fcimb-14-1343834

TYPE Review
PUBLISHED 01 February 2024

DOI 10.3389/fcimb.2024.1343834
Alternative in-vivo models

OPEN ACCESS of mucormycosis
EDITED BY
Georgios Chamilos, Jakob Scheler and Ulrike Binder*
University of Crete, Greece
REVIEWED BY
Department of Hygiene, Microbiology and Public Health, Division of Hygiene and Medical
Eman A. Gouda M. Youssef, Microbiology, Medical University Innsbruck, Innsbruck, Tirol, Austria
Lundquist Institute for Biomedical Innovation,
United States
Congyue Peng,
Clemson University, United States Mucormycosis is still regarded a rare fungal infection, but the high incidences of
COVID-associated cases in India and other countries have shown its potential
*CORRESPONDENCE
Ulrike Binder threat to large patient cohorts. In addition, infections by these fast-growing fungi
ulrike.binder@i-med.ac.at are often fatal and cause disfigurement, badly affecting patients’ lives. In
RECEIVED 24 November 2023 advancing our understanding of pathogenicity factors involved in this disease,
ACCEPTED 16 January 2024 to enhance the diagnostic toolset and to evaluate novel treatment regimes,
PUBLISHED 01 February 2024
animal models are indispensable. As ethical and practical considerations typically
CITATION favor the use of alternative model systems, this review provides an overview of
Scheler J and Binder U (2024) Alternative
in-vivo models of mucormycosis.
alternative animal models employed for mucormycosis and discusses
Front. Cell. Infect. Microbiol. 14:1343834. advantages and limitations of the respective model.
doi: 10.3389/fcimb.2024.1343834
COPYRIGHT
© 2024 Scheler and Binder. This is an open-
access article distributed under the terms of
the Creative Commons Attribution License KEYWORDS
(CC BY). The use, distribution or reproduction
mucormycosis, alternative model organisms, Galleria mellonella, Drosophila
in other forums is permitted, provided the
original author(s) and the copyright owner(s) melanogaster, Caenorhabditis elegans, Danio rerio, Bombyx mori
are credited and that the original publication
in this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
1 Introduction
Mucormycosis is a severe, quickly progressing and highly necrotizing fungal infection,
that is life-threatening particularly for immunodeficient patients (Gupta et al., 2023). There
is one term that maybe describes this disease best, which is heterogeneity. This
heterogeneity in the pathogens causing these infections, in the forms of disease and the
repertoire of underlying diseases is associated with drawbacks in understanding
mucormycosis (Figure 1). Among the Mucormycetes, an ancient and very diverse group
of fungi, species belonging to the order Mucorales are involved in causing severe infections
in human (Roden et al., 2005; Hoffmann et al., 2013). Worldwide, the majority of infections
is attributed to the genera Rhizopus, Lichtheimia and Mucor (Figure 1A). Depending on
geography and/or underlying disease, the species distribution varies and other genera such
as Apophysomyces, Saksenaea — both often associated with cutaneous mucormycosis (Al-
Zaydani et al., 2015; Sigera et al., 2018; Chander et al., 2021; Gupta et al., 2022; Planegger
et al., 2022) — Cunninghamella or Rhizomucor are reported (Gomes et al., 2011; Raju et al.,
2020; Hallur et al., 2021; Schober et al., 2021). Mucorales are ubiquitously found in the
environment and share common features like the coenocytic thallus and a cell wall
containing chitosan. On the other hand, this complex fungal group exhibit species-
specific differences in important mechanisms such as invasion pathways, host defense
mechanisms, cell wall and plasma membrane composition, spore size, prevalence of
Frontiers in Cellular and Infection Microbiology 01 frontiersin.org

Scheler and Binder 10.3389/fcimb.2024.1343834
A B C
FIGURE 1
Pathogenic agents (A), various forms of disease (B) and risk factors (C) in mucormycosis.
