Host insect species of Ophiocordyceps sinensis: a review

Abstract Ophiocordyceps sinensis (≡ Cordyceps sinensis) is one of the most valued medicinal fungi in China, used for its invigorating effects in strengthening the body and restoring energy. The fungus parasitizes larvae of moths and converts them into sclerotia from which the fungus fruiting body grows. Since the late 1950s, considerable effort has been devoted to the study of host insects related to the fungus. In the present paper, the research history of insect species associated with Ophiocordyceps sinensis is briefly reviewed and an extensive literature survey is presented. Ninety-one insect names, spanning 13 genera, related to host insects of Ophiocordyceps sinensis are investigated. The relationships between the reported insect species and Ophiocordyceps sinensis are analyzed. Fifty-seven of these are considered as recognizable potential host species of the fungus distributed throughout the Tibetan Plateau, whilst eight are considered as indeterminate hosts and 26 as non-hosts. Among the names of recognizable potential host insects, three are invalid (nomen nudum) and require further study. This work provides basic information for management of the insect resources and for the conservation and sustainable use of Ophiocordyceps sinensis.


Introduction
Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora is an ascomycete fungus, which is also known as the Chinese Caterpillar Fungus or "Dong Chong Xia Cao" (winter worm, summer grass) in Chinese, or "Hia Tsao Tong Tchong" and "Hea Tsaon Tsong Chung" in early English translations (Pegler et al. 1994). The fungus parasitizes larvae of moths belonging to the order Lepidoptera, especially Hepialus/Thitarodes. The infected larva is converted into a sclerotium covered by the intact exoskeleton of the insect to withstand the winter, which is regarded as "winter worm". In the late spring or summer of the next year, a clavate stroma of the fungus grows from the sclerotium and emerged from the ground appearing as a herb, which is regarded as "summer grass" (Pegler et al. 1994, Yao 2004. As a valued Chinese herb and tonic, O. sinensis has a long history of use and a high reputation of value both in China and abroad. In Traditional Chinese Medicine (TCM), the fungus is believed to nourish the lungs and kidneys (Wu 1757). It has also been shown in recent studies to have multiple pharmacological effects, including immunomodulating , hypocholesterolemic (Koh et al. 2003), hypoglycemic (Zhang et al. 2006), anti-tumor , anti-oxidation (Dong and Yao 2008) and anti-aging (Ji et al. 2009) activities.
The natural product of O. sinensis for medicinal use is actually a combination of the fungus and an insect larva. The fungus parasitizes underground dwelling larvae of moths and converts them into sclerotia, from which the fruiting body of the fungus grows (Pegler et al. 1994, Wang 1995, Yao 2004. Ophiocordyceps sinensis is endemic to the Tibetan Plateau, with a distribution covering five provinces in China, i.e., Gansu, Qinghai, Sichuan, Tibet and Yunnan. It may be found in alpine meadow and shrub habitat from an altitude of 3000 m up to the snow-line (Wang 1995, Yao 2004. The natural production of the fungus is limited owing to its strict host-specificity, confined geographic distribution and over exploitation by humans in recent decades. It is therefore currently listed as an endangered species under the second class of state protection (State Forestry Administration and Ministry of Agriculture 1999).
Insect host species of O. sinensis belong to the family Hepialidae (Lepidoptera) (Chu et al. 2004). Since the late 1950s, much effort has been devoted to study the insect species related to the fungus in China. In 1958, researchers from the Institute of Zoology, Chinese Academy of Sciences, began their investigation in some parts of Qinghai and Sichuan provinces. The first report on host insects of O. sinensis in China was on Hepialus armoricanus Oberthür (=Thitarodes armoricanus Oberthür, Chu 1965) and then followed by studies of biological characteristics of the moth (Chen et al. 1973). The research on Hepialidae diversity and taxonomy grew rapidly in China during the 1980s, leading to a series of publications of new taxa, including four new genera, 71 new species and one subspecies (see Chu and Wang 1985a, b;Liang et al. 1988;Wang 1990;Wu 1992;Li et al. 1993;Fu et al. 1991Fu et al. , 2002Yang 1993Yang , 1994Yang et al. 1991aYang et al. , 1992aLiang 1995;Yang and Jiang 1995;Shen and Zhou 1997;Yan 2000;Wang et al. 2001;Chu et al. 2004;Zhang et al. 2007;Tu et al. 2009). A number of attempts have been made to summarize the insect species associated with O. sinensis and various numbers of host species were recorded, e.g., five by Yin (1987); eleven by Yin et al. (1991) without a name list; two, 22 and 23 by Jiang (1989Jiang ( , 1991Jiang ( , 2001; 22 by Chen and Jin (1992); 20 by Long (1992); 37 by Liu et al. (1995); 19 by Li (1996); 38 by Dong and Luo (1996); 31 by Wang et al. (1996) and 37 by Yang (1998). Recently, Chu et al. (2004) published the volume on Hepialidae and Epiplemidae in the Fauna Sinica, in which seven genera and 82 species or subspecies of Hepialidae in China were listed, and 14 species in six genera were believed to be hosts of O. sinensis but the names were not given in that list. However, in two other recent publications (Liu et al. 2005(Liu et al. , 2006, 66 and 69 insect names were listed respectively as the hosts of the fungus. However, all these accounts, except Chu et al. (2004), provided only the number or a list of insect names without any relevant information to determine whether they are hosts of the fungus or not. Therefore, the number of insect host species of O. sinensis and the relationship between those insects and the fungus remain unclear. To clarify this situation, an extensive survey of the literature on the host of O. sinensis was carried out to gather all the insect names related to the fungus in the literature and to analyze the relationship between the insect species and the fungus. The results of this work are reported here.
Recently, a global inventory of the suborder Exoporia, comprising Mnesarchaeoidea and Hepialoidea, was presented by Nielsen et al. (2000), in which the systematic position of many taxa was checked and adjusted. Nielsen et al.'s classification system for Hepialidae is adopted in this study.

