INTRODUCTION
Some microbial parasites can manipulate the behavior of their infected hosts to aid transmission. This manipulation can create novel behaviors not present in uninfected hosts (
1). For example, infected ants climb to elevated locations and bite vegetation (
2). This behavior assists in the formation of the fruiting body and positively impacts post-mortem spore transmission (
1–3). Likewise,
Ophiocordyceps sinensis-infected ghost moth larvae are manipulated to move to the soil surface where they die with their heads up. A fruiting body then grows from the head of the caterpillar, which eventually leads to conidia that disperse (
4). Controllable behaviors benefit the next fungal infection cycle but molecular mechanisms underlying behavioral alterations in insects during their interactions with fungi are poorly understood (
5).
O. sinensis has been nominated as the national fungus of China and the flagship species of the Tibetan Plateau (
6,
7). Due to its uniqueness and market demand, much effort has been devoted to its artificial cultivation to produce Chinese cordyceps, a valuable traditional Chinese medicine (
8). However, the artificial cultivation of
Cordyceps is difficult due to low infection and mummification rates, long life cycles, and unexpected infections (
9). Few studies have analyzed the interaction process between
O. sinensis and host larvae. Once it invades the insect hemocoel,
O. sinensis uses the host larva body as a developmental substrate. Fungal cells in the host hemocoel can undergo morphological changes from blastospores to prehyphae, and ultimately to hyphae (
10). Unlike other parasitic fungi that rapidly kill their host,
O. sinensis can coexist with host larvae for an extended time (
11). Some host larvae can tolerate
O. sinensis growth
in vivo for periods greater than 1 year (
6). The
in vivo development and morphological changes of
O. sinensis have previously been documented, but there is little information on how
O. sinensis manipulates the mummification behavior of infected larvae (
10).
Parasitic fungi can control infected host brains and manipulate their behavior to facilitate spore dispersal (
5,
12). For example,
Ophiocordyceps hijacks the central nervous system of infected ants which leave their nests, climb up vegetation, and bite onto the leaves (
1,
13). This behavioral manipulation could be caused by immune and neural stress responses, as well as by apoptosis (
1). Although
O. sinensis infection can cause behavioral changes in
Thitarodes xiaojinensis larvae, it is unknown how
O. sinensis controls the host brain and mediates the mummification process.
Studies of phenotypes, genomes, transcriptomes, and metabolomes have been conducted on the underlying mechanisms of behavioral manipulation by fungi (
14–16). For instance, selected metabolites are closely associated with the host
Camponotus castaneus manipulation by
Ophiocordyceps kimflemingiae zombie ant fungus (
5,
14). In the present study, we evaluated the metabolic profile of
T. xiaojinensis host larvae after
O. sinensis infection. The alteration of lipids among infected, mummified, and control larvae was also studied using lipidomic analysis. A targeted neurotransmitter quantification of the brain was conducted to determine the underlying mechanism of
O. sinensis-induced mummification. Altered lipid-related metabolites, such as phosphatidylcholine (PC), were identified in infected and mummified larvae (ML). Among them, decreased neurotransmitter acetylcholine (ACh) and elevated choline were identified in both infected and mummified larvae brains, suggesting their importance in the mummification of host larvae after
O. sinensis infection. The aberrant activity of acetylcholinesterase (AChE) and relative mRNA expression of
ACE2 (acetylcholinesterase) in the mummified larvae brain indicate that the altered transformation of ACh and choline could mediate brain dysfunction. In addition, caspofungin treatment inhibited the mummification of infected larvae and the activity of AChE.
MATERIALS AND METHODS
Insect rearing and fungal infection
T. xiaojinensis larvae used in this study were obtained from the alpine meadows of Xiaojin County, Sichuan Province, China, and maintained in a laboratory at 15°C and 80% relative humidity (RH) for three generations. Larvae were fed with carrots.
O. sinensis was also isolated from fresh Chinese cordyceps obtained from the alpine meadows in Xiaojin County, Sichuan Province, China. The
O. sinensis fungi (100% matched
O. sinensis genomes, Table S1) were expanded in a dextrose peptone medium at 18℃. For inoculation, 5th instar
T. xiaojinensis larvae (head capsule 2–2.5 mm, body length 0.28–3.3 cm, weight 0.20–0.26 g) were inoculated with a 2-µL blastospores suspension (3 × 10
6 blastospores/μL), transferred to plastic cups, and fed individually in a rearing chamber (15°C, 80% RH) (
16).
