Neurotransmitter acetylcholine mediates the mummification of Ophiocordyceps sinensis-infected Thitarodes xiaojinensis larvae

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 × 10⁶ blastospores/μL), transferred to plastic cups, and fed individually in a rearing chamber (15°C, 80% RH) (16).

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).

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.

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).

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.

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.

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.

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.

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.

Morphological changes of T. xiaojinensis larvae were found after fungal infection. No obvious differences were observed between the 8th instar infected larvae (IL-8) and the uninfected controls (UIL-8) (Fig. 1A). This suggests that no symptoms occurred during the chronic infection process before mummification. When the host larvae entered the mummification stage, the ML became stiff compared to IL and UIL (Fig. 1A). Since the host larvae were invaded by O. sinensis, the appearance and morphological changes of O. sinensis in host hemolymph were observed (Fig. 1B). Compared to the controls (UIL-8), growing numbers of fungi were detected in infected larvae at the 6th and 8th instars (IL-6 and IL-8) after infection treatment. Morphotype transformations of O. sinensis were found from budding blastospores (fusiform) (IL-6) to capsular blastospores (IL-8) and prehyphae (ML). The number of prehyphae increased significantly. These data suggest that T. xiaojinensis was successfully infected by O. sinensis. In addition, the number and morphology of O. sinensis were closely related to the behavioral manipulation of infected larvae.

Identification of altered metabolites was used to study the interaction between O. sinensis and the host larvae. The LC-MS/MS data identified DAMs that distinguished uninfected (UIL-8) and infected larvae (IL-8). Compared to uninfected larvae, lipid-related metabolites were identified, including fatty acyls (lipids) and glycerophospholipids (lipids) (Table S2). Among them, 28 DAMs were closely associated with lipid metabolism (Fig. 1C). Compared to uninfected larvae, 11 metabolites were downregulated in larvae after fungal infection, while 17 metabolites were upregulated. Among the glycerophospholipids, elevated levels of choline and PC were found in infected larvae. In contrast, glycerophosphocholine was decreased after infection treatment. This finding suggests that metabolites, particularly lipids, can mediate the interaction between O. sinensis and the host larvae.

Lipidomic analysis was applied to identify the difference between infected larvae and controls to further illustrate the role of lipids in the mummification process of host larvae. The hierarchical clustering of DAMs among the control (UIL-8), infected (IL-8), and mummified larvae showed differences in Cre (ceramide), TAGs (triglycerides), PC, PE (phosphatidylinositol), DGTS (glycerol dialkyl glycerol tetraethers), and FA (fatty acid) (Fig. 2A). Compared to UIL and IL, PC and PE decreased in ML. However, PC and PE increased in IL compared to UIL. This finding suggests that PC/PE might be associated with the mummification process of the infected host larvae. The difference of PC in the ML was also confirmed (Fig. 2B). FA was present at high levels in the infected larvae before mummification as well as being higher than in the controls.

Based on the alterations of lipids, KEGG pathway analysis was applied to identify the most influential pathways involving mummification. KEGG pathway enrichment analysis showed that differentially abundant lipids (DALs) were closely associated with biosynthesis of secondary metabolites, metabolic pathways, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, linoleic acid metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, and autophagy (Fig. S1A). Among these pathways, linoleic acid metabolism, arachidonic acid metabolism, and glycerophospholipid metabolism are critical in lipid metabolism. The importance of glycerophospholipid and GPI-anchor biosynthesis metabolism was also confirmed by the interaction network of lipids (Fig. 2C). Given the critical role of glycerophospholipid in cell signaling and fungal pathogenicity, this study focused on glycerophospholipid metabolism-related pathways during the mummification process. PC plays an important role in glycerophospholipid metabolism (Fig. S1B). This finding was consistent with previous identification of altered lipid-related metabolites.

Fungi can control the nervous system and manipulate the behavior of host larvae, so we investigated the brain alterations of mummified larvae. Observation of paraffin sections (H&E staining) showed the distribution of hyphal bodies in the brains of infected (IL-6 and IL-8) and mummified larvae (Fig. 3A; Fig. S2). The morphology (fusiform) of hyphal bodies was observed by magnification of H&E stained brain tissue (Fig. S2A). We observed that the longer the infection duration, the greater the number of hyphal bodies in the interstitium of the brain, particularly in mummified host larvae. Hyphal bodies were observed in the brain parenchyma of T. xiaojinensis larvae. The appearance and morphological changes of O. sinensis in brain hemolymph were observed (Fig. S3). Compared to the controls (UIL-8), large numbers of hyphal bodies were detected in IL-6 and IL-8 after infection treatment, particularly in mummified larvae. Compared to the controls, shrinkage of the brain parenchyma occurred in infected and mummified host brains. These findings demonstrate that the brains of infected larvae were invaded by the fungus and suggest that the injured host brain could alter the host behavior. Thus, the O. sinensis-infected brain plays a critical role in the mummification process of T. xiaojinensis larvae.

