Parasitol Res DOI 10.1007/s00436-016-5122-2
ORIGINAL PAPER
Morphological and molecular characterization of Myxobolus sheyangensis n. sp. (Myxosporea: Myxobolidae) with intralamellar sporulation in allogynogenetic gibel carp, Carassius auratus gibelio (Bloch) in China X. H. Liu 1 & S. Yuan 2 & Y. L. Zhao 1 & P. Fang 3 & H. Chen 3 & J. Y. Zhang 1
Received: 12 April 2016 / Accepted: 10 May 2016 # Springer-Verlag Berlin Heidelberg 2016
Abstract Allogynogenetic gibel carp is one of the most important freshwater cultured species in China. However, myxosporidiosis represents a severe threat to prevent the sustainable development of aquaculture of this species. During the investigation of myxosporean diversity of reared allogynogenetic gibel carp in East China, a new myxosporean with typical characteristics of Myxobolus was found from 169 out of 210 (80.4 %) examined samples, designated as Myxobolus sheyangensis n. sp. by combing comparative analysis of morphological and molecular data. The diagnostic features of this species are reminiscent with Myxobolus pyramidis Chen, 1958 as follows: round or ellipsoidal, grayish white, cyst-like polysporous plasmodia, averaging 219.3 ± 11.9 (98.7–421.7) × 158.4 ± 9.7 μm (79.9–191.8) in size; spores flat-pear shaped in frontal view with tapering anterior and rounded posterior ends and lemon-shaped in sutural view, averaging 11.0 ± 0.31 (10.5–11.9) × 10.2 ± 0.25 (9.2– 10.7) × 6.3 ± 0.23 μm (5.9–6.9) in size; and two equal pyriform polar capsules averaging 5.5 ± 0.3 (4.5–6.1) × 3.4 ± 0.26 μm (2.9–4.0) in size with convergent longitudinal axes and polar filaments wounded in seven to eight coils, perpendicularly to the longitudinal axis of the polar capsules. Spore
* J. Y. Zhang
[email protected] 1
Fish Diseases Laboratory, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 430072 Wuhan, China
2
Jiangsu Agri-Animal Husbandry Vocational College, 225300 Taizhou, Jiangsu, China
3
Center of Controlling Diseases of Aquatic Animals, Fisheries Technology Extension Center of Jiangsu Province, 320106 Nanjing, China
surface was smooth and two spore valves were symmetrical, with a thin and straight sutural ridge. Occasionally, abnormal spores with typical Henneguya-like caudal appendage and atypical papillary caudal appendage could be observed. The sporogenesis is asynchronous. Histopathological investigation showed that the plasmodia developed inside the capillary network of gill lamellae, belonging to the intralamellar vascular type, and no significant inflammatory responses were provoked by the infection. Homology search by BLAST showed that the newly obtained sequence did not match any available sequences in GenBank. Phylogenetic analysis of the aligned sequences indicated that M. sheyangensis n. sp. positioned in a clade composed of Myxobolus species infecting the gill of several freshwater cyprinid fish. Keywords Myxobolus sheyangensis n. sp., Carassius auratus gibelio . Intralamellar sporulation . Henneguya-like caudal appendages
Introduction Allogynogenetic gibel carp Carassius auratus gibelio (Bloch) has been becoming one of the most popular freshwater cultured species in the mainland of China since it was artificially selectively bred by its special dual reproduction modes of gynogenesis and syngenesis in the early 1980s (Gui and Zhou 2010). Its annual production was beyond 2 million tons in 2011 (Wang et al. 2011). However, epizootic diseases have been causing severe economic and environmental problems with the rapid development of intensive aquaculture of this species. Virosis, bacteriosis, and parasitosis have been frequently reported to result in the outbreak of mortality of reared allogynogenetic gibel carp of the whole culture cycle (Wang et al. 2001; Xi et al. 2011; Zhang et al. 2010a, b; Luo et al.
