D0L:10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis 7 become integrated.Subsequently,the linked mycelium ger- compared with the DCXC growing in natural conditions. minates and yields hyphae.When the hemocoelom of the However,the shortage is that the complete artificial cultiva- larva is full of the hyphae,the polypide will get hard and turn tion mode is costly into a sclerotium,and then the sclerotium sprouts a stroma to become the DCXC (Zeng et al.,2006).Yu (2005)added the The mode of semi-natural cultivation spore suspension of Hirsutella sinensis into feeds or directly As to the generation and infection of the fungi for larvae,semi- inoculated on larvae and found that Hirsutella sinensis could natural cultivation of the DCXC is similar to the complete be attacked by means of the epidermis of host insects or artificial cultivation mode,but it is different from the latter buccal cavity,which is called as live parasitism.After where the infected larvae grow.In the complete artificial infecting the larva,the mycelium will grow for a certain time. cultivation mode,the infected larvae are placed in the When the epidermis of the larva is full of mycelium,its head laboratory to artificially feed,while in the semi-natural will yield a stroma (Yu,2005). cultivation mode,the infected larvae are released back into Mycotoxin is a secondary metabolite which is produced by nature to grow freely.After 3-5 years,the DCXC can come out eumycophyta.Some studies suggested that mycotoxin does in the natural environment.The semi-natural cultivation mode not only restrain the immune system of insects by can make the best of natural resources and reduce the cost of hemocoelom injection,but also affects the normal functions artificial cultivation of the DCXC.However,the survival rate of vasa mucosa and midgut (James et al.,1993).At the same of the infected larvae is unstable in this mode,which is time,it also destroys the physiological equilibrium of the host vulnerable to factors in the natural environment such as insects and disturbs the ecdysis and metamorphosis of insects. weather.soil and food conditions (Li et al..2006). Furthermore,the mycotoxin can make the muscle of the insects paralyzed and even cause them to die (Parry,1995; Proposal Song Li,2002;Vilcinskas Wedde,1997).Understanding of the mechanisms and processes that insect pathogenic Protection of the DCXC resources by artificial mycotoxin secretes into the blood cavity and through the body cultivation fluid enter into the cells to produce toxic effects is still very Increase the quantity of breeding larvae limited.Moreover,different insect pathogenic mycotoxins may have different unique mechanisms of action.The The DCXC is a parasitic complex of the moth larva of mechanism of fungal spores attacking the moth larvae has Hepialus and fungus,and its output depends on the quantity of not been reported in China.The studies regarding insect the larvae underground and the infection situation of them.In pathogenic mycotoxins are used as a reference for us to general,the more the infected larvae is,the larger the output of understand and find the mechanism of the DCXC fungi to DCXC (Hu et al..2005).Therefore.artificial intervention first attack the host insects. tends to increase the quantity of the larvae.The moth larvae of Hepialus spp.are sensitive to the natural environment,and Artificial cultivation of the DCXC have no choice but to live in suitable grassland for natural breeding.However,natural breeding is very limited for the Four conditions need to be realized before artificially output of the larvae.The technology artificially generating the culturing the DCXC,which include: moth larvae of Hepialus spp.has been developed,and we can (1)isolation and generation of the fungi. use it to increase the output of larvae.Furthermore,complete (2)generation of the host larvae of Hepialus spp., artificial cultivation technology can be employed to generate a (3)infection mechanisms of fungal spores, great number of Hepialus spp.larvae with high quality and S (4)simulation of growth environment,especially including resistance capacity (Gao et al.,1991). illumination,temperature and humidity in the air and Besides increasing the output of the larvae,we should soil.Both the fungi and the insects are an alternation of strengthen the prevention and control of disease and pests for generations,and they thus require the exacting growth the Hepialus spp.larvae.So far,there exist pests such as ants. conditions.So the artificial cultivation of the DCXC is butterflies and birds and they often prey on the larvae.The not easy to achieve.Up to now,artificial cultivation of larvae may die from some disease or fungus infection.Also, the DCXC can be divided into two modes,and its growth the overgrazing of cattle and sheep may cause the larvae being process is summarized as Figure 1(b) trampled and eaten by mistake.Therefore,it is very scarce that the moth larvae of Hepialus spp.are infected by The mode of complete artificial cultivation H.sinensis and finally change into the DCXC,and the Complete artificial cultivation mode of the DCXC is that the successful rate of the DCXC emerging from the larvae is whole culture process of it is carried out under artificial <1/1000.Thus,both the increase of the larvae and the conditions.First.the fungi and larvae producing the DCXC prevention of natural enemy and human damage can effect- are,respectively,generated artificially in the laboratory. ively improve the growth of the DCXC and protect the Second,the larvae are infected by the fungi through artificial precious resources of the DCXC. inoculation and the infected larvae are placed in laboratory conditions for artificial feeding.After 1-2 years,the DCXC Increase the number of breeding fungi can arise under the artificially cultivated conditions.The The quantity of the DCXC resources is closely related to the complete artificial cultivation mode can not only improve the number of H.sinensis.In order to prevent the larvae from survival rate of the larvae,but also shorten the growth period the infection of other fungi,the only thing to do is to increase RIGH T S L I N Kbecome integrated. Subsequently, the linked mycelium ger￾minates and yields hyphae. When