoan species can be confidently identified as M. pronini. Compared with the previous reports on M. pronini, this study documents, for the first time, goldfish as a host, intestines and gallbladders as the sites of infection, and Shandong Province as the geographic distribution of this myxozoan species.

1. Introduction

Myxozoans are a group of economically and ecologically important endoparasitic cnidarian [1,2]. A high species diversity of myxozoans has been documented, with more than 2600 myxozoan species recorded over the world [3]. Most myxozoans primarily infect fish, with a lower incidence of infection in amphibians, reptiles, birds, and mammals [4]. Although the majority of myxozoans do not pose a significant threat to fish hosts, some are important parasitic pathogens that cause the mass mortality of wild and farmed fish or significant economic losses [4]. Among these, the well-known species are Myxobolus cerebralis Hofer, 1903, which causes whirling disease in salmonid fish [5], Ceratonova shasta Noble, 1950, known for its high virulence and mortality rates nearing 100% in susceptible fish hosts [6], and M. honghuensis Liu, Whipps, Gu, Zeng, Huang, 2012, which infects pharynx of gibel carp Carassius auratus gibelio Bloch, 1782, and results in significant host mortality [7].

China is one of the countries with the richest myxozoan species diversity in the world, with approximately 700 myxozoan species documented [8,9,10]. In 1998, a book on one of the series of Fauna Sinica, named “Myxozoa: Myxosporea”, was published, which summarized 575 myxozoan species infecting freshwater fish in China. Due to the historical and technical limitations, the myxozoan species summarized in this book were identified solely by spore morphology [8]. One of the consequences is that many myxozoan species with similar morphological characteristics from various organs of different fish hosts were identified as the same species, which leads to a number of homonyms and cryptic species [11,12]. In the last two decades, the important roles of host/organ/tissue specificity and molecular sequence analysis have been gradually recognized in myxozoan species identification [11,13,14,15]. Therefore, an integrated approach combining spore morphology, host/organ/tissue specificity, and molecular sequence characteristics has been widely accepted for myxozoan species identification [4]. In China, a number of novel myxozoan species have been identified and numerous instances of misidentification and confusion have been corrected using this integrated approach [10,11,16,17,18,19,20]. However, the majority of surveys on myxozoan species diversity have predominantly focused on fish in aquaculture water bodies, with comparatively less attention given to fish in natural water bodies. In addition, investigations on myxozoan species diversity in ornamental fish remain limited as well [18].

To establish baseline data on myxozoan species diversity, an investigation was undertaken to examine myxozoan infections in fish from natural water bodies and ornamental fish in China. In this study, a myxozoan species was concurrently isolated from these two sources, which was described based on morphological and molecular characteristics.

2. Materials and Methods

2.1. Fish Collection

Sixty gibel carp C. auratus gibelio (13.3–17.3 cm in total length) and twenty-two goldfish C. auratus Linnaeus, 1758 (6.6–12.5 cm in total length) were collected from the Lake Weishan Wetland and Chengyang Fish Market in Qingdao city, Shandong Province, China, respectively. All the fish specimens were transported live to the laboratory, where they were anesthetized with tricaine methanesulfonate (MS222, SIGMA, Kawasaki, Japan) prior to dissection. Subsequently, the various external and internal organs, including gills, skin, fins, muscle, liver, spleen, kidney, intestine, heart, gallbladder, and swim bladder, were examined for myxozoans with the naked eye and light microscopy. All the fish specimens were treated in accordance with the guidelines of the Laboratory Animal Administration Law of China, with the permit number SD2007695 approved by the Ethics Committee of the Laboratory Animal Administration of Shandong Province, China.

2.2. Morphological Examination

To examine the suspected myxozoan plasmodia, the plasmodia were ruptured on slides to release spores. The fresh spores were measured and photographed using an Olympus BX53 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) fitted with an Olympus DP74 camera and differential interference contrast. Morphological and morphometric data were obtained from 30 randomly selected fresh spores according to Lom and Arthur [21]. The spore dimension presented by the mean ± standard deviation (SD) followed by the range in parentheses was given in micrometers (μm) unless stated otherwise. The spores were fixed with 10% formalin and deposited in the Aquatic Animal Parasitology Laboratory, School of Marine Science and Engineering, Qingdao Agricultural University.

