Reintroducing Akanthomyces ampullifer: providing genetic barcodes, culture, and updated description for the dipteran pathogen rediscovered in Germany

Abstract

The genus Akanthomyces (Ascomycota, Hypocreales) includes entomopathogenic species known to infect a variety of insects and spiders. In this study, we present the first isolate of A. ampullifer characterized by molecular methods, found on dead bodies of the common cave limoniid Limonia nubeculosa (Diptera) in the subterranean spaces of southwestern Germany. In total, seven specimens exhibited distinctive morphological traits that, when compared with historical records, confirm their identification as A. ampullifer―particularly noted for its affinity to dipteran hosts. Absent from culture collections and molecular repositories, this species has eluded detailed scientific documentation using modern methods. Our research bridges this knowledge gap, providing the first genetic identification barcodes of five genes, living culture, cultivation requirements, and an updated description. This overlooked fungus is phylogenetically most closely related to the species A. pyralidarum, A. laosensis, and some other species mostly associated with adult moths. It demonstrates a unique morphological signature with monoblastic phialides forming a layer on the surface of synnemata and produces long, cylindrical, chain-forming conidia. It prefers lower temperatures, exhibiting an inability to grow at 25 °C, coupled with notably slow growth in culture.

1. Introduction

In 2020, during an exploration of subterranean environments in Baden-Württemberg, Germany, we encountered cadavers of the dipteran species Limonia nubeculosa Meigen (Limoniidae, Diptera; formerly Tipulidae; also commonly known as crane flies) exhibiting fungal colonization. Genetic analysis positioned this fungus within the genus Akanthomyces Lebert (Cordycipitaceae, Hypocreales, Ascomycota), although it could not be precisely identified at the species level. A specific ecology and thorough phenotypic comparison with descriptions from historical records closely aligned our isolate with A. ampullifer (Petch) Mains. This species is known to parasitize Diptera species and, until now, has not been represented in culture nor characterized by sequence data.

The genus Akanthomyces was established by H. Lebert in 1858 with A. aculeatus Lebert as the type species. It encompasses entomopathogenic species infecting both spiders and various insects (mainly lepidopteran adult moths), mycoparasitic species, e.g., A. lecanii (Zimm.) Spatafora, Kepler & B. Shrestha, endophytes and saprotrophs. Some are able to survive in soil (Hodge, 2003; Hsieh et al., 1997; Mains, 1950; Nicolleti & Becchimanzi, 2020; Wang et al., 2023; etc.). Entomopathogenic species can be highly specialized or have a wide range of hosts, e.g., A. muscarius (Petch) Spatafora, Kepler & B. Shrestha. The highest species diversity is found in subtropical and tropical regions, especially in China and Southeast Asia (Wang et al., 2024).

The genus Akanthomyces is morphologically characterized by phialides producing conidia in chains, unlike the similar genus Hymenostilbe Petch, which has polyblastic phialides producing single conidia. In several species, a torrubielloid sexual morph is produced. In the studies by Hsieh et al. (1997) and Tzean et al. (1997), a key to 13 species of Akanthomyces is provided. Recently, the taxonomy of Cordycipitaceae has undergone considerable changes (Kepler et al., 2017). Some species of the genus Lecanicillium W. Gams & Zare were reassigned to the genus Akanthomyces, which has taxonomic priority. On the other hand, some Akanthomyces species have been transferred to the new genus Hevansia Luangsa-ard, Hywel-Jones & Spatafora. Members of this genus are morphologically similar to Akanthomyces, forming an akanthomyces-like asexual morph, parasitizing spiders, but forming a separate phylogenetic lineage (Kepler et al., 2017).

In recent years, many new species of Akanthomyces were introduced (Aini et al., 2020; Chen et al., 2022; Mongkolsamrit et al., 2018; Wang et al., 2023, 2024). The most recent study (Wang et al., 2024) includes 31 species with available DNA sequences and provides overview of morphological characteristics and ecological data for 35 species.

Now, the genus contains around 40 species, including A. ampullifer which is in Index Fungorum and MycoBank databases incorrectly listed under “current name” Hymenostilbe ampullifera Petch (Retrieved May 30, 2024, from www.indexfungorum.org, Identifier 344000; www.mycobank.org, MB#344000). This information in the databases requires revision.

