Altered Proteomic Polymorphisms in the Caterpillar Body and Stroma of Natural Cordyceps sinensis during Maturation
et al. (2014) Altered Proteomic Polymorphisms in the Caterpillar Body and Stroma of Natural Cordyceps
sinensis during Maturation. PLoS ONE 9(10): e109083. doi:10.1371/journal.pone.0109083
Altered Proteomic Polymorphisms in the Caterpillar Body and Stroma of Natural Cordyceps sinensis during Maturation
Yun-Zi Dong 0
Li-Juan Zhang 0
Zi-Mei Wu 0
Ling Gao 0
Yi-Sang Yao 0
Ning-Zhi Tan 0
Jian-Yong Wu 0
Luqun Ni 0
Jia-Shi Zhu 0
Raffaella Balestrini, Institute for Sustainable Plant Protection, C.N.R., Italy
0 1 Pharmanex Beijing Clinical Pharmacology Center , Beijing , China , 2 Department of Applied Biology and Chemistry Technology, Hong Kong Polytechnic University , Hung Hom, Kowloon, Hong Kong, 3 Shenzhen TCM Pharmacy and Molecular Pharmacology Kay Laboratory , Hong Kong Polytechnic University , Shenzhen, Guangdong , China , 4 Department of Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, CA, United States of America, 5 NS Center for Anti-Aging Research , Provo, UT , United States of America
Objective: To examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem. Methods: The surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). Results: SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%-4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster. Conclusion: Proteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs.
Funding: This study was supported by the Pharmanex Cordyceps sinensis Research Fund. The funder provided support in the form of full or partial salaries for
the authors [YZD, LJZ, ZMW, LG, YSY, NZT, JSZ], the purchase of research materials and the payment of SELDI-TOF MS service fees. The funder did not have any
additional role in the study design, data collection and analyses, decision to publish or preparation of the manuscript. The specific roles of these authors are
articulated in the author contributions section.
Competing Interests: The Pharmanex Cordyceps sinensis research fund was provided as a special supply for Cordyceps sinensis-related research by Nu Skin
Enterprises USA. The Pharmanex Beijing Clinical Pharmacology Center was one of the Nu Skin Research Labs and was closed in Dec. 2013. The co-authors [YZD,
LJZ, ZMW, LG, YSY, NZT] were staff scientists of Nu Skin Research Labs prior to its closure. Co-author JSZ was an employee of Nu Skin USA and left the company on
Oct. 1, 2013 (currently an Adjunct Professor at Hong Kong Polytechnic Univ.). Co-authors LJZ and ZMW are currently employed by other institutes, and their jobs
are completely unrelated to C. sinensis research. The remaining co-authors [YZD, LG, YSY, NZT] are currently unemployed, and JSZ remains in an adjunct position
at the university. The majority of the work related to this paper was performed before the lab closure. The authors continued working on the project (e.g., data
analyses, manuscript writing/revising, submitting/resubmitting) without salary support from Nu Skin after leaving the company in Oct. or Dec. 2013. This does not
alter the authors adherence to PLoS ONE policies on sharing data and materials.
. These authors contributed equally to this work.
For centuries, Cordyceps sinensis has been used as a precious
medicinal product in China and other Asian countries and features
a broad spectrum of health benefits, including anti-aging and
lifespan-extension effects . (Note: The Latin name Cordyceps
sinensis (Berk.) Sacc. is used for both the teleomorph/holomorph
of C. sinensis fungus and the wild product indiscriminately ,.
The fungus was re-named Ophiocordyceps sinensis (Berk.) Sung
et al. ; however, the Latin name for the wild product has
remained unchanged. Because a consensus Latin name for the
wild product has not been reached by mycological and TCM
botanical taxonomists, we have temporarily used the term O.
sinensis to refer to the fungus/fungi and continued to use the name
C. sinensis to refer to the wild product.) Mycological and
molecular approaches have demonstrated that C. sinensis
comprises more than 90 intrinsic fungi from more than 37 genera and
at least 6 O. sinensis genotypes . Although an
anamorphteleomorph connection between Hirsutella sinensis and O. sinensis
has been proposed based on the aggregation of indirect evidence
,, integrated analyses have demonstrated large
dissimilarities between the random amplified polymorphic DNA (RAPD)
polymorphisms of H. sinensis and of the C. sinensis ascocarp and
no studies to date have truly satisfied Kochs postulates by
describing the successful artificial induction of C. sinensis sexual
fruiting bodies and ascospores ,,. However,
there has been no direct evidence to either approve or reject the
Paecilomyces hepiali hypothesis for the O. sinensis anamorph .