2.3. DNA Extraction, Amplification and Sequencing

The isolated myxozoan plasmodia were fixed in absolute ethanol. Genomic DNA was subsequently extracted from the ethanol-fixed plasmodia using the Universal Genomic DNA Kit (CWBio, Shanghai, China) following the manufacturer’s protocol for animal tissue. The SSU rDNA sequences of the myxozoan species were amplified utilizing the primer pairs ERIB1/ERIB10 [22] and MyxospecF/MyxospecR [23]. The PCR reaction was carried out in a 25 μL reaction mixture, which contained 0.5 μL (10 μM) of each primer, 1 μL genomic DNA, 12.5 μL 2 × AceTaq^® Master Mix (Vazyme, Nanjing, China), and 10.5 μL double-distilled water. The PCR cycle consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 60 s, 55 °C for 60 s (ERIB1/ERIB10)/45 s (MyxospecF/MyxospecR), and 72 °C for 2 min, and a final extension at 72 °C for 7 min. The PCR products were separated using a 1% agarose gel and subsequently sequenced bidirectionally employing an ABI PRISM^® 3730XL DNA sequencer (AppliedBiosystems Inc., Foster, CA, USA). The resulting contiguous sequences were assembled using the SeqMan™ module within the Lasergene software suite version 7.1.0 (DNAStar, Madison, WI, USA). The newly acquired SSU rDNA sequences were aligned with sequences from the GenBank database using MAFFT v.7 with the L–INS–i algorithm [24]. Final sequence comparisons were edited and visualized utilizing Bioedit v.7 [25] and TBtools [26].

2.4. Secondary Structure Prediction of SSU rRNA Sequence

Using the secondary structure of the SSU rRNA sequence of Spherospora truttae Fischer-Scherl, El-Matbouli, Hoffmann, 1986 (AM410773), as a reference model [27], the secondary structures of V4, V6, and V7 regions of the SSU rRNA sequences of Myxobolus pronini Liu, Batueva, Zhao, Zhang, Zhang, Li, Li, 2016, were predicted. A multiple sequence alignment of the SSU rRNA sequences of M. pronini was performed using MAFFT version 7.490 [24]. The aligned sequences were subsequently compared with the reference sequence with MEGA version 7.0 [28] to identify the variable regions. A secondary structure prediction of the variable regions was conducted utilizing RNA Structure version 5.2, adhering to the principle of minimum free energy, with all parameters maintained at their default settings [29,30]. The predicted secondary structures were manually adjusted using RNAViz version 2.0 [31].

2.5. Phylogenetic Analyses

To assess the phylogenetic relationship of the newly identified myxozoan species with existing taxa, eight newly obtained sequences and forty-three myxozoan sequences from the Myxobolus clade were subject to phylogenetic analyses. Myxidium cuneiforme Fujita, 1924, and Zschokkella candia Kalatzis, Kokkari, Katharios, 2015, were chosen as outgroup taxa. A sequence alignment was performed using MAFFT version 7.490 [24] using default parameters and automatic selection of the appropriate alignment strategy, with a final alignment length of 1257 bp. Maximum likelihood (ML) analysis was conducted using IQ–TREE version 2.0 [32] with 10,000 ultrafast bootstrap replicates, employing the GTR + I + G model as determined by jModeltest 2 [33] under the Akaike Information Criterion (AIC). A Bayesian inference (BI) analysis was performed using MrBayes version 3.2.7 [34] with the GTR + I + G model selected via the AIC. Markov Chain Monte Carlo (MCMC) simulations were executed for 2 × 106 generations, with sampling every 100 generations and a burn-in of 5000 trees. The remaining trees were utilized to compute posterior probabilities employing a 50% majority rule consensus approach. Phylogenetic trees were visualized using MEGA version 7 [28] or Figtree version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 13 January 2025)).

