The study combined measurements of microscopic spores with small-subunit ribosomal DNA analysis, host information, infection location and geographic origin to distinguish the parasite from previously named species. The investigation examined 28 B. argentatus specimens collected by a commercial fishing boat from coastal waters of the East China Sea near Xiamen, Fujian Province, on 23 July 2017. The fish were 19 to 22 centimetres long and were identified through their morphology and DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene.

Researchers inspected the skin, fins, gills, muscles, hepatopancreas, intestine, spleen, heart, gallbladder and urinary bladder, along with bile, blood and urine. Six of the 28 fish were infected, corresponding to a prevalence of 21.4 percent. The parasite was found in the gallbladder, and no vegetative stages were observed in the examined material.

Under a microscope, the mature myxospores of M. branchiostegi were oval, with blunt anterior and posterior ends in both valvular and sutural views. Two equal-sized, pear-shaped polar capsules occupy the upper half of each spore and are arranged in an inverted V, with their anterior thirds closely appressed. Each capsule contains a polar tubule coiled four to six turns, and a single sporoplasm fills the intracapsular cavity beneath the capsules.

Measurements were based on 25 mature spores. Mean spore length was 9.21 ± 0.52 micrometres, with a range of 8.3 to 9.8 micrometres, and mean width was 7.40 ± 0.60 micrometres, ranging from 6.2 to 8.0 micrometres. The polar capsules averaged 4.40 ± 0.34 micrometres long and 2.58 ± 0.17 micrometres wide, with respective ranges of 3.7 to 4.8 and 2.3 to 2.9 micrometres.

Those measurements placed the new species among several Myxobolus taxa that can look similar under a microscope. M. branchiostegi most closely resembled M. arrabonensis, M. cerveirensis, M. labrosus, M. squamalis and M. urinaris, but the differences extended across several characters. It also lacked the distinct sutural markings reported in M. cerveirensis and M. labrosus.

Host and infection site supplied further distinctions: the known congeners occur in the gill filaments of Chondrostoma nasus, the intestine of Chelon ramada, the urinary bladder of Chelon labrosus, the scales of Oncorhynchus mykiss, or the urinary bladder of Mugil cephalus. M. branchiostegi instead occurs in the gallbladder of B. argentatus in China, whereas those five species have been reported from Hungary, Portugal or the United States. The molecular comparison separated M. branchiostegi from its closest known relative, M. iwagiensis.

The researchers amplified a partial small-subunit ribosomal RNA gene from parasite spores and deposited the resulting 1,848-base-pair sequence in GenBank under accession PV866587. A BLASTn search found no identical myxozoan sequence. The highest sequence similarity was 92.0 percent to M. iwagiensis, with a genetic distance of 0.0653.

Every other compared Myxobolus species had less than 80 percent similarity and a genetic distance greater than 0.3 from the new species. M. iwagiensis also has markedly larger spores and unequal polar capsules, while M. branchiostegi has equal-sized capsules. The two species also differ in their hosts, infection sites and reported environments: M. iwagiensis was described from peripheral nerves and beside gill-arch cartilage in Oryzias latipes from a brackish reservoir in Japan, whereas the new species was found in the gallbladder of a marine fish from the East China Sea.

Maximum-likelihood and Bayesian-inference analyses produced consistent tree topologies. Based on 30 small-subunit ribosomal RNA gene sequences, including the new sequence and sequences selected from a BLASTn search, both methods placed M. branchiostegi and M. iwagiensis together as sister taxa. This marine and brackish-water pairing formed one subclade within a larger Myxobolidae lineage.

The analyses supported the family Myxobolidae as monophyletic, with Bayesian posterior probability of 1.00 and maximum-likelihood bootstrap support of 100, but did not recover either the genus Myxobolus or Henneguya as monophyletic. Myxidium cuneiforme and Zschokkella nova were used as outgroups, while 1,000 bootstrap replicates and 10 million Bayesian generations were used to assess support in the respective analyses. The trees also showed a recurring association between related parasites and the taxonomic orders of their fish hosts.

Closely related members often occurred in groups associated with the same host order. For example, two Henneguya species infecting cichliform fishes were recovered together, as were Myxobolus species associated with characiform hosts. M. cerebralis and M. squamalis, both parasites of salmoniform fishes, were also sister taxa.

The pattern was not universal. Some parasites infecting siluriform fishes appeared in distant branches, and M. figueirae was positioned among Characiformes-associated taxa despite infecting a siluriform host. The authors describe host switching or expansion of host range as a possible explanation for such exceptions, but the tree itself does not establish the history of any individual host association.

Aquatic habitat also corresponded with major parts of the phylogeny. Marine, brackish-water and freshwater myxobolid species formed well-supported groupings, while the newly described marine parasite clustered with brackish-water M. iwagiensis rather than with the predominantly freshwater species elsewhere in Myxobolus. The authors consider a possible sequence of habitat transitions from marine to brackish-water and then freshwater environments, potentially aided by historical marine transgressions, but explicitly note that this scenario remains untested.

The study therefore identifies a phylogenetic pattern and a hypothesis to examine, rather than demonstrating a definitive direction of habitat change. Geography may have influenced the relationships recovered in the trees, although the analysis does not establish geographic proximity as a cause of genetic similarity. M. branchiostegi from the East China Sea was recovered beside M. iwagiensis from Japan, regions linked hydrologically and suggested as possible shared habitats or dispersal corridors.

Other examples included Brazilian myxosporean lineages associated with a species from Peru and Brazilian representatives clustering with a Peruvian Henneguya species. The authors note that Brazil and Peru share interconnected freshwater systems in the Amazon Basin. These distributions are consistent with, but do not prove, roles for marine connections, river-basin connectivity and host relationships in the phylogeographic structure of myxozoans.

The new species was formally distinguished through convergent evidence from spore morphology, ribosomal DNA, host identity, infection site and locality. Syntypes were deposited in the collection of the Animal Biology Key Laboratory of the Chongqing Education Commission at Chongqing Normal University, and the specific name branchiostegi derives from the generic name of B. argentatus. The study illustrates why microscopy alone may be insufficient for identifying Myxobolus: these parasites have simplified, sometimes convergent spore forms, and some spore measurements can vary.

The combined approach used here provides a more defensible basis for documenting a new species and placing it within the evolutionary history of a parasite group whose members live within their fish hosts.

Subject of Research: Taxonomic and phylogenetic characterization of Myxobolus branchiostegi infecting Branchiostegus argentatus in the East China Sea

Article Title: Taxonomy and phylogeny of Myxobolus branchiostegi sp. nov. ( Myxozoa , Myxosporea ) infecting the gallbladder of Branchiostegus argentatus Cuvier, 1830 from the East China Sea

Article References: Xiong, Y., Zhao, Y., Chen, X., Wang, M., Peng, J., Cui, X., & Yang, C. (2025). Taxonomy and phylogeny of Myxobolus branchiostegi sp. nov. (Myxozoa, Myxosporea) infecting the gallbladder of Branchiostegus argentatus Cuvier, 1830 from the East China Sea. Zoosystematics and Evolution, 101(4), 2357-2367. https://doi.org/10.3897/zse.101.170695

Image Credits: AI Generated

DOI: 10.3897/zse.101.170695

Keywords: Myxobolus branchiostegi, Myxozoa, fish parasites, Branchiostegus argentatus, gallbladder infection, SSU rDNA, parasite taxonomy, phylogenetics, East China Sea, cnidarian parasites, Taxonomy, phylogeny