Order Laboulbeniales Research Articles

New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales)

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Hyperparasitism in bat flies (Diptera: Streblidae): new records and interaction networks in the Neotropics

Hyperparasitism is defined as the interaction where one parasite is infected by another parasite. In bat flies (Streblidae and Nycteribiidae), both hyperparasites and microparasites (bacteria, viruses, fungi, and arthropods such as mites) have been documented. Fungi belonging to the order Laboulbeniales are microscopic parasites of a wide diversity of arthropod hosts. Three genera exclusively target bat flies: Arthrorhynchus, which parasitizes species within Nycteribiidae in the Eastern Hemisphere, while genus Gloeandromyces and Nycteromyces parasitize Streblidae in the Western Hemisphere. Among the hyperparasitic arthropods, mites of family Neothrombidiidae, particularly the monospecific genus Monunguis, are known to parasitize bat flies. Here we present the first records of the hyperparasites Monunguis streblida and Gloeandromyces pageanus f. polymorphus parasitizing Streblidae bat flies in Colombia and a summary of these hyperparasitic interactions in the Neotropics. We detected fungi and mites parasitizing bat flies that were collected in the Magdalena River Basin, Colombia, in field expeditions in 2018, 2022, and 2023. We identified 17 bat flies and two species of hyperparasites, specifically M. streblida and the fungi Gloeandromyces. Our search for reports of these interactions in the Neotropics revealed that seven species of Trichobius (Streblidae) are parasitized by M. streblida, whereas Paratrichobius longicrus (Streblidae) is parasitized by Gloeandromyces pageanus f. polymorphus. These interactions have been reported in 11 countries, but our records are the first of M. streblida and Laboulbeniales fungi parasitizing bat flies in Colombia. So far, a total of 14 species of fungi and one species of mite have been associated with 19 species of bat flies, which in turn, are linked to 15 species of Neotropical bats.

The paradoxical rarity of a fruit fly fungus attacking a broad range of hosts.

Understanding the factors that determine the realized and potential distribution of a species requires knowledge of abiotic, physiological, limitations as well as ecological interactions. Fungi of the order Laboulbeniales specialize on arthropods and are typically thought to be highly specialized on a single species or closely related group of species. Because infections are almost exclusively transmitted through direct contact between the hosts, the host ecology, to a large extent, determines the distribution and occurrence of the fungus. We examined ~20,000 fruit flies (Diptera: Dacinae) collected in Malaysia, Sulawesi, Australia, and the Solomon Islands between 2017 and 2019 for fungal infections and found 197 infected flies across eight different Bactrocera species. Morphology and 1,363 bps of small subunit (18S) DNA sequences both support that the infections are from a single polyphagous fungal species Stigmatomyces dacinus—a known ectoparasite of these fruit flies. This leads to the question: why is S. dacinus rare, when its hosts are widespread and abundant? In addition, the hosts are all Bactrocera, a genus with ~480 species, but 37 Bactrocera species found sympatric with the hosts were never infected. Host‐selection does not appear to be phylogenetically correlated. These results suggest a hidden complexity in how different, but closely related, host species vary in their susceptibility, which somehow limits the abundance and dispersal capability of the fungus.

Subterranean millipedes, some patterns and adaptations

Millipedes (class Diplopoda) are a large group of arthropods mostly living in the soil. More than 10,000 species have been described, but this is surely just a mere fraction of the actually existing number of living species. Many lineages of millipedes have invaded caves and other subterranean habitats, not the least in southern Europe. In spite of their many legs, millipedes are in general poor dispersers, and their present-day distributions often reflect very old biogeographical patterns. The monotypic genus Cantabrodesmus Mauriès, 1971, of uncertain family placement and only known from a few caves in N Spain, is an extreme example of this. The genus Thalassisobates Verhoeff, 1908, provides an example of “ecological disjunction”: One species is truly littoral and occurs along the coasts of Europe and eastern N America, whereas the two other species of the genus were collected in a cave in S Spain and in MSS on the island of La Gomera, Canary Islands, respectively, in both cases more than 500 m above sea level. Similar ecological disjunctions (littoral/subterranean) are known from pseudoscorpions (genus Paraliochthonius Beier, 1956) and isopods (genera Halophiloscia Verhoeff, 1908, and Littorophiloscia Hatch, 1947). Subterranean millipedes often exhibit classical troglomorphisms, but there are also some not-so-classical adaptations, such as modification of the mouthparts for a semiaquatic/hygropetric mode of life. Such modifications have apparently arisen at least seven times independently in unrelated lineages belonging to four millipede families. Millipedes are often hosts to epibiotic fungi of the order Laboulbeniales. Available evidence, although scant, suggests a difference between cave and epigean millipedes in the distribution patterns of the fungi on the hosts: Laboulbeniales on epigean millipedes tend to grow on body parts which come into contact during copulation, whereas those on cave millipedes tend to be scattered along the millipede body, suggesting transfer via the substrate.