P. hepiali, H. sinensis and several mutant genotypes of O. sinensis
have been found to naturally coexist in the ascocarps and
ascospores of natural C. sinensis, and the fungal complex showed
a 39-fold enhancement of its infection potency over that of pure H.
sinensis . Other researchers have thus hypothesized that C.
sinensis is an integrated micro-ecosystem with differential
expressions by multiple intrinsic fungi in its compartments and have
identified a culture-dependent microbial community or mycobiota
in natural C. sinensis along with evidence of possible symbiotic
interactions among the component fungi ,. We
have previously reported dynamic changes in the differential
fungal expression of at least 6 O. sinensis genotypes during C.
sinensis maturation ,. However, no previous studies
have compared the proteomes of C. sinensis and H. sinensis (the
proposed anamorphic fungus of O. sinensis) or reported global
changes in the macrocosmic proteomic polymorphisms in C.
sinensis compartments during maturation. In contrast to the
microcosmic studies that have focused specifically on individual
protein species, we used the surface-enhanced laser desorption/
ionization time-of-flight mass spectrometry (SELDI-TOF MS)
protein chip technique in this study to macrocosmically profile the
changes in proteomic polymorphisms in the C. sinensis caterpillar
body and stroma during maturation . We also examined
the similarities and cluster relationships between the proteomic
polymorphisms of C. sinensis and those of the mycelial
fermentation products H. sinensis Bailing and P. hepiali Cs-4.
Materials and Methods
Collection of C. sinensis
Fresh C. sinensis specimens were purchased in a local market
(Latitude 30u049N, Longitude 101u959E) in the Kangding County
of Sichuan Province, China. Governmental permission was not
required for C. sinensis purchases in local markets, and the
collections of C. sinensis specimen sales by local farmers fall under
the governmental regulations for traditional Chinese herbal
products. Premature C. sinensis features a plump caterpillar body
(sclerotium) and a short stroma ranging from 1.0 to 2.0 cm in
length (Figure 1). Mature C. sinensis features a less plump
caterpillar body and a long stroma with a length of.5.0 cm and
an expanded portion densely covered with ascocarps close to the
stroma tip. All fresh C. sinensis specimens were washed thoroughly
on site in running water with gentle brushing, soaked in 0.1%
mercuric chloride for 10 min for surface sterilization and washed
again 3 times with sterile water. The specimens were immediately
frozen in liquid nitrogen for transportation and storage prior to
further processing in the lab in Beijing .
Sample preparations for proteomic profiling
Ten C. sinensis specimens at each maturation stage were used in
this study. The caterpillar bodies and stromata from the premature
and mature C. sinensis specimens and the mycelial fermentation
products H. sinensis Bailing (Bailing capsule, Lot #040811,
#050403 and #051103, Zhejiang American-Sina Pharmaceutical
Company, Hangzhou, Zhejiang, China) and P. hepiali Cs-4
(Jinshuibao capsule, Lot #JX12931, #20040608 and #20051020,
Jiangxi TCM Pharmaceutical Company, Nanchang, Jiangxi,
China) were individually ground into powder in liquid nitrogen.
To evaluate the proteomic polymorphisms of the samples as
group-averages at each maturation stage to minimize the influence
of individual variations due to sampling and the lack of a more
accurate method to measure the samples maturation status, the
powders (0.5 g each) of the C. sinensis compartment samples were
pooled according to their compartment origins and maturational
stages to form the following testing samples: premature stroma,
premature caterpillar body, mature stroma and mature caterpillar
body. Based on the pre-test results with insignificant variations in
overall proteomic similarity, 0.95 for the 3 H. sinensis Bailing
samples and 0.96 for the 3 P. hepiali Cs-4 samples, the powders of
H. sinensis Bailing (Lot #051103) and P. hepiali Cs-4 (Lot
#20051020) were selected for the formal study. The powder
samples were dissolved in 600 ml of tris-glycine buffer (pH 8.3) and
centrifuged at 14,000 rpm for 5 min at 4uC. The sample
supernatants were used for proteomic profiling.
