Bacterial biofilms on thalli of Laboulbeniales: a community uncovered

Abstract

Bacterial biofilms have been described on a number of fungal taxa. These microbial communities are of interest both from an ecological and a biotechnological point of view, as they have been shown to play a role in biodegradation and biosynthesis. This study is the first to show the presence of biofilms on thallus surfaces of Laboulbeniales, an order of fungi that have biotrophic associations with arthropod hosts. Scanning electron microscopy micrographs show an abundance of bacterial biofilms on thalli of three species: Laboulbenia collae associated with Paranchus albipes (Carabidae), L. flagellata associated with Limodromus assimilis (Carabidae), and Hesperomyces virescens s.l. associated with Harmonia axyridis (Coccinellidae). These bacterial communities were mainly found on the thalli, and only in small quantities on the arthropod integument. We suggest genetics and metabolomics approaches to investigate possible interactions between Laboulbeniales fungi and the biofilms. Our work has laid a foundation for future research on biofilms on Laboulbeniomycetes.

Full text

Sydowia 74 (2022) 335
DOI 10.12905/0380.sydowia74-2022-0335 Published online 21 January 2022
Bacterial biolms on thalli of Laboulbeniales:
a community uncovered
Maarten Lubbers1,2, Gerda E.M. Lamers1, André De Kesel3, Oldrˇich Nedveˇd4,5, Menno Schilthuizen1,6,7 &
Danny Haelewaters2,4,5,*
1 Institute of Biology Leiden, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands
2 Research Group Mycology, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
3 Meise Botanic Garden, Nieuwelaan 38, 1860 Meise, Belgium
4 Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 C
ˇeské Budeˇjovice, Czech Republic
5 Biology Centre of the Czech Academy of Sciences, Institute of Entomology, Branišovská 31, 370 05 C
ˇeské Budeˇjovice,
Czech Republic
6 Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands
7 Taxon Expeditions B.V., Rembrandtstraat 20, 2311 VW Leiden, The Netherlands
* e-mail: danny.haelewaters@gmail.com
Lubbers M., Lamers G.E.M., De Kesel A., Nedveˇd O., Schilthuizen M. & Haelewaters D. (2022) Bacterial biolms on thalli of
Laboulbeniales: a community uncovered. – Sydowia 74: 335–342.
Bacterial biolms have been described on a number of fungal taxa. These microbial communities are of interest both from an
ecological and a biotechnological point of view, as they have been shown to play a role in biodegradation and biosynthesis. This
study is the rst to show the presence of biolms on thallus surfaces of Laboulbeniales, an order of fungi that have biotrophic
associations with arthropod hosts. Scanning electron microscopy micrographs show an abundance of bacterial biolms on thalli
of three species: Laboulbenia collae associated with Paranchus albipes (Carabidae), L. agellata associated with Limodromus
assimilis (Carabidae), and Hesperomyces virescens s.l. associated with Harmonia axyridis (Coccinellidae). These bacterial com-
munities were mainly found on the thalli, and only in small quantities on the arthropod integument. We suggest genetics and
metabolomics approaches to investigate possible interactions between Laboulbeniales fungi and the biolms. Our work has laid
a foundation for future research on biolms on Laboulbeniomycetes.
Keywords: Bacterial–fungal interactions, ectoparasitic fungi, scanning electron microscopy, symbiosis.
Biolms can be found on a plethora of biotic
surfaces, such as epithelial cells and fungal la-
ments (Gaddy & Actis 2009), and abiotic surfaces,
such glass and stainless steel (Marques et al. 2007).
These multicellular communities are secured by
self-produced extracellular biopolymers, providing
structure and protection (Seneviratne et al. 2008).
These interactions are what make biolms physio-
logically distinct from planktonic cells of the same
species (Costerton et al. 1995). Biolms have been
investigated extensively from an ecological and a
biotechnological point of view, as they have been
shown to play a role in biodegradation and biosyn-
thesis (Dobbins et al. 1992, Oppermann-Sanio &
Steinbüchel 2002). Biolm production is not only
restricted to bacterial taxa; it has also been ob-
served in green algae with cyanobacteria (García-
Meza et al. 2005), fungi (Reichhardt et al. 2016), and
other groups of organisms (van Wolferen et al. 2018).
Furthermore, biolms often comprise multiple spe-
cies interacting with both each other and their sur-
roundings (Davey & O’toole 2000).
Bacterial–fungal interactions have been studied
in a large number of taxa (Deveau et al. 2018). For
instance, soil fungi were found to have various bac-
terial associates (Warmink & van Elsas 2009). These
communities can consist of up to several hundreds
of bacterial species (Deveau et al. 2018). Bacterial
communities also play an important role in the li-
chen symbiosis, fullling essential functions such as
nutrient supply, resistance against biotic stress fac-
tors, and detoxication of metabolites (Grube et al.
2015). Filamentous fungi can harbor distinguished
sets of microbiomes for various differentiated tis-
sues, such as ascomata, basidiomata, and mycorrhi-
zae (Zagriadskaia et al. 2013, Deveau et al. 2016,
El-Jurdi & Ghannoum 2017). Bacterial microbiota
associated with fungi have also been found to con-
tribute to the biology of the host. For instance, when
fungi were treated with antibiotics suppressing or
altering bacterial communities, various processes,
such as mycelial growth and secondary metabolite
production, were impaired (Vahdatzadeh et al. 2015,
Schulz-Bohm et al. 2017).

