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

Abstract

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.

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

“…This inference was also supported by the presence of Carnobacterium in Thitarodes sp. unfertilized eggs and larvae [ 21 , 39 , 55 ]. Carnobacterium is a genus of Lactic Acid Bacteria (LAB), and C. maltaromaticum is frequently isolated from natural environment and foods [ 56 ].…”

“…It can also out-compete the pathogen and modulate the autochthonous midgut microbiota of Atlantic cod Gadus morhua [ 58 ]. More colonies of C. maltaromaticum were also detected in the gut of T. xiaojinensis larvae uninfected with O. sinensis than those injected with O. sinensis [ 21 ]. Therefore, whether C. maltaromaticum could be used as a probiotic bacterium to improve the growth of the ghost moth larvae needs further study.…”

“…The laboratory-reared ghost moth larvae were derived from the pupae collected from the above three geographic locations and reared at 9~13 °C in Guangzhou, China according to the described methods [ 21 , 41 ]. The hatched larvae were offered the roots of Potentilla anserine as food to obtain 5th instar larvae (average fresh weight = 0.57 ± 0.07 g) for gut microbial isolation.…”

“…In response to O. sinensis infection, only a proportion of infected larvae could turn into stiff worms and become Chinese cordyceps even though the larvae were sampled from the same site or reared at the same conditions [ 17 , 18 ]. It appeared that the immune system and gut microbiota are involved in the pathogenicity of O. sinensis fungus [ 19 , 20 , 21 ].…”

“…Given the important role of the gut microbes, researchers attempt to identify microbes from the gut of the ghost moth larvae, hoping to find beneficial microbes for the artificial rearing ghost moth larvae and promoting the infection and mummification rate by O. sinensis . However, only 8 bacterial and 3 fungal genera were isolated from the wild T. gonggaensis larvae [ 39 , 40 ] and 11 bacterial and 3 fungal genera were isolated from the laboratory-reared T. xiaojinensis [ 21 ] by the culture-dependent method. Furthermore, only Carnobacterium and Pseudomonas were common in the wild T. gonggaensis larvae and the laboratory-reared T. xiaojinensis .…”