different spore types or yeast-hyphae dimorphism (Ribes et al., overload. Elevated iron levels by deferoxamine therapy, traumata or
2000; Richardson, 2009; Li et al., 2011). For example, Rhizopus use of ineffective antifungals such as voriconazole leading to
species have been shown to be phagocytized by alveolar breakthrough infections are additional factors putting patients at
macrophages which leads to inhibition of germination, although risk. Also, health care associated cases, in which spores were
the spores stay viable, whereas Mucor spores are able to germinate transmitted via contaminated material like mouth spatulas or
within the cell resulting in macrophage cell death (Voelz et al., 2015; bandages have been reported, as well as mucormycosis (often
Andrianaki et al., 2018; Ló pez-Muñoz et al., 2018). gastrointestinal) in neonates or malnourished patients (Jeong
The different forms of mucormycosis (Figure 1B) strongly et al., 2019; Sharma et al., 2023). Diabetes was shown to be an
reflect the route of infection of the pathogenic agent, the spores. underlying disease in one third of mucormycosis patients in the so
Inhalation of spores results in rhino-orbital-cerebral forms or far biggest retrospective study comprising more than 900 patients
pulmonary forms, which comprise the highest percentage of (Roden et al., 2005). A more recent survey from Greece that
mucormycosis cases. Species dominating clinical samples of these included data from 108 cases found a lower percentage (15.9%)
patients are naturally those that produce airborne spores, such as attributed to diabetes. Interestingly, the same study showed, that
Rhizopus and Rhizomucor spp. Species of the genus Mucor or more than 20% of the cases involved immunocompetent persons
Lichtheimia produce wet sporangia that are set free in droplets that suffered from cutaneous or soft tissue infections after traumatic
This fact could explain why these fungi are more often associated events (Drogari-Apiranthitou et al., 2023). The mucormycosis
with major trauma, including burn wounds (Walther et al., 2019; outbreak in India among COVID-19 patients, which led to 40
Gupta et al., 2023). In these cutaneous infections, transmission takes 000 CAM (COVID-associated mucormycosis) cases, has shown
place via direct contact with contaminated material. This can be that an interplay of several host and iatrogenic factors led to the
wooden splints, as it has been observed in patients exposed to high emergence of CAM. Prakash and co-authors have extensively
natural disasters like tsunamis or tornadoes, but also clinical reviewed factors leading to the CAM outbreak (Prakash et al., 2021).
material such as contaminated bandages. Rarely, even very small Hyperglycemia, either because of pre-existing diabetes mellitus or
traumata such as animal scratches or insect bites have been the dysfunctional insulin production because of virus-damaged
entry portal for spores in patients developing a cutaneous form of pancreatic cells, or as a consequence of corticosteroid therapy,
disease (Skiada et al., 2022). Ingestion of contaminated foods or were found to be the main risk factors. Additionally, as known in
drinks has been described as the reason for gastrointestinal diabetic ketoacidosis, many patients showed high ferritin levels,
mucormycosis, which is more prevalent in Asia than in other consequently leading to impaired iron homeostasis. Irritation in
continents. All forms of disease share a fulminant progression iron homeostasis and higher free iron levels were enhanced by the
due to fast, abundant growth, angioinvasion, which facilitates viral infection, which causes hemoglobin to release iron (Prakash
dissemination of the disease, and extensive tissue necrosis. The et al., 2021). A recent meta-analysis comparing data of 958 CAM
high likelihood of dissemination together with the limited treatment cases from 45 countries, which were mainly middle and low-income
options explain a higher percentage of secondary cutaneous forms countries, also identified corticosteroid use and diabetes as the
when compared to other, non-mucormycete fungal infections major risk factors. Additionally, older age, an Aspergillus
(Cornely et al., 2019; Skiada et al., 2020; Clark et al., 2023). coinfection or the use of tocilizumab were determined as players
Predisposing factors are in part the same as for other invasive in causing CAM (Özbek et al., 2023).
fungal infections (Figure 1C): mainly neutropenia, followed by Whether mucormycosis is associated with COVID-19 or not,
hematological malignancies, organ, and stem cell transplantation the therapeutic problem is the same. Mucormycetes spread very fast
or long-term corticosteroid use. Other risk factors, more prominent in host tissue and their angioinvasive character ultimately causes
in mucormycosis than in other infections, comprise diabetic thrombosis and tissue necrosis, which makes surgery in addition to
ketoacidosis in poorly controlled diabetic mellitus patients or iron antifungal treatment necessary in many cases. Unfortunately, this

group of fungi exhibits intrinsic resistance to commonly used the years, many alternative hosts have been used and optimized
antifungal drugs like short-tailed azoles and elevated minimum to study pathogenicity factors or efficacy of antifungal treatment. In
inhibitory concentration (MICs) against polyenes are common as the following chapters, the principles of the individual model and
well, resulted in unacceptable high mortality rates even with recent findings obtained with each are summarized.
treatment (Cornely et al., 2019). In order to develop novel
treatment strategies, a better understanding of the disease is
essential. Especially in mucormycosis, where, except for CAM 3 Invertebrate models
cases in India, the patient number is low and therefore patient
samples are rarely available and clinical trials are also limited by the 3.1 Caenorhabditis elegans
great heterogeneity, animal models are of great importance.
