Isolation , identification , culture and production of adenosine and cordycepin from cicada larva infected with entomopathogenic fungi in Thailand

In this study, cicada larvae infected with entomopathogenic fungi were collected from Maha Sarakham Province in northeast Thailand. One strain of entomopathogenic fungi with cottony white cream colonies was isolated. Small subunit (SSU) rDNA, large subunit (LSU) rDNA, the elongation factor 1α (EF-1α) and the largest subunit of RNA polymerase II (rpb1) sequence analyses were used for identification of the isolate. A BLAST search in NCBI showed the sequence to be most similar to Cordyceps sp., Hirsutella sp., Ophiocordyceps longissima and O. longissima for SSU rDNA, LSU rDNA, EF-1α and rpb1, respectively. This correlated well with evidence from neighbor-joining trees, based on SSU rDNA, LSU rDNA, EF-1α and rpb1. Therefore, the isolate was classified as an anamorph strain of Ophicordyceps and assigned as Ophiocordyceps longissima isolate Cod-MK1. The O. longissima isolate Cod-MK1 was found to grow best in HCGA medium, compared with six other synthetic media. Moreover, the isolate was shown to develop numerous synnemata (stroma-like stalks) and conidia, when cultured in the applied HCGA medium at 25 to 28°C for 60 to 90 days. The content of adenosine was observed only in the extract from dried mycelia at 31.68 g/g. The content of cordycepin from dried mycelia was 335.65 g/g; lower than those of the extract from dried stroma-like stalks (366.14 g/g). Therefore, the induced culture from this study could be used for the production of adenosine and cordycepin in O. longissima isolate Cod-MK1.


INTRODUCTION
The genus Cordyceps is a group of entomopathogenic fungi that form fruiting bodies and live mainly on insects and other arthropods (Wu et al., 2011;Zhu et al., 2011).Many Cordyceps species are parasites of the cicada larvae (Wang et al., 2012).The infection process begins when spores of the fungus geminate and invade the body of the cicada larvae.Consumption of nutrients by the fungal hyphae eventually results in the death of the host.The internal tissue of the cicada larvae becomes *Corresponding author.E-mail: aphidech_sangdee@yahoo.com.
replaced by a mass of mycelium and is transformed into a sclerotium.After that, the fruiting body was developed on the surface of the host (Webster, 1980).
Many fungi belonging to Cordyceps have been used as food and herbal medicines in Asia (Kuo et al., 2005).In China, some Cordyceps spp.have successfully been used in immunity modulation, fatigue resistance, longevity elongation, and other functions (Nam et al., 2006).C. sinensis has been widely used as a general tonic for protecting and improving lung and kidney functions (Leung et al., 2009), as a roborant, a sedatives, and a supplementary therapy for jaundice, opiumism, tuberculosis and cancer (Nam et al., 2006).Also, C. militaris and C. ohioglossoides have been used in Japan for their anticarcinogenic and immunomodulatory activities (Ohmori et al., 1986;Kiho et al., 1996).
There are reported to be over 300 species of Cordyceps worldwide, more than 80 of which have been identified in Korea (Nam et al., 2006).In the last few years, PCR has successfully been used to systematically investigate Cordyceps species.PCR techniques based on particular regions of the fungal genome have been used for fungal taxonomic grouping, establishing evolutionary relationships, and functional properties (Kuo et al., 2005).Moreover, these techniques have been particularly useful in discriminating fungi at interspecific and intraspecific levels (Matsuda and Takamatsu, 2003;Skouboe et al., 1999).
Biochemical studies of Cordyceps spp.have shown they contain many bioactive compounds with different effects on the human body (Zhu et al., 1998;Weng et al., 2002;Holliday and Cleaver, 2008;Paterson, 2008;Das et al., 2010).Two common difficulties encountered in such studies are the slow growth rate and different morphological characteristics of Cordyceps spp.(Nam et al., 2006).Moreover, the quantity of bioactive compounds and growth rate can vary depending on artificial conditions and Cordyceps species (Liu et al., 2001;Dong and Yao, 2005;Masuda et al., 2006;Hung et al., 2009).Therefore, the present study sought to identify entomopathogenic fungi from cicada larvae in Thailand using the PCR technique with a particular genome, to understand its culture for optimum mycelial growth and conidia production, and to determine adenosine and cordycepin production from this fungus.

