Morphological and phylogenetic resolution of Conoideocrella luteorostrata (Hypocreales: Clavicipitaceae), a potential biocontrol fungus for Fiorinia externa in United States Christmas tree production areas.

The entomopathogenic fungus Conoideocrella luteorostrata has recently been implicated in natural epizootics among exotic elongate hemlock scale (EHS) insects in Christmas tree farms in the eastern United States. Since 1913, C. luteorostrata has been reported from various plant feeding Hemiptera in the southeastern United States, but comprehensive morphological and phylogenetic studies of U.S. populations are lacking. The recovery of multiple strains of C. luteorostrata from mycosed EHS in North Carolina provided an opportunity to conduct pathogenicity assays and morphological and phylogenetic studies to investigate genus- and species-level boundaries among the Clavicipitaceae. Pathogenicity assays confirmed that C. luteorostrata causes mortality of EHS crawlers, an essential first step in developing this fungus as a biocontrol. Morphological studies revealed that conidia aligned with previous measurements of the Paecilomyces-like asexual state of C. luteorostrata, with conidiophore morphology consistent with historical observations. Additionally, a Hirsutella-like synanamorph was observed in select C. luteorostrata strains. In both a four-locus, 54-taxon Clavicipitaceae-wide phylogenetic analysis including D1-D2 domains of the nuclear 28S rRNA region (28S), elongation factor 1 alpha (EF1-α), DNA-directed RNA polymerase II subunit 1 (RPB1), and DNA-directed RNA polymerase II subunit 2 (RPB2) and a two-locus, 38-taxon (28S and EF1-α) phylogenetic analysis, all three Conoideocrella species were resolved as strongly supported monophyletic lineages across all loci and both methods (maximum likelihood and Bayesian inference) of phylogenetic inference except for 28S for C. tenuis. Despite the strong support for individual Conoideocrella species, none of the analyses supported the monophyly of Conoideocrella with the inclusion of Dussiella. Due to the paucity of RPB1 and RPB2 sequence data, EF1-α provided superior delimitation of intraspecies groupings for Conoideocrella and should be used in future studies. Further development of C. luteorostrata as a biocontrol against EHS will require additional surveys across diverse Hemiptera and expanded pathogenicity testing to clarify host range and efficacy of this fungus.

Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi.

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. This is in part due to their large genome sizes and difficulty in obtaining sequenceable material. In this manuscript, we leveraged a recently-isolated, laboratory-tractable Entomophthora muscae isolate and improved long-read sequencing to obtain a largely-complete entomophthoralean genome. Our E. muscae assembly is 1.03 Gb, consists of 7,810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with other available (transcriptomic and genomic) datasets from entomophthoralean fungi, we provide new insight into the biology of these understudied pathogens. We offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex. Our findings suggest that substantial taxonomic revision is needed to define species within this group and we provide recommendations for differentiating strains and species in the context of the existing body of E. muscae scientific literature. We show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity, despite the presence of machinery to defend against transposable elements(RNAi). In addition, we find that E. muscae and its closest allies are enriched for M16A peptidases and possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We find that E. muscae has an expanded group of acid-trehalases, consistent with trehalose being the primary sugar component of fly (and insect) hemolymph. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Developmental genetic underpinnings of a symbiosis-associated organ in the fungus-farming ambrosia beetle Euwallacea validus.

Mutualistic interactions between organisms often mediate the innovation of traits essential to maintain the relationship. Yet our understanding of these interactions has been stymied due to various hurdles in studying the genetics of non-model animals. To understand the genetic mechanisms by which such traits develop, we examined the function of genes breathless (btl), trachealess (trh), and doublesex in the development of a novel fungus-carrying organ (mycangium) that facilitates an obligate relationship between fungus-farming ambrosia beetles and specific fungal partners. Gene knockdown by RNA interference and subsequent micro-computed tomography visualization suggest btl and trh are required for initiation of mycangia and that tubulogenesis may have been co-opted for early mycangial development.

A new species of Illacme from southern California (Siphonophorida, Siphonorhinidae).

