Life Cycle Of Several Freshwater Myxozoans And Taxonomy Of Actinosporean Collective Groups

Myxozoans are a group of metazoan endoparasites mainly infecting fish throughout the world.While most myxozoans are not highly pathogenic for their fish hosts,some cause severe disease and many others cause economic losses in aquaculture.However,there is no effective measure to prevent and control the myxosporidiosis at present.The life cycle of myxozoans usually involves the alternation between myxosporean stage in fish and actinosporean stage in annelids.The myxozoan life cycles are of great significance to the prevention and control of myxosporidiosis.Currently,about 50 myxozoan species are confirmed to follow two-host life cycle pattern,and most myxozoan life cycles are unknown,thus preventing the establishment of myxosporidiosis control measures.In the present dissertation,we identified the actinospores belonging to six collective groups in freshwater culture ponds using morphological,histologial and molecular methods.Of these,five actinosporean types were first reported,and one was first found in China.Also,four were redescribed.Moreover,six myxozoan life cycles were elucidated.The main results are summarized as follows:1.Aurantiactinomyxon Janiszewska,1957(1)Aurantiactinomyxon type 2 of Zhao et al(2016): Spores possessed one trefoilshaped spore body,three triangular caudal processes and three polar capsules.The sporoplasm contained 32 germ cells.Morphological comparison showed that the current aurantiactinomyxon type was a novel morphotype.Based on the 18 S r DNA sequence analysis,Aurantiactinomyxon type 2 of Zhao et al(2016)showed 99.2%–99.9% similarity to Thelohanellus kitauei,suggesting that Aurantiactinomyxon type 2 of Zhao et al(2016)is the actinosporean stage in the life cycle of T.kitauei.(2)Aurantiactinomyxon type of Zhao et al(2017): Spores possessed one triangular spore body,three leaf-like caudal processes and three polar capsules.The sporoplasm contained 32 germ cells.Morphological comparison showed that the aurantiactinomyxon type was a novel morphotype.Ultrastructural observations of the aurantiactinomyxon type showed that the development was asynchronous between pansporocysts,but synchronous within a pansporocyst.The formation of sporoblast and the development of sporogonic stage were also described and discussed.The meiosis was confirmed due to the occurrence of polar bodies in sporoblast.The presence of two to five central cells in sporoblasts but only one sporoplasm cannot support the theory of endogenous budding.The 18 S r DNA sequences of the current aurantiactinomyxon type corresponded to that of Thelohanellus testudineus,suggesting that Aurantiactinomyxon type of Zhao et al(2017)is the actinosporean stage in the life cycle of T.testudineus.(3)Aurantiactinomyxon type A: Spores possessed one spherical spore body,three leaflike caudal processes and three polar capsules.The sporoplasm contained 8 germ cells.On the basis of DNA sequence,the current aurantiactinomyxon type showed 99.9% identity to Zschokkella sp.(DQ118776)infecting the afferent bile duct of goldfish Carassius auratus,suggesting that Aurantiactinomyxon type A is the alternating stage of Zschokkella sp.(DQ118776).(4)Aurantiactinomyxon type B nov.: Spores possessed one spherical spore body,three elongated,leaf-like caudal processes and three polar capsules.Number of germ cells in sporoplasm was not determined.Morphological comparison showed that the aurantiactinomyxon type was a novel morphotype.At the DNA level,Aurantiactinomyxon type B nov.shared 99.0% identity to Zschokkella parasiluri,suggesting that Aurantiactinomyxon type B nov.is the actinosporean stage of Z.parasiluri.(5)Aurantiactinomyxon type C: Aurantiactinomyxon type C1 nov.possessed one spherical spore body,three elongated,leaf-like caudal processes which were curved downwards.Number of germ cells in sporoplasm was not determined.Morphological comparison showed that the aurantiactinomyxon type was a novel morphotype.Aurantiactinomyxon type C2 possessed one spherical spore body,three wide,short,leaf-like caudal processes which were slightly curved downwards.The sporoplasm contained 8 germ cells.The DNA sequence of Aurantiactinomyxon type C1 nov.was 99.6% identical with that of Aurantiactinomyxon type C2,suggesting that they were conspecific.BLAST analysis showed that Aurantiactinomyxon type C presented 98.1%–98.5% identity with Zschokkella sp.(DQ118776).The myxosporean stage of Aurantiactinomyxon type C is still unknown to date.