Isolation of entomopathogenic fungi
Ten cicada larvae infected with entomopathogenic fungi were collected from mixed deciduous forest, Maha Sarakham province in northeast Thailand.Isolation was carried out using the tissue transplanting technique.The cicada samples were washed with sterile distilled water and sectioned into two pieces.The inner tissue of cicada larvae were cut (5 x 5 mm 2 ), surface sterilized by dipping in 1% sodium hypochloride for 2 min, and rinsed several times with sterile distilled water before being transferred onto the surface of potato dextrose agar (PDA).The mycelium growing out of the cicada larvae tissue was sub-cultured on potato dextrose agar (PDA) and incubated at 25-28 o C for further study.

Identification of entomopathogenic fungi
The isolate was cultured in PDA at 25 to 28°C for 20 days.Mycelia harvested from the PDA were homogenized with liquid nitrogen in a mortar and pestle.The mycelium powder was transferred to a microcentrifuge tube, and genomic DNA was extracted using the plant DNA extraction kit handbook (Vivantis, Malaysia).DNA samples were checked on 1% agarose gel electrophoresis and stored at -20°C.

Medium screening for fungal growth
The synthetic media potato dextrose agar (PDA), Sabouraud dextrose agar (SDA), malt extract agar (MEA), yeast malt agar (YMA), Czapek Dox agar (CDA) and oat meal agar (OMA), and composed medium, homogenized died cricket glucose agar (HCGA), were prepared for culturing the fungal isolate Cod-MK1.The HCGA medium was prepared by dissolving 20g homogenized died cricket 1:2 (w/v) in distilled water, adding 3 g glucose and 1.5 g agar, then adjusting the final volume to 100 mL.Mycelial disks of the isolate (7 mm in diameter) were cut from colony margins and transferred to the center of each of the seven media.All the inoculated media were incubated at 25 to 28°C.After 20 days, the colony diameter was measured for mycelial growth.The experiment was performed in triplication to confirm reproducibility of results.Duncan's multiple range test was performed to check the growth differences.Data were assessed using the SPSS software version 14.0.

Induction of synnemata and conidia on applied HCGA medium
The fungal isolate Cod-MK1 was cultured on HCGA medium at 25 to 28°C for 20 days, after which mycelial disks were cut from colony margins and transferred to sterilized applied HCGA medium (containing 20g Thai Jasmine rice and 25 mL homogenized died cricket 1:2 (w/v) in distilled water) in an 8 oz cylindrical bottle.All of the inoculated media were incubated at 25 to 28°C and observed daily.
Table 1.Cordyceps and related species and their NCBI accession numbers used in this study.

Preparation of the entomopathogenic fungi mycelia
The seed culture of fungal isolate Cod-MK1 was cultured in 50 mL PDB medium on a rotary shaker at 28°C and 150 rpm for 7 days, after which 1 mL of seed culture was transferred to 25 mL of the induce culture.The induce culture was prepared by dissolving 0.5 g homogenized died cricket 1:2 (w/v) in distilled water, adding 2% glucose, 0.9% yeast extract, 1.5% peptone, 0.3% K 2 HPO 4 , and 0.4% CaCl 2, and then adjusting the final volume to 25 mL.The induce culture was incubated at 25 to 28°C without shaking for 14 days.The mycelium on the surface of the induce culture was collected and dried at 50°C overnight.

Extraction and determination of adenosine and cordycepin
The stroma-like stalks and mycelia of the fungal isolate Cod-MK1 were ground into powder.Then, 0.5 g of fungal isolate MK-1 powder was added into 5 mL methanol-water (50/50, V/V) in 15 mL centrifuge tube, followed by sonication with a High Intensity Ultrasonic Processor (Model VCX 750, USA).This was performed on ice for a total of 5 min in 15 s bursts with 5 s gaps for cooling.The sonicated solutions were centrifuged (Tomy MX-301, Japan) at 9100 g for 5 min and filtered through a 0.2 m filter prior to HPLC analysis.

Isolation of entomopathogenic fungi
The colony on PDA that developed from the inner tissue of cicada larvae grew to 20 mm in diameter under 25 to 28°C within 30 days and assigned as the Cod-MK1 isolate.This isolate produced cottony colonies on PDA with white cream color on the ventral surface and the reverse of the colonies.Mycelium on the medium was tightly, sterile, and 3.5 to 4 m in diameter.Moreover, it produced white cream synnemata or cotton-like hyphae on the cicada larvae tissue.