The millipede fauna inhabiting deep soil are poorly known. They are small and threadlike, slow moving, lacking pigmentation, and rarely encountered due to their obscure underground way of life. One family, the Siphonorhinidae, encompasses four genera and 12 species in a fragmentary distribution in California, southern Africa, Madagascar, the Malay Archipelago, and Indo-Burma. The family is represented in the Western Hemisphere by a single genus, Illacme Cook & Loomis, 1928 from California, with its closest known relative, Nematozoniumfilum Verhoeff, 1939, from southern Africa. A new species of this family is documented from soil microhabitats in the Los Angeles metropolitan area, Illacmesocal Marek & Shear, sp. nov. Based on this discovery and the recent documentation of other endogean millipede species, we show that these grossly understudied subterranean fauna represent the next frontier of discovery. However, they are threatened by encroaching human settlement and habitat loss, and conservation of this species and other subterranean fauna is of high importance.

Staining and Scanning Protocol for Micro-Computed Tomography to Observe the Morphology of Soft Tissues in Ambrosia Beetles.

Advances in imaging technology offer new opportunities in developmental biology. To observe the development of internal structures, microtome cross-sectioning followed by H&E staining on glass slides is a common procedure; however, this technique can be destructive, and artifacts can be introduced during the process. In this protocol, we describe a less invasive procedure with which we can stain insect samples and obtain reconstructed three-dimensional images using micro-computed tomography, or micro-CT (µCT). Specifically, we utilize the fungus-farming ambrosia beetle species Euwallacea validus to observe the morphology of mycangia, a critical internal organ with which beetles transport fungal symbionts. Not only this protocol is ideal to observe mycangia, our staining/scanning procedure can also be applied to observe other delicate tissues and small organs in arthropods. Graphical abstract.

Histologic findings of Massospora cicadina infection in periodical cicadas (Magicicada septendecim).

Massospora cicadina, an obligate fungal pathogen in the subphylum Entomophthoromycotina (Zoopagomycota), infects periodical cicadas (Magicicada spp.) during their adult emergence and modifies their sexual behavior to maximize fungal spore dissemination. In this study, 7 periodical cicadas from the Brood X emergence in 2021 infected by M. cicadina were histologically examined. In 7 of 7 cicadas, fungal masses replaced the posterior portion of the abdominal cavity, effacing portions of the body wall, reproductive organs, alimentary tract, and fat bodies. No appreciable inflammation was noted at the intersections of the fungal masses and host tissues. Fungal organisms were present in multiple morphologies including protoplasts, hyphal bodies, conidiophores, and mature conidia. Conidia were clustered into eosinophilic membrane-bound packets. These findings help uncover the pathogenesis of M. cicadina by suggesting there is evasion of the host immune response and by providing a more in-depth description of its relationship with Magicicada septendecim than previously documented.

Metagenome-Assembled Genomes of Bacteria Associated with Massospora cicadina Fungal Plugs from Infected Brood VIII Periodical Cicadas.

We report six metagenome-assembled genomes (MAGs) associated with Massospora cicadina strain MCPNR19 (ARSEF 14555), an obligate entomopathogenic fungus of periodical cicadas. The MAGs include representatives of Pantoea, Pseudomonas, Lactococcus, and one potential new Chryseobacterium species. Future research is needed to resolve the ecology of these MAGs and determine whether they represent symbionts or contaminants.

An Improved 1.5-Gigabase Draft Assembly of Massospora cicadina (Zoopagomycota), an Obligate Fungal Parasite of 13- and 17-Year Cicadas.

A 1.488-Gb draft genome sequence was assembled for the fungus Massospora cicadina, an obligate parasite of periodical cicadas. The M. cicadina genome has experienced massive expansion via transposable elements (TEs), which account for 92% of the genome.

Deoxybuzonamine Isomers from the Millipede Brachycybe lecontii (Platydesmida: Andrognathidae).

Millipedes (Diplopoda) are well known for their toxic or repellent defensive secretions. Here, we describe (6aR,10aS,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-anti-trans-deoxybuzonamine (1a)] and (rel-6aR,10aR,10bR)-8,8-dimethyldodecahydropyrrolo[2,1-a]isoquinoline [trans-syn-cis-deoxybuzonamine (1b)], two isomers of deoxybuzonamine found in the chemical defense secretions of the millipede Brachycybe lecontii Wood (Colobognatha, Platydesmida, Andrognathidae). The carbon-nitrogen skeleton of these compounds was determined from their MS and GC-FTIR spectra obtained from the MeOH extract of whole millipedes, along with a subsequent selective synthesis. Their structures were established from their 1D (1H, 13C) and 2D NMR (COSY, NOESY, multiplicity-edited HSQC, HSQC-TOCSY, HMBC) spectra. Additionally, computational chemistry (DFT and DP4) was used to identify the relative configurations of 1a and 1b by comparing predicted 13C data to their experimental values, and the absolute configuration of 1a was determined by comparing its experimental specific rotation with that of the computationally calculated value. This is the first report of dodecahydropyrrolo[2,1-a]isoquinoline alkaloids from a platydesmidan millipede.