Obviously, mammalian models are considered the gold standard 3.1.1 General aspects, advantages
and many have been established for studying mucormycosis. and disadvantages
Jacobsen has given a comprehensive review on that topic recently The nematode C. elegans occurs commonly in soil, decaying
explaining infection routes, mimicking of the various forms of plant matter as well as inside the gut of some invertebrates. These
disease and discussing advantages and limitations (Jacobsen, environments are naturally abundant in microbes, representing also
2019). Practical difficulties and ethical restrictions put a limit to the main food source that C. elegans feeds on. In the laboratory
the use of mammalian models for many laboratories. Furthermore, setting, worms are cultivated on agar plates or in liquid culture with
high throughput screenings or comparison of a large strain set is Escherichia coli OP50 as their nutritional source. This bacterial
impossible. Especially in the case of mucormycosis, representing strain is a biofilm formation defective mutant and uracil auxotroph
great heterogeneity in the species involved or the antifungal (Arata et al., 2020). The worms are typically maintained at
resistance profile, such high throughput assays are immensely temperatures ranging from 15 to 25°C (Schulenburg and Fé lix,
important. Therefore, this review will focus on alternative model 2017). The advantages of using this model organism, relevant for
systems that have been exploited so far for mucormycosis studies, testing all kinds of fungi, are its small size, transparent body, short
highlighting novel alternative model animals or adapted methods life cycle and cost-effectiveness in large-scale cultivation.
established in the last three to four years to avoid repetition of what Furthermore, its genome is completely sequenced and well
has already been reviewed in detail by Jacobsen (2019). The annotated and its genetic manipulation, particularly through RNA
alternative animal models presented in the review have interference, is well established (Fire et al., 1998; Lamitina, 2006).
significantly contributed to fostering our understanding of Additionally, machine learning solutions enable automated high-
previously undescribed virulence factors of Mucorales, some of throughput screenings to evaluate worm survival following
which are unique to this group of fungi (Patiño-Medina et al., 2018; infection with pathogens or when testing the toxicity and efficacy
Patiño-Medina et al., 2019a; Wurster et al., 2020; Alejandre- of antimicrobial agents (Wählby et al., 2012). In these screenings,
Castañeda et al., 2022; Itabangi et al., 2022; Trieu et al., 2022; the shape of C. elegans in liquid culture, as observed in bright field
Szebenyi et al., 2023). Moreover, these models have unveiled microscopy images, is assessed, with sinus-shaped worms
pathogen recognition and immune evasion strategies of considered being alive and straight ones considered dead (Marsh
Mucoralean fungi that are relevant for interactions with the and May, 2012; Wählby et al., 2012; Singulani et al., 2018).
human host (Chamilos et al., 2008a; Hoffmann et al., 2013; However, when studying fungal infections, C. elegans presents
Inglesfield et al., 2018; Ló pez-Muñoz et al., 2018; Hassan et al., two main disadvantages. First, due to their small size (with adult
2021; Itabangi et al., 2022). animals averaging at around 1 mm in length), the worms cannot be
infected with a defined inoculum size. Instead, E. coli OP50 is
simply replaced with the relevant infective agent as the food source
2 General aspects of for C. elegans (Muhammed et al., 2012). Additionally, C. elegans
alternative models worms cannot withstand temperatures higher than 25°C for
extended periods. For studying virulence factors of fungal
What are the expectations of an alternative animal model? pathogens in-vivo, it is crucial to mimic human host temperatures
Independent of the infection, alternative hosts are expected to be if aiming to mimic invasive forms of disease. This temperature
easily obtained, either by breeding or by commercial availability in constraint can be a limitation when studying certain virulence
high numbers, easy to handle by the operators and less cost factors, such as temperature-dependent morphological switches
intensive than mammalian models, which allows for high (Klein and Tebbets, 2007; Marsh and May, 2012).
throughput screening. Good correlation in the outcome to While C. elegans has been previously employed as a model for
mammalian models are a prerequisite and ideally, different routes studying fungal infections, as comprehensively reviewed by
of infection and different forms of disease can be investigated. The Arvanitis et al. (2013); Fusco-Almeida et al. (2023); Singulani
use of alternative infection models is in accordance with the 3R et al. (2018); Jacobsen (2019) noted its absence in studying
policy of replacement, reduction, and refinement of animal mucormycosis. Recent publications from the group of Vı́ctor
utilization in research (Tannenbaum and Bennett, 2015). Over Meza-Carmen at Universidad Michoacana de San Nicolá s de

Hidalgo adopted the model for investigating the virulence of Mucor 3.2 Drosophila melanogaster
circinelloides (now classified as M. lusitanicus) knockout strains as
well as the toxicity of secreted metabolites in their respective cell- 3.2.1 General aspects, advantages
free supernatants. and disadvantages
The fruit fly, D. melanogaster, has been a pivotal and intensively
3.1.2 The use of C. elegans for studying studied model organism in biological research since the beginning
virulence potential of the 20th century. Originally hailing from tropical regions, D.