Limonia Meigen is a genus with rich diversity, including over 200 species. Some species are considered a neglected component of subterranean environments (Lunghi et al., 2020). Limonia nubeculosa (common cave limoniid) is West- and East Palaearctic species. It was chosen as the cave organism of the year 2019 in Germany by German Speleological Society. It often aestivates in underground shelters during the hot summer season (Vogel & Zaenker, 2019).

In this work, we document the features of A. ampullifer, a rarely recorded fungus and the only representative of the genus known to parasitize crane flies. The species was identified based on typical ecology, as well as micro- and macroscopic features observed from natural material and living culture. Additionally, we are publishing valuable sequence data obtained from the culture, which can be used for identifying this species in future studies.

2. Materials and methods

2.1. Localities, isolation, and morphological observations

Seven dead bodies of L. nubeculosa infected by a fungus were collected from six localities in Germany, Baden-Württemberg, in artificial underground tunnels and spaces (Fig. 1; Table 1). These specimens were collected by B. Kunz in Jan and Feb 2020 on the walls of underground spaces. The herbarium items, including the dead bodies of L. nubeculosa and dried cultures, are stored in the Herbarium of Charles University, Prague (PRC). The living isolate is maintained at the Culture Collection of Fungi (CCF), Department of Botany, Faculty of Science, Charles University, Prague (Czech Republic) and at the Westerdijk Fungal Biodiversity Institute, Utrecht (The Netherlands).

Fig. 1. Localities in Baden-Württemberg (Germany) where samples of Limonia nubeculosa infected by Akanthomyces ampullifer were collected.

Table 1. Studied specimens of Limonia nubeculosa infected by Akanthomyces ampullifer from various localities in Germany.

Specimen No.	Date of collection	Locality	Latitude	Longitude	Preserved items
BK 2	30.01.2020	old railway tunnel, Wertheim	49.7592	9.5260	PRCa 9220
BK 3	30.01.2020	old railway tunnel, Wertheim	49.7592	9.5260	PRC 9219
BK 6	30.01.2020	old ice cellar, Bronnbach	49.7134	9.5436	PRC 9222
BK 10	06.02.2020	old tunnel for gypsum mining, Forchtenberg	49.2891	9.5645	PRC 9225
BK 24	08.02.2020	storage cellar, Laun	49.0766	10.1238	PRC 9221
BK 29	09.02.2020	Nuns’ Grotto, Gaildorf	49.0137	9.7858	PRC 9223
BK 39	17.02.2020	vineyard tunnel, Buchhorn	49.1970	9.3124	CCFb 6648 CBSc 150079 PRC 9218 PRC 9224

^a PRC - Herbarium of Charles University, Prague, Czech Republic.

^b CCF - Culture Collection of Fungi, Charles University, Prague, Czech Republic.

^c CBS - Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

The fungus was isolated from the dead body of an infected L. nubeculosa on potato dextrose agar (PDA; Samson et al., 2010). Colony diameters were measured after 6 wk at 10, 15, 20, and 25 °C on oat agar (OA; Samson et al., 2010), PDA, and Sabouraud maltose agar (SABM: pepton 10 g, maltose 20 g, MgSO₄.7H₂O 1 g, KH₂PO₄ 1 g, agar 20 g, water 1 L). Three replicates were used. For long-term preservation, yeast malt agar (YMA; Samson et al., 2010) was used.

Microscopic characters were examined from fungal growth on cadavers of crane flies as well as from a culture: 6-wk-old colonies on OA at 15 °C. These conditions were used because they induced the fastest and most abundant sporulation compared to other temperatures and media examined. They were mounted on slides with lactic acid including cotton blue. An Olympus BX51 microscope with an Olympus DP72 camera was used for observation (maximum magnification 1600×) (Olympus, Tokyo, Japan). Photomicrographs and measurements were made with the QuickPHOTO MICRO 3.0 (PROMICRA, Prague, Czech Republic) and Helicon Focus 5.0 (HeliconSoft, Kharkiv, Ukraine) software. For each structure, 20-50 measurements were performed.