3. Results

3.1. Plasmodia of Myxobolus pronini

In the present study, M. pronini was found to infect various organs of gibel carp and goldfish. In gibel carp, the presence of round or irregularly shaped, white plasmodia of different sizes (0.2–6.4 mm in diameter) was predominantly noted in the intestine (Figure 1a–d) and liver (Figure 1e–g). In addition, plasmodia were observed on the outer wall of the gallbladder (Figure 1h) and freely within the abdominal cavity of a small number of gibel carp. Occasionally, plasmodia were concurrently detected in the intestine, liver, and gallbladder of the same gibel carp (Figure 1j). In goldfish, a singular round and white plasmodium (8.6 mm in diameter) was found free within the abdominal cavity (Figure 1i).

3.2. Spores of Myxobolus pronini

Spores from different organs and hosts were identical in shape (Figure 2). The spores were elongated obovate in frontal view with the blunt anterior ends wider than the posterior ends. Two pyriform polar capsules were equal in size. A polar filament was coiled with five to seven turns. In addition, a small number of abnormal spores with caudal appendages were observed in plasmodia from various organs of gibel carp (Figure 3). Morphometric analysis showed that spores of different isolates were generally of similar size. Although some indicators of spores showed obvious differences, there was an overlap in the morphometric data of different isolates. All the morphometric data of spores are given in

Table 1. Morphometric data of Myxobolus pronini collected in the present study with its other records (all measurements in μm). SL: spore length, SW: spore width, PCL: polar capsule length, PCW: polar capsule width, SI: site of infection. ”-”: not sequenced. The characteristics of spore shape (elongated obovate) and polar capsule shape (pyriform) are identical among all isolates.

SL	SW	PCL	PCW	SI	Host	Source	Accession Number
15.1 ± 0.3 (14.3–16.2)	10.1 ± 0.1 (9.6–10.8)	5.4 ± 0.6 (4.3–6.7)	3.1 ± 0.1 (2.2–3.6)	Abdominal cavity	C. auratus gibelio	[35]	KU524889
14.7 ± 0.2 (13.8–15.6)	9.6 ± 0.7 (9.0–13.3)	5.3 ± 0.2 (4.8–5.6)	3.0 ± 0.1 (2.9–3.4)	Visceral serous membranes of liver	C. auratus gibelio	[35]	KU524890
13.86 ± 0.8 (12.2–15.39)	9.83 ± 0.5 (8.57–10.51)	5.69 ± 0.42 (4.8–6.47)	3.36 ± 0.21 (2.79–3.73)	Abdominal cavity, gallbladder	C. auratus gibelio	[36]	MH329619
16.8 ± 0.4 (16.0–17.9)	9.5 ± 0.4 (8.7–10.2)	6.0 ± 0.3 (5.6–6.8)	3.4 ± 0.2 (3.1–3.8)	Intestine	C. auratus gibelio	Present research	PP840598
15.2 ± 0.5 (14.4–16.4)	9.5 ± 0.5 (8.5–10.5)	5.8 ± 0.4 (5.0–6.4)	3.4 ± 0.2 (3.1–3.8)	Intestine	C. auratus gibelio	Present research	PP840599
16.5 ± 0.2 (16.1–16.9)	10.6 ± 0.4 (9.9–11.2)	5.8 ± 0.3 (5.3–6.3)	3.6 ± 0.2 (3.3–3.9)	Intestine	C. auratus gibelio	Present research	PP840600
14.1 ± 0.4 (13.3–15.0)	9.5 ± 0.5 (8.8–10.2)	4.9 ± 0.4 (4.0–5.5)	3.3 ± 0.3 (2.7–3.9)	Intestine	C. auratus gibelio	Present research	PP840601
13.0 ± 0.8 (11.4–14.3)	9.5 ± 0.5 (8.3–10.6)	5.3 ± 0.5 (4.4–6.3)	3.1 ± 0.4 (2.3–3.9)	Intestine	C. auratus gibelio	Present research	-
13.6 ± 0.5 (12.2–14.4)	9.2 ± 0.5 (8.4–10.3)	5.1 ± 0.3 (4.6–5.6)	3.3 ± 0.2 (2.8–3.7)	Intestine	C. auratus gibelio	Present research	-
15.0 ± 0.8 (13.6–16.5)	9.4 ± 0.5 (8.0–10.4)	5.8 ± 0.3 (5.0–6.3)	3.2 ± 0.2 (2.9–3.5)	Gallbladder wall	C. auratus gibelio	Present research	PP840602
15.0 ± 0.3 (14.5–15.6)	10.2 ± 0.3 (9.6–10.7)	5.9 ± 0.2 (5.4–6.4)	3.4 ± 0.2 (3.1–4.0)	Liver	C. auratus gibelio	Present research	PP840603
13.5 ± 0.4 (12.9–14.6)	9.7 ± 0.4 (8.8–11.0)	5.4 ± 0.3 (4.8–6.1)	3.3 ± 0.2 (3.0–3.6)	Liver	C. auratus gibelio	Present research	PP840604
14.2 ± 0.8 (12.9–15.7)	9.1 ± 0.3 (8.7–10.0)	5.1 ± 0.4 (4.1–5.7)	3.2 ± 0.2 (2.8–3.6)	Liver	C. auratus gibelio	Present research	-
13.3 ± 0.8 (12.0–15.3)	9.6 ± 0.6 (8.4–10.7)	4.9 ± 0.4 (4.2–5.6)	3.1 ± 0.3 (2.6–3.7)	Abdominal cavity	C. auratus gibelio	Present research	-
12.8 ± 0.8 (11.2–14.3)	9.5 ± 0.6 (8.4–10.5)	4.8 ± 0.4 (4.2–5.6)	2.9 ± 0.3 (2.3–3.5)	Abdominal cavity	C. auratus	Present research	PP840605