Patiño-Medina et al. utilized the model to study the role of melanogaster has expanded its habitat to temperate zones. In the
ADP-ribosylation factors (Arf) in the virulence of M. lusitanicus, wild, these flies feed on fermenting fruits, where they engage in
demonstrating increased virulence potential in both the knockout mating and lay their eggs. Possessing a highly conserved innate
strains and their supernatants (Patiño-Medina et al., 2018). immune system, D. melanogaster facilitates the transferability of
Similarly, the group used the model animals to investigate the experimental findings to mammalian hosts (Younes et al., 2020;
role of Arf-like proteins (Arl1 and Arl2), that are involved in vesicle Michael Harnish et al., 2021). Moreover, the utilization of a broad
trafficking and tubulin assembly, in virulence of the same fungus. number of genetic tools for genome manipulation has rendered it a
They observed higher virulence of sporangiospores of arl1 and arl2 highly tractable and precisely manipulable model organism, aiding
deletion strains and higher virulence of their cell-free supernatants in uncovering the functions of conserved genes in the human
when incubating them with C. elegans (Patiño-Medina et al., genome and deciphering recognition of fungal infections via the
2019a). According to the authors, these results collectively suggest Toll-pathway (Lemaitre et al., 1996; Bellen and Yamamoto, 2015;
that harmful proteins secreted by the fungus are responsible for Troha and Buchon, 2019).
increased virulence, also in the supernatant. However, the exact One of the main limitations of the model is the limited
protein(s) in question still need to be identified. In another tolerability of temperatures higher than 32°C for the completion
investigation employing the same approach, Dı́az-Pé rez et al. of its life cycle, resulting in experiments usually performed at 29°C
explored the fermentative metabolism of M. lusitanicus and (Schou et al., 2017; Wurster et al., 2019a). Additionally, due to its
linked increased levels of secreted acetaldehyde in the knockout diminutive size (with adult female flies measuring around 3 mm),
strain to enhanced virulence in the nematode model (Dı́az-Pé rez infecting them with a defined inoculum requires a microinjector.
et al., 2020). Similarly, the group investigated the role of For the study of fungal infections, anesthetized adult flies are
siderophores, specifically rhizoferrin, in the virulence of M. pricked with a stainless steel needle dipped into a defined fungal
lusitanicus. As reported by Alejandre-Castañeda et al., the spore solution or exposed directly to the sporulating fungus on its
deletion of the rhizoferrin encoding gene (rfs) resulted in culture plate (Troha and Buchon, 2019).
decreased virulence of the cell-free supernatant and reduced In studying mucormycosis, adult D. melanogaster flies have
virulence of the sporangiospores compared to the wild type been employed to show the significance of pathogen recognition for
(Alejandre-Castañeda et al., 2022). In 2019 and 2021, Patiño- the development of infection. One of the breakthrough
Medina et al. conducted experiments to determine whether the understanding in immunology against Mucorales was by a study
medium on which sporangiospores of M. lusitanicus are produced in Drosophila, which highlighted significant difference in
would influence their virulence (Patiño-Medina et al., 2019b; recognition and host response between R. oryzae and A.
Patiño-Medina et al., 2021). When yeast extract-peptone-glucose fumigatus. Phagocytosis of R. oryzae spores and damage to
(YPG) medium was supplemented with native blood serum, the hyphae was shown to be a lot lower than to A. fumigatus, which
virulence of the sporangiospores increased, leading to an elevated explained the establishment of invasive disease even in
mortality rate of C. elegans when compared to sporangiospores immunocompetent flies, while these would be resistant to
grown on standard YPG (Patiño-Medina et al., 2019b). infection with A. fumigatus (Chamilos et al., 2008a; Chamilos
Additionally, they demonstrated that sporangiospores produced et al., 2008b). Further, flies have been utilized to explore novel
from rice showed similar rates of virulence against C. elegans. antifungal approaches (Chamilos et al., 2006; Lewis et al., 2013;
Using rice as a growth medium allowed for more cost-effective Mircus et al., 2015; Bellanger et al., 2016; Ben Yaakov et al., 2016), to
mass production of sporangiospores compared to YPG-medium investigate the effects of pretreatment with azoles (Lamaris et al.,
(Patiño-Medina et al., 2021). 2009; Wurster et al., 2019b) and to examine the influence of fungal
In 2022, Richter et al. utilized C. elegans and another nematode, iron metabolism on virulence (Chamilos et al., 2008a; Pongas et al.,
Aphelenchus avenae, to investigate the potential nematodocidial 2009). As these works have been extensively reviewed by Jacobsen
effects of the toxin rhizoxin, produced by endosymbiotic bacteria of (2019), the focus here is on more recent publications.