336 Sydowia 74 (2022)
Lubbers & al.: Biolms on Laboulbeniales
Laboulbeniales (Ascomycota: Laboulbeniomy-
cetes) are characterized by a biotrophic lifestyle on
arthropods, determinate growth, lack of an asexual
stage, high species richness, and intractability to
culture (Haelewaters et al. 2015, 2021b). One major
difference compared with other members of Asco-
mycota is that these fungi do not form hyphae. In-
stead, they form thalli, 3-dimensional, multicellular
units of 1000s of cells that can be observed via
standard light microscopy approaches (Blackwell et
al. 2020). Laboulbeniales are associated with a
broad spectrum of species within three arthropod
subphyla: Chelicerata, Myriapoda, and Hexapoda
(Haelewaters et al. 2021a).
Light microscopy is the most popular technique
for morphological and developmental studies of
Laboulbeniales. Other techniques that thus far have
been used to visualize structures of Laboulbeniales
are scanning and transmission electron microscopy
(SEM, TEM) (Weir & Beakes 1996, Garcés et Wil-
liams 2004, Harwood et al. 2006, Santamaría et al.
2014, Tragust et al. 2016, Reboleira et al. 2018) and
X-ray microtomography (Perreau et al. 2021). Tra-
gust et al. (2016) investigated the mode of attach-
ment and possible penetrating structures of La-
boulbeniales associated with ants. SEM has been
used to identify Hesperomyces virescens based on
morphological characters (Garcés & Williams 2004,
Harwood et al. 2006) and study its developmental
stages (Weir & Beakes 1996). Finally, rotational
scanning electron (rSEM) micrographs were used
for a more advanced visualization of Thaxterimyces
baliensis on its millipede host (Reboleria et al. 2018).
Thus far, possible bacterial–fungal interactions
have never been studied in Laboulbeniales
Material and methods
Infected ground beetles (Coleoptera: Carabidae),
and ladybirds (Coleoptera: Coccinellidae) were ob-
served using SEM (Tab. 1). Firstly, fresh carabids
were collected on marshes of the Scheldt river in a
wooded freshwater tidal area in Hingene (Bornem,
Belgium) on 8 June 2021. The specimens were sent
alive to the Institute of Biology at Leiden Universi-
ty on 10 June and killed using ethyl acetate vapor
(Herome Cosmetics, Almere, The Netherlands) on 14
June. Fresh specimens of Harmonia axyridis infect-
ed by Hesperomyces virescens s.l. were collected on
Philadelphus coronarius plant hosts (Cornales: Hy-
drangeaceae) in C
ˇeské Budeˇjovice (Czech Republic)
on 8 June 2021, sent on 14 June, and killed on
24 June. Secondly, specimens of Limodromus assi-
milis infected by Laboulbenia agellata and of Ha-
lyzia sedecimguttata infected by Hesperomyces ha-
lyziae were stored in 70–99 % ethanol for a pro-
longed period of time before the next step.
First, all specimens were placed for 1 h in a xa-
tion solution (2 % v/v glutaraldehyde 2 % v/v for-
maldehyde in 1M sodium cacodylate). Next, the
host specimens stored in ethanol were transferred
from xation to a critical point dryer specimen
holder and dehydrated using 90 % acetone (Honey-
well, Muskegon, MI, USA). After incubation for
10 min at room temperature, the critical point dryer
specimen holder was placed in 100 % acetone for
1 h. The medium was refreshed twice to remove all
remaining ethanol. Freshly collected host specimens
were transferred from xation to 70 % acetone for
1 h. After refreshing, the specimens were stored
overnight in 70 % acetone. The specimens were then
placed in a critical point dryer specimen holder and
washed in three different acetone concentrations
(3×20 min in 80 % acetone, 3×20 min in 90 % ace-
tone, 3×20 min in 100 % acetone). After incubating
for 10 min at room temperature, the critical point
dryer specimen holder was placed in 100 % acetone
for 1 h. The metal column was then dried with car-
bon dioxide in a Bal-Tec CPD 030 critical point
dryer (Bal-Tec, Delft, The Netherlands).
Each specimen was attached with double-sided
tape to a metal holder. Specimens were subsequent-
Tab. 1. Specimens examined during this study using scanning electron microscopy.
Specimen label Host species Laboulbeniales species Transport to lab
D. Haelew. 3033
D. Haelew. 3654
D. Haelew. 3655
D. Haelew. 3322
D. Haelew. 3657
D. Haelew. 3664
Halyzia sedecimguttata
Harmonia axyridis
Harmonia axyridis
Limodromus assimilis
Limodromus assimilis
Paranchus albipes
Hesperomyces halyziae
Hesperomyces virescens s.l.
Hesperomyces virescens s.l.
Laboulbenia agellata
Laboulbenia agellata
Laboulbenia collae
Ethanol
Fresh
Fresh
Ethanol
Fresh
Fresh