Scanning electron microscopy (SEM): A piece of synnema with conidia was fixed in osmium tetroxide (Merck, Darmstadt, Germany) vapours for 7 d at 5-10 °C and gold coated in a Bal-Tec SCD 050 sputter coater (BAL-TEC Inc., Balzers, Liechtenstein). SEM micrographs were performed on a JEOL JSM-IT800 microscope (JEOL Ltd. Tokyo, Japan) in the Laboratory of Electron Microscopy, Faculty of Science, Charles University, Prague, Czech Republic.

2.2. Molecular studies

2.2.1. DNA extraction, amplification, and sequencing

The ZR Fungal/Bacterial DNA Kit^TM (ZYMO RESEARCH, Irvine, California, USA) was used to isolate genomic DNA from 14-d-old colonies grown on MEA (Oxoid Ltd., Basingstoke, UK). The internal transcribed spacer region (ITS1-5.8S-ITS2 cluster) was amplified and sequenced with primers ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990); the partial large subunit (LSU) ribosomal DNA region was amplified and sequenced with the primers NL1 and LR6 (Vilgalys & Hester, 1990; O’Donnell, 1993); the partial translation elongation factor 1-α (tef1-α) was amplified and sequenced with the primers EF1-983F and EF1-2218R (Rehner & Buckley, 2005); the partial RNA polymerase II largest subunit (RPB1) was amplified and sequenced with the primers RPB1-AFasc and RPB1-6R1asc (Hofstetter et al., 2007); and the partial RNA polymerase II 2^nd largest subunit (RPB2) was amplified and sequenced with the primers RPB2-5F2 and fRPB2-7cR (Liu et al., 1999; Sung et al., 2007). The PCR protocol by Hubka et al. (2018) was followed. PCR amplicons were purified using EXOSAP PP-218 L (Jena Bioscience, Jena, Germany) and sequenced using both terminal primers by Sanger sequencing Bioanalyzer 2100 (Agilent, Santa Clara, USA) at BIOCEV, Czech Republic. The obtained DNA sequences were assembled and deposited into the GenBank database under accession numbers listed in Table 2.

Table 2. Specimens of Akanthomyces used in phylogenetic analysis and their respective accession numbers for DNA sequences.