SELDI-TOF mass spectrometry
The supernatants prepared above were diluted in PBS to a
concentration of 200300 nM before application to a
normalphase biochip and analysis on a PBS-II protein chip reader
(SELDI-TOF MS; BioSpace Ciphergen Biosystems, Fremont, CA,
USA) . The SELDI-TOF MS experiments were
performed at the Universities Confederated Institute for Proteomics
at the School of Life Sciences, Beijing Normal University, Beijing,
China. In brief, different proteins captured on the surface of
protein chips were collected through SELDI-TOP mass
spectrometry using a laser power of 215 (sensitivity 9; molecule size
where the similarity of the 2 densities d1k and d2k is defined as the
common portion of their values.
The second ZUNIX equation (2) is suitable for comparing the
proteomic polymorphisms in more mass spectrometry
chromatograms, where dik$0, i = 1,2,,n, k = 1,2,,m and the
description is as follows:
range: 060,000 Da). Following mass calibration, total ion current
normalization and baseline subtraction, the molecular size ranges
of proteins were manually selected for analyses, and the intensities
(peak heights) were extracted using ProteinChip software
(Ciphergen proteinchip 3.0.2).
Across-chromatogram normalization of densities of
The SELDI-TOF MS chromatograms were scanned with
Quantity One software (Bio-Rad Laboratories, Inc., Hercules,
CA, USA). To conduct integrated proteomic profiling on the basis
of chromatographic tracing at the molecular weight segments, the
band trace quantities (OD*mm) of all protein bands in all
chromatograms were normalized using the maximal
chromatographic tracing scales for each molecular weight segmented tracing
panel as the reference factor (Figure 2). The relative intensity/
density was defined as the scanned band trace quantity (OD*mm)
multiplied by the difference between the maximum scale n on
the vertical axis of each chromatographic tracing panel and the
baseline scale if the trace baseline was not exactly zero.
Similarity computation for proteomic polymorphisms
ZUNIX equations (http://www.ebioland.com/ZUNIX.htm;
Beijing Bioland Technology, 2013) were used for similarity
computations with band intensities/density weighting 
while considering (i) mismatched protein bands and (ii) matched
protein bands with dissimilar intensities/densities. The following
ZUNIX equation (1) was used to compare the polymorphisms of 2
mass spectrometry chromatograms: dik$0, i = 1,2, k = 1,2, , m.
We defined the measure of similarity as follows:
Density-weighted cluster analysis for the polymorphic
For the mismatched protein species, a missing band at the given
molecular weight location in a MS chromatogram was assigned a
score of 0. The digital density data of all matched and unmatched
protein bands in the compared chromatograms were ranked and
arbitrarily assigned scores of 19 according to the ranks of their
densities in 2 or more compared chromatograms . The
digital data scores were entered into PAUP 4.0B (Swofford, 2002;
Sinauer Asso. Inc, Sunderland, MA, USA) to construct cluster
trees (semi-quantitative density-weighted neighbor-joining distance
method; bootstrap = 1000). In addition to the semi-quantitative
algorithm provided by PAUP 4.0B, a fully quantitative cluster
analysis was also performed with a parametric hierarchical
clustering analysis (density-weighted furthest neighbor Pearson
correlation average linkage distance method) in SPSS 10.1 (SPSS
Inc., Chicago, IL, USA; Note: no bootstrap strategy was provided
in the software package).
Comparison of the protein fingerprint chromatograms of
premature and mature C. sinensis
Figure 3 displays the SELDI-TOF MS chromatograms for the C.
sinensis protein species at the 2 maturation stages in a molecular
weight range of 0 to.60,000 Daltons. Using the density-weighted
ZUNIX equation (1), a percentage similarity of 57.9% was observed
between the protein fingerprint polymorphisms of pooled
premature and mature C. sinensis samples, thus indicating altered protein
expression during C. sinensis maturation.
