Morphological description and molecular identification of Myxobolus dajiangensis n. sp. (Myxozoa: Myxobolidae) from the gill of Cyprinus carpio in southwest China

New species

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: D94613DA-E0C5-43A2-824B-9A6848A7C4C0. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Sample collection

Nine common carp, C. carpio, were collected from Chenjiaqiao Farmer’s Market, Shapingba District, Chongqing, and Golden Wharf Farmer’s Market, Bijiang District, Tongren, Guizhou, China, respectively. The fish measured between 11.6 cm to 15.9 cm in length. All fish were immediately transported to the laboratory for the parasitological examination. This study was approved by the Animal Care and Use Committee of the Key Laboratory of Animal Biology of Chongqing (Permit number: Zhao-20191010-01) and performed according to the recommendations in the Guide for the Care and Use of Animals at the Key Laboratory of Animal Biology of Chongqing, China.

Fish examination and morphological identification

The tissues and organs of the fish, including the gills, muscle, liver, intestine, and other organs were dissected. Samples were observed by the naked eyes and microscopy. Several small cysts were isolated from the infected gill lamellae (Fig. 1). The cysts were pierced with a fine needle to release the myxospores, which were then diluted with distilled water. The specimens were treated and identified as previously described (Zhao, Ma & Song, 2001). The morphological structure of the myxospores was observed, photographed, and determined using a Leica DM6000B microscope (Leica Microsystems, CMS GmbH, Germany) at 1,000× magnification. CorelDRAW 11 and Photoshop CS6 were used to illustrate our findings.

Histological analysis

The infected gills were fixed in Bouin’s solution for 24 h, then dehydrated in a graded ethanol series, hyalinized in xylene, and embedded in paraffin wax. Tissue sections (5–6 µm) were stained by hematoxylin and eosin (H & E).

DNA extraction, polymerase chain reaction (PCR), and sequencing

Genomic DNA of fresh myxospores from cysts was extracted using a DNeasy Blood & Tissue Kit (QIAGEN, Düsseldorf, Germany) following the manufacturer’s instructions. The SSU rDNA primers were as follows: 18E -5′CTG GTT GAT CCT GCC AGT-3′ (Hillis & Dixon, 1991) and 18R - 5′CTA CGG AAA CCT TGT TAC G-3′ (Whipps et al., 2003). PCR was performed using a Veriti™ 96-Well Thermal Cycler (Applied Biosystems, Singapore) in a 20 µL reaction system consisting of 7.2 µL dd H₂O, 6 µL 2 × Taq Master Mix, 0.5 µL each primer, and 5.8 µL genomic DNA. Briefly, after an initial denaturation at 94 °C for 90 s, the amplifications were carried out with 35 cycles of 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 2 min, followed by an extra extension at 72 °C for 5 min and held at 12 °C. 3 µL PCR amplicon was subjected to electrophoresis on 1% agarose gel stained with GoldView™ Nuclear Staining Dyes. PCR products were purified with the DNA Agarose Gel Extraction Kit (OMEGA Bio-Tek, Norcross City, GA, USA) and inserted into a pMD18-T vector (TAKARA, Otsu, Japan). The target nucleotide sequences were sent to the TSINGKE (Chongqing, China) for bidirectional sequencing.

Molecular and phylogenetic analysis

The pairwise sequence similarities were calculated using BLASTn from GenBank. Multiple sequence alignments and divergences were carried out using MEGA6 with default parameters (Tamura et al., 2013). The predicted secondary structures, based on free energy minimization, were constructed for the selected species using RNA structure 5.2 with default settings. The obtained structures were displayed and manually adjusted using RNAViz 2.0 (Zhang et al. 2015). Three hypervariable regions of SSU rRNA (V4, V6 and V7) were selected to study their variability.

A total of 59 valid SSU rDNA sequences of Myxobolus species with over 94% similarity from Cyprinidae fishes in GenBank were selected and used for phylogenetic relationship analysis. Kudoa quadricornis (Whipps et al., 2003) and Kudoa alliaria Shulman et Kovaleva, 1979 were used as outgroups. A Bayesian inference (BI) tree was conducted using MrBayes 3.12 (Ronquist & Huelsenbeck, 2003). Maximum likelihood (ML) analyses were performed with RAxML HPC software (Stamatakis, Hoover & Rougemont, 2008) with 1,000 bootstrap replicates using the GTR + G model. Trees were initially examined in FigTree v1.4.2, then edited and annotated in Adobe Photoshop. The secondary structure model was available at the European ribosomal RNA database (Peer et al., 2000).

Taxonomic summary

Type host: Cyprinus carpio

Site of infection: gill lamellae

Prevalence of infection: 100% (5/5) in Chongqing; 25% (1/4) in Guizhou.

Type locality: Chenjiaqiao, Shapingba District, Chongqing (29°30′N, 106°27′E), and Bijiang District, Tongren, Guizhou (27°71′N, 109°19′E), China.