Rhizopus microsporus (Richter et al., 2022). Their study revealed
that the secondary metabolite exhibits toxic effects towards both 3.2.2 The use of D. melanogaster for studying
organisms. Furthermore, they showed that A. avenae, actively feeds virulence factors
on endosymbiont-free R. microsporus whereas endosymbiont- Additionally, the fly model is used to study genetically modified
harboring fungus is lethal towards the nematode through Mucorales strains. Ibragimova et al. created uracil auxotrophic
starvation or exposure to the toxin. strains of Lichtheimia corymbifera by disrupting the pyrG-gene

via CRISPR-Cas9 and evaluated their pathogenicity in comparison among the tested fungi, it did not significantly influence the
to the original strain in the D. melangoster model (Ibragimova et al., mortality of 3rd instar larvae (Zhao et al., 2017). This could
2020). Szebenyi et al. investigated the role of spore coat proteins possibly be linked to the low amount of 1-octen-3-ol M.
(CotH) in virulence of M. lusitanicus by employing the D. racemosus produced in comparison to the more toxic Aspergillus
melanogaster model as well as the G. melonella and the diabetic strains that exhibited higher secretion of this VOC. While the
ketoacidosis mouse model (Szebenyi et al., 2023). They profiling of VOCs from patient’s breath may provide a new tool
demonstrated that mutants with CRISPR-Cas9 mediated gene for noninvasive diagnostics of fungal diseases (Acharige et al.,
disruptions in CotH3 or CotH4 protein encoding genes exhibited 2018; Licht and Grasemann, 2020), there is still a lack of research
reduced virulence in the mouse and fly models, but only for CotH4 on the role of VOCs in the pathogenicity of Mucoralean species
in G. mellonella. Both proteins are important for temperature and their specific VOC profile for diagnosis. A study as the one
adaptation and cell wall development. CotH4 additionally plays a carried out by Li et al., investigating breath samples of 53 patients
role in spore wall formation. with chronic pulmonary aspergillosis, are of great importance (Li
Exploring the impact of tornadic shear stress as exerted on et al., 2021). For pulmonary mucormycosis, is will be a great
spores during natural catastrophes like tornados, Wurster et al. challenge to collect a similar number of samples due to the high
compared the influence of such mechanical stress events on the variability of pathogens causing the disease. Here, alternative
virulence of different mold species (Wurster et al., 2020). Employing model systems, like the already employed Drosophila model, will
the D. melanogaster model, they observed a transient significantly contribute in the development of such VOC based
hypervirulence only in species belonging to the order Mucorales. diagnostics tools.
After ruling out an interfering influence of shear-stress challenged
spores of R. arrizhus on the immune capacity of the host’s
phagocytic cells, the authors demonstrated that the increased 3.3 Galleria mellonella larvae
virulence of stressed spores resulted from soluble factors secreted
by the fungus. The inhibition of hypervirulence by adding 3.3.1 General aspects, advantages
calcineurin inhibitors during or after shear-stress exposure and disadvantages
highlighted the pivotal role this pathway plays in pathogenicity. G. mellonella (the Greater Wax moth) is a member of the order
The pathway’s relevance was further underlined by exposing M. Lepidoptera and the family Pyralidae, which are found globally.