Comparison of the polymorphic protein chromatograms
of the caterpillar bodies and stromata of premature and
mature C. sinensis
Figure 4 displays the SELDI-TOF MS chromatograms for the
C. sinensis caterpillar body and stroma protein moieties at the 2
maturational stages in a molecular weight range of 0 to.60,000
Daltons. Using ZUNIX equation (2), an overall percentage
similarity of 3.1% was observed between the proteomic
polymorphisms for all C. sinensis caterpillar body and stroma samples at
both maturation stages. Using ZUNIX equation (1) for pairwise
comparisons, the calculated similarities from Figure 4 were 57.4%
and 42.8% between the proteomic polymorphisms of the 2
maturation stages in the stromata or caterpillar bodies,
respectively, but were much lower (10.0%18.4%) for the
acrosscompartment pair comparisons (Table 1). These similarities
indicate major differences in the proteomic profile within the C.
sinensis caterpillar body and stroma resulting from large
differences in the compositions of the multiple intrinsic fungi from
more than 37 genera and at least 6 mutant O. sinensis genotypes
together with the transcription and translation of their fungal
genes. In contrast to the large between-compartment differences in
the proteomic profile, the within-compartment differences were
moderate across the C. sinensis maturation stages.
Comparison of the C. sinensis, H. sinensis and P. hepiali
sample protein fingerprint chromatograms in multiple
molecular weight segments
The above-described results for the C. sinensis proteins are
displayed integrally from molecular weights of 0 to.60,000
Daltons, as shown above in Figures 3 and 4. To increase the
chromatographic resolution, Figure 5 displays 7 panels of the
segmented SELDI-TOF MS chromatograms of all protein species
in the C. sinensis caterpillar bodies and stromata at the 2 maturation
stages as well as of the commercial mycelial fermentation products
H. sinensis Bailing and P. hepiali Cs-4. Figure 5-A presents protein
species in the molecular weight range from 0 to 5,000 Daltons,
Figure 5-B ranges from 5,000 to 10,000 Daltons, Figure 5-C ranges
from 10,000 to 15,000 Daltons, Figure 5-D ranges from 15,000 to
20,000 Daltons, Figure 5-E ranges from 20,000 to 30,000 Daltons,
Figure 5-F ranges from 30,000 to 40,000 Daltons and Figure 5-G
ranges from 40,000 to.60,000 Daltons.
The segmented mass spectrometry chromatograms shown in
Figure 5 indicate large polymorphic differences between the
protein profiles of the C. sinensis compartments at each of the 2
maturation stages, and the complex protein expression patterns
resulting from multiple intrinsic fungi across the C. sinensis
compartments underwent differential maturational fungal
expression changes. The mass spectrometry chromatograms also indicate
large overall differences between the proteomic polymorphisms of
C. sinensis and those of the fermentation products H. sinensis
Bailing and P. hepiali Cs-4.
Polymorphic similarities in the protein fingerprints of the
C. sinensis compartments at 2 maturational stages and
the mycelial fermentation products H. sinensis Bailing
and P. hepiali Cs-4
The densities of all protein species in all 7 mass spectrometry
chromatogram panels shown in Figure 5 were normalized using
the mass spectrometry tracing scales described in the Methods
section and illustrated in Figure 2. The normalized densities were
subjected to polymorphic similarity calculations with ZUNIX
similarity equation (1) , to examine the similarities between
the protein profiles of the C. sinensis compartment and those of
the mycelial products H. sinensis Bailing and P. hepiali Cs-4. As
shown in Table 2, the proteomic polymorphism similarities were
low (0.51%1.04%) between the H. sinensis Bailing and C.
sinensis protein profiles and were 2.8- to 4.8-fold higher (1.92%
4.34%) between the P. hepiali Cs-4 and C. sinensis compartments
than between the H. sinensis Bailing and C. sinensis
Density distributions of all protein bands and
determination of the scoring cutoff value for the
After normalization, the scanned band trace quantities
(OD*mm) of approximately 1,900 protein bands were sorted for
arbitrary scoring in preparation for the cluster construction using
the semi-quantitative density-weighted algorithm (Figure 6).
Density-weighted cluster analysis of the protein
fingerprints of C. sinensis and the mycelial products H.
sinensis Bailing and P. hepiali Cs-4
The highest density value in Figure 6 was divided by 9 to obtain
the critical cut-off values for a semi-quantitative density grouping.
Each density was assigned a score from 1 to 9 according to the
above-mentioned cutoff values, and all arbitrarily assigned scores
were used for the cluster construction according to the
densityweighted algorithm provided by PAUP 4.0B software .
Figure 7 displays a cluster tree that was constructed with the
density-weighted neighbor-joining algorithm (bootstrap = 1000).
