Establishment of an Agrobacterium tumefaciens-Mediated Transformation System for Hirsutella sinensis.

Ophiocordyceps sinensis (Berk.) is a complex is formed by Hepialidae larvae and Hirsutella sinensis. Infestation by H. sinensis, interaction with host larvae, and fruiting body development are three crucial processes affecting the formation of O. sinensis. However, research on the molecular mechanism of O. sinensis formation has been hindered by the lack of effective genetic transformation protocols. Therefore, Agrobacterium tumefaciens-mediated transformation (ATMT) was adopted to genetically transform two H. sinensis strains and optimize the transformation conditions. The results revealed that the most suitable Agrobacterium strain for H. sinensis transformation was AGL1, and that the surfactant Triton X-100 could also induce ATMT, although less effectively than acetosyringone (AS). In addition, the endogenous promoters of H. sinensis genes had a stronger ability to drive the expression of the target gene than did the exogenous promoter. The optimal transformation conditions were as follows: AS and hygromycin B concentrations of 100 µM and 50 µg/mL, respectively; A. tumefaciens OD600 of 0.4; cocultivation at 18 °C for 24 h; and H. sinensis used within three passages. The results lay a foundation for the functional study of key regulatory genes involved in the formation of O. sinensis.

Host Range Expansion of an Endemic Insect Herbivore is Associated With High Nitrogen and Low Fibre Content in Exotic Pasture Plants.

Endemic moth species of the genus Wiseana spp. (Hepialidae) have become serious pests of introduced pasture plants in New Zealand. The original native host plants of these moths have not been confirmed. This study investigated the performance (survival, development time, weight gain) of three Wiseana species on seven putative host plants: five native and two exotic species. The aim was to identify native hosts for the three Wiseana species and to compare their performance on native plants and exotic pasture plants. The chemical composition of the seven putative host plants was investigated to compare native and exotic plant chemistries, and to test for associations between plant characteristics and performance of selected Wiseana species. Carbon, nitrogen, silica and fibre contents were measured for each plant species; primary metabolite composition was determined by gas chromatography-mass spectrometry. For the three moth species, increased survival and weight gain were significantly associated with high nitrogen and low fibre contents in one exotic host plant, white clover (Trifolium repens), although one species, W. umbraculata, did not complete development to adult on any of the plants tested, including clover. Two exotic plants (T. repens, Lolium perenne × Lolium multiflorum), and two native plants (Aciphylla squarrosa and Festuca actae) supported W. copularis development to the adult stage, but only one exotic (T. repens) and one native (F. actae) species supported complete development of W. cervinata. Exotic and native plant species had distinct metabolite profiles, but there was no significant association between metabolite composition and Wiseana performance. We conclude that W. copularis and W. cervinata, but not W. umbraculata, have expanded their host range, because of their ability to use both native and new hosts. No evidence was found for a host shift, i.e., a loss of performance on the ancestral host compared with the new host.

Adaptive advantages of appearance: predation, thermoregulation, and color of webbing built by New Zealand's largest moth.

Prey are often difficult to locate visually, which may help them avoid predators. However, an animal's appearance might also evolve in response to the abiotic environment. Here, we investigate the processes that determine the appearance of silk webbing built by New Zealand's largest endemic moth Aenetus virescens (Lepidoptera: Hepialidae), whose larvae burrow into the trunks of native trees. Larvae cover tunnel entrances with silk webbing, detritus, and epiphytes, giving them a similar appearance to tree bark. First, we conducted spectral analyses of webbing and background bark in avian tetrahedral color space to test whether webbing made larvae less visible to predatory parrots. Next, we manipulated the spectral contrast of webbing and background bark and assessed its effect on predation by parrots for over 2 yr. Last, we measured the effect of webbing on tunnel temperatures and quantified how temperatures within tunnels affected larval growth. Results indicate that webbing made larvae less visible to predatory parrots. However, webbing contrast to background bark did not affect predation by parrots. Instead, webbing increased temperatures within tunnels and facilitated more rapid larval growth. Overall results indicate that the appearance of organisms that are difficult to locate visually may not always result from selection by predators.

Mitogenomics, phylogeny and morphology reveal two new entomopathogenic species of Ophiocordyceps (Ophiocordycipitaceae, Hypocreales) from south-western China.