the hemocoelom of the larva is full of the hyphae, the polypide will get hard and turn into a sclerotium, and then the sclerotium sprouts a stroma to become the DCXC (Zeng et al., 2006). Yu (2005) added the spore suspension of Hirsutella sinensis into feeds or directly inoculated on larvae and found that Hirsutella sinensis could be attacked by means of the epidermis of host insects or buccal cavity, which is called as live parasitism. After infecting the larva, the mycelium will grow for a certain time. When the epidermis of the larva is full of mycelium, its head will yield a stroma (Yu, 2005). Mycotoxin is a secondary metabolite which is produced by eumycophyta. Some studies suggested that mycotoxin does not only restrain the immune system of insects by hemocoelom injection, but also affects the normal functions of vasa mucosa and midgut (James et al., 1993). At the same time, it also destroys the physiological equilibrium of the host insects and disturbs the ecdysis and metamorphosis of insects. Furthermore, the mycotoxin can make the muscle of the insects paralyzed and even cause them to die (Parry, 1995; Song & Li, 2002; Vilcinskas & Wedde, 1997). Understanding of the mechanisms and processes that insect pathogenic mycotoxin secretes into the blood cavity and through the body fluid enter into the cells to produce toxic effects is still very limited. Moreover, different insect pathogenic mycotoxins may have different unique mechanisms of action. The mechanism of fungal spores attacking the moth larvae has not been reported in China. The studies regarding insect pathogenic mycotoxins are used as a reference for us to understand and find the mechanism of the DCXC fungi to attack the host insects. Artificial cultivation of the DCXC Four conditions need to be realized before artificially culturing the DCXC, which include: (1) isolation and generation of the fungi, (2) generation of the host larvae of Hepialus spp., (3) infection mechanisms of fungal spores, (4) simulation of growth environment, especially including illumination, temperature and humidity in the air and soil. Both the fungi and the insects are an alternation of generations, and they thus require the exacting growth conditions. So the artificial cultivation of the DCXC is not easy to achieve. Up to now, artificial cultivation of the DCXC can be divided into two modes, and its growth process is summarized as Figure 1(b). The mode of complete artificial cultivation Complete artificial cultivation mode of the DCXC is that the whole culture process of it is carried out under artificial conditions. First, the fungi and larvae producing the DCXC are, respectively, generated artificially in the laboratory. Second, the larvae are infected by the fungi through artificial inoculation and the infected larvae are placed in laboratory conditions for artificial feeding. After 1–2 years, the DCXC can arise under the artificially cultivated conditions. The complete artificial cultivation mode can not only improve the survival rate of the larvae, but also shorten the growth period compared with the DCXC growing in natural conditions. However, the shortage is that the complete artificial cultiva￾tion mode is costly. The mode of semi-natural cultivation As to the generation and infection of the fungi for larvae, semi￾natural cultivation of the DCXC is similar to the complete artificial cultivation mode, but it is different from the latter where the infected larvae grow. In the complete artificial cultivation mode, the infected larvae are placed in the laboratory to artificially feed, while in the semi-natural cultivation mode, the infected larvae are released back into nature to grow freely. After 3–5 years, the DCXC can come out in the natural environment. The semi-natural cultivation mode can make the best of natural resources and reduce the cost of artificial cultivation of the DCXC. However, the survival rate of the infected larvae is unstable in this mode, which is vulnerable to factors in the natural environment such as weather, soil and food conditions (Li et al., 2006). Proposal Protection of the DCXC resources by artificial cultivation Increase the quantity of breeding larvae The DCXC is a parasitic complex of the moth larva of Hepialus and fungus, and its output depends on the quantity of the larvae underground and the infection situation of them. In general, the more the infected larvae is, the larger the output of DCXC (Hu et al., 2005). Therefore, artificial intervention first tends to increase the quantity of the larvae. The moth larvae of Hepialus spp. are sensitive to the natural environment, and have no choice but to live in suitable grassland for natural breeding. However, natural breeding is very limited for the output of the larvae. The technology artificially generating the moth larvae of Hepialus spp. has been developed, and we can use it to increase the output of larvae. Furthermore, complete artificial cultivation technology can be employed to generate a great number of Hepialus spp. larvae with high quality and resistance capacity (Gao et al., 1991). Besides increasing the output of the larvae, we should strengthen the prevention and control of disease and pests for the Hepialus spp. larvae. So far, there exist pests such as ants, butterflies and birds and they often prey on the larvae. The larvae may die from some disease or fungus infection. Also, the overgrazing of cattle and sheep may cause the larvae being trampled and eaten by mistake. Therefore, it is very scarce that the moth larvae of Hepialus spp. are infected by H. sinensis and finally change into the DCXC, and the successful rate of the DCXC emerging from the larvae is 51/1000. Thus, both the increase of the larvae and the prevention of natural enemy and human damage can effect￾ively improve the growth of the DCXC and protect the precious resources of the DCXC. Increase the number of breeding fungi The quantity of the DCXC resources is closely related to the number of H. sinensis. In order to prevent the larvae from the infection of other fungi, the only thing to do is to increase DOI: 10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis 7 Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 07/22/13 For personal use only