Identification of the fungal isolate Cod-MK1
DNA fragments of approximately 600 to 700 bp were amplified by PCR using primers NS1 and NS2.The fragment contained the 5' end of small subunit rDNA (SSU rDNA).A fragment of SSU rDNA consisting of 661 nucleotides was submitted to GenBank (accession number JQ922263).A BLAST search in NCBI (www.ncbi.nih.gov/blast)showed this sequence to be most similar to Cordyceps sp.97003 (AB027329, 95%), Metacordyceps yakusimensis (AB044632, 95%), Ophiocordyceps sobolifera (EF468972, 94%), C. sobolifera (DQ838796, 94%) and O. sobolifera (AB027328, 94%).A phylogenetic tree was generated from 14 aligned sequences with an equal character.This tree showed that the fungal isolate did not locate in the same clade with other reference fungal sequences (Figure 1).However, the fungal isolate sequence was closely related with C. sobolifera and O. sobolifera.

Medium screening for fungal growth
Seven media were screened to determine the optimum growth medium for the fungal isolate Cod-MK1.Colonies of the isolate grew to 69.5±0.5, 67.5±0.5 and 66.5±0.5 in diameter on the HCGA, MEA and YMA media, respectively, over 20 days under 25 to 28°C.Growth on HCGA was fast, with significant difference greater than that on the other media.On the other hand, growth on OMA and SDA media was particularly slow and significantly slower than on CDA and PDA media (Figure 5) The mycelium obtained on the PDA, HCGA, OMA and SDA media agglutinated tightly and was of white cream color.The mycelium obtained on the YMA, MEA and CDA media was loose but had the same white cream color (Figure 6).

Induction of synnemata and conidia on applied HCGA medium
The sterilized applied HCGA medium was inoculated with the fungal isolate Cod-MK1 and incubated at 25 to 28°C.Synnemata formation first occurred on the surface of the medium after 45 to 60 days cultivation.Numerous synnemata were observed after 90 days.Conidia varied in size from 2 to 3 x 8 to 10 µm, and were produced by the mature synnemata or mycelium after over 100 days incubation (Figure 7).

Determination of adenosine and cordycepin in the fungal isolate Cod-MK1
HPLC analysis showed peaks of adenosine and cordycepin at the retention times of 9.57 min and 14.12 min, respectively.The extract from dried mycelia of the fungal isolate Cod-MK1 showed two peaks corresponding to adenosine and cordycepin (9.43 and 13.86 min) and the extract from dried stroma-like stalks showed one peak of cordycepin at a retention time of 13.84 min.Standard adenosine concentration ranging from 1 to 25 g/mL and cordycepin concentration ranging from 6.25 to 75 g/mL were used to establish the linear curve between concentration and peak area.The regression equations were calculated as Y = 290.04X-90.025(R 2 = 0.9992) for adenosine and Y = 46.739X-85.697(R 2 = 0.9991) for cordycepin.These were established in triplicate.The content of adenosine was observed in the extract from dried mycelia at 31.68 g/g.The content of cordycepin from dried mycelia was 335.65 g/g; lower than the extract from dried stroma-like stalks (366.14 g/g).