Similar to the percentage similarity results shown in Table 1, the
caterpillar body samples formed 1 clade and the stroma samples
formed another clade; these 2 clades then formed a C. sinensis
cluster. The mycelial fermentation product H. sinensis Bailing
formed an isolated clade that was separated from the C. sinensis
cluster by the clade formed by P. hepiali Cs-4.
Figure 4. SELDI-TOF MS protein chromatograms to examine the protein fingerprints (molecular weight: 0 to.60,000 Daltons) and
proteomic polymorphisms of the caterpillar bodies and stromata of premature and mature C. sinensis.
Table 1. Percentage similarities in the total protein profiles of the caterpillar bodies and stromata of the premature and mature C.
sinensis, computed with the density-weighted ZUNIX equation (1).
Although the PAUP 4.0B software offered the advantage of
constructing cluster trees according to the bootstrap value
(bootstrap = 1000), the program used only semi-quantitative
algorithms. A fully quantitative, density-weighted algorithm
included in the SPSS 10.1 software package was also employed
to construct a cluster tree . As shown in Figure 8, the C.
sinensis sample clade formation pattern for C. sinensis samples
generated using the fully quantitative algorithm was similar to that
generated via semi-quantitation, as shown in Figure 7. The
caterpillar body and stroma clades joined to form a C. sinensis
cluster with a greater rescaled distance in the cluster tree that was
indicative of large differences in polymorphic protein expression
between the C. sinensis caterpillar bodies and stromata and
reflective of the low similarity observed between the C. sinensis
compartments in Table 1. The mycelial fermentation products H.
sinensis Bailing and P. hepiali Cs-4 formed a clade with a greater
rescaled distance and were thus situated outside of the C. sinensis
C. sinensis is one of the most valued Chinese medicinal
products. This organism grows only in areas of high elevation on
the Qinghai-Tibetan Plateau and features a complex life cycle.
Studies have reported that C. sinensis comprises more than 90
intrinsic fungal species from more than 37 genera and at least 6
genotypes of O. sinensis fungi . Of these, the most abundant
culturable fungi are Pseudogymnoascus roseus in the sclerotia and
Figure 5. SELDI-TOF MS protein chromatograms to examine protein polymorphisms in the stroma and the caterpillar body
specimens of premature and mature C. sinensis and the mycelial fermentation products H. sinensis Bailing and P. hepiali Cs-4. Total
proteins were extracted from the caterpillar bodies or stromata of natural C. sinensis at 2 maturation stages and from the mycelial products H. sinensis
Bailing (BL) or P. hepiali Cs-4. Panels A, B, C, D, E, F and G present proteins with molecular weights ranging from 05,000, 5,00010,000, 10,00015,000,
15,00020,000, 20,00030,000, 30,00040,000 and 40,000 to.60,000 Daltons, respectively.
Table 2. Percentage similarities in proteomic polymorphisms between the C. sinensis compartment samples at 2 maturational
stages and H. sinensis Bailing (BL) and P. hepiali Cs-4.
(P. hepiali Cs-4 vs. H. sinensis BL)
cortices and Penicillium chrysogenum in the stromata, as reported
by Zhang et al. . Previously, we reported that C. sinensis
maturation was associated with dynamic changes in the intrinsic
fungal species and mutant O. sinensis genotypes along with
significant changes in the RAPD molecular marker
polymorphisms and component chemicals ,,. The fungal
background of C. sinensis becomes even more complex when
nonculturable fungal species are considered . These findings
reflect the altered fungal expression of multiple intrinsic fungi and
support the hypothesis that C. sinensis is an integrated
microecosystem of multiple intrinsic fungi, as proposed by Liang et al.
. Density-weighted algorithms for similarity computations and
cluster constructions were used in this study to analyze the mass
spectrometry chromatograms of polymorphic proteomes, the
downstream transcription/translation products of multiple fungal
genomes. We observed different proteomic profiles with
similarities of 10.0% between the premature caterpillar bodies and
stromata and 17.8% between the mature caterpillar bodies and
stromata of C. sinensis (cf. Figure 4; Table 1), consistent with the
mycological and molecular observations of diverse fungal
populations in the two C. sinensis compartments . However,
considerably great proteomic polymorphism similarities of 42.8%
and 57.4% were observed within the C. sinensis caterpillar body
and stroma, respectively, at the 2 C. sinensis maturation stages (cf.