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2013; Yuan et al. 2015; authors’ unpublished data). Among parasitic diseases, myxosporidiosis was one of the most important diseases. During our ongoing survey on myxosporean diversity of cultured allogynogenetic gibel carp in Yancheng city, Northern Jiangsu province, East China, since 2009, more than 50 species of Myxobolus, Thelohanellus, Henneguya, Myxidium, Cholormyxum, Zschokella, Mitraspora, Hoferellus, Unicauda, and Sphaerospora have been found (Yuan et al. 2015, authors’ unpublished data), some of which were previously described in the monograph of Chen and Ma (1998). Among them, Myxobolus honghuensis, Myxobolus wulii, Thelohanellus wuhanensis, and Thelohanellus wangi were proved to be the etiological agents of severe pharyngeal myxosporidiosis, liver myxosporidiosis, skin myxosporidiosis, and gill myxosporidiosis of allogynogenetic gibel carp, respectively (Wang et al. 2001; Xi et al. 2011; Zhang et al. 2010b; Yuan et al. 2015). To reduce the economic losses caused by these myxosporidiosis, large amounts of pesticide and chemical disinfectant were applied by the local fish farmers, which was thought to be harmful to the fish immunity and then possibly responsible for the recent outbreak of hematopoietic necrosis associated with cyprinid herpesvirus at some extents (Wang et al. 2012). Myxobolus Bütschli, 1882 is the most speciose myxozoan genus, and approximately 850 species have been reported worldwide, although some of them were described solely upon simple morphological features of spores (Ali et al. 2002; Eiras et al. 2005; Molnár 2011; Eiras et al. 2014; Abdel-Ghaffar et al. 2005, 2015). The late taxonomic criteria integrating the spore morphology, exact location of sporulation, tissue specificity, host specificity, and molecular data have replaced the traditional methods to be widely applied for the taxonomy of myxosporean parasites (Fiala et al. 2015). Here, a new Myxobolus species dwelling in the capillary network of gill lamellae of allogynogenetic gibel carp, which is reminiscent of M. pyramidis, was described by applying the late taxonomic criteria, and its phylogenetic position was discussed.
Molecular methods In total, 210 specimens of gibel carp from six adjacent culture ponds in Sheyang county (33° 78′ 26″ N, 120° 25′ 27″ E), Yancheng city, East China, ranging from 12.6 to 15.9 cm in body length were captured by gill nets and examined on the spot from May to July of 2014. Strict parasitological examination was performed as follows. After checking the visible myxosporean-like pseudocysts by naked eye and dissection microscopy (Olympus SZ51), gross microscopic examination of the gills, liver, spleen, kidney, brain, spinal cord, intestine, muscle tissues, and gall bladder was conducted by preparing fresh wet preparations. Once the occurrence of myxosporean
plasmodia or spores has been established, the infected tissues were preserved in 10 % neutral buffered formalin for histopathological analysis and the isolated plasmodia in 95 % ethanol for extracting the genomic DNA. Plasmodia containing myxospores with similar morphological features with M. pyramidis were placed on the slides and photographs taken. Morphological and morphometrical features of the released spores were analyzed according to Lom and Arthur (1989) by measuring 50 mature spores from three individual plasmodia using an Olympus BX 53 microscope equipped with an ocular micrometer. Line drawings were made based on the digitized images. All measurements were given in micrometers (μm) unless otherwise indicated. Lugol’s iodine (2 %) was used to visualize iodinophilic vacuole and a drop of black Indian ink to observe the possible mucous envelopes. The hardness of spore valves of M. pyramidis and the concerned species was compared by pressing spore preparations under coverslips and counting the number of ruptured spores among 200 randomly selected spores of three visual fields under light microscope. For histopathological analysis, formalin-fixed infected gill tissues containing plasmodia were dehydrated and embedded in paraffin wax. Sections of 5–6 μm were stained with hematoxylin and eosin (H & E) and observed under light microscopy