Diversity And Evolution Of Myxosporea Venom-related Genes And Gene Cloning And Molecular Simulations Of MH-Kunitz

Myxozoa is a group of endoparasitic cnidarians with simple morphology and a tiny body.Most myxozoans not only infect fishes but also parasitize amphibians,birds,and mammals.Myxozoans are widely distributed in freshwater,marine,and terrestrial habitats,with more than 2,600 species reported.The adaptive evolution has drawn great interest from researchers,but the driving force and molecular mechanisms are still unclear.As one of the cornerstone species of metazoan,carrying out research on its phylogeny and molecular evolution and exploring the myxozoa adaptive evolution are of great significance for elucidating the evolution of metazoans.Myxozoans are responsible for many fish diseases,resolving their endoparasitic and pathogenic mechanisms can provide a theoretical basis for disease control.In this study,we sequenced five pathogenic Myxobolidae species and conducted a comparative analysis in combination with some public cnidarians datasets.We analyzed the diversity and composition patterns of myxozoa venom-related genes,resolved the phylogenetic relationships and evolutionary patterns of some venom families,cloned and molecularly simulated the MH-Kunitz.The major results are as follows:1.Diversity,composition and evolution patterns of venom-related genes in Myxozoa.Through a customized bioinformatics pipeline,we mined venom-related genes from19 myxozoan transcriptomes/genomes(including five newly sequenced transcriptomes)and four free-living cnidarian transcriptomes.We found that Myxozoa lost most of the cnidarian venom families,such as type I and III K~+channel blockers,all Na~+channel blockers,JFT and MACPF.However,Kunitz,CRISP,and M12B were retained in almost all species,and PLA2 and Actinoporins were also present in some myxozoans.The number of venom-related genes identified in Myxozoa(4-88)was less than that in free-living cnidarians(88-611).We further compared the differences in venom composition patterns of Endocnidozoa,Anthozoa,and Medusozoa.Our results showed that Endocnidozoa contained the most hemorrhagic toxins,accounting for more than 63%,but the Anthozoa consisted mainly of enzymes(64.7%),while the Medusozoa was rich in cytolysis(36.8%).We conducted phylogenetic analyses on Kunitz,CRISP,PLA2,Actinoporin,and M12B.We found that myxozoan venom genes rarely clustered with cnidarians,they even clustered with other toxic organisms,for instance,the Kunitz genes of T.kitauei and K.iwatai clustered within a branch with spiders.We found that the Kunitz conserved domain is duplicated in myxozoans and free-living cnidarians,although not in other toxic organisms,and the duplication occurred twice in myxozoans.We detected domain recruitment in M12B and domain divergence in CRISP,which may lead to neofunctionalization.The selection analysis showed Kunitz,M12B and CRISP were under purifying selection and a few instances of gene-specific or lineage-specific episodic positive selection.We also interpret this discrete positive selection as concomitant with increased potency and/or specificity of a particular toxin or species.2.MH-Kunitz gene cloning and function prediction.An MH-Kunitz gene was cloned from the M.honghuensis,the ORF of MH-Kunitz was 429 bp and predicted to encode 143 amino acids.Bioinformatics analysis revealed that the protein encoded by the MH-Kunitz was a secreted protein with a transmembrane helix,a signal peptide,and five disulfide bonds.Conserved domain analysis showed that MH-Kunitz consists of two KU domains,further confirming that it was a Kunitz protein.Phylogenetic analysis showed that MH-Kunitz was clustered with Conkunitzin-S1 and Sd PI.We further constructed the 3D structures of MH-Kunitz,Conkunitzin-S1,and Sd PI,and carried out molecular simulations.Our results showed the structural similarity,protein complex score,and stability of MH-Kunitz/Sd PI were better than those of Conkunitzin-S1.Therefore,we speculated that MH-Kunitz may function as a protease inhibitor just like Sd PI.In conclusion,the diversity and quantity of venom-related genes have been greatly reduced in the endoparasitic adaptive evolution of Myxozoa,but the"core"venom genes are still retained;The venom-related genes of myxozoans are dominated by divergent evolution,and there are differences in phylogeny and selection pressure from free-living cnidarians;The myxozoans venom gene families may get neofunctionalization to adapt to the endoparasitic lifestyle through domain duplication,recruitment,and divergence;The MH-Kunitz may have protease inhibitory activity and play a role in parasite invasion of the host.