From the depths: Heterocaucaseuma deprofundum sp. nov., the world's deepest-occurring millipede (Diplopoda, Chordeumatida, Anthroleucosomatidae) from caves in the western Caucasus.

We describe a new anthroleucosomatid millipede, Heterocaucaseuma deprofundum sp. nov., found in the second and third deepest caves of our planet, Krubera-Voronja and Sarma, in Abkhazia, western Caucasus. A brief discussion on its taxonomy and relationships within the Caucaseuma complex and genus Heterocaucaseuma is provided, together with new faunistic records on other three species of Heterocaucaseuma. Notes on the distribution and troglomorphy of the genus Heterocaucaseuma are added. Ecological information and data on the subterranean community of the Krubera-Voronja Cave are updated. We also discuss the vertical distribution of the new species in caves and the presence of an ectoparasitic fungi of the order Laboulbeniales. This new species is currently the world's deepest-occurring millipede, also representing one of the largest known chordeumatidans.

Host age determines parasite load of Laboulbeniales fungi infecting ants: Implications for host-parasite relationship and fungal life history

Arthropod-parasitic fungi of the order Laboulbeniales are known to exhibit specialization to individual host taxa in most cases. Some species exhibit ecological specificity to multiple, often unrelated hosts in certain microhabitats; and often position specificity to different host body parts. The myrmecophilous Rickia wasmannii (Ascomycota: Laboulbeniales) infects Myrmica species (Hymenoptera: Formicidae) (host specificity), and occasionally other arthropod inquilines inside the ant nest (ecological specificity). An effect of the position of infection on the thallus densities has also been reported. Another determinative factor that may also exist in the Rickia-Myrmica host-parasite system, the chronological age of ant worker hosts, has also been linked to parasite load. Comprehensive studies on the age-related infection intensity, however, are still lacking. Here we investigated whether the level of infection correlates with the age of the M. scabrinodis host consistently. We found that older hosts exhibited higher parasite load, even though the infection level of the different colonies varied widely. The results highlight that the level of R. wasmannii infections are strongly influenced by host individual and host colony factors.

A crush on small fungi: An efficient and quick method for obtainingDNAfrom minute ascomycetes

AbstractWe have developed a reliable technique for extractingDNAfrom single microscopic fungal thalli, including efficient cell disruption and transfer of cell content for subsequent polymerase chain reaction (PCR). The technique was primarily developed for members of the ascomycete order Laboulbeniales, which are minute fungi with tough cell walls that are exceedingly difficult to disrupt with standard extraction techniques.Our method makes routine amplification ofDNAfrom single thalli possible, even from small species or poorly developed individuals.DNArelease is accomplished in an entirely mechanical manner using an arbor press fitted with custom‐made components. This approach has eliminated additional treatment such as laborious freeze‐thaw cycles, enzymes, or lysing agents.The overallPCRsuccess rate of 89% is comparable to or better than alternative protocols that make use of substantially larger amounts of fungal tissue. From 97% of the successfulPCRs a total of 156 sequences from four gene regions were produced.Being able to restrictDNAextractions to a single thallus is critical to all genetic studies requiring data at the level of individual, e.g. population genetics. As all researchers working with minute uncultivable organisms in many respects face the same problems (effective handling of the material, small quantities ofDNAetc.), the methodology described here has a potential to be widely applicable. Necessary custom‐made components can be manufactured at fairly low cost by any precision‐tool workshop using our detail drawings.

Parasites of parasites of bats: Laboulbeniales (Fungi: Ascomycota) on bat flies (Diptera: Nycteribiidae) in central Europe