as above. The isolated plasmodia of this new myxosporean was used for genomic DNA extraction using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany), following the manufacturer’s recommended protocol for animal tissue. The gDNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific) at 260 nm. The partial sequence of the SSU ribosomal DNA (rDNA) gene was amplified using a primer pair, MyxospecF (Fiala 2006) and 18R (Whipps et al. 2003), in a 25-μl reaction volume comprising 30 ng template DNA, 1× PCR mixture (CWBiotech, China), and 10 pmol of each primer. Amplification parameters were as follows: initial denaturation for 4 min at 94 °C; 35 cycles of 1 min at 94 °C, 50 s at 46 °C, and 90 s at 65 °C; and a terminal extension of 10 min at 65 °C. The PCR products were electrophoresed in 1.2 % agarose gels in Tris–borate–EDTA buffer gel stained with 1 % ethidium bromide and then purified with a PCR purification kit (CWBiotech, China). The purified product was cloned into PMD18-T vector system (Takara, Japan) and then sequenced with the ABI BigDye Terminator v 3.1 Cycle Sequencing Kit with an ABI 3100 Genetic Analyzer. Two replicate infected lamellae sampled from two different ponds were sequenced to compare the possible sequence variation. Two contiguous DNA sequences were assembled and deposited in GenBank with accession numbers KU313484 and KU313485. BLAST searches were conducted to find the species with high similarity to those of the present species. Selected Myxobolus sequences were aligned with Clustal X (Thompson et al. 1997) using default settings. Phylogenetic analysis of the aligned sequences, excluding the gaps, was
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conducted by the neighbor-joining (NJ) method, the maximum parsimony (MP) method, the maximum likelihood (ML), and the Bayesian method, which were implemented in the MEGA5 computer package (Tamura et al. 2013), PAUP* 4.0b1 (Swofford 2003), PhyML (Guindon et al. 2010), and MrBayes 3 (Ronquist and Huelsenbeck 2003), respectively. Parsimony analysis used the heuristic search algorithm and tree bisection–reconnection (TBR) branch swapping. NJ analysis of genetic distances was calculated with Kimura 2 model parameter (Tamura et al. 2013) to construct a NJ tree. Optimal evolutionary models for ML and Bayesian analysis were selected by jModelTest using the Akaike information criteria and identified as the general time-reversible model (GTR + I + G) (Posada 2003). Bootstrap support values were calculated with 1000 replicates. Two independent runs were conducted with four chains for two million generations for Bayesian analysis. Phylogenetic trees were sampled every 100 generations. The first 25 % of the samples were discarded from the cold chain. Trees were initially examined in TreeView X (Page 1996) and edited and annotated in Adobe Illustrator (Adobe Systems Inc.).
Results The plasmodia were generally found in the gill filaments of 169 (80.4 %) out of the 210 examined allogynogenetic gibel carp. Generally, only one plasmodium was found per infected fish, however, for some specimens, several plasmodia (up to ten) could be obeserved in the gill filaments of host. And one gill filament was exclusively habituated by only one plasmodium. The plasmodium usually located in the middle part of gill filaments (Fig. 1a) and almost occupied the whole infected gill lamellae (Figs. 1b and 4). Comparison of fresh spores, infection site, and histological examination proved identity of gill infections in all examined specimens. Morphologically comparisons with published descriptions of Myxobolus species indicated that the present species is very similar with M. pyramidis Chen, 1958 (Table 1). However, the results of sequence and phylogenetic analysis based on partial SSU Fig. 1 Photomicrographs of infections of Myxobolus sheyangensis n. sp: a showing a single plasmodium located inside the lamellae, scale bar = 100 μm, and b showing mature spores in frontal view, scale bar = 10 μm; inset showing mature spores in sutural view, scale bar = 10 μm
rDNA gene sequences clearly distinguished it from M. pyramidis and other morphological similar Myxobolus species with sporulation in the gills of cyprinid fish.
