Introduction

Parasitism is defined as the relationship between two organisms where one organism, the parasite, benefits while the other organism, the host, is harmed by the relationship1. Typically, the parasite extracts nourishment from the host and thereby reduces its fitness1. A parasite itself can be infected with another parasite, which defines the latter as a hyperparasite2. Understanding parasitism, especially hyperparasitism, requires a complex multidisciplinary approach involving ecology, evolution and behaviour of the three participants in the interaction2,3,4,5.

Laboulbeniales have long been considered ectoparasites of living arthropods, where they can be found on the external surface of their cuticle6,7. Laboulbeniales hosts must combine important properties: (i) successive generations of adult hosts should overlap in time because transmission occurs mainly during copulation, because the vast majority does not live on eggs or larval stages of their host; (ii) their populations must be large and stable; and (iii) they must inhabit moist environments8. Studies on Laboulbeniales have mostly been taxonomic, with a very recent emergence of phylogenetic analysis9,10,11,12,13, and a few recent studies have provided insights into the interaction of Laboulbeniales and their hosts, and the environment2,14,15,16,17.

Morphological studies on Laboulbeniales have focused on the external part of the fungus, the thallus. A recent paper on the histopathology of the genus Rickia started a debate on the absence of the haustorial structures in some genera of Laboulbeniales18, whether this is a secondary loss remains unknown.

We investigate the presence and reveal the structure of Laboulbeniales haustoria in situ in their hosts, using the novel visualization technique based on micro-CT, and also on scanning electron microscopy (SEM).

Results

Micro-computed tomography (µCT)

A 3D reconstruction based on µCT of Arthrorhynchus nycteribiae on a male Penicillidia conspicua, shows host cuticle with attached thalli, attached to an intersegment membrane (Fig. 1 and Supplementary Video S1), and a group of four thalli (of which one is broken at the base) of Arthrorhynchus nycteribiae. At the base of each thallus the cuticle (with a diameter of 25–30 µm) is penetrated by a circular hole with a diameter of 22–28 µm (n = 4). From this penetration a cylindrical lumen, which we interpret as the haustorium extends 45–71 µm (n = 4) into the host’s tissue. Inside the host’s body cavity this structure, the haustorium, tapers distally and gives rise to several side branches. In contrast, at the base of the four thalli of Rickia gigas shown in (Fig. 1 and Supplementary Video S2), no such penetration into the host’s cuticle is visible in the µCT-data.

Figure 1
figure 1

Comparison of the haustoriate Laboulbeniales Arthrorhynchus nycteribiae (a–g) on the abdomen of the batfly Penicillidia conspicua (SR-Nyct85) and the non-haustoriate Rickia gigas on the leg of the millipede Tropostreptus hamatus (h–l). (a–c,i,h) Segmentation based on µCT data. (d–g,j–l) Virtual sections based on µCT data. (a) External view. (b) Penetration of the hosts cuticle (transparent). (c) Haustorium, internal view. (d) Transverse section trough the fungi and the host’s cuticle. (e) Transverse section trough the host’s cuticle underneath the base of the thallus. (f) Section trough the host’s cuticle underneath the base of the thallus, plane as indicated in (d). (g) Section trough the host’s tissue and haustorium, plane as indicated in (d). (h) External view. (i) Penetration of the hosts cuticle (transparent). (j) Transverse section trough the fungi and the host’s cuticle. (k) longitudinal section through the host’s leg. (l) Cross section trough the host’s leg. Cu host’s cuticle, Ex exterior, Ha haustorium, Pe penetration of cuticle, Th thallus, Ti host’s tissue. Red arrows indicate the base (foot) of the thalli. Figure plate prepared with Gimp 2.10.6 (GIMP Development Team, https://www.gimp.org) and Inkscape 1.0 (Inkscape Developers, https://inkscape.org).