Methods
Based upon an exhaustive literature search, a total of 4793 publications related to Cordyceps/Ophiocordyceps and Hepialus/Thitarodes, in either English or Chinese, were gathered. Those publications relevant to host insects of O. sinensis, including reports on taxonomy, checklists, fauna, biological characteristics, ecology and geographical distribution were examined for information about these insects. All the insect names associated with O. sinensis were assessed based on the following criteria to determine their relationship with the fungus. Taxa which met both of the following requirements were considered as recognizable potential insect host species of O. sinensis: (1) The distribution areas of the insect overlapped that of O. sinensis, which was determined on the basis of field collections made by this research group during the years 2000−2010, examination of herbarium specimens, and another exhaustive literature analysis carried out in this laboratory (Li et al. in press). (2) The insect was reported from an altitude above 3000 m on the Tibetan Plateau. However, stem-boring insects were excluded as hosts of the fungus, even if they were hepialid and distributed above 3000 m within the distribution areas of O. sinensis, because the fungus infects only subterranean rootboring insects. Species of root-borers lacking altitude information were considered as indeterminate hosts of O. sinensis requiring further confirmation, despite the overlap of distribution areas with O. sinensis. Species falling in both of the following circumstances were deemed not to be host insects of O. sinensis: the distribution of the insect was outside that of O. sinensis and below an altitude of 3000 m.

Results
A total of 91 names in 13 genera of Hepialidae were found in the literature search. They are listed in alphabetical order in Table 1, together with geographic distribution, altitude, main references and the relationship with O. sinensis as determined by this study. Insect names used in the references, if different from that in Nielsen et al. (2000), are also given. There are 67 names in the references being combined in different genera by Nielsen et al. (2000) and a total of 71 species were originally described from China. Twenty four species described in the literature were not included in Nielsen et al. (2000).