Three novel Ambrosia Fusarium Clade species producing multiseptate "dolphin-shaped" conidia, and an augmented description of Fusarium kuroshium.

The Ambrosia Fusarium Clade (AFC) is a monophyletic lineage within clade 3 of the Fusarium solani species complex (FSSC) that currently comprises 19 genealogically exclusive species. These fungi are known or predicted to be farmed by adult female Euwallacea ambrosia beetles as a nutritional mutualism (Coleoptera: Scolytinae; Xyleborini). To date, only eight of the 19 AFC species have been described formally with Latin binomials. We describe three AFC species, previously known as AF-8, AF-10, and AF-11, based on molecular phylogenetic analysis of multilocus DNA sequence data and comparative morphological/phenotypic studies. Fusarium duplospermum (AF-8) farmed by E. perbrevis on avocado in Florida, USA, is distinguished by forming two morphologically different types of multiseptate conidia and brownish orange colonies on potato dextrose agar (PDA). Fusarium drepaniforme (AF-10), isolated from an unknown woody host in Singapore and deposited as Herb IMI 351954 in the Royal Botanic Gardens, Kew, UK, under the name F. bugnicourtii, is diagnosed by frequent production of multiseptate sickle-shaped conidia. Fusarium papillatum (AF-11), isolated from mycangia of E. perbrevis infesting tea in Kandy, Sri Lanka, forms multiseptate clavate conidia that possess a papillate apical cell protruding toward the ventral side. Lastly, we prepared an augmented description of F. kuroshium (AF-12), previously isolated from the heads or galleries of E. kuroshio in a California sycamore tree, El Cajon, California, USA, and recently validated nomenclaturally as Fusarium. Conidia formed by F. kuroshium vary widely in size and shape, suggesting a close morphological relationship with F. floridanum, compared with all other AFC species. Maximum likelihood and maximum parsimony analyses of a multilocus data set resolve these three novel AFC species, and F. kuroshium, as phylogenetically distinct based on genealogical concordance. Given the promiscuous nature of several Euwallacea species, and the overlapping geographic range of several AFC species and Euwallacea ambrosia beetles, the potential for symbiont switching among sympatric species is discussed.

Ecology: Fungal Mimics Dupe Animals by Transforming Plants.

Many fungi transform host tissues to benefit their own reproduction. A recent study investigates a fungus that converts its plant host's reproductive tissues into ornate flower mimics. These 'pseudoflowers' present complex cues that may enlist insects to facilitate fungal dispersal.

Morphological and Phylogenetic Resolution of Diplodia corticola and D. quercivora, Emerging Canker Pathogens of Oak (Quercus spp.), in the United States.

In Mediterranean Europe and the United States, oak species (Quercus spp.) have been in various states of decline for the past several decades. Several insect pests and pathogens contribute to this decline to varying degrees, including Phytophthora cinnamomi, Armillaria spp., various insect defoliators, and, in the United States, the oak wilt pathogen Bretziella fagacearum. More recently, two emerging canker pathogens, Diplodia corticola and D. quercivora, have been implicated in causing dieback and mortality of oak species in Europe and in several regions in the United States. In 2019, a fungal survey was conducted in the Mid-Atlantic region of the eastern United States, including Maryland, Pennsylvania, Virginia, and West Virginia, to determine the range and impact of D. corticola and D. quercivora on forest health in the United States. A total of 563 oak trees between red and white oak family members were evaluated across 33 forests spanning 18 counties. A total of 32 Diplodia isolates encompassing three Diplodia spp. were recovered from 5,335 total plugs collected from the 13 of 18 sampled counties. Recovered Diplodia species included D. corticola, D. quercivora, and D. sapinea, as well as Botryosphaeria dothidea, a closely related canker pathogen in the Botryosphaeriaceae. Both D. corticola and D. sapinea were recovered from red and white oak family members, whereas D. quercivora was exclusive to white oak family members and B. dothidea to red oak family members. Of these species, D. corticola was most frequently isolated, followed by D. quercivora, D. sapinea, and B. dothidea. Overall, mortality was low across all sampled counties, indicating that these fungi, at the levels that were detected, are not widely inciting oak decline across the region, but probably are acting opportunistically when the environment is conducive to disease. To better understand the relationships between D. corticola and potentially their geographic origins, a multigene phylogenetic study and corresponding morphological study were conducted. A total of 49 Diplodia isolates from Spain, France, Italy, and the United States were assessed. Across all isolates and geographic regions, D. corticola formed a strongly supported monophyletic clade sister to D. quercivora and included two strongly supported subclades, one that included isolates from Spain and California and a second that included isolates from Italy, Maryland, and West Virginia. Both subclades also exhibited overlapping spore measurements. These results support D. corticola as a cosmopolitan pathogen, native to both Europe and the United States, with the possibility of secondary introductions.