Myxobolus sheyangensis n. sp. (Figs. 1, 2, 3, and 4, Table 1)
Morphology of spores Fresh spores (light microscopy, n = 50) are flat-pear shaped in frontal view with tapering anterior and rounded posterior ends and lemon-shaped in sutural view, 11.0 ± 0.31 (10.5–11.9) long, 10.2 ± 0.25 (9.2–10.7) wide, and 6.3 ± 0.23 (5.9–6.9) thick. Sutural ridge was straight and thin. Mucus envelope, iodinophilic vacuole, distinct intercapsular appendix, and sutural edge marking were not observed. Spore surface was smooth and spore valves were symmetrical. Two equal-sized elongate pyriform polar capsules were 5.5 ± 0.3 (4.5–6.1) long and 3.4 ± 0.26 (2.9–4.0) wide, with convergent long axes, occupying over half of spores. Polar filament coiled perpendicularly to the long axis of the polar capsule with seven to eight turns (Figs. 1b and 2). Some spores have typical Henneguya-like caudal appendage and atypical papillary caudal appendage (Fig. 3), occupying about 5 % of total spores per plasmodium. Additionally, spore valves of M. pyramids are more fragile than those of M. sheyangensis sp. n (21.6 vs 5.0 % of the rate of ruptured spores). Vegetative stages Round or ellipsoid cyst-like plasmodium localized in the capillary network of gill lamellae. Sporogenesis asynchronous. Typical host: Allogynogenetic gibel carp Carassius auratus gibelio (Bloch) (Cyprinidae). Typical locality: Sheyang county, Yancheng city, East China (33° 78′ 26″ N, 120° 25′ 27″ E). Site of infection: Gill lamellae. Date of sampling: May to July of 2014. Prevalence: 169 out of the 210 examined (80.4 %) infected.
Fig. 2 Schematic drawing of myxospores of Myxobolus sheyangensis n. sp. a In frontal view. b In sutural view. Scale bar = 5 μm
Type material: Syntype specimens of spores in glycerin gelatin, H & E-stained tissue section, and their digitized photos were deposited in the Laboratory of Fish Diseases, Institute of Hydrobiology, Chinese Academy of Sciences (accession no. MTR20150609). The partial SSU rDNA sequences were deposited in GenBank under the accession numbers KU313484 and KU313485. Etymology: The species is named after its typical locality of distribution.
Data not available d
Minimum–maximum
Mean c
b
Mean ± SD
Pathogenicity No remarkable clinical external signs were observed for all examined fish. Histopathological examination revealed that a single plasmodium developed in the capillary network of gill lamellae and occupied almost all of its volume. The neighboring two lamellae were compressed by the severely extended gill lamellae caused by the infection at some extents, but other lamellae in the infected gill filament have not been significantly affected. No remnants of capillaries of the infected lamellae could be observed at the late developmental stages. The host responses to the infection were characterized by local proliferation of connective tissue and collagen fibers forming a concentric membrane to encapsulate the plasmodium, and some atrophic epithelial cells of gill lamellae remained around this membrane. No distinct inflammatory responses were observed, even in the local infection focus. Considering the low infection intensity and unremarkable pathological responses, this infection has no significant negative effect on the health of host fish (Figs. 1a and 4).