Figure 4; Table 1). The differences in the across and
withincompartment similarities between the proteomic profiles might
possibly be derived from 2 major factors: (1) differential protein
expression of the multiple fungal genomes (multiple mutant O.
sinensis genotypes and the multiple intrinsic mesophilic and
psychrophilic fungi), of which part or all undergo maturational
alterations, and (2) protein species from the dead bodies of the C.
sinensis ghost moth larvae, which are not merely a group of
Figure 7. Integral cluster tree of all the proteomic chromatograms constructed with the semi-quantitative density-weighted
algorithm. H.sinensis BL refers to the H. sinensis Bailing mycelial product, P.hepiali Cs-4 refers to the P. hepiali Cs-4 mycelial product, and
Caterpillar refers to the caterpillar body. Each protein species from all proteomic chromatograms in Figure 5 was assigned a score of 19 based on
its density rank among the densities of all compared protein species; the missing protein band at the same molecular weight was assigned a score of
0. All protein species from the chromatograms in Figure 5 were entered into the cluster construction using the neighbor-joining distance method
(bootstrap = 1000).
nutrients for fungal growth but also as a part of the species complex,
along with all of the previously reported small chemical components
,, contribute to the overall pharmacology of the natural
medicinal product and partly explain the various therapeutic
potencies of premature and mature C. sinensis that have been
identified by traditional Chinese medicine quality grading system.
Figure 8. Integral cluster tree of all proteomic chromatograms constructed with the fully quantitative density-weighted algorithm.
H. sinensis BL refers to the H. sinensis Bailing mycelial product; Caterpillar or Caterpillar refers to the caterpillar body. All protein species from all
chromatograms in Figure 5 were entered into the cluster construction, using the furthest neighbor (Pearson correlation average linkage) method of
hierarchical cluster analysis.
Density-weighted algorithms for similarity computations and
cluster constructions were used to compare RAPD molecular
marker polymorphisms in previous studies of C. sinensis .
Although density-unweighted arithmetic methods have been
widely used in literature, these methods are only suitable for the
analyses of all-or-none data. The density-weighted arithmetic
methods used in this proteomic study are more mathematically
general and sufficiently sensitive to capture all of the detailed
information regarding dynamic changes in proteins expressed by
the various intrinsic fungi during C. sinensis maturation .
These algorithms provide scientists with accurate analytical means
with which to trace changes in the proteomic and molecular
marker polymorphisms in natural C. sinensis.
In this first study testing the proteomic polymorphisms of
different compartments of wild C. sinensis during maturation, the
overall proteomic polymorphisms were compared in the pooled
samples of 10 C. sinensis specimens of each maturation stage. The
height of the C. sinensis stroma (cf. Figure 1) has been taken as the
standard for the potency-quality grading of natural C. sinensis on
the market. Such a common practice for potency grading can be
explained by a C. sinensis mycology expert that the premature C.
sinensis with a short stroma grows asexually, whereas the
longstroma C. sinensis with the formation of the ascocarp portion (the
expanded portion close to the tip of the stroma) primarily grows
sexually (personal communication with Prof. YL Guo). According
to the comments of Prof. Guo regarding the asexual-sexual growth
of C. sinensis, our previous molecular systematic studies
demonstrated that the maturation of wild C. sinensis is a continuous
biological course along with the weather during spring. However,
there is no existing accurate method thus far to measure samples
maturation status. We also found previously large differences in
the fungal activity of H. sinensis, biomasses of the fungus-specific
DNA species, and small organic chemicals in wild C. sinensis
during maturation, indicating large differences in fungal
expression during C. sinensis maturation ,,.