From Hashtag to High School: How Viral Tweets Are Inspiring Young Scientists To Embrace STEM.

Social media is an increasingly important professional tool for scientists. In particular, scientists use their social media profiles to communicate science and build communities with like-minded scientists and nonscientists. These networks include journalists who can amplify social media science communication, disseminating it to new audiences on- and offline. Our experience with an outreach project where Peeps marshmallows were inoculated with diverse fungi, which we called #FungalPeeps, has demonstrated that these networks can be an effective conduit between researchers and high school students. Following popular science journalism, #FungalPeeps, a project initiated at West Virginia University, inspired a mycology research project in Notre Dame High School in San Jose, California. Herein, we describe how this connection between academia, journalists, and the high school classroom happened, and how everyone involved benefited from this educational collaboration. We further suggest ways that modern social media networks could be leveraged to incorporate more such practical learning experiences into progressive science curricula to better cultivate young STEM scientists.

Animal-associated fungi: Editorial.

Of 1882 fungal species described in 2019, only 3.5% were animal-associated. This percentage is representative of the poor understanding we have of this group of fungi, which are ephemeral, sometimes inconspicuous, and difficult to access, while often requiring specialized methods for their study. Following a two-session symposium on animal-associated fungi during the 2019 Annual Meeting of the Mycological Society of America, this special issue presents the work of 61 researchers in 16 countries. Twelve articles cover animal-associated fungi among Ascomycota, Basidiomycota, and Neocallimastigomycota-describing 29 new species, presenting new evolutionary hypotheses, and unearthing new ecological data.

Characterization of mating type genes in heterothallic Neonectria species, with emphasis on N. coccinea, N. ditissima, and N. faginata.

Neonectria ditissima and N. faginata are canker pathogens involved in an insect-fungus disease complex of American beech (Fagus grandifolia) in North America commonly known as beech bark disease (BBD). In Europe, both N. ditissima and N. coccinea are involved in BBD on European beech (Fagus sylvatica). Field observations across the range of BBD indicate ascospores to be the dominant spore type in the environment. Several studies report a heterothallic (self-sterile) mating strategy for Neonectria fungi, but one study reported homothallism (self-fertility) for N. ditissima. As such, investigations into mating strategy are important for understanding both the disease cycle and population genetics of Neonectria. This is particularly important in the United States given that over time N. faginata dominates the BBD pathosystem despite high densities of nonbeech hosts for N. ditissima. This study utilized whole-genome sequences of BBD-associated Neonectria spp. along with other publicly available Neonectria and Corinectria genomes and in vitro mating assays to characterize mating type (MAT) locus and confirm thallism for select members of Neonectria and Corinectria. MAT gene-specific primer pairs were developed to efficiently characterize the mating types of additional single-ascospore strains of N. ditissima, N. faginata, and N. coccinea and several other related species lacking genomic data. These assays also confirmed the sexual compatibility among N. ditissima strains from different plant hosts. Maximum likelihood phylogenetic analyses of both MAT1-1-1 and MAT1-2-1 sequences recovered trees with similar topology to previously published phylogenies of Neonectria and Corinectria. The results of this study indicate that all Neonectria and Corinectria tested are heterothallic based on our limited sampling and, as such, thallism cannot help explain the inevitable dominance of N. faginata in the BBD pathosystem.