lusitanicus (former M. circinelloides f. lusitanicus) strains with loss- Naturally, the larvae are a pest of bee hives, and they are bred as bait
of-function mutations in genes responsible for calcineurin protein for fishing or reptile food (Jafari et al., 2010). Over the last 20 years,
synthesis to shear-stress. In these strains, shear-stress led to no the larvae gained an important role as model host to study microbial
significant higher virulence, due to the inability of these strains to infections. This model has been employed globally for dozens of
synthesize calcineurin. Lee et al. previously demonstrated the fungal species, including species of the Mucorales (Curtis et al.,
avirulence of the regulatory B subunit deletion strain in G. 2022). The structural and functional similarities of the insect
mellonella model (Lee et al., 2013). Additionally, Vellanki et al. immunity and the innate immune system of mammals help to
demonstrated that when exposing macrophages to calcineurin gain comparable results to murine models. Fungi are recognized via
lacking mutants phagosome maturation was higher compared to pathogen-associated patterns (PAMPS) which are homologous to
the wild type, underlining the importance of calcineurin to support those in mammals, and both a humoral and cellular response is
immune evasion (Vellanki et al., 2020). Conversely, their ability to activated. Six different types of hemocytes have been described
cause damage to endothelial cells was lower when compared to the circulating in Galleria hemolymph that orchestrate to prevent
wild type. fungal growth by phagocytosis, nodulation, agglutination,
encapsulation, and secretion of antifungal peptides (Trevijano-
3.2.3 Translational aspects Contador and Zaragoza, 2019). It has been shown by the
The Drosophila model has been used to study the detrimental Kavanagh group that hemocytes have a comparable mechanism
toxic effects of volatile organic compounds (VOCs), a possible to the NADPH oxidase complex of human neutrophils, and
contributing factor to fungal pathogenicity (Bennett and Inamdar, produce superoxide and their activity could be diminished by
2015; Zhao et al., 2017). Macedo et al. demonstrated that 1-octen- mycotoxins such as gliotoxin or fumagillin (Bergin et al., 2005;
3-ol decreased survival and inhibition of locomotion in adult D. Fallon et al., 2011). The reasonable size of the larvae makes it easy to
melanogaster flies due to toxic effects of the compound on inoculate a defined amount of spores, which in most protocols is
mitochondria, causing inflammation and apoptosis (Rosowski done via the prolegs, which provide a natural entrance to the body,
et al., 2018). Almaliki et al. cultured different medically relevant and a thin needle can enter without harming the animal.
pathogenic fungi (excluding Mucorales) together with instar Importantly, Galleria larvae can be used over a wide temperature
larvae of D. melanogaster in a microhabitat, exposing them to range from 20-42°C, so they are ideal to mimic human body
fungal VOCs but not directly to the fungi (Almaliki et al., 2020). temperature (Fuchs et al., 2010). The larvae can be obtained
They observed development delays in insect metamorphosis when commercially or easily bred in an incubator if only small
exposed to pathogenic strains producing high concentrations of numbers are needed. This, and the low costs, are big advantages
VOCs, such as1-octen-3-ol. Conversely, Zhao et al. found, that and facilitate high throughput screenings. Unfortunately, the
although Mucor racemosus emitted the highest amount of VOCs different origin of larvae represent one of the main limitations of

this model, rendering the comparison of results between deletion had no impact on virulence, although the mutant was
laboratories difficult. similarly attenuated in growth (Trieu et al., 2022). Iron acquisition
Results are obtained rapidly and a number of experimental is crucial for pathogenicity also in Mucorales, as M. lusitanicus that
endpoints and readouts are possible, which have been summarized is not able to produce rhizoferrin, the mucormycete siderophore,
in details by Curtis et al. (2022). Larval survival, histopathological was shown to be avirulent. Moreover, increased virulence was
examination, determination of fungal burden and defining the rate observed for the same species when spores were generated on
of melanization are the most commonly applied readouts, either to serum containing agar plates which goes in hand with higher
compare virulence potential of a high number of strains or to mitochondrial activity (Patiño-Medina et al., 2023).