Morphological observations of fungus and larvae
Infected 6th to 8th instar T. xiaojinensis larvae and mummified larvae (N = 8) were selected for observation. The morphology of host larvae and the hemolymph of control, infected, and mummified larvae were observed under microscopy (Olympus X71).
LC-MS/MS analysis
Infected 8th instar (IL-8) and uninfected larvae (UIL-8) were collected for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (nine samples in each pool). Samples were lyophilized to constant weight and then crushed into a fine powder. Samples (100 mg) were then extracted with 0.6 mL of 80% methyl alcohol (Aladdin, HPLC grade), vortexed for 62 s, and ground twice at 50 Hz. The product was then sonicated for 15 min and centrifuged at 12,000 rpm for 10 min at 4°C to obtain the supernatant. The supernatant (200 µL) was determined on a Thermo-Fisher UPLC system (Thermo-Fisher, U3000) equipped with a mass spectrometer (Thermo-Fisher, QE Plus). Mobile phase A consisted of 40% water, 60% acetonitrile, and 10 mmol/L ammonium formate. Mobile phase B consisted of 10% acetonitrile and 90% isopropanol, which was combined with 50 mL of 10 mmol/L ammonium formate for every 1,000 mL mixed solvent. The analysis was conducted with an elution gradient as follows: 0–12.0 min, 40%–100% B; 12.0–13.5 min, 100% B; 13.5–13.7 min, 100%–40% B; 13.7–18.0 min, 40% B. The column temperature was 55°C. The auto-sampler temperature was 4°C, and the injection volume was 2 µL (positive) or 2 µL (negative). The QE mass spectrometer was used for its ability to acquire MS/MS spectra in data-dependent acquisition mode in the control of the acquisition software (Xcalibur 4.0.27, Thermo). In this mode, the acquisition software continuously evaluated the full scan MS spectrum. The electrospray ionization (ESI) source conditions were set as follows: sheath gas flow rate as 30 Arb, aux gas flow rate as 10 Arb, capillary temperature 320°C (positive), 300°C (negative), full MS resolution as 70,000, MS/MS resolution as 17,500, collision energy (CE) as 15/30/45 in normalized collision energy mode, spray voltage as 5 kV (positive) or −4.5 kV (negative) (
17–19).
The raw data files were converted to files in mzXML format using the “msconvert” program from ProteoWizard. Peak detection was first applied to the MS1 data. The CentWave algorithm in XCMS was used for peak detection with the MS/MS spectrum, and lipid identification was achieved through a spectral match using the LipidBlast library.
Target determination of neurotransmitter phenotype
The brains and whole host bodies were collected from infected, mummified, and control host larvae (IL-8, ML, and UIL) for determination of the neurotransmitter phenotype. After samples were thawed and ground, 20 mg of the sample was mixed with 500 µL of 70% methanol/water. The sample was vortexed for 3 min at 2,500 rpm and then centrifuged at 12,000 r/min for 10 min at 4°C. We introduced 300 µL of the supernatant into a new centrifuge tube and placed the supernatant in a −20°C refrigerator for 30 min. Then, the supernatant was centrifuged at 12,000 r/min for 10 min at 4°C. After centrifugation, we transferred 200 µL of the supernatant for further LC-MS analysis.
The sample extracts were analyzed using an LC-ESI-MS/MS system (UPLC, ExionLC AD,
https://sciex.com.cn/; MS, QTRAP 6500+ System,
https://sciex.com). The analytical conditions were as follows: high performance liquid chromatography (HPLC): column, Waters Acquity UPLC HSS T3 C18 (100 mm × 2.1 mm i.d., 1.8 µm); solvent system, water with 0.1% formic acid (A), acetonitrile with 0.1% formic acid (B). The gradient was started at 5% B (0 min), increased to 95% B (0–8 min), 95% B (8–9.5 min), and finally ramped back to 5% B (9.6–12 min); flow rate, 0.35 mL/min; temperature, 40°C; injection volume, 2 µL. AB 6500+ QTRAP LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in both positive and negative ion modes was used and controlled by Analyst 1.6 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature, 550°C; ion spray voltage (IS) 5,500 V (positive), −4,500 V (negative); curtain gas (CUR) was set at 35.0 psi; declustering potential (DP) and CE for individual multiple reaction monitoring (MRM) transitions was done with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the neurotransmitters eluted within the period.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was applied to identify the most influential pathways involved in the mummification process. For KEGG enrichment analysis, all the differentially abundant metabolites (DAMs) were mapped to KEGG pathways in the KEGG database (
https://www.kegg.jp/kegg/). The significance of each pathway was estimated based on the
P value (
P < 0.05).