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Scanning electron microscope (SEM)

We here present SEMs of the “gigantic” non-haustoriate species Rickia gigas. When a thallus of R. gigas is separated from the host cuticle, an horseshoe-shaped wall remains on the host, surrounding an amorphous structure in the middle (Fig. 2). This structure is mirrored by the foot. On the underside of the foot of the fungus, a similar horseshoe-shaped rim, or impression of a wall, is evident. Within this outer rim/wall a smaller circular structure is evident, which surrounds an amorphous substance in the middle. The inner circle attaches to the foot’s outer margin in a position corresponding to the opening in the horseshoe-shaped ring wall (Fig. 3). SEM images of Arthrorhynchus—a Laboulbeniales species with haustoria—were previously shown2.

Figure 2
figure 2

Rickia gigas on the cuticle of the millipede Tropostreptus hamatus, SEM image, thallus and detail of the inner part of the foot. Scale bars: 10 μm in overview, 1 μm in detail image.

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Figure 3
figure 3

SEM image of detached Rickia gigas on the cuticle of the millipede Tropostreptus hamatus. Scale bars: 10 μm in overview image, 1 μm in detail image.

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Discussion

The micro-CT results from Arthrorhynchus agree perfectly with the previously known light microscope and transmission electron microscope images2. This emphasizes that microtomography is a good technique to visualize the type of fungal attachment to the host and especially the penetration of the cuticle, apart from the study of thallus in amber fossils17. As Jensen et al. (2019) demonstrated the presence of a haustorium in Arthrorhynchus using scanning electron microscopy, we are confident that the lack of penetration and haustorium in Rickia found by micro-CT is real. This is also in agreement with results from the scanning electron microscopical investigation of the attachment sites of R. gigas, which exhibits no indication of penetration and are very similar to those of R. wasmannii previously shown18.

Despite the absence of a haustorium, and hence without any obvious means of obtaining nutrition, Rickia gigas is quite a successful fungus, being often abundant on several species of Afrotropical millipedes of the family Spirostreptidae10. It was originally described from Archispirostreptus gigas, and Tropostreptus (= ‘Spirostreptus’) hamatus20, and was subsequently reported from several other Tropostreptus species19.

A further challenge for Laboulbeniales growing on millipedes is that infected millipedes, in some species even adults, may moult, shedding the exuviae with the fungus, as has been observed by us on an undescribed Rickia species on a millipede of the genus Spirobolus (family Spirobolidae).

The question of how non-haustoriate Laboulbeniales obtain nutrients has been discussed by several authors18, including staining experiments using fungi of the non-haustoriate genus Laboulbenia on various beetles21. Whereas the surface of the main thallus was almost impenetrable to the dye applied (Nile Blue), the smaller appendages could sometimes be penetrated21. The dye injection into the beetle elytra upon which the fungi were sitting, actually spread from the elytron into the fungus, thus indicating that in spite of the lack of a haustorium, the fungus is able to extract nutrients from the interior of its host21.

Such experiments have not been performed on Rickia species, but the possibility that nutrients may pass from the host into the basis of the fungus cannot be excluded. For this genus, or at least R. gigas, there may, however, be an alternative way to obtain nutrients: the small opening in the circular wall by which the thallus is attached to the host may allow nutrients from the surface of the millipede or from the environment to seep into the foot of the fungus. However, further experiments are needed in order to evaluate this hypothesis. Moreover, we should not exclude a potential role of primary and secondary appendages in Laboulbeniales nutrition, as we still do not understand exactly their functional role on the fungus life cycle11.

The predominant position of the Laboulbeniales on the host might be related to the absence or presence of a haustorium. Thus, the haustoriate species of the genus Arthrorhynchus are most frequently encountered in large numbers on the arthrodial membranes of the host’s abdomen, although some thalli are found on legs2,22. At the arthrodial membranes the cuticle is more flexible and therefore might be easier to penetrate by a parasite. Furthermore, most tissues providing/storing nutrition (e.g., fat body) are located within the abdomen. In contrast, non-haustoriate fungi as are often located on more stiff and sclerotized body-parts like the genus Rickia on the legs or body-rings of millipedes7,20,23 or the genus Laboulbenia on the elytra of beetles21,24. A reason for this might be that the non-haustoriate forms, which are only superficially attached to the host need a more or less smooth surface for adherence and can easily become detached from a flexible surface, which is movable in itself, like the arthrodial membrane, while the haustoriate forms are firmly anchored within the hosts’ cuticle.