Species	Straina	Host/Substrate	GenBank/ENA/DDBJ accession numbers					Reference
			ITS	LSU	tef1-α	RPB1	RPB2
A. aculeatus	HUA 186145	-	-	MF416520	MF416465	-	-	Kepler et al. (2017)
A. aculeatus	TS772	Lepidoptera; Sphingidae	KC519371	KC519370	KC519366	-	-	Sanjuan et al. (2014)
A. ampullifer	CCF 6648	Diptera; Limonia nubeculosa	PP437869	PP437868	PP436442	PP436443	PP436444	This study
A. araneicola	GY29011T	Araneae; spider	MK942431	-	MK955950	MK955944	MK955947	Chen et al. (2019)
A. araneogenus	GZUIF DX2T	Araneae; spider	MH978179	-	MH978187	MH978182	MH978185	Chen et al. (2018)
A. araneogenus	YFCC 1811934	Araneae; spider	OQ509518	OQ509505	OQ506281	OQ511530	OQ511544	Wang et al. (2024)
A. araneogenus	YFCC 2206935	Araneae; spider	OQ509519	OQ509506	OQ506282	OQ511531	OQ511545	Wang et al. (2024)
A. araneogenus	KY11571b	Araneae; spider	ON502848	ON502825	ON525447	-	ON525446	Chen et al. (2022)
A. araneogenus	KY11572	Araneae; spider	ON502821	ON502827	ON525449	-	ON525448	Chen et al. (2022)
A. araneosus	KY11341T	Araneae; spider	ON502826	ON502832	ON525443	-	ON525442	Chen et al. (2022)
A. attenuatus	CBS 170.76T	Lepidoptera; Carpocapsa pomonella	MH860970	OP752153	OP762607	OP762611	OP762615	Manfrino et al. (2022)
A. coccidioperitheciatus	NHJ 6709	Araneae; spider	JN049865	EU369042	EU369025	EU369067	EU369086	Kepler et al. (2012)
A. dipterigenus	CBS 126.27	Hemiptera; Icerya purchasi	AJ292385	KM283797	KM283820	KR064300	KM283862	Kepler et al. (2017)
A. dipterigenus	YFCC 2107933	Soil	OQ509520	OQ509507	OQ506283	OQ511532	OQ511546	Wang et al. (2024)
A. kanyawimiae	TBRC 7242	Araneae; spider	MF140751	MF140718	MF140838	MF140784	MF140808	Mongkolsamrit et al. (2018)
A. kanyawimiae	TBRC 7243	Unidentified	MF140750	MF140717	MF140837	MF140783	MF140807	Mongkolsamrit et al. (2018)
A. kunmingensis	YFCC 1708939	Araneae; spider	OQ509521	OQ509508	OQ506284	OQ511533	OQ511547	Wang et al. (2024)
A. kunmingensis	YFCC 1808940T	Araneae; spider	OQ509522	OQ509509	OQ506285	OQ511534	OQ511548	Wang et al. (2024)
A. laosensis	YFCC 1910941T	Lepidoptera; Noctuidae	OQ509523	OQ509510	OQ506286	OQ511535	OQ511549	Wang et al. (2024)
A. laosensis	YFCC 1910942	Lepidoptera; Noctuidae	OQ509524	OQ509511	OQ506287	OQ511536	OQ511550	Wang et al. (2024)
A. lecanii	CBS 101247	Hemiptera; Coccus viridis	JN049836	AF339555	DQ522359	DQ522407	DQ522466	Kepler et al. (2012)
A. lepidopterorum	GZAC SD05151T	Lepidoptera (pupa)	MT705973	-	-	-	MT727044	Chen et al. (2020b)
A. muscarius	CBS 455.70B	-	-	MH871560	-	-	-	Kepler et al. (2017)
A. neoaraneogenus	GZU1031LeaT	Araneae; spider	KX845703	-	KX845697	KX845699	KX845701	Chen et al. (2017)
A. neocoleopterorum	GY11241T	Coleoptera	MN093296	-	MN097813	MN097816	MN097812	Chen et al. (2020a)
A. neocoleopterorum	GY11242	Coleoptera	MN093298	-	MN097815	MN097817	MN097814	Chen et al. (2020a)
A. noctuidarum	BCC 36265T	Lepidoptera; Noctuidae	MT356072	MT356084	MT477978	MT477994	MT477987	Aini et al. (2020)
A. noctuidarum	BCC 47498	Lepidoptera; Noctuidae	MT356074	MT356086	MT477980	MT477996	MT477988	Aini et al. (2020)
A. noctuidarum	BCC 28571	Lepidoptera; Noctuidae	MT356075	MT356087	MT477981	MT478009	MT478006	Aini et al. (2020)
A. pissodis	CBS 118231T	Coleoptera; Pissodes strobi	-	KM283799	KM283822	KM283842	KM283864	Chen et al. (2020b)
A. pseudonoctuidarum	YFCC 1808943T	Lepidoptera; Noctuidae	OQ509525	OQ509512	OQ506288	OQ511537	OQ511551	Wang et al. (2024)
A. pseudonoctuidarum	YFCC 1808944	Lepidoptera; Noctuidae	OQ509526	OQ509513	OQ506289	OQ511538	OQ511552	Wang et al. (2024)
A. pyralidarum	BCC 28816T	Lepidoptera; Pyralidae	MT356080	MT356091	MT477982	MT478000	MT478007	Aini et al. (2020)
A. pyralidarum	BCC 32191	Lepidoptera; Pyralidae	MT356081	MT356092	MT477983	MT478001	MT477989	Aini et al. (2020)
A. sabanensis	ANDES-F 1023	Hemiptera; Pulvinaria caballeroramosae	KC633237	-	KC633267	KC875222	-	Kepler et al. (2017)
A. sabanensis	ANDES-F 1024	Hemiptera; Pulvinaria caballeroramosae	KC633232	KC875225	KC633266	-	KC633249	Kepler et al. (2017)
A. subaraneicola	YFCC 2107937T	Araneae; spider	OQ509527	OQ509514	OQ506290	OQ511539	OQ511553	Wang et al. (2024)
A. subaraneicola	YFCC 2107938	Araneae; spider	OQ509528	OQ509515	OQ506291	OQ511540	OQ511554	Wang et al. (2024)
A. sulphureus	TBRC 7248T	Araneae; spider	MF140758	MF140722	MF140843	MF140787	MF140812	Mongkolsamrit et al. (2018)
A. sulphureus	TBRC 7249	Araneae; spider	MF140757	MF140721	MF140842	MF140786	MF140734	Mongkolsamrit et al. (2018)
A. sulphureus	YFCC 1710936	Araneae; spider	OQ509529	OQ509516	OQ506292	OQ511541	OQ511555	Wang et al. (2024)
A. thailandicus	TBRC 7245T	Araneae; spider	MF140754	-	MF140839	-	MF140809	Mongkolsamrit et al. (2018)
A. tortricidarum	BCC 72638T	Lepidoptera; Tortricidae	MT356076	MT356088	MT478004	MT477997	MT477992	Aini et al. (2020)
A. tortricidarum	BCC 41868	Lepidoptera; Tortricidae	MT356077	MT356089	MT477985	MT477998	MT478008	Aini et al. (2020)
A. tuberculatus	HUA 186131	Lepidoptera (adult moth)	-	MF416521	MF416466	-	-	Kepler et al. (2017)
A. uredinophilus	KACC 44066	Rust	-	KM283784	KM283808	KM283830	KM283850	Park et al. (2016)
A. uredinophilus	KACC 44082T	Rust	-	KM283782	KM283806	KM283828	KM283848	Park et al. (2016)
A. uredinophilus	KUN 101466	Insect	MG948305	MG948307	MG948315	MG948311	MG948313	Park et al. (2016)
A. uredinophilus	KUN 101469	Insect	MG948306	MG948308	MG948316	MG948312	MG948314	Park et al. (2016)
A. waltergamsii	TBRC 7251	Araneae; spider	MF140747	MF140713	MF140833	MF140781	MF140805	Mongkolsamrit et al. (2018)
A. waltergamsii	TBRC 7252T	Araneae; spider	MF140748	MF140714	MF140834	MF140782	MF140806	Mongkolsamrit et al. (2018)
A. waltergamsii	YFCC 883	Araneae; spider	OQ509530	OQ509517	OQ506293	OQ511542	OQ511556	Wang et al. (2024)
A. xixiuensis	HKAS 125851T	Lepidoptera (moth)	OP693461	OP693481	OP838888	OP838890	OP838892	Liu et al. (2024)
A. xixiuensis	XX21081764	Lepidoptera (moth)	OP693460	OP693480	OP838887	OP838889	OP838891	Liu et al. (2024)
A. zaquensis	HMAS 246915T	Fungi; Ophiocordyceps sinensis	MT789699	MT789697	MT797812	MT797810	-	Wang et al. (2023)
A. zaquensis	HMAS 246917	Fungi; Ophiocordyceps sinensis	MT789698	MT789696	MT797811	MT797809	-	Wang et al. (2023)
Akanthomyces sp.	YFCC 945	Soil	OQ509531	-	OQ506294	OQ511543	OQ511557	Wang et al. (2024)
Samsoniella aurantiac	TBRC 7271T	Lepidoptera	MF140764	MF140728	MF140846	MF140791	MF140818	Mongkolsamrit et al. (2018)
S. inthanonensisc	TBRC 7915T	Lepidoptera (pupa)	MF140761	MF140725	MF140849	MF140790	MF140815	Mongkolsamrit et al. (2018)