a
LS length of the spore, WS width of the spore, TS thickness of the spore, LPC length of the polar capsule, WPC width of the polar capsule, CPF coils of the polar filament, NF number of folds of the spore valve, SS shape of the spore in frontal view, H host, IF infection sites, Ref. references
Chen and Ma (1998) Gills 3 – 10.3 (10.0–10.5) 11.5 (11.0–12.0) M. solidus
8.5 (8.4–8.6)
6.9 (6.7–7.0)
3.9 (3.5–4.2)
Spherical
Chen and Ma (1998) Chen and Ma (1998) Gills Gills 4–7 – 7–8 5–7 9.3 (8.4–9.6) 9.1 (8.4–9.6) 11.0 (10.2–13.2) 10.4 (9.6–11.0) M. linghuensis M. changshigensis
7.0 (6.0–7.2) 6.0–
4.9 (4.8–5.4) 4.9 (4.6–5.4)
2.8 (2.6–3.4) 2.9 (2.4–3.1)
Oval round Oval round
Chen and Ma (1998) Gills 7–8 7–8 9.5 (8.0–11.0) 10.5 (9.0–12.0)
M. follius
M. dogieli
6.5 (6.0–7.0)
5.0 (4.0–6.0)
3.5
Oval round
Chen and Ma (1998) Gills 4–8 7–8
Pyriform
Zhang et al. (2006) Gills 0 5–6
Pyriform
Present study Gills
Carassius auratus gibelio Carassius auratus gibelio Carassius auratus gibelio Carassius auratus gibelio Aristichthys nobilis Carassius auratus gibelio Carassius auratus gibelio Flat-pear shaped 0 7–8
3.4 ± 0.26 (2.9–4.0) 3.5 ± 0.2 (3.0–4.1) 3.6 (3.4–3.7) 5.5 ± 0.3 (4.5–6.1) 5.5 ± 0.7 (4.5–6.3) 5.6 (5.2–7.2) 10.2 ± 0.25 (9.2–10.7) 10 ± 0.9 (9.2–11.5) 9.7 (9.0–10.8) 11.0 ± 0.31a (10.5–11.9)b 10.5 ± 1.1 (9.6–12.0) 12.0c (10.8–12.2) Myxobolus sheyangensis sp. n. M. pyramidis
6.3 ± 0.23 (5.9–6.9) 6.1 ± 0.2 (5.8–6.3) 7.2–d
NF CPF WPC LPC TS WS LS Species
Table 1
Comparison of Myxobolus sheyangensis sp. n. with the most closely morphological related species of Myxobolus Bütschli, 1882
Ref. IF H SS
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Molecular analysis Partial SSU rRNA gene sequences were obtained from two positive clones with 1722 and 1725 bp, respectively, after trimming the ambiguous positions in the two sequencing ends, which are 99.4 % similar (9/1725-nt differences). Sequence similarity search showed that the most similar sequence to them is that of Myxobolus koi (FJ841887; 95 % with 100 % coverage), Myxobolus pyramidis (HQ613411, 95 % with 100 % coverage),
Parasitol Res Fig. 3 Photomicrographs of some abnormal spores of Myxobolus sheyangensis n. sp. with caudal appendage of different type: a showing the short typical Henneguya-like caudal appendage (arrow), scale bar = 5 μm, and b showing papillary caudal appendage (arrow), scale bar = 5 μm
M. wulii (HQ613412, 95 % with 100 % coverage), and Myxobolus toyamai (LC010116. 95 % with 98 % coverage), all of which infect the gill of cyprinid fish and were recorded in China. Phylogenetic analysis by the applied four methods produced trees of similar topological structure although with different support values at some branch nodes. Regardless of algorithm, M. sheyangensis n. sp. localized at the basal
Fig. 4 LM images of histopathological sections of the gill filaments harboring a mature plasmodium of Myxobolus sheyangensis n. sp., stained with H & E: a showing the plasmodium locating inside of the gill lamellae of allogynogenetic gibel carp, scale bar = 100 μm, and b showing the plasmodium encapsulated by a thin intact concentric layer of connective tissue (white arrow) and extruded two neighboring lamellae (black arrow), scale bar = 50 μm
position of cyprinid gill-infecting Myxobolus clade with robust bootstrap support, rather than forming a sister relationship with M. pyramidis alone, which have same host, infection site, and spore morphology with the new species. Infection site, genetic affinity of fish host, and geographical distribution provided distinct evolutionary signals to refer the phylogenetic relationships of selected Myxobolus species (Fig. 5). Remarks Strict morphological comparisons showed that M. sheyangensis n. sp. is very similar with M. pyramidis, which is also w ith intralamellar sporulation in allogynogenetic gibel carp, although tiny differences pertaining to the turns of polar filaments (5–6 vs 7–8) and thickness of spore valves can be discerned for these two species. But, histopathological analysis clearly revealed that M. sheyangensis n. sp. belonged to intralamellar vascular type, which was different from intralamellar epithelial type of M. pyramidis. Sequence