Immulectin-2 from the ghost moth, Thitarodes xiaojinensis (Lepidoptera: Hepialidae), modulates cellular and humoral responses against fungal infection.

C type-lectins constitute a large family of pattern recognition receptors, playing important roles in insect immune defenses. Thitarodes xiaojinensis larvae showed distinct immune features after Ophiocordyceps sinensis, Cordyceps militaris, or Beauveria bassiana infection. Based on transcriptome and immunoblot analysis, we found that immulectin-2 (IML2) was induced after T. xiaojinensis larvae were infected by C. militaris or B. bassiana but maintained at a low level after larvae injected with O. sinensis or Ringer's buffer. Recombinant IML2 (rIML2) could promote melanization, encapsulation, phagocytosis, and hemocyte aggregation in vitro. RNA interference with IML2 induced a significant reduction in the transcript levels of various antimicrobial peptides. Importantly, we found that the abundance of O. sinensis blastospores coated with rIML2 dramatically decreased in the host hemolymph. Overall, this study demonstrated that T. xiaojinensis IML2 modulates cellular and humoral responses to entomopathogenic fungi, broadening our view of the immune interaction between O. sinensis and its host.

Wu PP ，Shu RH ，Gao XX ，Li MM ，Zhang JH ，Zhang H ，Qin QL ，Zou Z ，Meng Q ... -  《-》

Distinct Responses of Thitarodes xiaojinensis β-1,3-Glucan Recognition Protein-1 and Immulectin-8 to Ophiocordyceps sinensis and Cordyceps militaris Infection.

Melanization and encapsulation are prominent defense responses against microbes detected by pattern recognition receptors of their host insects. In the ghost moth Thitarodes xiaojinensis, an activated immune system can melanize and encapsulate the fungus Cordyceps militaris However, these responses were hardly detected in the host hemolymph postinfection of another fungus Ophiocordyceps sinensis The immune interaction between O. sinensis and the host remains largely unknown, which hinders the artificial cultivation of Chinese cordyceps. We found that T. xiaojinensis β-1,3-glucan recognition protein-1 (βGRP1) was needed for prophenoloxidase activation induced by C. militaris Failure of βGRP1 to recognize O. sinensis is a primary reason for the lack of melanization in the infected host. Lyticase or snailase treatment combined with binding and immunofluorescence detection showed the existence of a protective layer preventing the fungus from βGRP1 recognition. Coimmunoprecipitation and mass spectrometry analysis indicated that βGRP1 interacted with immulectin-8 (IML8) via binding to C. militaris IML8 promotes encapsulation. This study suggests the roles of T. xiaojinensis βGRP1 and IML8 in modulating immune responses against C. militaris Most importantly, the data indicate that O. sinensis may evade melanization by preventing βGRP1 recognition.

Meng Q ，Wu PP ，Li MM ，Shu RH ，Zhou GL ，Zhang JH ，Zhang H ，Jiang H ，Qin QL ，Zou Z ... -  《-》

Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis.

Ophiocordyceps sinensis is an entomopathogenic fungus that infects ghost moth larva, forming the most valuable and rare traditional Chinese medicine, Chinese cordyceps. Our knowledge of the basic morphology and developmental biology of Chinese cordyceps is limited. In this study, morphological and ultrastructural observations of O. sinensis development in the hemocoel of Thitarodes xiaojinensis were obtained by multiple light and electron microscopy techniques, and the host immune reaction activities were determined. Our results indicated that fungal cells in the host hemocoel underwent morphotype transformations from blastospores to prehyphae to hyphae in sequence. The fusiform yeast-like blastospores were the initial cell type present in the host hemocoel and remained for 5 months or more; the encapsulation reaction and phenoloxidase activity of T. xiaojinensis hemolymph were inhibited during this period. When larvae entered the last instar, the blastospores switched to prehyphae and expanded throughout the host tissues, and then hyphae germinated from the prehyphae and mycelia formed, which finally led to host death. Considering the distinct differences between blastospores and hyphae, we identified prehyphae, which play important roles in fungal expansion, hyphae germination, and fusion formation among filaments. Notably, the elongation of prehyphae was strongly presumed to occur through fission but without separation of the two sister cells, in contrast to blastospore budding. During the morphotype transformation, the amount and composition of lipid droplets changed greatly, suggesting their important roles in these events. Overall, we provide a morphological and ultrastructural characterization of O. sinensis vegetative development within the hemocoel of T. xiaojinensis, identify and name the prehypha fungal cell type in entomopathogenic fungi for the first time, and conclude that O. sinensis infection causes sustained immunosuppression in T. xiaojinensis.

Li M ，Meng Q ，Zhang H ，Ni R ，Zhou G ，Zhao Y ，Wu P ，Shu R ，Qin Q ，Zhang J ... -  《-》

Knockdown of Thitarodes host genes influences dimorphic transition of Ophiocordyceps sinensis in the host hemolymph.

The Chinese cordyceps, a unique parasitic complex of Thitarodes/Hepialus ghost moths and Ophiocordyceps sinensis fungus in the Tibetan Plateau, is a highly valuable biological resource for medicine and health foods in Asian countries. Efficient system for artificial cultivation of Chinese cordyceps relies on understanding the gene functions involved in the induction of growing blastospores into hyphae in the larval hemolymph of insect host, during O. sinensis infection. Transcriptome analysis and ribonucleic acid interference (RNA interference) method were employed to identify the key differentially expressed genes and to demonstrate their functions in Thitarodes xiaojinensis. Key larval genes critical for O. sinensis blastospore development or filamentation were identified. Nine of the 20 top upregulated genes encoded cuticles proteins, indicating that these proteins highly activated when the larval hemolymph was full of blastospores. Small interfering RNA (siRNA) knockdown of five larval genes such as Flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3 significantly stimulated the dimorphic transition from blastospores to prehyphae in O. sinensis in the larval hemolymph after 120 h after injection. The expressions of these genes determined by quantitative real-time PCR were suppressed in various levels from 38.64% to 91.54%, compared to the controls. These results demonstrated that injection of the siRNAs of key upregulated genes into the larval hemolymph containing high load of blastospores caused the gene silence in T. xiaojinensis larvae and induced the fungal transition from blastospores to prehyphae, providing novel knowledge on the regulation of O. sinensis fungal dimorphism by Thitarodes host and cues for further study of Thitarodes biology and commercial cultivation of Chinese cordyceps.

Sun T ，Jin Y ，Rao Z ，Liyan W ，Tang R ，Zaryab KM ，Li M ，Li Z ，Wang Y ，Xu J ，Han R ，Cao L ... -  《Frontiers in Cellular and Infection Microbiology》

Infection of Ophiocordyceps sinensis Fungus Causes Dramatic Changes in the Microbiota of Its Thitarodes Host.

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.

Wu H ，Rao ZC ，Cao L ，De Clercq P ，Han RC ... -  《Frontiers in Microbiology》