^a Ex-type strains/specimens are designated with a superscript T

^b ex-type of A. tiankengensis

^c outgroup

2.2.2. Phylogenetic analysis

The sequences of five loci of the Akanthomyces members were retrieved from previous studies and respective accession numbers and references are listed in Table 2. Alignments of the regions were performed using the FFT-NS-i option implemented in the MAFFT online (Katoh et al., 2019). The alignments were trimmed, concatenated, and then analysed using Maximum likelihood (ML) method in IQ-TREE v. 2.2.2.6 (Minh et al., 2020) with nodal support determined by ultrafast bootstrapping (BS) with 100,000 replicates. The dataset contained 59 taxa and a total of 4,083 characters of which 1,099 were variable and 874 parsimony-informative. Partitioning schemes and best-fit substitution models (Bayesian information criterion) were as follows: the TIM3+F+G4 model was proposed for the ITS region; TNe+I+G4 model for the LSU rDNA; TN+F+I+G4 model for the tef1-α gene; TNe+G4 model for the RPB1 gene; and TNe+G4 model for the RPB2 gene. The trees were rooted with clade containing Samsoniella inthanonensis Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard and S. aurantia Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard. The final alignment and tree is available from the DRYAD digital repository (https://doi.org/10.5061/dryad.cz8w9gjck).