Therefore, we designed this study with 2 special sample arrangements of
the test materials to minimize variations in individual specimens:
(1) the selection of C. sinensis specimens with the clear
morphological characters shown in Figure 1, i.e., very premature
C. sinensis with a short possible stroma (1-2 cm) and mature C.
sinensis with a long stroma (.5 cm) and with definite formation of
the ascocarp portion; and (2) assessment of the pooled samples (10
specimens at each maturation stage; stromata and caterpillar
bodies separated from the same specimens). In addition to the
examination of maturational and compartmental group
differences in proteomic polymorphism within a C. sinensis population, it is
possible that there are individual differences in some degree in
proteomic polymorphism within a C. sinensis population and
within maturation groups. These individual differences are likely
due to differences such as in the instar and nutrition status of the
larvae of ghost moths within the family Hepialidae at the time of
fungal infection, the growth location and environment (e.g.,
elevation and temperature, strength of plateau wind and sunshine
in the growth area, amount of snow in winter and rain in spring,
soil fertility, surrounding vegetation), the total weight and length of
the C. sinensis specimens, and the weight ratio and height ratio of
the caterpillar body and stroma of individual C. sinensis
specimens. This study design of pooling samples, however, is
limited regarding the exploration of such individual variations at
each estimated maturation stage. However, there may also be
population differences among the C. sinensis specimens collected
from different production areas, likely due to the different species
of larvae of ghost moths within the family Hepialidae, differences
in latitude, possibly different local soil fungal flora or mycobiota
and other environment factors. All these considerations should
encourage future studies to further explore variations in the
molecular and proteomic polymorphisms and chemical profiles
among the individual C. sinensis specimens collected within a
production area or among the C. sinensis populations from various
production areas. Perhaps prior to the future comparison of
individual specimens, an accurate method for determining C.
sinensis maturation stages may need to be established with the
combined use of morphological characters and molecular markers
to distinguish proteomic variations due to slightly different
maturation stages of C. sinensis specimens or due to true
differences in protein expression in individual specimens at the
same maturation stage. To this end, fungal biomass ratios, for
example, GC-biases vs. AT-biases of O. sinensis, may serve as a
molecular marker to assist the morphological characterization
when determining the C. sinensis maturation status ,[19
H. sinensis has been proposed as an anamorph of O. sinensis;
natural C. sinensis is considered a single fungus product .
These hypotheses were proposed based on the aggregation of
indirect evidence, such as morphological findings for the isolates
from natural C. sinensis, ITS sequencing and results from
microcycle conidiation of ascospores under particular culture
conditions ,. No scientific studies to date have truly
satisfied Kochs postulates, which have demonstrated the
successful artificial induction of the C. sinensis sexual fruiting body and
ascospores ,,,. Shen et al.  reported
extremely slow growth (approximately 2 cm after 7 months) of
artificial Cephalosporium dongchongxiacae (; H. sinensis;
,) fruiting bodies and observed regular, fine and deep twills
on the surfaces of long, conically shaped fruiting bodies. The
overall appearance of the artificial fruiting bodies, unfortunately,
was distinct from that of natural C. sinensis, which has a long,
round and cylindrical stroma with vertical fine wrinkles, as
described in the Chinese Pharmacopoeia. Shen et al.  also
reported the production of ascospores from one of the artificial C.
dongchongxiacae fruiting bodies that featured no morphological
formation of a C. sinensis-like ascocarp, the sexual organ of C.
sinensis, thus indicating an overall teleomorphic morphology
distinct from that of natural C. sinensis. Shen et al. 
characterized in their paper the unique teleomorphic features of
C. dongchongxiacae, and the results actually negated the
anamorph-teleomorph connection between C. dongchongxiacae
(; H. sinensis) and O. sinensis in accordance with Kochs
postulates. In addition to the dramatic dissimilarities in the RAPD
molecular marker polymorphisms between the C. sinensis
ascocarp and H. sinensis, the drastically different proteomic
polymorphisms in C. sinensis and H. sinensis might not support
the single-fungus hypothesis of C. sinensis or the hypothesis of
an anamorph-teleomorph connection between H. sinensis and O.
sinensis ,,, (cf. Figure 5 and Table 2). Based
on these microcosmic and macrocosmic studies, Liang et al. 
hypothesized that C. sinensis is an integrated micro-ecosystem
with varying compositions of multiple intrinsic fungi. In fact, the
coexistence of these multiple fungi has been demonstrated in the
culture-dependent and independent microbial communities or
mycobiota present in natural C. sinensis, along with evidence of
symbiotic interactions among the component fungi; these likely
represent the key biological actions essential to the natural or
artificial production of sexual fruiting bodies and ascospores [17
Studies of C. sinensis have detected several groups of chemical
components, including carbohydrates; galactomannan;
nucleosides; proteins, polypeptides, oligopeptides, polyamines, and
diketopiperazines (cyclo-dipeptides); non-hormone sterols; fatty
acids and other organic acids; and vitamins and inorganic
elements ,. Other compounds such as verticiol, acid
deoxyribonuclease, myriocin, 3-deoxyadenosine (coydycepin) and
cordysinins AE have also been detected . Chemical
constituent fingerprinting techniques have been used together with
similarity comparisons and cluster constructions in C. sinensis
studies to demonstrate the high similarity of C. sinensis samples
collected from different production areas ,,. A
cluster analysis of these small organic chemicals via capillary
electrophoresis technology demonstrated that several mycelial
fermentation products were situated in different clades outside of
the cluster containing the natural C. sinensis samples collected
from different production areas on Qinghai-Tibetan Plateau .