Sydowia 74 (2022) 337
Lubbers & al.: Biolms on Laboulbeniales
Fig. 1. Bacterial biolms on Laboulbenia agellata infecting Limodromus assimilis (D. Haelew. 3657). A. Two mature thalli.
B. Perithecium wall covered with bacteria. C. Close-up of biolm present on the perithecium. Abbreviations: a appendages, i in-
tegument, p perithecium.
Fig. 2. Bacterial biolms on Laboulbenia collae infecting Paranchus albipes (D. Haelew. 3664). A. Two mature thalli. B. Perithe-
cium covered with bacteria. C. Close-up of biolm present on the perithecium. Abbreviations: a appendages, p perithecium,
tf thallus foot.

338 Sydowia 74 (2022)
Lubbers & al.: Biolms on Laboulbeniales
Fig. 3. Bacterial biolms on Hesperomyces virescens infecting Harmonia axyridis (D. Haelew. 3654). A. Five mature thalli. B. Per-
ithecium covered with bacteria. C. Close-up of biolm. Abbreviations: a appendages, p perithecium, tf thallus foot.
Fig. 4. Laboulbenia agellata infecting Limodromus assimilis. A–B. Thalli on a freshly killed and xed host specimen (D. Haelew.
3657). C–D. Thalli on a host (D. Haelew. 3322) stored in ethanol.

Sydowia 74 (2022) 339
Lubbers & al.: Biolms on Laboulbeniales
ly loaded in a Q150T Plus turbomolecular pumped
coater (Quorum Technologies Ltd, East Sussex, UK)
and coated with a layer of platinum–palladium of
20-nm thickness. Finally, the specimens were ob-
served in a JSM-7600F Schottky eld emission
scanning electron microscope (JEOL, Zaventem,
Belgium) at 5.0 kV.
Results
SEM micrographs revealed the presence of bac-
terial biolms on thallus surfaces of Laboulbeni-
ales. All observed bacteria had a rod-shaped mor-
phology with an average length of 2 µm. Biolms
were clearly present on the thalli, but barely on the
insect integument itself (Figs. 1–3, 4A–B, 5A). The
biolms were only observed on the surface of thalli
attached to insects that had been collected alive,
killed, and then quickly stored in xation chemi-
cals. Biolms were observed on all parts of the thal-
lus, although most abundant on the perithecium.
On specimens that had been stored in ethanol, bio-
lms were absent (Figs. 4C–D, 5B).
Discussion
This study is the rst to show the presence of
biolms on Laboulbeniales. Richards & Smith
(1955) did mention gram-negative, bipolar-staining,
short-rod bacteria in the interior of mature perithe-
cia of Herpomyces stylopygae (Herpomycetales).
One reason that biolms have not been reported
previously may be that these rod-shaped bacteria
are too small to observe with a compound micro-
scope, traditionally the most common microscope in
use for Laboulbeniales research. However, biolms
have also not been observed in previous electron
microscopy research (Weir & Beakes 1996, Harwood
et al. 2006, Garcés & Williams 2004, Tragust et al.
2016, Santamaría et al. 2014, Reboleira et al. 2018).
Biolms, if they were present, were most likely
eradicated because the infected insects were stored
in ethanol. Previous research has shown the effec-
tiveness of ethanol on removing biolms (Peters et
al. 2013). This was also the case for our initial scans,
which were taken of specimens that had been pre-
served in ethanol for longer than a year. It was ap-
Fig. 5. Hesperomyces spp. infecting ladybirds. A. Thalli of H. virescens s.l. on freshly killed and xed Harmonia axyridis (D.
Haelew. 3655). B. Thalli of H. halyziae on Halyzia sedecimguttata stored in ethanol (D. Haelew. 3033). Abbreviations: a append-
ages, p perithecium.