BackgroundBat flies (Streblidae and Nycteribiidae) are among the most specialized families of the order Diptera. Members of these two related families have an obligate ectoparasitic lifestyle on bats, and they are known disease vectors for their hosts. However, bat flies have their own ectoparasites: fungi of the order Laboulbeniales. In Europe, members of the Nycteribiidae are parasitized by four species belonging to the genus Arthrorhynchus. We carried out a systematic survey of the distribution and fungus-bat fly associations of the genus in central Europe (Hungary, Romania).ResultsWe encountered the bat fly Nycteribia pedicularia and the fungus Arthrorhynchus eucampsipodae as new country records for Hungary. The following bat-bat fly associations are for the first time reported: Nycteribia kolenatii on Miniopterus schreibersii, Myotis blythii, Myotis capaccinii and Rhinolophus ferrumequinum; Penicillidia conspicua on Myotis daubentonii; and Phthiridium biarticulatum on Myotis capaccinii. Laboulbeniales infections were found on 45 of 1,494 screened bat flies (3.0%). We report two fungal species: Arthrorhynchus eucampsipodae on Nycteribia schmidlii, and A. nycteribiae on N. schmidlii, Penicillidia conspicua, and P. dufourii. Penicillidia conspicua was infected with Laboulbeniales most frequently (25%, n = 152), followed by N. schmidlii (3.1%, n = 159) and P. dufourii (2.0%, n = 102). Laboulbeniales seem to prefer female bat fly hosts to males. We think this might be due to a combination of factors: female bat flies have a longer life span, while during pregnancy female bat flies are significantly larger than males and accumulate an excess of fat reserves. Finally, ribosomal DNA sequences for A. nycteribiae are presented.ConclusionsWe screened ectoparasitic bat flies from Hungary and Romania for the presence of ectoparasitic Laboulbeniales fungi. Arthrorhynchus eucampsipodae and A. nycteribiae were found on three species of bat flies. This study extends geographical and host ranges of both bat flies and Laboulbeniales fungi. The sequence data generated in this work contribute to molecular phylogenetic studies of the order Laboulbeniales. Our survey shows a complex network of bats, bat flies and Laboulbeniales fungi, of which the hyperparasitic fungi are rare and species-poor. Their host insects, on the other hand, are relatively abundant and diverse.

Bringing Laboulbeniales into the 21st century: enhanced techniques for extraction and PCR amplification of DNA from minute ectoparasitic fungi.

Laboulbeniales is one of the most peculiar orders of Ascomycota. These fungi are characterized by an ectoparasitic life-style on arthropods, determinate growth, lack of an asexual stage, high species richness, and intractability to culture. The order Laboulbeniales, sister to Pyxidiophorales, has only recently been assigned a separate class, the Laboulbeniomycetes, based on very few ribosomal DNA sequences. So far, DNA isolations and PCR amplifications have proven difficult. Here, we provide details of isolation techniques and the application of commercially available kits that enable efficient and reliable genetic analyses of these fungi. We provide 43 newly generated Laboulbeniales ribosomal DNA sequences, among which are the first published sequences for species in the genera Gloeandromyces, Herpomyces, Laboulbenia, Monoicomyces, and Polyandromyces. DNA extractions were possible using from 1 to 30 thalli from hosts preserved in ethanol (70-100 %). In two cases, we successfully isolated DNA from thalli on dried insect collections. Laboulbeniales molecular systematics could be substantially enhanced through these improved methods by allowing more complete sampling of both taxa and gene regions.

Highly disjunct and highly infected millipedes – a new cave-dwelling species of Chiraziulus (Diplopoda: Spirostreptida: Cambalidae) from Iran and notes on Laboulbeniales ectoparasites

Chiraziulus is a highly disjunct, hitherto monotypic genus of cambalid millipedes, geographically isolated in Iran by more than 7000 km from its presumed closest relatives in East Asia and North America. Recent fieldwork in caves of Iran has provided several specimens of this genus, allowing the description of Chiraziulus troglopersicus sp. nov. The intraspecific variability of the type species, C. kaiseri Mauriès, 1983, is illustrated with scanning electron micrographs. Chiraziulus is characterized by exceedingly long microtrichose gonopod flagella which from their insertion points on the posterior face of the anterior gonopod coxites first point distad instead of basad or basad-posteriad as in most other flagelliferous Cambalidea (and Julida), then traverse a groove on the mesal surface of the anterior gonopod coxites, making a full (360°) loop. The same feature is also illustrated for the first time in the genus Cambala. The patterns and prevalence of the infection with a species of ectoparasitic fungus of the genus Rickia (order Laboulbeniales) in the type material of C. kaiseri is described. An updated review of the cave-adapted fauna of Iran is given.

Redescription of Lusitanipus alternans (Verhoeff, 1893) (Diplopoda, Callipoda, Dorypetalidae) and ecological data on its Laboulbeniales ectoparasites in caves.