Deposition of type materials: Syntype specimens (No. 202101) were deposited at the Collection Center of Type-specimens, Chongqing KLAB, Chongqing Normal University, China.

Etymology: The species was named after the main river flowing through the collection locality, Tongren City, Guizhou, China.

Vegetative stage: Many spherical whitish cysts, 0.2–0.8 mm in diameter, were located in the gill filaments (Fig. 1).

Morphological description: All the myxospores were mature. Vegetative spores were not observed in this study. The mature myxospores were elongated and pyriform in the frontal view, tapered towards an acuminate anterior end, rounded posterior end, and leptosomatic in the sutural view (Fig. 2). The myxospores were 14.8 ± 0.4 (13.9–15.6) µm in length, 8.0 ± 0.5 (7.3–9.1) µm in width (n = 60), and 5.5 µm in thickness (n = 1). The two polar capsules were slightly unequal in length. The length of the larger one was 8.0 ± 0.4 (7.1–8.8) µm, the smaller one was 7.4 ± 0.4 (6.1–8.0) µm, and the width of both polar capsules was 2.5 ± 0.2 (2.0–3.2) µm (n = 60). Polar filaments were coiled with approximately 10 turns (ranging from 9 to 11) and perpendicularly situated to the longitudinal axis of the polar capsule. The aspect ratio of the myxospore was 1:0.5, and the aspect ratio of the polar capsule was 1:0.3. Moreover, the ratio of myxospore length to polar capsule length was 1:0.5. The ratio of the myxospore width to polar capsule width was 1:0.3. The structures, including the intercapsular appendix, mucous envelope, iodinophilous vacuole and sutural edge marking were not observed (Fig. 2).

Histology

No remarkable external clinicopathological features were observed for the examined fish. Host responses to the infection led gill lamellae to expand to accommodate the plasmodia (an outer tissue consisting of epithelium cells and pillar cells). An abundance of plasmodia was observed in the secondary lamellae and a large plasmodium in the gill lamellae pushed and deformed the gill lamellae on both sides (Figs. 1 and 3). Any pseudoeosinophil cells and distinct inflammatory responses were not observed at the infection sites.

Molecular and phylogenetic analysis

Two SSU rDNA sequences with 1,885 nt and 1,969 nt were successfully obtained from Chongqing and Guizhou, respectively. These sequences were deposited in GenBank, and Table 1 lists the accession numbers. The alignment results indicated that the present species had a 100% similarity with M. koi (FJ710800), which was also derived from the gills of C. carpio. There was a similarity of less than 98% with other myxosporeans, including a 97.76% similarity with M. tanakai (LC228236), a 97.37% similarity with M. parakoi (MH196558), a 97.33% similarity with M. koi (MH196560), a 97.32% similarity with M. koi (FJ841887), a 97.03% similarity with M. koi (FJ725077), and a 96.87% similarity with M. koi (KT240127).

Compared with other species similar in morphology, the secondary structures of SSU rRNA of our specimens coincided with those of M. koi (FJ710800). However, they were not the same as those of SSU rRNA of M. koi parasites (MH196560, FJ841887, KT240127, and KJ725077), M. parakoi, M. tanakai and Myxbolus orissae (Haldar, Samal & Mukhopadhyaya, 1996) (Fig. 4). The left internal bulges of H23_1-2 in the V4 of the present species were the same as those of M. parakoi, but bigger than those of M. koi parasites (MH196560, FJ841887, KT240127, and KJ725077), M. tanakai and M. orissae. The second internal bulges of H23_1-2 in the present species were smaller than those of M. koi parasites (MH196560, FJ841887, KT240127, and KJ725077), M. tanakai, and M. orissae, except for M. parakoi. The third internal bulges of H23_1-2 in the studied species and M. koi (FJ710800) were smaller than those of other similar species in morphology. The lateral bulges of H23_1-2 showed no difference with these of M. parakoi and M. tanakai but differed from those of M. koi parasites (MH196560, FJ841887, KT240127, and KJ725077) and M. orissae. There was an internal bulge in H43_3 in the V7 of the present species and M. koi (FJ710800) but not in others. The biggest difference of H43_4 in the V7 between the present species and other similar species was the right internal bulge and a hairpin loop. H29 in the V6 and H43_5 in the V7 of the present species were identical with those of other species except for M. orissae.

In the present study, the phylogenetic results of ML and BI showed highly similar topology, which were incorporated into one tree based on the ML tree (Fig. 5). The myxospore morphology in this phylogenetic tree was closely correlated to their phylogenetic relationships. The species with pyriform spores sharing a tapering anterior end were clustered together in Clade I, and those possessing ellipsoidal spores with blunt anterior ends were clustered in Clade II. The two sequences from Guizhou and Chongqing in this study and M. koi (FJ710800) were first clustered together, forming a sister relationship with M. parakoi, and joined with M. tanakai to form a small subclade. The other M. koi parasites (MH196560, FJ841887, KT240127, and KJ725077) branched independently and formed a sister relationship with M. orissae to compose another small subclade. Then the above mentioned two subclades were clustered together in Clade I.