.

3.3. Sequence and Phylogenetic Analyses

In the current study, seven SSU rDNA sequences were obtained from specimens collected from the intestine (length/accession number: 1949 bp/PP840598, 1992 bp/PP840599, 2074 bp/PP840600, 2104 bp/PP840601), liver (length/accession number: 1994 bp/PP840603, 1959 bp/PP840604), and gallbladder (length/accession number: 1610 bp/PP840602) of gibel carp. Additionally, a sequence with 1952 bp (accession number: PP840605) was acquired from specimens collected from the abdominal cavity of goldfish. BLAST analysis revealed that these SSU rDNA sequences in the present study exhibited 99.19–100% identity with M. pronini (MH329619, OM678575, OM678576, KU524890, KU524889). To assess intraspecific sequence variations, all available SSU rDNA sequences of M. pronini were subjected to sequence analysis, resulting in the identification of 22 variable sites distributed across 6 M. pronini isolates (Figure 4). The secondary structure analysis of SSU rRNA sequences showed that the V6 region’s structure was conserved across various M. pronini isolates (Figure 5). In contrast, structural variations were detected in the E23–3 section of the V4 region and the E43–2 section of the V7 region. Specifically, although all isolates exhibited one lateral process and one internal ring in the E23–3 section of the V4 region, the positions of the lateral process of three isolates (KU524890, PP840600, and PP840604) are different from that of other isolates. Furthermore, in the E43–2 section of the V7 region, most isolates possessed a large internal loop of 10 bases and a small internal loop of 8 bases, while an isolate from the intestine (PP840601) had two internal loops, both comprising 8 bases and an isolate. (KU524890) from Liu et al. (2016) had a larger internal loop of 12 bases. The ML analysis confirmed the clustering patterns of the Bayesian analysis in several cases with different bootstrap values. Phylogenetic analyses indicated that all M. pronini isolates clustered together, forming a distinct branch within the Carassius-infecting clade (Figure 6).

• Taxonomic summary:
• Host: Gibel carp Carassius auratus gibelio, goldfish C. auratus.
• Host size: 13.3–17.3 cm in total length (gibel carp), 6.6–12.5 cm in total length (goldfish).
• Site of infection: Intestine (gibel carp), liver (gibel carp), gallbladder (gibel carp), abdominal cavity (gibel carp, goldfish).
• Prevalence: 33.3% (20/60, gibel carp), 4.5% (1/22, goldfish).
• Locality: Lake Weishan Wetland (gibel carp), Chengyang Fish Market in Qingdao City (goldfish), Shandong Province, China.
• Sample preservation: Accession no. MTR202205201, MTR202303121.