The Portuguese endemic callipodidan millipede Lusitanipus alternans (Verhoeff, 1893) is redescribed, based on abundant new material collected in caves of central Portugal. The species is illustrated with comprehensive SEM images. The relationships of the genus Lusitanipus Mauriès, 1978 to other callipodidan genera of the subfamily Cyphocallipodinae Verhoeff, 1909 are discussed. The major part of the studied material is highly infected with ectoparasitic fungi of the order Laboulbeniales, we also include information about the infection in the subterranean ecosystem.

Rickia wasmannii increases the need for water in Myrmica scabrinodis (Ascomycota: Laboulbeniales; Hymenoptera: Formicidae)

The order Laboulbeniales (Fungi, Ascomycota) is a little-studied group of microscopic ectoparasites of invertebrates, mostly insects. The effects of Laboulbeniales species on their hosts are mostly unknown. Rickia wasmannii Cavara, 1899 is a common Laboulbeniales fungus occurring in Europe and is currently known to be a parasite of at least eight Myrmica ant species. R.wasmannii serves as a good model organism for Laboulbeniales–host interactions, as this species covers the host in a very high density, and infected host individuals can be easily collected in high numbers. The effect of R. wasmannii on the survival rate of its most common host species, Myrmica scabrinodis Nylander 1846, was therefore investigated in a laboratory experiment on an individual level. To enhance the results, environmental stresses were simulated by depriving infected and uninfected workers of water and food. The survival of individuals was recorded hourly until the death of the last individual. Infected specimens were significantly more sensitive to the withdrawal of food and water than uninfected specimens. When we tested for water consumption, we found that infected ants spent more time consuming water than uninfected ants. Therefore, it is possible that infected ants must replace the loss of water. Based on these results, R. wasmannii substantially decreases the chances and time of survival of infected individuals, at least in resource-limited environments, which suggest that R. wasmannii has a negative effect on its host.

Rare and new Labonlbeniales from Poland. VI

The author describes two new genera of fungi from the order <i>Laboulbeniales: Tavaresiella</i> gcn.n., similar to <i>Symplectromyces</i> Thaxter, with the type species <i>T. hebri</i> sp. n., and <i>Triceromyces</i> gen.n., related to <i>Distolomyces</i> Thaxler, with type species <i>T. balazucii</i> sp. n.; both are parasites of <i>Hebrus</i> spp. (<i>Heteroptera</i>). A new species on <i>Sphaerosoma globosum</i> (<i>Coleoptera, Endomychidac</i>), <i>Euphoriomyces rossii</i> sp.n. is also described, and the localities of seven other species new for Poland are presented.

Effects of the ectoparasitic fungus Rickia wasmannii on its ant host Myrmica scabrinodis: changes in host mortality and behavior

Social animals like ants, although seemingly well-armed against parasites because of the protective nature of the social system, are nevertheless exposed to several parasitic organisms, among them fungi of the order Laboulbeniales (Ascomycetes). Rickia wasmannii is one such ectoparasitic fungus that lives on different species of the ant genus Myrmica in Europe. The fungus attaches to the outer layer of the cuticle, while appearing on the surface of the hosts as a setae-like structure. There is little information on the effects of the fungi on the host. Usually it is regarded as neutral, in spite of its high abundance on the ants. We studied the impact of a R.wasmannii infection on the lifespan and grooming behavior of its main host M. scabrinodis in artificial nests in laboratory conditions for 2 months. The lifespan of infected individuals was significantly reduced in comparison with the lifespan of uninfected ants. The frequency of allogrooming was significantly higher in infected nests, while only a marginally significant positive effect could be detected in the case of autogrooming. Increased grooming may either be an effective aspect of host defense or, alternatively, a pathogen-induced behavioral change that enhances pathogen transmission. This study thus provides the first documented evidence of the potential effects of R. wasmannii on its ant hosts.

Incidence of infection of carabid beetles (Coleoptera: Carabidae) by laboulbenialean fungi in different habitats