difference (5 %) further supports the separation of these two species. Beside M. pyramidis, Myxobolus follius, Myxobolus linghuensis, Myxobolus changshigensis, Myxobolus solidus, and Myxobolus dogieli recorded in the gill of cyprinid in China are also similar with this new species in spore morphology. But, the sutural edge marking and mucous membrane at the posterior end of M. follius spores make it different from the present new species. Additionally, eggplant-shaped polar capsules of M. follius also differ from pyriform polar capsules of M. sheyangensis n. sp. M. dogieli can differentiate from the present species by its grey yellow smaller plasmodia and seven to eight sutural markings at the spores’ posterior end. Smaller polar capsules and different host can differentiate M. linghuensis from this new species. M. changshigensis is different from M. sheyangensis n. sp. by its slightly small polar capsules, fewer polar filament coils (5–7 vs. 7–8), and ovoid spores. Thick sutural ridges and occasionally occurrence of three circular folds surrounding the anterior part of spore of M. solidus distinguish it from the present species.
Parasitol Res Fig. 5 Phylogenetic tree generated by Bayesian analysis of the aligned partial SSU rRNA gene sequences of Myxobolus sheyangensis n. sp. and related cyprinid-infecting Myxobolus species, rooted at Ceratonova shasta. GenBank accession numbers are given adjacent of species names. Support values are listed at the branching points. The described species here are highlighted in bold
Discussion Morphologically similar species found in different sites of the same fish and same sites of different fish species, especially those with close genetic relationships, were generally identified to be conspecific in previous publications (Donec and Shulman 1984; Chen and Ma 1998), which resulted in some synonyms (Ali et al. 2003, 2007; Zhang et al. 2010a; Zhao et al. 2013). Moreover, the recognition of plasticity of spore morphology and inconsistence of molecular data-based phylogeny and spore morphology-based classification severely challenge the traditional taxonomy of myxosporean, especially at species and genus levels (Fiala 2006; Jirků et al. 2007; Guniter and Adlard 2010). So, a holistic approach integrating morphological characters of myxospores, tissue tropism, life cycle, host affinity, and SSU rDNA sequence has been widely accepted to replace the traditional methods for myxosporean taxonomy and species identification depending upon spore morphology alone during the past 20 years (Molnár et al. 2010; Gábor et al. 2012; Fiala et al. 2015). In the present work, this approach was used to describe a new Myxobolus species, M. sheyangensis n. sp., infecting the capillary network of gill
lamellae of reared allogynogenetic gibel carp in East China. Morphologically, this new species is almost undistinguished from M. pyramidis (Chen and Ma 1998; Zhang et al. 2006). Additionally, both of them develop inside the gill lamellae of same fish host. But, strict histopathological analysis could show that M. pyramidis developed between the epithelial layer of gill lamellae and the vascular capillary was compressed to one side of the plasmodium, distinctly differing from this new species with intravascular developmental pattern. Indeed, some histozoic myxosporeans could be found to infect several different organs, such as Myxobolus turpisrotundus (Zhang et al. 2010a), but generally preferring same tissue for plasmodia development. Up to this point, tissue tropism is of more importance than organ specificity for identification of myxosporean species. However, Thelohanellus kitauei, a common parasite infecting the intestine of common carp, was recently found in the skin of the same fish host, but no plasmodium occurred in this new recorded infected organ (Zhai et al. 2016). This habitat shift represents a possible way of adaptive radiation of a certain myxosporean species, but this transitional species should be correctly assigned based on the current taxonomic criteria. Almost 100 % sequence
