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Gut Bacterial and Fungal Communities of the Wild and Laboratory-Reared Thitarodes Larvae, Host of the Chinese Medicinal Fungus Ophiocordyceps sinensis on Tibetan Plateau.

ABSTRACT: By employing a culture-dependent and -independent 16S rRNA and ITS gene high-throughput sequencing analyses, comprehensive information was obtained on the gut bacterial and fungal communities in the ghost moth larvae of three different geographic locations from high-altitude on Tibet plateau and from low-altitude laboratory. Twenty-six culturable bacterial species belonging to 21 genera and 14 fungal species belonging to 12 genera were identified from six populations by culture-dependent method. Carnobacterium maltaromaticum was the most abundant bacterial species from both the wild and laboratory-reared larvae. The most abundant OTUs in the wild ghost moth populations were Carnobacteriaceae, Enterobacteriaceae for bacteria, and Ascomycota and Basidiomycota for fungi. Larval microbial communities of the wild ghost moth from different geographic locations were not significantly different from each other but significant difference in larval microbial community was detected between the wild and laboratory-reared ghost moth. The larval gut of the wild ghost moth was dominated by the culturable Carnobacterium. However, that of the laboratory-reared ghost moth exhibited significantly abundant Wolbachia, Rhizobium, Serratia, Pseudomonas, and Flavobacterium. Furthermore, the larval gut of the wild ghost moth had a significantly higher abundance of Ophiocordyceps but lower abundance of Candida and Aspergillus than that of the laboratory-reared ghost moth.

SUBMITTER: Liu G

PROVIDER: S-EPMC8067570 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOphiocordyceps sinensis (syn. Cordyceps sinensis), endemic to alpine regions on the Tibetan plateau, is one of the most valuable medicinal fungi in the world. Huge commercial demand has led to excessive harvest and a dramatic decline in its numbers. The diversity of terrains and climates on the Tibetan Plateau and the broad insect host range (more than 50 species in the family Hepialidae) may have resulted in substantial intraspecific genetic diversity for this fungus. The objective of this study was to evaluate the population distribution of O. sinensis from geographically diverse regions of the Tibetan Plateau based on nrDNA ITS and MAT1-2-1 gene sequences. Understanding of the genetic diversity and genesis of O. sinensis will provide important information for the evolution and conservation of this fungus.ResultsSignificant sequence variations in the ITS and MAT1-2-1 genes (27 and 23 informative sites, eight and seven haplotypes, respectively) were observed. Phylogenetic analysis based on ITS sequences, MAT1-2-1 sequences, or their combined data set, clustered isolates from northern regions in one clade (clade I), whereas isolates from southern regions were dispersed in all four clades (clade I-IV). Single-strand conformation polymorphism (SSCP) analyses of 2639 ITS clones from seven samples revealed 91 different SSCP patterns that were subsequently sequenced. ITS heterogeneity was found in XZ-LZ07-H1 (Nyingchi population), and 17 informative sites and five haplotypes were detected from 15 clones. The five haplotypes clustered into three clades (clade I, II, and IV).ConclusionsSignificant genetic divergence in O. sinensis was observed and the genetic diversification was greater among southern isolates than that among northern isolates. The polymorphism of nrDNA ITS sequences suggested that O. sinensis spread from a center of origin (the Nyingchi District) to southern regions and subsequently to northern areas. These results suggest that southern populations are important reservoirs of genetic diversity and should be taken into account in conservation programs.

Project description:The Chinese cordyceps is a unique and valuable parasitic complex of Thitarodes/Hepialus ghost moths and the Ophiocordyceps sinensis fungus for medicine and health foods from the Tibetan Plateau. During artificial cultivation of Chinese cordyceps, the induction of blastospores into hyphae is a prerequisite for mummification of the infected Thitarodes larvae. To explore the microbial involvement in the induction of mycelia-blastospore transition, the microbiota of the hemolymph and gut from Thitarodes xiaojinensis larvae with or without injected O. sinensis blastospores were investigated by culture-dependent and -independent methods. Twenty-five culturable bacterial species and 14 fungal species, together with 537 bacterial operational taxonomic units (OTUs) and 218 fungal OTUs, were identified from the hemolymph and gut of samples from five stages including living larvae without injected fungi (A) or with high blastospore load (B), mummifying larvae without mycelia coating (C), freshly mummifying larvae coated with mycelia (D), and completely mummified larvae with mycelia (E). Two culturable bacterial species (Serratia plymuthica, Serratia proteamaculans), and 47 bacterial and 15 fungal OTUs were considered as shared species. The uninfected larval hemolymph contained 13 culturable bacterial species but no fungal species, together with 164 bacterial and 73 fungal OTUs. To our knowledge, this is the first study to detect large bacterial communities from the hemolymph of healthy insect larvae. When the living larvae contained high blastospore load, the culturable bacterial community was sharply inhibited in the hemolymph but the bacterial and fungal community greatly increased in the gut. In general, high blastospore load increased bacterial diversity but sharply decreased fungal diversity in the hemolymph and gut by OTUs. The bacterial loads of four culturable species (Chryseobacterium sp., Pseudomonas fragi, S. plymuthica, S. proteamaculans) increased significantly and O. sinensis and Pseudomonas spp. became dominant microbes, when the infected larvae became mummified, indicating their possible involvement in the larval mummification process. The discovery of many opportunistic pathogenic bacteria in the hemolymph of the healthy larvae, the larval microbial diversity influenced by O. sinensis challenge and the involvement of dominant bacteria during larval mummification process provide new insight into the infection and mummification mechanisms of O. sinensis in its Thitarodes hosts.