Hematoxylin-eosin staining of host brain
The host larvae brains were fixed in a 4% paraformaldehyde fixative for more than 24 h and then treated with 75% ethanol. Paraffin embedding was performed by automated instrumentation (JB-P5, Wuhan Junjie Electronics Co., Ltd., Wuhan, China). Embedded tissues were cut into 4 µm sections on a Leica microtome (RM20016) and mounted on glass slides. H&E (hematoxylin and eosin) staining was conducted using a standard protocol. Images were collected and analyzed by microscope. The morphology of the fungus was observed by magnification of the H&E stained brain tissue.
Quantitative real-time PCR
The total RNA of larvae brains was extracted using the TRIzol (Invitrogen) method, and the RNA concentration was determined using a NanoDrop-2000 spectrophotometer (Thermo Scientific). RNA was then reverse transcribed into cDNA using Transcriptor cDNA Synth. kit 2 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Reaction mixtures contained 2.5 µL template DNA, 12.5 µL master mix, 1.25 µL primer mix (0.25 µM for each primer), and ribonuclease-free water to a final volume of 25 µL. Real-time quantitative PCR (RT-qPCR) was performed using primers specific for ACE2 and RPS3 (Ribosomal Protein S3) (Sangon, Shanghai, China), based on the FS Essential DNA Green Master (Roche). Primer sequences were as follows: ACE2 forward, 5′-ACTGCAATGACATGTTGAGCG-3′, reverse, 5′-TACTTTACGCATCGTACTAGCACG-3′; RPS3 forward, 5′-GAGAATTGGCTGAAGATGGT-3′, reverse, 5′-GAGGTCAACTCCCTGATACG-3′. The reaction conditions included an initial denaturation step at 95°C for 5 min. Amplification was carried out for 40 cycles with denaturation at 95°C for 30 s, followed by annealing at 62°C for 45 s, and extension at 72°C for 10 s. A final extension was carried out at 72°C for 1 min. Reactions were performed in triplicate. The results were analyzed according to the 2−ΔΔCT method and normalized by RPS3.
Determination of AChE activity
AChE activity in the host brain was determined spectrophotometrically in a 96-well microtiter plate using a modified Ellman’s assay as previously described (
20,
21). The brain was first homogenized, and centrifuged at 2,500 rpm for 10 min, and then the supernatants were used for determination. AChE activity was calculated from the quotient between brain AChE activity and protein content. The results are expressed as nmol/min/g of protein.
Treatment of infected larvae with caspofungin
Infected 8th instar host larvae, including larvae at the early infection stage (fusiform O. sinensis) and larvae at the late infection stage (capsular O. sinensis), were treated with caspofungin, an antifungal drug (N = 100 for each group). Caspofungin (BrightGene Bio-Medical Technology Co., Ltd.), with concentrations of 2.2 mg/mL and 3.4 mg/mL, was prepared with sterile water as the solvent. The 20 µL solution (sterile water or caspofungin) was injected into the larvae body through stomata using a customized microinjection system (Zhonglou, model 0.33 mm, Instrument Factory of West China Medical University). The mummification rate of the host larvae and the number of hyphal bodies in the hemolymph were determined. Morphological changes of the host larvae were also observed.
Statistical analysis
All assays were performed with at least three independent biological replicates. The results are presented as the mean ± standard deviation (SD). One-way analysis of variance and an unpaired, two-tailed Student’s t-test were performed using GraphPad Prism version 10.0 software to analyze the significance of differences between treatment and controls. Differences with a P value <0.05 were considered statistically significant for all treatments.
DISCUSSION
Manipulation of host behavior can benefit parasite transmission.