3. Results

3.1. Molecular studies and phylogeny

In order to achieve reliable identification and infer genus-wide phylogeny, we utilized sequences of five loci: ITS and LSU rDNA, tef1-α, RPB1, and RPB2. DNA sequences were available for at least 29 accepted Akanthomyces species, with their accession numbers listed in Table 1. In the best scoring ML tree based on five loci (Fig. 2), the strain CCF 6648 is sister to A. pyralidarum Aini, Luangsa-ard, Mongkols. & Thanakitp. (BS 97%). These two species form a robust, well-supported clade (BS 100%) with A. laosensis Hong Yu bis & Y. Wang, A. tortricidarum Aini, Luangsa-ard, Mongkols. & Thanakitp., A. xixiuensis X. C. Peng & T. C. Wen, A. noctuidarum Aini, Luangsa-ard, Mongkols. & Thanakitp., A. pseudonoctuidarum Hong Yu bis & Y. Wang, A. aculeatus and A. tuberculatus (Lebert) Spatafora, Kepler & B. Shrestha.

Fig. 2. The best scoring maximum likelihood tree calculated from ITS, LSU, tef1-α, RPB1, and RPB2 loci sequences showing species relationships within the genus Akanthomyces and the position of A. ampullifer CCF 6648. Maximum likelihood bootstrap supports are attached to nodes, with only values >70% shown; lower supports are indicated with a hyphen; ex-type strains and type specimens are designated by a superscript T; the tree is rooted with Samsoniella species.

Akanthomyces ampullifer displays distinctive sequences for all amplified genes, which were notably different from those of other known species with sequences available in databases. Using the BLAST similarity search (blastn algorithm), the ITS rDNA region of strain CCF 6648 showed the greatest similarity (94.2%) with A. xixiuensis specimen HKAS 125851^T (OP693461) and A. aculeatus strain TS772 (KC519371; 93.1%); the LSU region showed the greatest similarity with A. dipterigenus (Petch) Spatafora, Kepler, Zare & B. Shrestha CBS 126.27 (KM283797; 99.1%) and A. laosensis YFCC 1910941^T (OQ509510; 98.9%); the tef1-α gene showed the greatest similarity with A. pyralidarum strain BCC 28816^T (MT477982; 97.9%) and A. laosensis YFCC 1910941^T (OQ506286; 96.9%); the RPB1 gene showed the greatest similarity with A. laosensis YFCC 1910941^T (OQ511535; 96.2%) and A. pyralidarum strain BCC 28816 (MT478000; 94.5%); and the RPB2 gene showed the greatest similarity with A. pyralidarum strain BCC 28816^T (MT478007; 93.6%) and A. laosensis YFCC 1910941^T (OQ511549; 95.4%).

3.2. Taxonomy - updated description

Akanthomyces ampullifer (Petch) Mains [as ‘ampullifera’], Mycologia 42(4): 573 (1950); MycoBank MB 344000; Figs. 3, 4, 5, 6, 7

Basionym: Hymenostilbe ampullifera Petch, Trans. Br. Mycol. Soc. 21(1-2): 55 (1937)

Type: FH 727 deposited at Farlow herbarium (USA, Massachusetts, Cambridge)

Fig. 3. Cadavers of Limonia nubeculosa infected by Akanthomyces ampullifer. A: Specimen BK 2. B: Specimen BK 24. C: Specimen BK 6. D: Specimen BK 39. E: Specimen BK 3. Bars 5 mm. Photo B. Kunz.

Fig. 4. Colonies of Akanthomyces ampullifer on several agar media (Petri dish 6 cm). A: SABM, 8 wk, 15 °C (incipient sporulation at center of colony). B: PDA, 8 wk, 15 °C (formation of young synnemata). C: YMA, 9.5 mo, 10 °C (mature synnemata). Bars 10 mm. Photo A. Kubátová.

Fig. 5. Akanthomyces ampullifer (herbarium specimen PRC 9222). A, B: Parts of synnema from Limonia nubeculosa with phialides and conidia. Phase contrast. Bars 10 μm. Photo A. Kubátová.