Whereas the vast majority of the more than 2000 described species of Laboulbeniales show no sign of host penetration, haustoria have been reported from some other genera18, including Trenomyces parasitizing bird lice25,26, Hesperomyces growing on coccinellid beetles and Herpomyces on cockroaches (formerly a Laboulbeniales and now in the order Herpomycetales10), with pernicious consequences on the hosts’ fitness18,27. Micro-CT studies on these genera could help to understand the host penetration. In order to fully understand how Laboulbeniales obtain nourishment, although other approaches are, also needed—for the time being it remains a mystery how the non-haustoriate Laboulbeniales sustain themselves.

Methods

Specimens used

All specimens were obtained from the collection of the Natural History Museum of Denmark. Arthrorhynchus nycteribiae (Peyr.) Thaxt. on Penicillidia conspicua Speiser, 1901, a male bat fly, infected on the dorsal part of the abdomen, 21.07.2018, Igrejinha de Soídos, Algarve, Portugal, L. Rodrigues & S. Reboleira leg. (ref. SR-Nyct85); Rickia gigas Santam., Enghoff & Reboleira on Tropostreptus hamatus (Demange, 1977), a male millipede heavily infected on the legs, Udzungwa Mountains Natural Park, Sanje Chini camp, 598 m, 17-20.01.2014, Thomas Pape leg.

Specimens were initially examined under a binocular stereomicroscope Leica M165C, and measurements were made with the software Leica Application Suite V4.12. Fungal thalli were dissected and mounted on temporary slides in glycerine for morphological taxonomic study, following the standard methodology8, under light microscopy in a Leica DM2500 microscope with Differential Interference Contrast (DIC).

Micro-computed tomography (µCT)

Specimens for micro-computed tomography (µCT) were transferred to 100% ethanol, stained for 24 h in 3% Iodine-solution and washed subsequently with 100% ethanol. These specimens were then critical point dried (CPD) with a Leica EM CPD 300, mounted in a pipette tip and scanned using a SKYSCAN 1272 (Bruker microCT) with the following scanning parameters: source voltage = 25 kV, source current = 130 μA, exposure = 4000 ms, rotation of 360° in rotational steps of 0.2°, frame averaging = 7, random movement = 15, filter = none, pixel size = 0.8 µm. Post-alignment, ring-artefact reduction, beam-hardening correction and reconstruction was performed in NRecon 1.7.1.6 (Bruker microCT). The image stack was modified using Fiji ImageJ 1.50e28 (https://www.imagej.net). Volume rendering and measurements were performed in Drishti Version 2.6.329 (https://www.github.com/nci/drishti), and segmentation of the thallus and the haustorium was done in ITKSnap 3.8.030 (http://www.itksnap.org), within the host the lumen supposedly created by the haustorium was segmented. Smoothing, rendering and animation was performed in MeshLab 1.3.331 (https://www.meshlab.net) and Blender 2.77 (Blender Foundation; https://www.blender.org). Figures were prepared in GIMP 2.10.6 (GIMP Development Team; https://www.gimp.org) and Inkscape 1.0 (Inkscape Developers; https://inkscape.org). The generated µCT-data is deposited on Zenodo under doi: 10.5281/zenodo.4737626.

Scanning electron microscopy (SEM)

Specimens for SEM were transferred to 100% ethanol, critical point-dried in a Tousimis Autosamdri 815 Series A critical point dryer, mounted on an aluminium stub, coated with platinum/palladium and studied under a JEOL JSM-6335F scanning electron microscope.