Genetic Differentiation Of Eopycna Repanda And Eopycna Coelestia And Distribution And Transovarial Transmission Of Their Symbionts(Hemiptera: Cicadidae)

Cicadas(Hemiptera: Cicadidae)have been known as the important pests of numerous trees,which feed exclusively on plant sap and lay eggs in the plant twigs.Plant sap is relatively abundant in carbohydrates,but nutritionally deficient in nitrogenous nutrients.Therefore,cicadas are dependent on obligate symbionts for provisioning of vitamins and essential amino acids that are extremely unbalanced or limited in the diet.It has been reported that Candidatus Sulcia muelleri(hereafter referred to as Sulcia)is harbored in the bacteriomes of cicadas,whereas Candidatus Hodgkinia cicadicola”(hereafter referred to as Hodgkinia)is only harbored in bacteriomes of some cicada species.Hodgkinia is absent in the majority of cicadas,in which yeast-like fungal symbiont(YLS)is harbored in the fat bodies of the Hodgkinia-free cicadas.Previous studies reported that some cicada species may be associated with facultative symbionts such as Rickettsia and Arsenophonus,in addition to the obligate symbionts.Although bacterial communities of alimentary canal and some other organs of a few cicada species have been investigated,knowledge is quite limited about the distribution and transovarial transmission of symbionts in cicadas.Eopycna is one of the important groups of the Platypleurini of Cicadinae,which currently consisted of two species distributed in China,Eopycna repanda(Linnaeus,1758)and Eopycna coelestia(Distant,1904).E.repanda is endemic to the high-altitude mountainous areas(1500–3500 m)in Central and southwest China,whereas E.coelestia is primarily distributed in the relatively low-altitude areas(300–1000 m)in southern China.The mountain-habitat preference and low dispersal ability of E.repanda and E.coelestia represent ideal species for the studies on population differentiation of sibling species.In this study,we explored the population differentiation of E.repanda and E.coelestia and evolutionary relationships of symbionts using integrated methods including morphological and phylogenetic methods.The distribution and transovarial transmission of symbionts in E.repanda and E.coelestia were explored based on high-throughput sequencing,RFLP-based cloning approach,histological and fluorescence microscopy.Additionally,we also investigated the distribution and transovarial transmission of symbionts in other five representative cicadas.The distribution and transovarial transmission of symbionts among E.repanda,E.coelestia and other five cicadas were compared.The main results are as follows:(1)Population differentiation of E.repanda and E.coelestia and diversification of obligate symbionts at the intraspecific level.The phylogenetic trees of cicadas revealed that 12 species of Platypleurini were categorized into four clades.Both E.repanda and Eopycna sp.formed a well-supported clade sister to Platypleura kaempferi.Specifically,Platypleura badia and Platypleura nobilis formed a clade which is the sister group to E.coelestia.These suggest that both Eopycna and Platypleura were not monophyletic group.Populations of E.repanda and E.coelestia were categorized into four and five geographically distinct lineages,respectively.Different lineages of E.repanda and E.coelestia exhibit morphological variations of mesonotum,wings and male pygofer at the intraspecific level.Both E.repanda and E.coelestia exhibited slight differences in calling song structures among different populations of the same species.In contrast,calling song structures showed obvious differences between E.repanda and E.coelestia.Different clades of both E.repanda and E.coelestia were formed during the Pleistocene,which suggests that climatic changes and geological movements may play important roles in shaping the evolutionary history of these two species.Sulcia of E.repanda and E.coelestia were categorized into three and five lineages,respectively.The topological structures of Sulcia are generally similar to that of host cicadas at population level,which indicated that the diversification of Sulcia may be closely related to the genetic differentiation of host cicadas.