The prevalence of obligate parasitic fungi may depend partly on the environmental conditions prevailing in the habitats of their hosts. Ectoparasitic fungi of the order Laboulbeniales (Ascomycetes) infect arthropods and form thalli on the host's body surface. Although several studies report the incidence of infection of certain host species by these fungi, quantitative data on laboulbenialean fungus-host arthropod interactions at the host assemblage level are rarely reported. To clarify the effects of host habitats on infection by ectoparasitic fungi, the incidence of infection by fungi of the genus Laboulbenia (Laboulbeniales) of overwintering carabid beetles (Coleoptera: Carabidae) in three habitats, a riverside (reeds and vines), a secondary forest and farmland (rice and vegetable fields), were compared in central Japan. Of the 531 adults of 53 carabid species (nine subfamilies) collected in the three habitats, a Laboulbenia infection of one, five and one species of the carabid subfamilies Pterostichinae, Harpalinae and Callistinae, respectively, was detected. Three species of fungus were identified: L. coneglanensis, L. pseudomasei and L. fasciculate. The incidence of infection by Laboulbenia was higher in the riverside habitat (8.97% of individuals; 14/156) than in the forest (0.93%; 2/214) and farmland (0%; 0/161) habitats. Furthermore, the incidence of infection by Laboulbenia in the riverside habitat ranged from 0 to 33.3% and differed significantly in the ten microhabitats (riverbank, edge of track, tall reeds, kudzu vines, slope of a hollow, rotten wood, vine reeds, under stones, the shoulder of a terrace and marshy ground) where the carabid beetles overwintered. These results suggest that host habitats and microhabitats are closely associated with successful infection by laboulbenialean fungi.

A new species of Stigmatomyces from Baltic amber, the first fossil record of Laboulbeniomycetes

A fossil ascomycete was found attached to the thorax of a stalk eyed fly (Diopsidae: Prosphyracephala succini) in a fragment of Baltic amber. The fungus is assigned to the extant genus Stigmatomyces and described as S. succini sp. nov. This find is the first fossil record of the order Laboulbeniales. At the same time it constitutes the oldest record of a parasitic fungus on an insect. The palaeohabitat is discussed with regard to the find.

Laboulbeniales on beetles: host utilization patterns and species richness of the parasites

Fungi of the order Laboulbeniales are obligate ectoparasites that are easy to detect on the integument of their arthropod hosts. This apparency, and other characteristics that suit them for the role of ‘model’ group in the exploration of parasite biodiversity patterns, are discussed. Salient features of the general biology of these organisms are summarized and past patterns of species description reviewed. In recent years, much as in the fungi as a whole, roughly equal proportions of new species have been described from tropical and temperate regions. Comparison of the known Laboulbeniales mycobiotas of Europe, Asia and ‘tropical Asia’ reveals that most of the more significant parasite genera are more or less equally well represented in each region. Patterns of host utilization are also largely similar, with more than 50% of host species recorded from each region belonging to the two beetle families Carabidae and Staphylinidae. However, there are indications that parasite species that are not confined to a single host species have significantly broader geographical ranges than any of their hosts. The pattern of host utilization in the relatively fully-inventoried assemblage of beetles of a tropical forest in North Sulawesi is described, in terms of the taxonomic group membership, habitat affiliation and feeding biology of recorded hosts. On the basis of the Sulawesi dataset and other available data on host exploitation patterns at various spatial scales, we suggest that between 2 and 5%, i.e. between ca 40000 and ca 100000 of an estimated 2 million extant beetle species, are hosts of one or more species of Laboulbeniales. Known and predicted levels of host specificity imply that a global figure for Laboulbeniales species exploiting Coleoptera is likely to be between 10000 and 50000 species. A smaller number of Laboulbeniales species, probably no more than half the number associated with beetles, are to be found on other arthropod hosts. Ways in which these estimates might be refined are discussed.

Parasitism of Mallophaga by Trenomyces histophtorus

Despite current theory that fungi of the order Laboulbeniales are not injurious to their hosts, we found that Trenomyces histophtorus, a parasite of Mallophaga, invaded the body cavity of its host and produced an extensive rhizomycelium that resulted in the loss of fat body and skeletal muscle in these insects.

MORPHOLOGY OF IMMATURE STAGES OF EUZODIOMYCES LATHROBII THAXTER AND THE TAXONOMIC POSITION OF THE GENUS EUZODIOMYCES

American Journal of BotanyVolume 38, Issue 7 p. 555-560 Article MORPHOLOGY OF IMMATURE STAGES OF EUZODIOMYCES LATHROBII THAXTER AND THE TAXONOMIC POSITION OF THE GENUS EUZODIOMYCES R. K. Benjamin, R. K. Benjamin Department of Botany, University of Illinois, Urbana, IllinoisSearch for more papers by this authorLeland Shanor, Leland Shanor Department of Botany, University of Illinois, Urbana, IllinoisSearch for more papers by this author R. K. Benjamin, R. K. Benjamin Department of Botany, University of Illinois, Urbana, IllinoisSearch for more papers by this authorLeland Shanor, Leland Shanor Department of Botany, University of Illinois, Urbana, IllinoisSearch for more papers by this author First published: 01 July 1951 https://doi.org/10.1002/j.1537-2197.1951.tb14858.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume38, Issue7July 1951Pages 555-560 RelatedInformation