(6)Aurantiactinomyxon type JD of Xi et al(2015): Spores possessed one trefoilshaped spore body,three triangular caudal processes and three polar capsules.The sporoplasm contained 32 germ cells.The aurantiactinomyxon type examined herein was considered to be the same type with Aurantiactinomyxon type JD reported by Xi et al(2015)based on morphological comparison and molecular analysis.BLAST analysis showed that Aurantiactinomyxon type JD of Xi et al(2015)herein shared 95.7%–95.8% identity to Thelohanellus wuhanensis.The myxosporean stage of Aurantiactinomyxon type JD of Xi et al(2015)is still unknown to date.2.Neoactinomyxum Granata,1922(1)Neoactinomyxum type A: Spores were trefoil-shaped in apical view,which possessed one spherical or subspherical spore body,three disc-like caudal processes and three polar capsules.The sporoplasm contained 32 germ cells.The 18 S r DNA sequences of the current neoactinomyxum type showed 99.2%–99.3% similarity to that of Thelohanellus wangi,suggesting that Neoactinomyxum type A is the actinosporean stage of T.wangi.(2)Neoactinomyxum type B nov.: Spores were rounded triangular in apical view,which possessed one triangular spore body,three narrow,crescent-shaped caudal processes and three polar capsules.The sporoplasm contained 32 germ cells.Morphological comparison showed that the neoactinomyxum type was a novel morphotype.Molecular analysis revealed that the 18 S r DNA sequences of Neoactinomyxum type B nov.did not match any available sequences in Gen Bank.The myxosporean stage of Neoactinomyxum type B nov.is still unknown to date.(3)Neoactinomyxum type A1 of Eszterbauer et al(2006): Spores were triangular in apical view,which possessed one spherical or subspherical spore body,three crescent-shaped caudal processes and three polar capsules.The sporoplasm contained 32 germ cells.The neoactinomyxum type examined herein was considered to be the same type with Neoactinomyxum type A1 reported by Eszterbauer et al(2006)based on morphological comparison and molecular analysis.BLAST analysis showed Neoactinomyxum type A1 of Eszterbauer et al(2006)herein presented 97.9% identity to T.wuhanensis.The myxosporean stage of Neoactinomyxum type A1 of Eszterbauer et al(2006)is unknown to date.3.Raabeia Janiszewska,1955Raabeia type of Myxobolus cultus: The raabeia-type actinospores were released from Branchiura sowerbyi and Bothrioneurum vejdovskyanum.Spores possessed one barrel-or flask-shaped spore body,three curved-upward caudal processes and three pyriform polar capsules.The sporoplasm contained 8 or 16 germ cells.The 18 S r DNA sequences of the raabeia-type actinospores showed 99.1%–99.9% similarity to that of Myxobolus cultus,suggesting M.cultus is the myxosporean stage of the raabeia-type actinospores examined in this study.Additionally,the sequence of Myxobolus wulii was obtained from DNA samples of the raabeia-type actinospores released from B.vejdovskyanum,suggesting the oligochaete population in the fish ponds is naturally infected with more than one myxospore.4.Hungactinomyxon Rácz,Eszterbauer et Molnár,2005Hungactinomyxon type of Rácz et al(2005): The hungactinomyxon-type actinospore is a complex consisting of eight echinactinomyxon-type spores.The hungactinomyxon type examined herein was considered to be the same type with Hungactinomyxon type reported by Rácz et al(2005)based on morphological comparison and molecular analysis.BLAST analysis showed that Hungactinomyxon type of Rácz et al(2005)herein shared 95.6%–95.7% identity with Myxobolus lentisuturalis.Therefore,the corresponding myxospore species is unknown to date.The hungactinomyxon-type actinospores were first discovered in China.5.Triactinomyxon ?tolc,1899Triactinomyxon type: Spores were anchor-shaped and possessed one barrelshaped spore body,one cylindrical style,three curved-upward caudal processes and three pyriform polar capsules.The morphology and morphometry of the three triactinomyxon-type samples(No.TAM-1,TAM-2 and TAM-3)indicated that TMA-1 and TMA-2 were the same type,while TMA-3 may be the other triactinomyxon-type considering the longer spore axis than TMA-1 and TMA-2.6.Guyenotia Naville,1930Guyenotia type of Eszterbauer et al(2006): Spores possessed one spherical spore body,three finger-like caudal processes and three polar capsules apically positioned on spore body.The sporoplasm contained 8 germ cells.The guyenotia type examined herein was considered to be the same type with Guyenotia type reported by Eszterbauer et al(2006)based on morphological comparison and molecular analysis.BLAST analysis showed that Guyenotia type of Eszterbauer et al(2006)herein presented 99.9% similarity with Zschokkella sp.(DQ118776).The genetic similarity between the current Aurantiactinomyxon type A and Guyenotia type of Eszterbauer et al(2006)was 99.9%,suggesting that they were conspecific and both represented the alternating stages of Zschokkella sp.(DQ118776).According to the result,the taxonomic criteria between guyenotia and aurantiactinomyxon collective groups were discussed and the validity of the taxonomic criteria was suspected.