Discussion
Through an extensive literature survey, all the Hepialidae species reported from China were listed and analyzed using detailed information on their geographic distribution, altitude and nomenclature. The relationships between the insect species and O. sinensis were clarified based on available information. The data provided here serve as a foundation for further investigations on the conservation biology of this endangered fungal species and its insect hosts. Species in different genera of Chinese hepialids can be divided into two categories according to the feeding strategy of the larvae (Chu and Wang 1985a, b;Chu et al. 2004): stem-borers (12 taxa) and root-borers (79 taxa). The stromata of O. sinensis are produced directly on the dead larvae of hepialids which were tunneling under the ground (Wang 1995, Yao 2004, Sung et al. 2007, and the host larvae of the fungus feed on plant roots underground (Chen et al. 1973, Shen et al. 1983, Wang 1995, Yao 2004. Therefore, the stem-borers, including nine in Endoclita, one in Palpifer and two in Sthenopis (Table  1), apparently can not be hosts of O. sinensis. The remaining 79 taxa found in this survey were categorized as potential hosts, indeterminate hosts, or non-hosts assessed based on the criteria described in the methods. Fourteen of the 79 root-borers were ruled out as hosts of O. sinensis, including 1 Bipectilus, 1 Gazoryctra, 2 Hepialiscus, 2 Hepialus, 4 Napialus, 1 Parahepialiscus and 3 Thitarodes species (Table 1), because they have not been reported from the distribution area of O. sinensis and were found below the elevation of 3000 m, either far away from the Tibetan Plateau (12 species), e.g., Bipectilus zhejiangensis from Zhejiang Province, Hepialus hainanensis from Hainan Province, etc., or on the Plateau (two species), e.g., Thitarodes xizangensis and T. zhangmoensis, which were found in Zhangmu Town in Tibet Autonomous Region, where the altitude range is from 1700 to 2400 m (People's Government of Tibet Autonomous Region, 2011) and no evidence for the occurrence of O. sinensis has been found (Li et al. in press).
Eight species, including 1 Hepialiscus, 4 Hepialus, 1 Thitarodes and 2 Triodia species (Table 1), are considered as indeterminate hosts of O. sinensis. While the distribution ranges of these species are within that of O. sinensis, they lack an altitude record and require further confirmation before being considered as potential hosts of O. sinensis, e.g., Hepialus yadongensis, Triodia sylvina, etc.
Fifty-seven taxa are recognized as potential hosts of O. sinensis, including 1 Bipectilus, 1 Endoclita, 1 Gazoryctra, 12 Hepialus, 2 Magnificus, 3 Pharmacis and 37 Thitarodes species (Table 1). The distribution ranges of these insects overlap that of O. sinensis. Altitude information for these insects was reported in three ways in the literature: (1) The altitude range of the insect was reported unambiguously above 3000 m, e.g., Thi-tarodes baimaensis, T. meiliensis, etc. (37 species). Among these, the lowest altitude of 3200 m was reported for T. cingulatus (Yang 1998). (2) The altitude range of the species was not specified, but the types were collected at an elevation above 3000 m, e.g., T. baqingensis, Magnificus jiuzhiensis, etc. (11 species). The lowest altitude of the type locality is at 3200 m for B. yunnanensis (Chu and Wang 1985a). (3) There is no data reported on the altitude range for the species or the type specimen, but the altitude of the recorded localities of the moth were above 3000 m, e.g., Hepialus gangcaensis, Pharmacis carna, etc. (nine species). The lowest altitude for the locality of this group is 3050 m for P. carna (Chu et al. 2004) in Luhuo County, Sichuan Province, where the occurrence O. sinensis was confirmed (Li et al. in press).
Three names of the recognizable potential host insects are invalid (nomen nudum) because no full description of the species was published in the literature, although the names appeared several times in various publications (Table 1). Among them, Thitarodes dongyuensis was described by Yang (1992) as 'Hepialus dongyuensis' and deemed as a nomen nudum in Nielsen et al. (2000), while Hepialus guidera and H. lagii were described by Yan (2001a, b) and recognized as nomen nudum in the present study. Further study is required to describe these species in full.
Species of Hepialus and Phassus described from China after 1984 have been transferred to Thitarodes and Endoclita respectively by Nielsen et al. (2000). Most of these species were described on the male genitalia and occasionally venation of one or very few individuals but not all morphological characteristics of the adult (Nielsen et al. 2000). However, the structure of the valve on male genitalia was still employed recently as the sole basis for classification in the revision of Chinese Hepialus by Zou et al. (2010). Further, disparate and incongruent regional taxonomies were regarded as developing rapidly for the Chinese Hepialidae (Nielsen et al. 2000), but the situation has not been changed much. As seen in this study, 24 names listed in Table 1 were not included in Nielsen et al. (2000). Two of them were described pre-2000 and apparently missed by Nielsen and his colleagues, while the remaining 22 were newly described after the year 2000 (Table  1). It seems that further study, especially robust phylogenetic hypotheses from molecular data, of these taxa is required to clarify their taxonomic status and generic placement.
Natural production of O. sinensis has been declining significantly over the last few decades while the market demands on the fungus have increased sharply in recent years. Clarification of the host insects of O. sinensis will provide basic information for management of the insect resources and for the conservation and sustainable use of the fungus. This work has gathered the available information on the host insects of O. sinensis and will lay a foundation for further studies of the relationship between the fungus and its hosts, especially their co-evolution (an ongoing research project based on DNA sequence analyses in this laboratory), and also for the cultivation of this valuable fungus for massive production.