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identity between these two T. kitauei isolates from skin and intestine can make this final decision. As such, 5 % sequence difference between M. sheyangensis n. sp. and M. pyramidis absolutely indicated that their genetic distances were beyond the intraspecific variation. Among species infecting genetically related fish host, tissue tropism has been proved to provide stronger evolutionary signals to infer the phylogenetic relationship of myxosporeans than spore morphology (Eszterbauer 2004; Fiala et al. 2015). In the present analysis, M. sheyangensis n. sp. clearly clustered with other gill lamellae-infecting species of cyprinid fish distributed widely and then formed a sister relationship with other non-gill-infecting species of cyprinid, rather than forming an independent branch with M. pyramidis alone. But, it was also distinctly discerned that spore morphology represented the difference of genotype at some extents under a short evolutionary time. In this gill-infecting Myxobolus clade, most of species with elongated pyriform spore, including M. koi, Myxobolus orissae, Myxobolus longisporus, M. toyamai, M. wulii, and Myxobolus ampullicapsulatus, except M. pyramidis firstly cluster together and then formed a sister relationship with flat-pear-shaped pyriform spores, M. sheyangensis n. sp. From another perspective, all species with taper anterior end of spores (all species with this character within this clade) were genetically separated from those with round anterior end of spore, like Myxobolus hearti, Myxobolus oralis, Myxobolus nielii, and M. turpisrotundus. This genetic affinity of Myxobolus species based on spore morphology was also previously reported (Zhang et al. 2010a; Liu et al. 2016). Moreover, the present analysis indicated that phylogenetic relationships of gill-infecting Myxobolus of cyprinid fish showed a distinct geographical pattern, synergistic with the host affinity. However, the predominant role that biological feature plays remains unknown. Additional biological difference between M. pyramidis and M. sheyangensis n. sp. is that spores of the former are more fragile than those of the latter. Although the function of hardness of spore valves remained unknown, resisting the hostile environments or facilitating the transmission, it was often encountered that myxospore valves of some species were hard, but others were very fragile (Zhang et al. 2010a, b; Liu et al. 2016; authors’ unpublished data). Additionally, some abnormal spores with Henneguya-like caudal appendages were observed inside the plasmodia of M. sheyangensis n. sp., which are different from M. pyramidis. Occurrence of this type of caudal appendages among species with different sites of sporulation development, such as gill, intestine, and oral cavity and habitat environment, e.g., freshwater and marine water (Liu et al. 2016), possibly represents that this character independently evolved several times during the evolutionary history of Myxobolus to Henneguya or vice versa. So, in our opinion, this transitional Myxobolus species with a few abnormal spores should not be suspected for their affinity, for spore
morphological features were generally described based on most of spores with normal morphology inside the plasmodia. The genus Henneguya should not be suppressed to avoid further confusion, for the morphological features of spore body of some Henneguya species are undistinguished from those of morphologically similar Myxobolus species, although these two genera are genetically closely related. Taking into account the infection intensity of M. sheyangensis n. sp., it could be thought to be harmless to fish host, like most of fish myxosporeans, although high infection prevalence occurred. The possible mechanisms of one gill filament habituated by only one individual plasmodium remained unknown. In summary, we provide here sufficient taxonomic data to describe a new species, M. sheyangensis n. sp., with intralamellar development of sporulation in allogynogenetic gibel carp.
Acknowledgments The present work was financially supported by the Jiangsu Province Fund of Sciences (BK2012240), Jiangsu Fishery Project (D2015-11), and Chinese Natural Sciences Fund (31411130191, 31472296).
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