Fig. 6. Akanthomyces ampullifer (strain CCF 6648, OA, 6 wk, 15 °C). A: Synnema with phialides and conidia, differential interference contrast (DIC). B: Conidia, DIC. C: Conidia, phase contrast. Bars 10 μm. Photo A. Kubátová.

Fig. 7. Akanthomyces ampullifer, SEM micrographs of conidia (CCF 6648) showing the conidial surface covered with elongated spindle-shaped formations oriented mostly longitudinally. A: 30000×. B: 50000×. Bars 1 μm. Photo J. Nebesářová.

Characteristics on natural substrate (crane fly, L. nubeculosa, Fig. 3): Synnemata of variable length (approx. 1-6 mm) growing from various parts of dead bodies as well as small mycelial tufts; mass of spores on their surface is whitish to light beige.

Characteristics in culture (isolate CCF 6648): Colonies growing very slowly on all media used (Table 3; Fig. 4), initially whitish, after several weeks forming whitish synnemata, which later turn beige. An amber-coloured exudate is often produced. Colony reverses gradually turn orange, rusty to brown. Colony diameters after 6 wk at different temperatures are given in Table 3. The optimal growth temperature appears to be 15-20 °C, with good sporulation observed at 10 °C. No growth was noted at 25 °C, corresponding to the occurrence of this fungus in cooler underground spaces.

Table 3. Colony diameters of Akanthomyces ampullifer CCF 6648 after 6 weeks on three agar media at different temperatures.

Temperature	Medium (mm)a
	OA	PDA	SABM
10 °C	5	8-9	11
15 °C	11-13	17	20
20 °C	17	15-16	24-25
25 °C	no growth	no growth	no growth

^a For media abbreviations and composition see Materials and Methods.

Micromorphology (Figs. 5, 6, 7): Synnemata are composed of parallel hyphae, with surface hyphae covered by a layer of flask shaped smooth phialides. Phialides arise at right angles from hyphae, sometimes in groups of 2-3 on short metulae, or on a sparsely branched conidiophore. Phialides ellipsoidal to cylindrical, tapering into a neck of various length. Conidia formed in chains, one-celled, cylindrical to elongately club-shaped, hyaline, and smooth. When observed by SEM, the conidial surface is covered with elongated spindle-shaped formations oriented mostly longitudinally, but rarely transversely (Fig. 7). The size of phialides and conidia on the natural substrate as well as in culture can be seen in Table 4.

Table 4. The size of phialides and conidia of Akanthomyces ampullifer based on available data.

Phialides (µm)	Conidia size (µm)	Source
On OAa: 10.7-15.1 × 2.8-3.7 (12.2 × 3.2 on average)	On OAa: 5.4-11.2 × 1.3-2.4 (8.6 × 1.8 on average)	This study
―	On L.n.b: 6.7-10.4 × 1.4-2.2 (8.5 × 1.8 on average)	This study
11-15 × 4-5	7-11 × (1.5)2-2.5	Petch (1937)
10-15 × 3-5	5.2-9 × 1.1-2	Mains (1950) & Koval´ (1974)
6.4-13.9(-15.5) × 3.6-4.4	6.0-11.5 × 2.1-3.2	Hsieh et al. (1997)

^a OA - oat agar.

^b L.n. - Limonia nubeculosa.

4. Discussion

Akanthomyces ampullifer was described by Petch (1937) on L. fusca Meigen collected by R. Thaxter in the USA. Other records come from Ukraine (Koval´, 1974) and Taiwan (Hsieh et al., 1997) (Table 5). The latter finding is peculiar as it originates from a spider. However, no further data on the occurrence of this species have been found. Similarly, no information was found in the available literature about the cultivation of A. ampullifer. Consequently, no living culture has been preserved and no sequence data have been deposited in GenBank.

Table 5. Available data on occurrence of Akanthomyces ampullifer

Country	Insect host	Reference
USA	Limonia fusca (Dicranomyia pubipennis, Limoniidae, Diptera); specimens FHa 727 (T), FH 6254	Petch (1937), Mains (1950)b
Ukraine	Larvae of Dicranomyia sp. (Limoniidae, Diptera)	Koval´ (1974)
Taiwan	Spider	Hsieh et al. (1997), Tzean et al. (1997)c
Germany	Limonia nubeculosa (Limoniidae, Diptera)	This study

^a FH - Farlow Herbarium, Massachusetts, USA.

