Fertilization Remodels the Microbial Community of the Thitarodes Eggs. From the years of 2006 to 2016, we had conducted a pilot investigation at Shergyla mountain to survey the density of Chinese cordyceps and host Thitarodes larvae [12], and ultimately had screened out three sampling sites (see 2.1) with different occurrence rates for our series of studies. To study the influence from environmental microbes on the occurrence of Chinese cordyceps, we had comparatively analyzed the external (soil) and internal microbial community on Thitarodes insects [12, 14]. For external soil habitat, both the bacterial and fungal community varied significantly among the three sampling sites [12]; for internal microbial analysis, we chose the unfertilized eggs in female Thitarodes moths as research sample, and found significant differences (p < 0.05, ANOSIM, Adonis, and MRPP analyses) of bacterial community among the three sites, while no significant differences were observed (p > 0.05) of fungal community [14]. In this study, the laid out fertilized eggs were analyzed. It’s surprising to find significant differences (P < 0.05) among the three sites in aspects of both bacterial and fungal communities (Table 2). Furthermore, unlike unfertilized eggs, α- and β- fungal diversities and the microbial composition of bacteria in the fertilized eggs varied regularly among different sites, which were totally different from that in unfertilized eggs. According to the life habit of Thitarodes, the Thitarodes moths mate immediately after eclosion from the pupae. During the mating process which lasts for 2.5 hours on average, semen ejected from the male Thitarodes moth is stored in the bursa copulatrix of the female, which is located near the ovipositor [17]. After the male Thitarodes leaves, the female moth begins to lay eggs to the ground, and these eggs are simultaneously fertilized with the stored semen through the micropyle [18]. To avoid the disturbance from the external habitat, we collected the fertilized eggs in asepsis condition (see 2.1). Thus, it can be inferred that fertilization may remodel the microbiome in the eggs. Nevertheless, another possibility is that the ovarian tissue firmly attached on the unfertilized eggs might be the reason for the irregular variety of the microbial community [14], which cannot be ignored.
The Microbiome in the Fertilized Eggs might Be Related with the Occurrence of Chinese cordyceps. Some intrinsic bacterial factors might be significant in the occurrence of Chinese cordyceps. Controversy has surrounded the anamorphs of O. sinensis since the 1980s. Currently, at least 37 genera and 90 fungal species related to the anamorphs of O. sinensis have been isolated [19–20]. Among these fungi, Hirsutella sinensis is the most widely accepted anamorph [21]. However, the H. sinensis strain isolated from the stroma of Chinese cordyceps could not induce the germination of stroma on healthy Thitarodes larvae in the laboratory [22]. Therefore, according to Koch's postulates, the pathogenicity of H. sinensis has not been proven. Recently, scientists proposed that Chinese cordyceps constitutes a complex and ordered microecological system instead of being a solitary organism, and hence, sole H. sinensis could not trigger the occurrence of Chinese cordyceps, which might be more likely induced by the synergy among H. sinensis and some other microorganim(s) [23]. Consistent with this inference, studies have shown that the synergy between H. sinensis and other related fungi, such as Paecilomyces hepiali and Aspergillus tennesseensis, enhances the potency of infection [24]. Among the reported fungal combinations, different combinations promote the formation of stiff worms to varying degrees, while stromata also do not germinate successfully. Thus, the definite microbial factors inducing the occurrence of Chinese cordyceps remain to be investigated.
Due to the long and complex life cycles of the larvae, systematic research on the colonized microorganisms of Thitarodes larvae is difficult to conduct. Furthermore, the intrinsic bacterial factors present in Thitarodes larvae are complex and vary with changes in the soil environment [7], which contains highly abundant microbial biomass [12]. To exclude interference from the soil environment, this study chose fertilized eggs as research sample and found that fertilization remodeled the microbial community of the eggs, and the microbial factors especially bacterial factors might play a more significant role in the occurrence of Chinese cordyceps, owing to the more significant differences revealed by α-diversity and β-diversity analyses (P < 0.05) among different groups. For the bacterial microbiome at site A (high Chinese cordyceps group), bacterial α-diversity was significantly higher than that of sites B (low Chinese cordyceps group) and C (null Chinese cordyceps group) indicated by Shannon and Simpson indices; 20 top families evenly distributed without special bias which occurred in sites B and C, and LEfSe analyses revealed much abundant biomarkers which specially enriched in site A than that of sites B and C. Thus, the bacterial community in fertilized Thitarodes eggs might be significant for the occurrence of Chinese cordyceps, and the high evenness of the community might aid for the occurrence of Chinese cordyceps.
In each microhabitat, microbial species is not an independent existence, and it lives in a complicated community with interactions among the other microbial species. These interactions mainly include positive (commensalism, mutualism) and negative (amensalism, parasitism or predation, and competition) relation [25]. Network analysis was applied to investigate these potential microbial interactions in the complex microbial communities [26]. Chinese cordyceps is essentially the outcome of the competition of the entomogenous fungus (H. sinensis) and the other microbial inhabitants in the host insects [13]. Thus, the microbial interactions would influence the infection, colonization, and growth of H. sinensis, which ultimately related with the occurrence of Chinese cordyceps. Co-occurrence patterns were explored to offer insight into microbial interactions. According to the intra-kingdom network analysis based on the positive and negative correlations among the 40 most abundant fungal genera (Table 3 and Fig. 5), there were more positive correlations and average degrees in the high Chinese cordyceps group (sites A). Furthermore, according to Inter-kingdom network analysis based on the positive and negative correlations among the different bacterial and fungal families, the number of positive correlations, negative correlations and the total links for sites A were also the highest (Table 3 and Fig. 6). These findings indicate that a closer correlation of the microbial community, especially closer fungal positive correlation, in the fertilized eggs might help for the colonization of O. sinensis. This discovery reinforced the above-mentioned theory that the synergy among H. sinensis and some other microorganim(s) which ultimately triggers the occurrence of Chinese cordyceps [23].