evaluate efficacy of antifungal drugs or combination thereof. With
all the advantages and methods developed for Mucorales in Galleria 3.3.3 Immunological aspects investigated by the
larvae, only a higher standardization of protocols is needed to make use of Galleria larvae
intra-laboratory comparisons easier. For example, a striking point, Two studies investigated the role of the spore surface layers or
when comparing the methods sections of individual manuscripts, is proteins in the recognition process, effect on phagocytosis leading
the great variability in inoculum size used even for the same strain. consequently to changes in virulence potential. In Lichtheima
Ranges differ between 2×103 spores – 107 spores per larva, leading corymbifera, spores were physically and chemically modified to
to more or less the same mortality rates. Reasons thereof comprise get rid of one cell wall layer after the other to determining the
the different larval batches that are used (in which larval size can influence of each layer in recognition and on the spore uptake by
vary a lot), potential differences in growth medium to obtain the macrophages. Surface modification led to an increased rate of
spores solutions, and in inoculum preparation, where no phagocytosis especially in those confronted with glucanolytic
standardized protocols are yet defined for filtration of spore enzymes or pronase E. In Galleria larvae a combination of the
solutions, storage, or freezing temperatures. Having comparable latter led to attenuated virulence potential (Hassan et al., 2021). A
data and deciphering the impact of variabilities in these simple study deciphering the role of cotH genes in M. lusitanicus, showed
methodological aspects might improve the quality of data obtained comparable data in larvae and a diabetic ketoacidosis (DKA) mouse
from Galleria experiments in the future. model. Of the five cotH genes that were deleted (Mucor has 17 cotH
The larval model has also been used frequently for studying genes altogether), two deletions, cotH3 and cotH4 caused
mucormycetes. Pioneering works have been reviewed in detail by attenuated virulence compared to the wild type strains. Contrary
Jacobsen already (Jacobsen, 2019). They comprise investigations of to Lichtheimia, where changes in the proteins of spore surfaces
inter-and intraspecies dependent virulence potential, correlating rendered them easier to phagocytize, Mucor lacking CotH proteins
differences in temperature adaptation, spores size, growth speed, were not affected in their uptake by macrophages, nor was the
oxidative stress tolerance, or if higher iron concentrations in the acidification of phagosomes impaired (Szebenyi et al., 2023).
growth medium affect virulence of spores (Schwartze et al., 2012;
Kaerger et al., 2015; Maurer M. et al., 2019). 3.3.4 The use of G. mellonella for studying
antifungal efficacies
3.3.2 The use of G. mellonella for studying Galleria larvae were also employed to test antifungal treatment
virulence factors efficacy against mucorales species. The first such study by Maurer
A recent study investigating the thermal adaptation of Mucor et al. investigated efficacy of a single dose of azoles and polyenes,
irregularis, once more, demonstrated that strains growing well in including a modified formulation of nystatin, nystatin-IL, and
high temperature also cause higher mortality rates in infected isavuconazole. The single application of nystatin-intralipid
larvae, which was underlined by higher fungal burden determined exhibited the best activity against Mucorales, followed by
by fluorescent staining of homogenized dead larvae (Zhang et al., posaconazole, while limited efficacy was seen for liposomal
2022). Deciphering virulence traits on a genetic level has been amphotericin B and isavuconazole in larvae. Furthermore, the
delayed in Mucorales due to unavailability of annotated genome effect on hemocyte density; was evaluated to rule out an
sequences and their reluctance to genetic modification. Luckily, this unspecific immune response caused by the drug. Importantly,
has improved tremendously in the last decade and, although it is pharmacokinetics of antifungals can, and probably should be
still more difficult than in other fungi, great tools and protocols have examined before concluding (in)efficacy of drugs. Maurer et al.
been established to delete or silence genes (Lax et al., 2022). To showed a linear increase in C mas and AUC 0-24 in larval
decipher the role of these genes for virulence, Galleria larvae have haemolymph which is similar to human serum, but differences
been employed. RNAi silenced mutants of M. circinelloides f. emerged regarding metabolism and half-life. These parameters are
lusitanicus (now M. lusitanicus) showed that phospholipaseD and relatively easy to study in the larvae and would contribute to a better
a myosin V family transporter are necessary to establish full comparison between larval and mammalian models (Astvad et al.,
virulence in both, Galleria larvae and mice (Trieu et al., 2017). 2017; Maurer E. et al., 2019). Recently, combinatorial treatment
The same group recently demonstrated that myosin II plays an regimen of voriconazole and either amphotericin B, posaconazole
important role in growth, sporulation and hyphal development in or caspofungin were evaluated in Galleria larvae increasing larval
Mucor. Interestingly, only disruption of one myosin II gene, survival in comparison to the single treatments. Of note, multiple
mcmyo2A, led to abrogation of pathogenicity, while mcmyo2B injections at 3 time points (2 h, 26 h and 50 h post infection) of the

antifungals drugs were carried out and no negative effect on the glabrata, Crpytococus neoformans and Aspergillus fumigatus
larvae by the multiple injections was observed (Macedo et al., 2019). (Matsumoto and Sekimizu, 2019).