Micro-computed tomography permits enhanced visualization of mycangia across development and between sexes in Euwallacea ambrosia beetles.

Symbiosis can facilitate the development of specialized organs in the host body to maintain relationships with beneficial microorganisms. To understand the developmental and genetic mechanisms by which such organs develop, it is critical to first investigate the morphology and developmental timing of these structures during the onset of host development. We utilized micro-computed tomography (µCT) to describe the morphology and development of mycangia, a specialized organ, in the Asian ambrosia beetle species Euwallacea validus which maintains a mutualistic relationship with the Ascomycete fungus, Fusarium oligoseptatum. We scanned animals in larval, pupal and adult life stages and identified that mycangia develop during the late pupal stage. Here we reconcile preliminary evidence and provide additional morphological data for a second paired set of structures, including the superior, medial mycangia and an inferior, lateral pair of pouch-like structures, in both late-stage pupae and adult female beetles. Furthermore, we report the possible development of rudimentary, or partially developed pairs of medial mycangia in adult male beetles which has never been reported for any male Xyleborini. Our results illustrate the validity of µCT in observing soft tissues and the complex nature of mycangia morphology and development.

Evolutionary relationships among Massospora spp. (Entomophthorales), obligate pathogens of cicadas.

The fungal genus Massospora (Zoopagomycota: Entomophthorales) includes more than a dozen obligate, sexually transmissible pathogenic species that infect cicadas (Hemiptera) worldwide. At least two species are known to produce psychoactive compounds during infection, which has garnered considerable interest for this enigmatic genus. As with many Entomophthorales, the evolutionary relationships and host associations of Massospora spp. are not well understood. The acquisition of M. diceroproctae from Arizona, M. tettigatis from Chile, and M. platypediae from California and Colorado provided an opportunity to conduct molecular phylogenetic analyses and morphological studies to investigate whether these fungi represent a monophyletic group and delimit species boundaries. In a three-locus phylogenetic analysis including the D1-D2 domains of the nuclear 28S rRNA gene (28S), elongation factor 1 alpha-like (EFL), and beta-tubulin (BTUB), Massospora was resolved in a strongly supported monophyletic group containing four well-supported genealogically exclusive lineages, based on two of three methods of phylogenetic inference. There was incongruence among the single-gene trees: two methods of phylogenetic inference recovered trees with either the same topology as the three-gene concatenated tree (EFL) or a basal polytomy (28S, BTUB). Massospora levispora and M. platypediae isolates formed a single lineage in all analyses and are synonymized here as M. levispora. Massospora diceroproctae was sister to M. cicadina in all three single-gene trees and on an extremely long branch relative to the other Massospora, and even the outgroup taxa, which may reflect an accelerated rate of molecular evolution and/or incomplete taxon sampling. The results of the morphological study presented here indicate that spore measurements may not be phylogenetically or diagnostically informative. Despite recent advances in understanding the ecology of Massospora, much about its host range and diversity remains unexplored. The emerging phylogenetic framework can provide a foundation for exploring coevolutionary relationships with cicada hosts and the evolution of behavior-altering compounds.

Natural history of the social millipede Brachycybe lecontii Wood, 1864.

The millipede Brachycybe lecontii Wood, 1864 is a fungivorous social millipede known for paternal care of eggs and forming multi-generational aggregations. We investigated the life history, paternal care, chemical defence, feeding and social behaviour of B. lecontii and provided morphological and anatomical descriptions, using light and scanning electron microscopy. Based on observations of B. lecontii from 13 locations throughout its distribution, we report the following natural history aspects. The oviposition period of B. lecontii lasted from mid-April to late June and the incubation period lasted 3-4 weeks. Only males cared for the eggs and subsequent care of juveniles was not observed. In one case, the clutches of two males became combined and they were later cared for by only one of the males. The defensive compound of B. lecontii is stored in large glands occupying a third of the paranotal volume and were observed only in stadia II millipedes and older. We observed B. lecontii feeding on fungi of the order Polyporales and describe a cuticular structure on the tip of the labrum that may relate to fungivory. We found that their stellate-shaped aggregations (pinwheels) do not form in the absence of fungus and suggest the aggregation is associated with feeding. We describe and illustrate a previously undescribed comb-like structure on the tibia and tarsi of the six anterior-most leg-pairs and measure the colour and spectral reflectance of the B. lecontii exoskeleton.