Project description:BackgroundOphiocordyceps sinensis (Berk.) is a well-known entomopathogenic and medicinal fungus. It parasitizes and mummifies the underground ghost moth larvae to produce a fruiting body named Chinese cordyceps. Specific for the fungus, O. sinensis experiences a biotrophic vegetative growth period spanning over 5 months. During this vegetative growth, it appears successively in the host hemocoel in three/four morphotypes, namely, the yeast-like blastospores (subdivided into proliferative (BP) and stationary phase (BS)), prehyphae (PreHy) and the hyphae (Hy). This peculiar morphogenesis has been elucidated through morphological and ultrastructural observations, but its molecular basis remains cryptic. In this study, transcriptome and metabolome profiling of BP, BS, PreHy and Hy stages were performed to characterize the key genes, metabolites, and signaling pathways that regulated the vegetative development of O. sinensis in Thitarodes xiaojinensis larva.ResultsThe molecular events and metabolic pathways that regulated different intracellular processes at various stages were examined. Cluster analyses of differentially expressed genes across the four stages revealed the stage specifically enriched pathways. Analysis of metabolome profiles showed that carbon metabolism and several amino acids biosynthesis were significantly perturbed during the tested development stages of O. sinensis in the host hemocoel. Genes homologous to Saccharomyces cerevisiae MAPK cascade were significantly up-regulated during the transition from blastospore to hypha. The up-regulation of Sho1, a regulator protein, suggested nutrient starvation act a role in activation of MAPK pathway and filamentous growth. In addition, up-regulation of several fatty acid synthesis genes and their corresponding products accumulation in the samples of BS might explain more lipid droplets were observed in BS than in BP. Coupled with the up-regulation of fatty acid degradation during PreHy and Hy stages, it is presumed that lipid accumulation and mobilization play important roles in filamentous development.ConclusionsThis is the first report comprehensively describing developmental transcriptomics and metabolomics of O. sinensis in vivo. Our findings provide new perspectives into the key pathways and hub genes involved in morphological changes of fungus developed in the hemocoel of its host, and are expected to guide future studies on morphogenesis and morphotype changes of entomopathogenic fungi in vivo.

Project description:BackgroundOphiocordyceps sinensis, which is only naturally found in the high-elevation extreme environment of the Tibetan Plateau, has been used in traditional Chinese medicine. Information concerning the evolutionary and geologic context of O. sinensis remains limited, however.MethodsWe constructed the high-quality genome of O. sinensis and provided insight into the evolution and ecology of O. sinensis using comparative genomics.ResultsWe mapped the whole genome of the anamorph/asexual form Hirsutella of O. sinensis using Illumina and PacBio sequencing technologies and obtained a well assembled genome of 119.2 Mbp size. Long-read Single Molecule Real Time (SMRT) sequencing technology generated an assembly with more accurate representation of repeat sequence abundances and placement. Evolutionary analyses indicated that O. sinensis diverged from other fungi 65.9 Mya in the Upper Cretaceous, during the uplift of the Tibetan Plateau. Gene family expansions and contractions in addition to genome inflation via long terminal repeat (LTR) retrotransposon insertions were implicated as an important driver of O. sinensis divergence. The insertion rate of LTR sequences into the O. sinensis genome peaked ~ 30-40 Mya, when the Tibetan Plateau rose rapidly. Gene Ontology (GO) enrichment analysis suggested that O. sinensis contained more genes related to ice binding compared to other closely related fungi, which may aid in their adaptability to the cold Tibetan Plateau. Further, heavy metal resistance genes were in low abundance in the O. sinensis genome, which may help to explain previous observations that O. sinensis tissues contain high levels of heavy metals.ConclusionsOur results reveal the evolutionary, geological, and ecological context for the evolution of the O. sinensis genome and the factors that have contributed to the environmental adaptability of this valuable fungus. These findings suggest that genome inflation via LTR retrotransposon insertions in O. sinensis coincided with the uplift of the Tibetan Plateau. LTRs and the specific genetic mechanisms of O. sinensis contributed to its adaptation to the environment on the plateau.