Ophiocordyceps encompasses over 300 species, demonstrating a wide range of morphological features, hosts and habitats within its species diversity. In this study, two novel species in Ophiocordyceps were revealed parasitising Hepialidae larva buried in soil. Ophiocordycepsalbastroma was morphologically characterised by white stromata, solitary and cylindrical conidiogenous cells and smooth ovoid or ellipsoidal conidia. Ophiocordycepsnigristroma was characterised by woody and dark brown stromata, monophialidic, swollen base and lageniform conidiogenous cells and smooth fusiform or oval conidia. The two new species formed a separate clade, respectively, based on the phylogenetic analyses of a combined dataset including nrSSU, nrLSU, rpb1, rpb2, and tef-1α, as well as a dataset of mitochondrial 14 protein coding genes (PCGs). They were all closely grouped with O.sinensis. The mitochondrial genomes of them were first reported. Their mitogenomes were all typical of circular molecules, with positive AT and GC skew, similar GC content, similar genetic composition, similar codon usage and conservative gene positions. However, the length of the mitogenomes varied. Changes in the length of the genes were the leading cause of changes in the length of mitochondrial genome of Ophiocordyceps. The discovery and identification of new Ophiocordyceps species and analysis their mitochondrial genomes may serve as foundations for phylogeny and diversity research within the genus Ophiocordyceps.

The complete mitochondrial genome of Ahamus yushuensis Chu et Wang 1985 (Lepidoptera: Hepialidae) and phylogenetic analysis.

The complete mitochondrial genome (mitogenome) of Ahamus yushuensis was determined in this study. This mitogenome is 15,336 bp and encodes 37 mitochondrial genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rrnL and rrnS). The A. yushuensis mitogenome has an A + T content of 82.2% and presents a positive AT-skew (0.052) and a negative GC-skew (-0.236). Twelve PCGs start with a typical ATN codon, whereas a single PCG uses CGA (coxI) as the initial codon. The maximum likelihood phylogenetic analysis based on the concatenated nucleotide sequences of 13 PCGs strongly supported the monophyletic relationship of A. yushuensis to the clade of Thitarodes damxungensis and A. yunnanensis.

Morphological notes on Gazoryctra sciophanes (Ferguson) and G. confusus (Edwards) (Lepidoptera: Hepialoidea: Hepialidae).

The genus Gazoryctra Hübner comprises 10 species in North America and four in northern Eurasia. The remaining diversity of North American Hepialidae is represented by four species of Sthenopis Packard, three species of Phymatopus Wallengren, and one species of Korscheltellus Börner (Nielsen et al. 2000; Grehan Knyazev 2019). The North American distribution of Gazoryctra extends between Alaska and southern Appalachians and southern Rocky Mountains (Grehan Mielke 2018). As with other North American Hepialidae, Gazoryctra is absent from much of the southern-central United States where there is ostensibly suitable habitat present as this genus is found in forested regions where it is believed to feed on roots or other organic matter (Schweitzer et al. 2011). This absence may be due to the lack of colonization following regression of inland seas that covered much of this region until the end of the Mesozoic (Grehan Mielke 2018).

New species of Endoclita (Lepidoptera: Hepialidae) and revived species status of E. kosemponis from Taiwan.

Two species of Endoclita C. . R. Felder, 1874 from Taiwan are evaluated for their taxonomic status: Endoclita meifenga Buchsbaum Grehan sp. n. is described as new for a unique male from the central mountains of Taiwan, and E. kosemponis (Strand, 1916), stat. rev. is elevated to species status. We show that the forewing markings and genitalic characteristics of E. meifenga sp. n. distinguish this species from all other Endoclita. The external appearance of E. kosemponis is similar to E. sinensis (Moore, 1877) but the two species show distinct differences in the male and female genitalia. Preliminary comparison of E. sinensis from Taiwan and Japan suggests they represent more than one species. A sample of the COI gene was sequenced for E. meifenga sp. n. and the primary types of E. meifenga sp. n. and E. kosemponis sp. n. are illustrated here for the first time. The taxonomic status of the non endemic Taiwanese E. davidi (Poujade, 1886) and E. sinensis needs future evaluation.

Complete mitochondrial genome of two Thitarodes species (Lepidoptera, Hepialidae), the host moths of Ophiocordyceps sinensis and phylogenetic implications.

Thitarodes (Lepidoptera, Hepialidae) is the only genus that hosts to the Ophiocordyceps sinensis, a traditional Chinese medicine considered as a powerful medicinal supplement. In this study, the complete mitochondrial genomes (mitogenomes) of two species, T. damxungensis and T. pui, have been sequenced, which are 15,928 bp and 15,362 bp in size respectively, and both contain 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNAs and an AT-rich region. Like other hepialoids, the gene arrangement of the mitogenomes of T. damxungensis and T. pui is identical to the ancestral arrangement but differs from those of other lepidopteran species on account of the different arrangements of trnM, trnI, and trnQ. The size of AT-rich region is 545 bp in T. damxungensis and 1030 bp in T. pui. Tandem repetition in the AT-rich region is responsible for the length difference of the A + T-rich region in both species. In Hepialidae, the phylogenetic study based on the dataset of the sequences that combined the protein-coding genes and RNA genes suggested that the species T. yunnanensis should still belong to the genus Thitarodes rather than Ahamns, which is different from the results based on the traditional phylogeny.