O. sinensis can infect
T. xiaojinensis larvae and manipulate the mummification process. This can lead to the formation of
Cordyceps (
4,
22). However, the interaction mechanism between
O. sinensis and its host at the early infection stage was previously not understood. In the present study, we investigated the underlying mechanisms of
O. sinensis-induced mummification of
T. xiaojinensis larvae.
Although no significant difference was found in the appearance of
T. xiaojinensis larvae after being infected by
O. sinensis, there were significant morphological differences in mummified larvae. This was consistent with previous studies showing that the host larvae could tolerate fungal proliferation during the chronic infection process lasting more than 1 year before mummification (
10,
11). Given that the insect hemocoel is the critical site for the proliferation of fungi,
O. sinensis was found in the hemocoel of
T. xiaojinensis larvae after infection. The results indicate that the infected
T. xiaojinensis larvae model was successfully established. In keeping with previous studies,
O. sinensis underwent morphotype transformations from budding blastospores to capsular blastospores and prehyphae (
10,
23). During the mummified stage, the number of prehyphae increased significantly, laying the foundations for hyphae germination. These findings indicate that the morphological changes of
O. sinensis are closely associated with the behavior of host larvae. The underlying mechanisms in
O. sinensis morphogenesis are presently unknown.
Previous metabolomic studies have identified metabolites associated with fungal infection and proposed links between these altered compounds and behavioral manipulation (
13–15). In the present study, a large number of DAMs were identified in infected larvae compared to controls. The result suggests that
O. sinensis infection mediated the metabolic alteration of
T. xiaojinensis larvae. Most of these DAMs were found to be lipid-related metabolites such as the alteration of glycerophosphocholine and choline. The elevated choline could be produced by the degradation of glycerophosphocholine by enzymatic activity (
24,
25). Previous studies have described the role of lipids as a key element of the insect defense system against fungal infection (
26–28). This finding suggests that lipid alteration could mediate the interaction between
O. sinensis and the host larvae after infection.
To illustrate the role of lipids in the mummification process of host larvae, lipidomic analysis was conducted to identify the DALs. Six types of lipids were identified as DALs in the control, infected, and mummified larvae. Among these DALs, elevated PC, PE, DGTS, and FA were found in infected larvae compared to control larvae. This suggests that O. sinensis infection can alter the lipid metabolism of host larvae. In contrast, PC and PE decreased in mummified larvae compared to infected and control larvae. The significant difference in PC/PE between mummified and infected larvae indicates that PC/PE is important in the behavior manipulation of infected host larvae. However, the exact roles of PC and PE in O. sinensis-induced larvae mummification are poorly understood. The KEGG enrichment and interaction network data also proved the importance of PC in the glycerophospholipid metabolism that was identified as a key pathway in the behavioral manipulation of host larvae. Elevated Cre and TAGs were also detected in mummified larvae compared to infected and control larvae. Further studies are urgently needed to determine their roles in the mummification process of host larvae. These findings indicate that lipid metabolism, particularly PC, is closely related to the behavioral manipulation of host larvae.
The behavioral manipulation of infected larvae can be affected by parasites infecting the nervous system of the host (
13,
14,
29). Previous studies have shown that fungus can invade the host nervous system (
29,
30), and the present study also found hyphal bodies in the brains of infected larvae. The number and morphology of
O. sinensis in the brain were closely associated with the stage of the infected host larva. This was consistent with the data from the present study, demonstrating that the morphological changes and number of
O. sinensis are closely associated with the behavior of host larvae. Fungal infection in the brain could cause neural damage, ultimately leading to neural and behavioral disorders (
31,
32).
O. sinensis infection could cause neural damage that helps mediate the mummification of host larvae. However, few studies have focused on the neural dysfunction and disorder of
O. sinensis-infected host larvae. Mechanistically,
O. sinensis-induced neural damage could be caused by the growth of
O. sinensis consuming nutrients from the host insect or by physical tissue destruction by the fungal mycelium (
33–35). Moreover,
O. sinensis infection might produce secondary metabolites, such as mycotoxins, presenting a toxicological challenge to host larvae (
36–38).
In the present study, neurotransmitter quantification in the brain was used to reveal the changes in the infected and mummified host brains. Altered levels of neurotransmitters in the infected and mummified host brains proved that
O. sinensis infection caused changes in host brains. For example, decreased ACh and elevated choline levels were detected in both infected and mummified larvae compared to controls. The neurotransmitter ACh can be hydrolyzed into choline by cholinesterase while choline can be used in ACh synthesis (
39–41). Thus,
O. sinensis infection could induce the hydrolyzation of ACh and the production of choline in the brain. Given that ACh exerts effects on the brain networks and involves behavior and cognition, the alteration of ACh could induce aberrant larval behavior (
42–47). Moreover, altered ACh and choline were also observed in the whole bodies of infected and mummified larvae. Neuron-derived factors may be responsible for maintaining the normal innervated skeletal muscle phenotype. For example, the neurotransmitter ACh could negatively regulate the neuromuscular junction (
48,
49). ACh derived from the presynaptic nerve terminal could bind to the ACh receptor tightly clustered on the surface of the muscle fiber and trigger an endplate potential. This could initiate a muscle action potential, and eventually lead to muscle contraction (
50). Therefore, altered levels of ACh in the mummified larvae might cause muscle contraction by regulating the neuromuscular junction. Further studies are needed to decipher the role of ACh in insect body mummification, and these might provide a novel perspective on insect nerve and muscle physiology.