4. Discussion

In order to identify the present Myxobolus species, it was compared with all previously reported Myxobolus species. The findings revealed that this myxozoan species was morphologically indistinguishable from M. pronini based on spore morphology, as detailed in Table 1. Although morphometric variations were noted among different isolates of M. pronini, there was a significant overlap in the morphometric data of these isolates. This overlap suggests that the observed variations are likely intraspecific in nature. Indeed, the intraspecific morphometric variations of M. pronini spores were previously documented in the initial research by Liu et al. [35]. In addition, intraspecific morphometric variations of myxospores have been documented in a number of myxozoan species [37,38,39]. Early research primarily relied on spore morphology for the identification of myxozoans, and these intraspecific morphometric variations have posed significant challenges for accurate species identification, often leading to the establishment of numerous synonyms [8,16].

To further characterize the present myxozoan species, sequence analysis of the SSU rDNA was conducted in this study. The results indicated that the SSU rDNA sequences of the myxozoan species exhibited a remarkably high degree of similarity (99.19–100%) with M. pronini, which falls within the range of intraspecific sequence variations previously documented for myxozoan species [40,41]. In addition, variable sites within the SSU rDNA sequences of different isolates of M. pronini were identified in this study. This analysis revealed a total of 22 variable sites, with a maximum of fifteen variable sites observed between any two sequences. These results challenge the previously held assumption that the number of base variations within intraspecies of the genus Myxobolus is less than 10 bases [42]. Recently, a secondary structure of the SSU rRNA sequence has been identified as a potent tool for distinguishing myxozoan species with closely related molecular sequences [43]. In the current study, the structure of the V6 region was observed to be conserved across various isolates of M. pronini, whereas structural variations were detected in the E23–3 section of the V4 region and the E43–2 section of the V7 region. These findings indicated that the evolution rate of secondary structures in different regions of SSU rRNA may be different. Phylogenetic analyses revealed that all M. pronini isolates clustered together, forming a distinct branch placed in the Carassius-infecting clade, which is consistent with previous research [35,36]. Based on the comprehensive morphological and molecular analysis, the myxozoan species collected from various organs and hosts in the present study can be confidently identified as M. pronini, despite the observed morphological and molecular sequence variations among different isolates.

In the initial description by Liu et al. [35], M. pronini isolates were obtained from gibel carp from China and Russia. More recently, this parasite was again collected from gibel carp in China [36]. In the present study, in addition to gibel carp, M. pronini was first found to infect goldfish. Notably, several myxozoan species, such as M. honghuensis [44], M. ampullicapsulatus Zhao, Sun, Kent, Deng, Whipps, 2008 [36], M. wulii Landsberg, Lom, 1991 [36], M. pyramidis Chen, 1958 [36], have been documented to parasitize both gibel carp and goldfish, possibly due to their close genetic relationship. However, the limited attention given to myxozoans in goldfish has resulted in the identification of only approximately 10 myxozoan species in goldfish [10], as opposed to the over 50 myxozoan species reported to infect gibel carp [45]. We hypothesize that with increased research into myxozoan diversity in goldfish, a greater number of myxozoan species infecting both gibel carp and goldfish will be identified in the future. In China, M. pronini was initially collected from a natural water body, Lake Taibai, in Hubei Province [35]. Recently, this parasite was reported in aquaculture ponds in Hubei and Liaoning Provinces [36]. The present study has identified an expanded geographical distribution of this parasite, encompassing additional natural and aquaculture water bodies in Shandong Province. These findings suggest that M. pronini is a widely distributed parasite across China, thereby elevating its potential threat to both wild and farmed fish. In the initial description, M. pronini was reported to infect the abdominal cavities and visceral serous membranes of the livers of gibel carp [35]. Subsequently, free spores of M. pronini were observed in the gallbladders of gibel carp [36]. In addition to these known infection sites, the current study identified the intestine and gallbladder wall as additional target organs for M. pronini. The presence of multiple infection sites suggests the necessity of examining the various internal organs of a host when detecting M. pronini in the future.

5. Conclusions

This study identified the myxozoan species infecting various organs of gibel carp and goldfish as M. pronini. Isolates of this parasite from different organs and hosts exhibited both morphometric and molecular variations. In comparison to previous reports on M. pronini, the present research offers novel insights into its fish host, infection site, and geographic distribution.