A targeted neurotransmitter quantification of the brain was used to illustrate the underlying mechanism of O. sinensis-induced mummification. Compared to uninfected larvae, decreased levels of choline, succinic acid, 3,4-dihydroxyphenylacetic acid, glutathione, betaine, gamma-aminobutyric acid, 3-hydroxybutyric acid, and tyrosine were detected in the infected and mummified host brains (Fig. 3B). In contrast, acetylcholine, epinephrine, lysine, glutamine, tryptophan, kynurenine, 3-phenylpyruvic acid, tryptamine, 3-methoxytyramine, and xanthurenic acid were significantly increased in the uninfected larvae. Consistent with previous alterations in lipid metabolites, an elevated choline level was detected in infected and mummified larvae compared to uninfected larvae. Compared to the control group, decreased ACh levels were found in infected and mummified larvae. Significant differences in the levels of 3-hydroxybutyric acid, tyrosine, 2-picolinic acid, betaine aldehyde chloride, ornithine, homogentisic acid, aspartic acid, 3-hydroxytyramine, n-acetylserotonin, methionine, metanephrine, and serotonin were also observed between infected and mummified larvae. Alterations of choline and ACh were also consistently found in the whole bodies of infected and mummified larvae, as evidenced by the decreased level of choline after O. sinensis infection (Fig. 3C). These data indicate that neurotransmitters mediate the behavioral manipulation of T. xiaojinensis larvae by O. sinensis.

Since altered ACh and choline were found in infected and mummified larvae, we studied their relationship in the brain. AChE is a hydrolytic enzyme that catalyzes the transformation of ACh to choline. Compared to the controls (28.24 ± 1.61), the activity of AChE was significantly increased in infected (34.12 ± 1.61, P < 0.0001) and mummified larvae (35.88 ± 2.46, P < 0.0001) (Fig. 3D). However, nonsignificant differences were observed between infected and mummified larvae. Subsequently, qRT-PCR was used to verify the expression of ACE2 after fungal infection. The results showed that the relative mRNA expression of ACE2 was significantly upregulated in infected (P < 0.01) and mummified (P < 0.05) larvae (Fig. 3E). Nonsignificant differences were found in the activity of choline acetyltransferase (ChAT, catalyzes the biosynthesis of acetylcholine) and the relative mRNA expression of ChAT in infected and mummified larvae, compared to the controls (data not shown). These data demonstrated that elevated levels of choline could originate from the degradation of ACh catalyzed by AChE. The choline could then help drive the behavioral manipulation of O. sinensis-infected host larvae.

To detail the role of O. sinensis in the mummification process of infected host larvae, infected 8th instar host larvae were treated with caspofungin. Although a nonsignificant difference was found between the control and the 25-day treated group, the percentage of hyphal bodies in the hemolymph of host larvae was significantly decreased after 50-day, 75-day, and 100-day caspofungin treatment (74.88 ± 5.95, 51.63 ± 7.64, 0.62 ± 0.68, P < 0.0001, respectively) (Fig. 4A and B). These data indicate that caspofungin can effectively treat O. sinensis infection in larvae. Subsequently, we observed the behavior of infected host larvae after caspofungin treatment. Compared to the controls (larvae at early infection stage: fusiform O. sinensis), mummification rates were significantly decreased at 25, 50, 75, and 100 days after low-level (2.2 mg) and high-level (3.4 mg) caspofungin treatment (both P < 0.01) (Fig. 4C). However, larval mortality occurred following high-level caspofungin treatment (data not shown). Similarly, decreased mummification rates were found in caspofungin-treated groups at 25, 50, 75, and 100 days compared to the control group (larvae at late infection stage: capsular O. sinensis) (both P < 0.01) (Fig. 4D). These data demonstrated that infected host larvae treated with caspofungin were unable to enter the mummification stage. Caspofungin exhibited dose-dependent effects. Moreover, the mummification rate of infected larvae treated with caspofungin at an early stage was lower than that of infected larvae at a late stage. This finding suggests that the development of O. sinensis in the host is closely associated with the mummification process of infected larvae.

To study the role of the neurotransmitter AChE in O. sinensis-induced mummification of host larvae, we monitored the activity of AChE after caspofungin treatment. Consistent with the above result that infected host larvae could not enter the mummification stage, we observed that the activity of AChE in uninfected and caspofungin-treated larvae was similar (Fig. 4E). Nonsignificant differences were also found in the levels of ACh and choline between uninfected and caspofungin-treated larvae (data not shown). These data suggest a critical role of AChE in the O. sinensis-induced mummification of host larvae.

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.