However, when analyzing the small chemicals of several natural C.
sinensis samples collected from Tibet or Qinghai Provinces and of
fermentation products (P. hepiali Cs-4 and H. sinensis Bailing)
through HPLC fingerprinting, the clades of natural C. sinensis
were much closer in rescaled distance to the clade containing the
P. hepiali Cs-4 products than to the clade containing the H.
sinensis Bailing products . In the proteomic fingerprint
analysis conducted in the current study, in which the bootstrap
strategy (bootstrap = 1,000) was used in the density-weighting
algorithm, the fermented products P. hepiali Cs-4 and H. sinensis
Bailing were situated in an isolated clade outside of the C. sinensis
cluster with the possibility that P. hepiali Cs-4 was closer to the C.
sinensis cluster than was H. sinensis Bailing, as shown in Figure 7.
This possibility was supported by the 2.8- to 4.8-fold higher
similarities of the proteomic polymorphisms between the P. hepiali
Cs-4 and C. sinensis compartments relative to those between the
H. sinensis Bailing and C. sinensis compartments (cf. Table 2).
The cluster relationship demonstrated by the semi-quantitative
neighbor-joining algorithm was validated using the fully
quantitative approach of the furthest neighbor algorithm without the
bootstrap strategy provided by the SPSS software (cf. Figure 8).
Local herbal farmers in the C. sinensis production areas of the
Qinghai-Tibetan Plateau have long recognized the temperature
dependency of the C. sinensis maturational features and believe
that eating worms in the winter and grass in the summer
provides tonic herbal properties. Changes in the therapeutic
properties of C. sinensis during its maturation have also been
recognized by the field of traditional Chinese medicine, in which
natural C. sinensis is graded accordingly. We have reported
maturational changes in the composition of multiple intrinsic
fungal species of C. sinensis and at least 6 genotypes of O. sinensis,
along with environmental changes (temperature, sunlight intensity,
snow/rain, moisture and plateau wind) on the Qinghai-Tibetan
Plateau ,. The altered fungal background of natural C.
sinensis at various maturation stages causes large variations in (i)
the RAPD molecular marker polymorphisms, (ii) the fingerprints
of small organic compounds, and (iii) proteomic polymorphisms in
the caterpillar bodies and stromata, as demonstrated in this and
previous studies ,,. The integration of the
component compounds that are differentially expressed in
different compartments of C. sinensis and differentially altered
during C. sinensis maturation constitutes the dynamic
pharmacological base that is responsible for the varying potencies of the
health benefits and therapeutic activities associated with C.
In conclusion, SELDI-TOF MS proteomic profiling was used to
macrocosmically detect the dynamic polymorphic alterations
among differentially expressed proteins in the different C. sinensis
compartments during maturation. The apparent proteomic
polymorphism dissimilarity between H. sinensis and C. sinensis
suggests different fungal backgrounds of these organisms and thus
might not support the single-fungus hypothesis of C. sinensis or
the hypothesis of an anamorph-teleomorph connection between
H. sinensis and natural C. sinensis. However, the findings from
this proteomic study, in corroboration with prior mycological and
molecular observations, support the integrated micro-ecosystem
hypothesis for natural C. sinensis.
The authors are grateful to Prof. P.Y. Xu, Mr. W. Chen, Ms. M. Yang and
Mr. Y.C. Zhou for their assistance during the collection of the wild C.
Conceived and designed the experiments: LJZ JSZ. Performed the
experiments: YZD LJZ. Analyzed the data: YZD LG YSY NZT LN.
Contributed reagents/materials/analysis tools: ZMW. Wrote the paper:
JSZ JYW. Formulated analytic algorithms: LN.
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