Re-characterization of Gymelloxes with a re-description of Gymelloxes terea from Central America (Lepidoptera: Hepialidae).

The hepialid genus Gymelloxes Viette, 1952 is characterized by, and differs from all other genera, by the male genitalia and a combination of characters. G. terea is redescribed due to the brevity of the original description. The male genitalia of Gymelloxes terea (Schaus, 1892) include two features that are unique within the Hepialidae - a posteriorly angled and digitiform tergal lobe, and two shallow, peg-like processes on the posterior margin of the saccus. The male phallus also exhibits minute sclerotized spicules or spots on the apex of an otherwise membranous tube. The external genitalia of the female includes a sclerotized antevaginalis with a broad, dorsally projecting, flat central margin. The species is included within the cibyrine clade of Hepialidae by the structure of the tergosternal connection and narrow spacing between Sc and R on the hindwing. The potential systematic significance of specialized similarities shared with other cibyrine Hepialidae is discussed. A lectotype of Dalaca terea Schaus, 1892 is here designated.

Antimicrobial peptide repertoire of Thitarodes armoricanus, a host species of Ophiocordyceps sinensis, predicted based on de novo transcriptome sequencing and analysis.

Ophiocordyceps sinensis is an entomopathogenic fungus that parasitizes the larvae of ghost moths. The fungus-caterpillar complex resulting from fungal parasitism is a medicinally and economically important traditional Chinese medicine famous for its tonic, aphrodisiac and immunomodulatory activity. Antimicrobial peptides (AMPs), which play predominant roles in responding to infection of pathogenic microorganisms in the immune system of ghost moths, may remain behind the fungus-caterpillar complex. Thitarodes armoricanus, as a widely distributed ghost moth on the Tibetan Plateau, is the first reported and one of the most common host insects of O. sinensis. However, there is little information about AMPs of T. armoricanus. In this study, high-throughput RNA-seq analysis and transcriptome assembly and annotation of T. armoricanus was performed to explore the AMP repertoire. A total of 41,464 unigenes were obtained by de novo assembly, and 20,080 unigenes including 385 immunity-related candidates were annotated. Among which, 16 AMP nucleotide sequences were identified. Based on their peptide structure, these were further classified as cecropin, defensin, attacin and gloverin AMPs. All identified AMPs are highly conserved in the Hepialidae family with distinct features as in other insect AMPs. In conclusion, this research offers insights into the AMP repertoire of T. armoricanus which gives opportunity for the further study of the AMP-related medical components of the fungus-caterpillar complex. The identified AMPs from T. armoricanus also provide valuable information for a better understanding of the immune mechanism of Thitarodes and the formation of the mysterious entomo-fungal combination.

The complete mitochondrial genome of Endoclita signifer (Lepidoptera, Hepialidae).

The ghost moth Endoclita signifer is a new wood-boring pest of eucalyptus in south of China. It infests dozens of native plant species, and causes severe damage to the plantations of exotic eucalyptus. We sequenced the complete mitochondrial genome (mitogenome) of E. signifer, which has the typical 37 mitochondrial genes of insects. Contrary to most of the Lepidoptera, the E. signifer mitogenome has the putative ancestral insect gene order. Atypical start codon (TTG) and incomplete stop codon (one T-nucleotide) were found for COX2. The E. signifer control region is 389 bp without tandem repeats, and two (TA)n stretches were observed.

Systematic analyses of Ophiocordyceps lanpingensis sp. nov., a new species of Ophiocordyceps in China.

A new species of Ophiocordyceps, Ophiocordyceps lanpingensis collected from Lanping County, Yunnan Province, southwestern China, was described based on morphological characteristic, ITS1-5.8S-ITS2 rDNA sequences analyses, 5-gene (nrSSU, nrLSU, tef-1α, rpb1 and rpb2) sequences analyses and MAT1-2-1 gene sequences analyses. This species was characterized by thinner stroma, smaller perithecium, thinner ascospore (multiseptate with short septation). The phylogenetic analyses based on the ITS, the 5-gene and MAT1-2-1 gene dataset showed O. lanpingensis had the closest evolution relationship with O. robertsii and O. sinensis, but still had obvious distances to them. Both morphological character and systematic analyses supported that O. lanpingensis was a new species of Ophiocordyceps.

Host insect species of Ophiocordyceps sinensis: a review.

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.