DISCUSSION
Appropriate culture medium nutrients are an important requirement for fungal growth.In this study, we successfully isolated the fungal isolate Cod-MK1 from the inner tissue of cicada larvae, but the growth rate was slow and culture took 30 days on the isolating medium.The slow growth rate on a culture medium lacking some special ingredients such as SDA, CDA and OMA was also found in this study.The growth rate of the fungal isolate Cod-MK1 on agar supplement with homogenized died cricket (HCGA medium) was faster than the other culture media.In addition, growth of the isolate on HCGA media was 1.7, 2.0, 2.5 and 1.5 times higher than on PDA, OMA, SDA and CDA media, respectively.These  results are in general agreement with those of Nam et al. (2006), who found that C. sphecocephala grew more slowly on PDA medium than PDBLA or PDBAA media containing of frozen dried honeybee larvae and honeybee adult.Supplementing media with mineral salts has also been shown to increase growth rate of C. cardinalis (Sung et al., 2010).
The fungal isolate Cod-MK1 was found to grow on applied HCGA.In this medium, the fungal isolate used Thai Jasmine rice as a carbon source, and homogenized died cricket as a nitrogen and mineral source.However, growth is slow.On this medium, numerous synnemata (stroma-like stalks) and conidia were only produced after 60 to 90 days incubation.Since Cordyceps species grow slowly, the isolating medium and culture medium should be supplemented with special ingredients to reduce isolation time, increase growth rate, and increase production of synnemata and conidia.Mycelium growth rate can also be increased by successive subculture (Sung et al., 2010).
In the present study, the phylogenetic relationship between the Cordyceps and related species based on SSU, LSU, EF-1α and rpb1 gene were also investigated.The SSU rDNA phylogeny shows that the fungal isolate Cod-MK1 was not located within a clade with the other 13 isolates used, but shared a closer relationship with O. sobolifera and C. sobolifera.Similar findings were obtained from phylogeny derived from the LSU rDNA data set.In the LSU rDNA tree, the fungal isolate Cod-MK1 did not nest to the other 13 isolates, but classified in a clade with H. stilbelliformis var.Myrmicarum (anamorph of Ophicordyceps genus).Moreover, the EF-1α and rpb1 phylogenetic tree showed that the fungal isolate Cod-MK1 sequences were closely related with O. longissima which 99% identity whereas other anamorphs and telomorphs of the genus Cordyceps showed a maximum identity of less than 95%.Based on the nucleotide sequence of four genes, insect host and fungal morphology, the fungus isolated from the inner tissue of the cicada larvae can be identified as an anamorph strain of Ophiocordyceps and assigned as O. longissima isolate Cod-MK1.These investigative tool resemble those of Chan et al. (2011), who used the sequence of the ITS region and three loci, nuclear ribosomal large subunit (nrLSU), elongation factor1α (EF-1α) and the largest subunit of RNA polymerase II (rpb1) to identify Cordyceps samples as C. gunnii.Therefore, the SSU, LSU, EF-1α and rpb1 primers represent beneficial tools for the classification and identification of Cordyceps species.
The ITS region, in particular, is very useful for species identification (Chen et al., 2001).This is because of the high diversity of the ITS region between species, as well as the homogeneity of the region within species.Indeed, the ITS region may be the most suitable marker to study the life cycles of Cordyceps sinensis, Hirsutella sinensis, Paecilomyces sp., Tolypocladium sp. and Stachybotrys sp. and confirm teleomorph-anamorph connection (Chen et al., 2001).Kuo et al. (2005) successfully used PCRsingle-strand conformation polymorphism (PCR-SSCP) of the ITS2 region for intraspecies classification of 17 Cordyceps isolates.
In this study, the contents of adenosine and cordycepin were measured using HPLC.The peaks corresponding to standard adenosine and cordycepin were identified.

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These results indicate that O. longissima isolate Cod-MK1 can produce adenosine and cordycepin especially in induced conditions resembling those of C. militaris (Huang et al., 2009;Xie et al., 2009).However, the concentrations of adenosine and cordycepin produced in this study were low.Large scale production of adenosine and cordycepin using the O. longissima isolate Cod-MK1 would therefore require optimization of conditions.
In conclusion, this study isolated an entomopathogenic fungus and based on SSU, LSU, EF-1α and rpb1 sequences analyses, identified it as belonging to the Ophiocordyceps genus.The isolate that grows best in HCGA medium, can develop numerous synnemata and conidia in applied HCGA medium, and can produce adenosine and cordycepin in induced condition.

Figure 1 .
Figure 1.Phylogenetic relationships of the fungal isolate Cod-MK1 with Cordyceps and related species based on partial SSU rDNA sequences.A Neighbor Joining tree (NJ tree) was constructed using Mega 4. The percentages expressed above the branches are frequencies with which a given branch appeared in 1000 bootstrap replications.

Figure 2 .
Figure 2. Phylogenetic relationships of the fungal isolate Cod-MK1 with Cordyceps and related species based on partial LSU rDNA sequences.A Neighbor Joining tree (NJ tree) was constructed using Mega 4. The percentages expressed above the branches are frequencies with which a given branch appeared in 1000 bootstrap replications.

Figure 3 .
Figure 3. Phylogenetic relationships of the fungal isolate Cod-MK1 with Cordyceps and related species based on partial sequencing of the elongation factor 1α (EF-1α).A Neighbor Joining tree (NJ tree) was constructed using Mega 4. The percentages expressed above the branches are frequencies with which a given branch appeared in 1000 bootstrap replications.

Figure 4 .Figure 5 .
Figure 4. Phylogenetic relationships of the fungal isolate Cod-MK1 with Cordyceps and related species based on partial sequencing of the largest subunit of RNA polymerase II (rpb1).A Neighbor Joining tree (NJ tree) was constructed using Mega 4. The percentages expressed above the branches are frequencies with which a given branch appeared in 1000 bootstrap replications.

Figure 6 .
Figure 6.Various colony morphologies of the fungal isolate Cod-MK1 on six synthetic media (A-F) and one composed medium (G) after 20 days incubation at ambient temperature.A = PDA; B = YMA; C = MEA; D = OMA; E = SDA; F = CDA; G = HCGA.