340 Sydowia 74 (2022)
Lubbers & al.: Biolms on Laboulbeniales
parent that the surface was completely clean aside
from organic, non-living debris (Fig. 4C–D). On the
specimens that were freshly collected, killed, and
quickly stored in xation chemicals however, the
biolms could be xed during the procedure with-
out losing structure.
Therefore, for future experiments on bacterial
associations with insect-associated fungi, we think
it better to directly x freshly-killed infected ar-
thropod specimens in xation chemicals. They
should not be stored in ethanol for the following
reasons. First, it takes a signicantly longer time to
dry the specimens as all ethanol must be replaced
by acetone prior to critical point drying. Otherwise,
drift might occur, meaning that micrographs at a
certain magnication are distorted by the motion of
the sample during image acquisition (Jin & Li 2015).
Second, ethanol can extract water and fat from
structures that in fresh specimens can be clearly
seen (Figs. 4C–D, 5B). We assume these ‘imploded’
structures are especially present in specimens
stored for prolonged time in ethanol. Third, as men-
tioned before, bacterial biolms can be washed by
ethanol through denaturation of proteins (Peters et
al. 2013).
Bacterial communities were predominantly
found on thallus surfaces, and only few bacteria
were found on the arthropod integument. The bac-
teria could potentially thrive on the thallus surface
beneting from enhanced dispersal and growth on
fungal exudates (Warmink & Van Elsas 2009, Loh-
berger et al. 2019). However, Laboulbeniales thalli
might also consume bacteria as has also been sug-
gested for, e.g., Agaricus bisporus and slime molds
(Castillo et al. 2011, Vos et al. 2017, Kertesz & Thai
2018). It would therefore be of interest to investi-
gate (i) whether these biolms consist of one or
multiple species; (ii) whether the species composi-
tion of the biolms may be dependent on the fun-
gus, arthropod host, or habitat; and (iii) whether
these bacteria represent mutualistic or pathogenic
species. Bacterial inoculation on Lysogeny broth
(LB) agar and Sanger sequencing or high-through-
put sequencing approaches focusing on the 16S of
the ribosomal RNA gene will be necessary to iden-
tify the species that form these biolms (e.g., Janda
& Abbott 2007, Chamberland et al. 2017). Besides,
these communities could produce certain antimi-
crobial molecules to inhibit growth of other bacte-
rial and fungal species; metabolomics approaches
could be employed to investigate possible bioactive
compounds produced by the bacteria.
Bacteria could perhaps even contribute to the
development of ascospores into mature thalli. Cul-
turing attempts of Laboulbeniales have thus far
been proven unsuccessful (reviewed in Haelewaters
et al. 2021a). Whisler (1968) was perhaps most suc-
cessful; he attempted growth on brain-heart-agar
medium but thalli of Stigmatomyces ceratophorus
only developed to a 20-cell stage, producing sper-
matia but not perithecia. For these culturing exper-
iments, perithecia were washed in several antibiotic
rinses after which the ascospores were squeezed out
(Whisler 1968). These rinses prevented bacterial
growth on the culture media but, conversely, may
have contributed to the unsuccessful growth. The
presence of a biolm on the mature perithecium
(Figs. 1–2, 5A), especially around the tip, seems a
convenient way to inoculate exiting ascospores with
bacteria—transferring bacteria from one genera-
tion to the next. Co-culturing approaches with bac-
teria have been shown necessary for growth and
fructication of cultivated mushrooms (Garbaye et
al. 1992; Noble et al. 2003, 2009). The alternative
possibility, the biolm bacteria being dependent on
the fungus, is less probable because the biolms ap-
pear easily on young, developing thalli from the en-
vironment and thus they might represent common
species and be separately culturable. Ectomycor-
rhizal fungi also have been described to modify and
select the microbiome that is associated with their
environmental niche (Li et al. 2017, Liu et al. 2021).
For Laboulbeniales, the bacteria could perhaps di-
rectly or indirectly provide nutrients necessary for
growth into mature thalli, a hypothesis that, we
think, merits further investigation. This can be more
important for those Laboulbeniales lacking a haus-
torium, for mining nutrients from the host’s integu-
ment (Tragust et al. 2016, Haelewaters & De Kesel
2020, Haelewaters et al. 2022).
Acknowledgements
Michał Gorczak (University of Warsaw) is
thanked for critically reviewing the manuscript.
This project was supported in part by the Research
Foundation–Flanders (junior postdoctoral fellow-
ship 1206620N to DH) and the Czech Science Foun-
dation (grant 20-10003S to ON).
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(Manuscript accepted 15 September 2021; Corresponding Edi-
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