(2)The distribution of symbionts in different tissues of E.repanda and E.coelestiaHigh-throughput sequencing analysis,RFLP analysis and diagnostic PCR amplification revealed that Sulcia and Hodgkinia were dominant in the bacteriomes and ovaries of E.repanda and E.coelestia.Arsenophonus was dominant in the bacteriomes and ovaries of E.repanda,and in the fat bodies and ovaries of E.coelestia.Rickettsia was detected in the salivary glands,filter chamber,conical segment,midgut,hindgut and testes of both cicadas.Histological and fluorescent microscopy revealed that Sulcia and Hodgkinia harbored in the bacteriomes and ovaries of E.repanda and E.coelestia,which are not present in the salivary glands,filter chamber,conical segment,midgut,hindgut,fat bodies and testes.In the bacteriomes of E.repanda and E.coelestia,Sulcia harbored in the peripheral bacteriocytes,whereas Hodgkinia occupied the central bacteriocytes.Arsenophonus harbored in the bacteriomes of E.repanda,and in the fat bodies of E.coelestia.(3)Transovarial transmission of symbionts in E.repanda and E.coelestiaFor E.repanda and E.coelestia,Sulcia were directly released from the peripheral bacteriocytes into the hemolymph,but Hodgkinia emigrated from central bacteriocytes through the multi-nuclear compartment into the hemolymph.Sulcia cells together with Hodgkinia cells in the bacteriomes of cicadas were gathered tightly before being released to the hemolymph,which become individual Sulcia or Hodgkinia cell in the hemolymph.Fluorescence and transmission electron microscopy revealed that Arsenophonus can be transovarially transmitted with Sulcia and Hodgkinia between generations of both cicadas.In E.repanda,Arsenophonus together with Sulcia and Hodgkinia were released from the bacteriomes into the hemolymph.In contrast,Arsenophonus of E.coelestia was released into the hemolymph based on disintegration of fat bodies.In both cicadas,Arsenophonus,Sulcia and Hodgkinia gathered around the posterior pole of terminal oocytes and then migrated through the cytoplasma of epithelial plug cells into the perivitelline space(viz.,space between epithelial plug cells and oolemma),and they finally formed a characteristic“symbiont ball” in each egg.For Tettigetta sp.,Sulcia and Hodgkinia harbored in the bacteriomes and additional symbiont(s)only harbored in some host nuclei of the fat bodies,and only Sulcia and Hodgkinia can be transovarially transmitted between generations.The transovarial transmission process of Sulcia and Hodgkinia of Te.sp.is generally similar to that of E.repanda and E.coelestia.For Karenia caelatata,Sulcia harbored in the bacteriomes,YLS harbored in the fat bodies,and additional symbiont(s)harbored in the fat bodies,bacteriomes,and some host nuclei of bacteriomes,and only Sulcia and YLS can be transovarially transmitted between generations.For Tanna sp.,Sulcia harbored in the bacteriomes and YLS harbored in the fat bodies,and additional symbiont(s)harbored in the fat bodies,bacteriomes,and some host nuclei of bacteriomes,and both Sulcia and YLS can be transovarially transmitted between generations.For Graptopsaltria tienta and Hyalessa maculaticollis,Sulcia harbored in the bacteriomes and YLS harbored in the bacteriome sheath cells and fat bodies,and both Sulcia and YLS can be transovarially transmitted between generations.Sulcia cells or Sulcia cells together with Hodgkinia cells(when harbored together)in the bacteriomes of cicadas were gathered tightly before being released to the hemolymph,while YLS cells in the bacteriomes of G.tienta and H.maculaticollis did not gather together before being released to the hemolymph.The results revealed that the distribution and transovarial transmission of symbionts were distinct among E.repanda,E.coelestia and other cicada species.In summary,we clarified phylogeny of E.repanda and E.coelestia and other Oriental Platypleurini species,population differentiation of E.repanda and E.coelestia,and evolutionary relationships of symbiont using integrated methods such as morphological and phylogenetic methods.Moreover,the distribution of symbionts in salivary glands,bacteriomes,fat bodies and digestive and reproductive organs of the E.repanda and E.coelestia were deeply uncovered.The distribution and transovarial transmission of symbionts in E.repanda and E.coelestia and other five representative cicadas were clarified using histological,ultrastructural and fluorescence microscopy.The results provide new insights into the phylogeny and population differentiation of cicadas and the codiversification of hosts and symbionts.