Project description:Ophiocordyceps sinensis is a widely known medicinal entomogenous fungus, which parasitizes the soil-borne larva of Thitarodes (Hepialidae, Lepidoptera) distributed in the Qinghai-Tibetan Plateau and its adjacent areas. Previous research has involved artificial cultivation of Chinese cordyceps (the fungus-caterpillar complex), but it is difficult to achieve large-scale cultivation because the coupling relation between the crucial microbes and their hosts is not quite clear. To clarify the influence of the internal microbial community on the occurrence of Chinese cordyceps, in this study, the unfertilized eggs of Thitarodes of different sampling sites were chosen to analyze the bacterial and fungal communities via 16S rRNA and ITS sequencing for the first time. The results showed that for bacteria, 348 genera (dominant genera include Wolbachia, Spiroplasma, Carnobacterium, Sphingobium, and Acinetobacter) belonging to 26 phyla (dominant phyla include Proteobacteria, Firmicutes, Tenericutes, Actinobacteria, Acidobacteria, and Bacteroidetes), 58 classes, 84 orders, and 120 families were identified from 1294 operational taxonomic units (OTUs). The dominant bacterial genus (Spiroplasma) may be an important bacterial factor promoting the occurrence of Chinese cordyceps. For fungi, 289 genera, mainly including Aureobasidium, Candida, and Cryptococcus, were identified, and they belonged to 5 phyla (Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, and Zygomycota), 26 classes, 82 orders, and 165 families. Eight bacterial OTUs and 12 fungal OTUs were shared among all of the detected samples and were considered as core species. Among them, Wolbachia, Spiroplasma, Carnobacterium, Aureobasidium, and Phoma may play important roles in helping the host larva to digest foods, adapt to extreme environments, or resist pathogens. On the other hand, the external (soil) microbial community was synchronously and comparatively analyzed. Comparative analysis revealed that external microbial factors might play a more significant role in the occurrence of Chinese cordyceps, owing to the significant differences revealed by α-diversity and β-diversity analyses among different groups. In summary, the results of this study may contribute to the large-scale cultivation of Chinese cordyceps.

Project description:The caterpillar fungus Ophiocordyceps sinensis is one of the most valuable medicinal fungi in the world, and it requires host insects in family Hepialidae (Lepidoptera) to complete its life cycle. However, the genetic diversity and phylogeographic structures of the host insects remain to be explored. We analyzed the genetic diversity and temporal and spatial distribution patterns of genetic variation of the host insects throughout the O. sinensis distribution. Abundant haplotype and nucleotide diversity mainly existed in the areas of Nyingchi, ShangriLa, and around the edge of the Qinghai-Tibet Plateau, where are considered as the diversity center or micro-refuges of the host insects of O. sinensis. However, there was little genetic variation among host insects from 72.1% of all populations, indicating that the host species composition might be relatively simple in large-scale O. sinensis populations. All host insects are monophyletic except for those from four O. sinensis populations around Qinghai Lake. Significant phylogeographic structure (NST>GST, P<0.05) was revealed for the monophyletic host insects, and the three major phylogenetic groups corresponded with specific geographical areas. The divergence of most host insects was estimated to have occurred at ca. 3.7 Ma, shortly before the rapid uplift of the QTP. The geographical distribution and star-like network of the haplotypes implied that most host insects were derived from the relicts of a once-widespread host that subsequently became fragmented. Neutrality tests, mismatch distribution analysis, and expansion time estimation confirmed that most host insects presented recent demographic expansions that began ca. 0.118 Ma in the late Pleistocene. Therefore, the genetic diversity and distribution of the present-day insects should be attributed to effects of the Qinghai-Tibet Plateau uplift and glacial advance/retreat cycles during the Quaternary ice age. These results provide valuable information to guide the protection and sustainable use of these host insects as well as O. sinensis.

Dataset Information

Gut Bacterial and Fungal Communities of the Wild and Laboratory-Reared Thitarodes Larvae, Host of the Chinese Medicinal Fungus Ophiocordyceps sinensis on Tibetan Plateau.

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