^b Petch (1937) and Mains (1950) examined identical specimens.

^c Hsieh et al. (1997) and Tzean et al. (1997) examined identical specimen.

In our cultivation attempts, only one culture was obtained from seven specimens. The culture itself grows very slowly and gradually loses the ability to form conidia during repeated passages. We believe that, compared to Beauveria species (Cordycipitaceae, Hypocreales), this fungus is a pathogen with a stronger affinity to the host and a weaker ability to grow as a saprotroph. This is probably also the reason why this fungus is not represented in culture collections, along with its slow growth and inability to grow at 25 °C. The detailed micromorphological description of A. ampullifer is given in Petch (1937), Mains (1950), and Hsieh et al. (1997). Our observations match to their data with minor variations (see Table 4). Petch (1937) did not mention the production of conidia in chains in his description. However, Mains (1950), who studied his samples, observed this conspicuous feature. Therefore, he reassigned this species from the genus Hymenostilbe (which produces single conidia on polyphialides) to Akanthomyces. We observed no differences in conidia size between natural specimens and culture, while the arrangement of phialides on the insect body was much more compact than in agar culture.

The studied Limonia samples were collected in Jan and Feb, suggesting that they had likely been in underground shelters since the summer, i.e., several months prior. During this period, some samples were also infected with another Akanthomyces/Lecanicillium species. These fungi were isolated from specimens BK 3, BK 6, BK 10, and BK 29 instead of the expected A. ampullifer. A growing axenic culture of A. ampullifer was only isolated from specimen BK 39.

Based on our observations, A. ampullifer appears to be a cold-loving species, unlike many others that prefer subtropical and tropical regions (Wang et al., 2024). Our discoveries of A. ampullifer in various locations lead us to conclude that it is an overlooked rather than a rare fungus. It may be easily overlooked because its host is not an economically important insect and because the fungal pathogen develops on it hidden in underground shelters.

Akanthomyces ampullifer is distinguished from other species by the following characteristics: (1) production of monoblastic phialides in a layer on the surface of synnemata, (2) long, cylindrical to elongated club-shaped conidia in chains, (3) preference for a Limonia host (Limoniidae, Tipuloidea, Diptera), (4) better growth at lower temperatures, inability to grow at 25 °C, and (5) very slow growth in culture in general. Other Akanthomyces species produce either lecanicillium-, simplicillium- or isaria-like asexual morphs, or torubiella-/cordyceps-like sexual morph, or have smaller conidia, conidia of a different shape, verrucose phialides or parasitize a different insect group. Akanthomyces aranearum (Petch) Mains, A. clavatus (Mains) K.T. Hodge [as “clavata”], and A. fragilis (Petch) K.T. Hodge have similarly sized conidia; however, A. clavatus forms ellipsoid to oblong conidia and parasitizes members of Gryllidae, while the other two species have verrucose phialides.

On Diptera, “on a fly attached to a living leaf”, Cephalosporium dipterigenum Petch (current name A. dipterigenus) was also described by Petch (1931). This species also has long conidia (5-9 × 1-1.5 µm), but it differs in the verticillate conidiophore branching and the arrangement of conidia in the heads compared to chains found in A. ampullifer. Phylogenetically, A. dipterigenus is clearly distinct from A. ampullifer, with a poorly resolved position and unclear affiliation to major phylogenetic clades within Akanthomyces (Fig. 2).

Disclosure

The authors declare no conflicts of interest. This study was conducted in accordance with the current laws in Czech Republic and Germany.

Acknowledgements

The authors acknowledge Miroslav Hyliš and Jana Nebesářová from the Viničná Microscopy Core Facility (VMCF of the Faculty of Science, Charles University) for their support and assistance in this work. The study was financed by the Institutional Support for Science and Research of the Ministry of Education, Youth and Sports of the Czech Republic. Contribution of Vit Hubka was supported by project Strategie AV21‘VP33 Houby kolem nás i v nás’ of the Czech Academy of Sciences and Czech Academy of Sciences Long-term Research Development Project (RVO: 61388971).

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