Wolbachia and Spiroplasma might Be Significantly Related with the Occurrence of Chinese cordyceps. In our previous study, we found Cordyceps-related fungi (10 genera in total) may enter the host Thitarodes as early as in the oocyte stage. Among them, Purpureocillium, which also belongs to the Ophiocordycipitaceae family [27], was detected in unfertilized egg samples from the Chinese cordyceps group, providing the possibility of maternal infection and enlarging the knowledge on the infection mechanism [23]. In addition, Purpureocillium was supposed to manipulate the behaviors of its host insect Edessa rufomarginata [28]. This interesting phenomenon bears a striking resemblance to the infected Thitarodes larvae. In this study, totally 10 genera of Cordyceps-related fungi were observed in the fertilized eggs. Coincidently, Purpureocillium also present preference in site A. The similar distribution presence of Purpureocillium in both unfertilized [14] and fertilized eggs from site A reinforced our previous inference that this fungus in Ophiocordycipitaceae family might be in assistance for the occurrence of Chinese cordyceps. More interestingly, as the anamorph of Chinsese cordyceps, H. sinensis hasn’t been detected in the soils [12], unfertilized [14] or fertilized eggs. The absence revealed that, H. sinensis, at least during the egg stage, is not predominant in the Thitarodes insect. And it might be from the result that it win the battle against the immune system of Thitarodes insect at laval stage, and ultimatley kill the host and become the biggest overlord of the whole microbiome. In addition, based on our investigations on the soil, unfertilized eggs and fertilized eggs, the Cordyceps-related fungi are in trace abundance in these samples. These results reinforced the discovery of Zhong, which revealed that most of the entomogenous fungi, including Cordyceps-related fungus and H. sinensis, prefer to colonize in plant roots and derive nutrition from plant sources in the absence of insect hosts [29]. While after uptake into the insect host (Thitarodes larvae) via feeding behaviors, for certain opportunity, H. sinensis would seize the opportunity.
Spiroplasma might aid for the occurrence of Chinese cordyceps. We had found that OTU4 (Spiroplasma) preferred to exist in the unfertilized egg samples of site A and was the dominant bacteria in some samples of site A [14] (Fig. 3b). While in the subsequent fertilized eggs, the abundance of OTU4 was very low (even could not ranked in top 40 OTUs (Fig. 2e), indicating that OTU4 may originated from the ovarian tissue of the mother which attached on the unfertilized eggs, and only a small part can enter into the fertilized eggs. Nevertheless, it still shows preference in site A and absent in sites B and C (Fig. 3c). Thus, Spiroplasma might play positive roles in the occurrence of Chinese cordyceps in the subsequent insect stages. However, the inference was based on the correlation between the abundance of Spiroplasma and potential Chinese cordyceps occurrence, and need to be verified by further experimental studies.
Wolbachia might be the inhibitory bacterium for the occurrence of Chinese cordyceps. In unfertilized eggs, OTU1 was the predominant bacterium and presented distribution preference in site B (predominant in 4 of 5 samples of site B, Fig. 3b), and occurred in minor amount in all of the rest samples including samples from sites A and C [14]. While in fertilized eggs, OTU1 ranked the overwhelming predominance in all of the samples from sites B and C. Interestingly, it was absent in the samples from site A (Fig. 3c). This study revealed that the mating behaviors would also introduce the Wolbachia to the fertilized eggs, owing to the distinct difference between the abundance in the eggs from site C before (in minor abundance) and after (the most predominant) fertilization. In contrast to site C, Wolbachia was detected in minor abundances in the eggs before fertilization at site A, while it was absent in the fertilized eggs at site A, the decrease might be caused by the inhibition from the Spiroplasma [30]. The strict distribution preference in low (site B) and null Chinese cordyceps group (site C) indicated that Wolbachia might play negative roles in the occurrence of Chinese cordyceps. Wolbachia is an intracellular Gram-negative bacterial genus that infects a vast range of arthropod species, probably making it the most prevalent endosymbiont in the world. Wolbachia has evolved the ability to cause reproductive alterations in its arthropod hosts, such as cytoplasmic incompatibility, parthenogenesis, feminization, and male killing [31]. More recently, Wolbachia has attracted concerns for its ability in increasing host fecundity, and immunity enhancement to protect against pathogens [32–33]. By coincidence, this study revealed that, except the predominant Wolbachia, the other bacteria are in minor abundance in sites B and C and the discovery reinforced the pathogen inhibition effect from Wolbachia. While in site A, without the inhibition from Wolbachia, the bacterial diversity and evenness are much higher (Table 1, Fig. 2e and 3c). It can be inferred that, in the subsequent larval stage, the growth and colonization of multiple bacteria create an appropriate living environment for the survive of H. sinensis, and ultimately induced the invasion and colonization of fungi H. sinensis, which triggers the occurrence of Chinese cordyceps.