AChE, a hydrolytic enzyme that catalyzes the transformation of ACh to choline, was found activated in the brains of infected and mummified larvae compared to the controls. This was consistent with elevated choline and decreased ACh in both infected and mummified larvae. Moreover, the mRNA relative expression of
AChE was also upregulated in the brains of infected and mummified larvae. This result shows that the alteration of ACh and choline could be caused by the over-hydrolysis of ACh catalyzed by AChE after
O. sinensis infection. Nonsignificant biosynthesis of ACh was found in the brain of
O. sinensis-infected host larvae. Thus,
O. sinensis-induced behavioral manipulation of host larvae could be mediated by disrupting the expression and the activity of
AChE. Interestingly, it is well established that a decrease in brain ACh levels is also implicated in the pathophysiology of cognitive dysfunction occurring in Alzheimer’s disease (AD) (
51,
52). The inhibition of the ACh catabolic enzyme, AChE, could contribute to an increase in ACh brain levels, thus providing a potentially therapeutic strategy for ameliorating cognitive dysfunction in AD (
52). In the present study, cognitive dysfunction might contribute to the behavioral manipulation of
O. sinensis-infected host larvae. To date, the underlying mechanisms that regulate the gene regulation of
AChE and the enzyme activity of AChE remain unknown. More studies are demanded to illustrate their underlying mechanisms. In addition to
O. sinensis infection in the host brain, the transformation of ACh to choline might be induced by the extracellular infection. For example,
Pseudomonas aeruginosa can acquire and metabolize choline from PC derived from the host membrane (
53,
54). High brain choline levels have been linked to an increase in neurological diseases such as depression (
55,
56).
To verify the role of
O. sinensis in the mummification process of infected host larvae, caspofungin was used to treat
O. sinensis-infected larvae. Caspofungin, an antifungal drug, was found to effectively treat
O. sinensis infection in larvae. Caspofungin exhibited time-dependent and dose-dependent manners on fungal survival (
57,
58). Although caspofungin holds great promise for treating
O. sinensis infection, a high-level caspofungin treatment may be toxic to host larvae and cause larval mortality. The caspofungin treatment of larvae infected with
O. sinensis significantly reduced the mummification rates. Similarly, caspofungin treatment exhibited time-dependent and dose-dependent effects on the mummification rates of infected larvae. Moreover, larvae in the early infection stage (fusiform
O. sinensis) were more sensitive to caspofungin treatment than larvae in the late infection stage (capsular
O. sinensis). These results indicate that the growth and development of
O. sinensis in the host were closely related to larvae mummification. Previous studies demonstrated that early treatment should be considered for neurodegenerative diseases at the early disease stages (
59–61).
Since a lower mummification rate was observed after caspofungin treatment, we evaluated the activity of AChE that mediates the hydrolysis of ACh. A nonsignificant difference in the activity of AChE between uninfected and caspofungin-treated larvae suggests that AChE was not hydrolyzed into choline. Presumably, infected larvae might not enter the mummification process because, after caspofungin treatment, AChE could maintain the host’s brain homeostasis. These findings indicate the importance of ACh in O. sinensis-induced mummification of host larvae.
Conclusion
Parasitic fungi can adaptively manipulate the behavior of the infected hosts to facilitate their transmission. In this study, the morphological changes of O. sinensis indicate that the development of O. sinensis in the host was closely associated with the mummification process of infected larvae. Altered metabolites, particularly lipid-related metabolites, were identified in infected and mummified larvae, suggesting that lipids play important roles in O. sinensis-mediated behavioral manipulation of host larvae. Meanwhile, the number and distribution of hyphal bodies in the larvae brain showed that O. sinensis invaded the host brain and caused neural damage, eventually leading to aberrant behavior. In this case, aberrant levels of ACh/choline, activity of AChE, and expression of ACE2 were observed in the brains of both infected and mummified larvae. The altered transformation between ACh and choline could cause brain dysfunction of mummified larvae. Since caspofungin treatment inhibited the activity of AChE and the mummification of infected larvae, our research highlights the critical role of ACh in the mummification of infected host larvae.