One of the limitations of in vitro antifungal susceptibility testing is,
that in standardized protocols, drug efficacy is tested against spores, 3.4.2 The use of B. mori for studying antifungals
a morphology rarely found on the site of infection. Lately, a single and screening virulence potential of strains
dose of amphotericin B (10 mg/kg) against spores or germlings of R. Moreover, Yu et al. devised an easy-to-use protocol for
arrhizus and L. corymbifera was shown to be efficient only against visualizing diseases progression of filamentous fungal infection
infections with spores but not germlings, where all larvae died 24 h and evaluating treatment results with antifungal agents (Yu et al.,
after infection, independently of receiving amphotericin B or not 2021). Post-infection with A. fumigatus and L. corymbifera, they
(Samdavid Thanapaul et al., 2023). dissected and stained larval organs with calcofluor white for the
Attempts to facilitate and speed up in-vivo antifungal visualization of fungal growth and microscopic examination. By
susceptibility testing have been made for Candida and Aspergillus optimizing their protocol, the authors could raise the maximum
species, in which luciferase reporter strains were used for in-vivo temperature for larval incubation to 34°C, reassembling conditions
detections of infections and monitoring of drug efficacy in either closer to those in the human host. Tominaga et al. utilized the
living larvae or the homogenized pulp of the infected animal silkworm infection assay to assess the dose-dependent mortality of
(Delarze et al., 2015; Vanhoffelen et al., 2023). In Mucorales infection with R. oryzae (Tominaga et al., 2018). Additionally, they
luciferase expressing strains have been generated in M. tested the efficacy of amphotericin B, as well as new, potentially
lusitanicus, but light emission was shown to be too weak for antifungal, compounds with moderate efficacies isolated from a hot
animal experiments in strains with non-codon optimized spring-inhabiting Penicillium sp. in the in-vivo model. Similarly,
luciferase. Luciferase expressing strains, once codon optimized, Kurakado et al. evaluated the efficacy of posaconzaole against eleven
will not only facilitate detecting fungal infections but could also R. oryzae isolates of different origins and examined variations in
shed light on in-vivo transcriptional regulation of virulence relevant mortality rates based on incubation temperature and inoculum size
genes in the future. Moreover, Galleria larvae definitely represent a (Kurakado et al., 2021). By reducing the inoculum size, they
very useful tool for prescreening assays enabling the reduction of increased the incubation temperature to 37°C and successfully
mouse studies needed for confirmation. demonstrated posaconzaole’s therapeutic effect in the silkworm
model. In addition, Panthee et al. isolated novel pathogenic
strains belonging to Mucor and Backusella spp from plant
3.4 Bombyx mori larvae biomass (Panthee et al., 2021). They demonstrated their
pathogenicity in immunocompromised mice and the silkworm
3.4.1 General advantages and disadvantages model, expanding the understanding of these strains’ virulence.
The silkworm, Bombyx mori, stands as a domesticated insect
species that has played a vital role in human economies for more
than 5000 years (Xu and O’Brochta, 2015). Similar to other 3.5 Mealworm (Tenebrio molitor)
invertebrate model organisms, the silkworm provides distinct
advantages in life science research due to its easy propagation, Finally, Canteri de Souza et al. introduced the mealworm,
small size and manageable maintenance. Additionally, extensive Tenebrio molitor, as an additional host for investigating fungal
genomic information has been scrutinized and a myriad of tools for infections (de Souza et al., 2015). Notably, this model can be
genetic manipulation have been successfully established (Toshiki maintained at temperatures up to 37°C. Fusco-Almeida et al. offer
et al., 2000; Xia et al., 2008; Wei et al., 2014; Xu and O’Brochta, a comprehensive review of its applications for medically relevant
2015; Baci et al., 2022). The silkworm, like other invertebrates, fungi, however, as of yet, it has not been applied for the study of
depends solely on its innate immune response, encompassing mucormycosis (Fusco-Almeida et al., 2023).
mechanisms such as melanization, phagocytic cells, antimicrobial
peptides and lysozymes, to combat microbial infections. Detailed
reviews on the immune strategies of this model organism can be 4 Vertebrate models
found in works by Chen & Lu (Chen and Lu, 2018) and Kausar et al.
(2019). In the realm of infection biology, precise concentrations of 4.1 Zebrafish (Danio rerio)
infectious agents can be administered via injection into the
hemolymph or into the midgut of the 5th instar larvae (Meng 4.1.1 General aspects, advantages
et al., 2017). This offers an advantage in contrast to the G. mellonella and disadvantages
model where infections are usually injected into the hemolymph The zebrafish, D. rerio, is naturally occurring in the river basins
without any organs specifically addressed (Matsumoto and of the northeastern Indian subcontinent and stands as one of the
Sekimizu, 2019). Due to the convenient size, organ extraction is most widely employed model organisms in biological research. Its
feasible, enabling the study of dissemination of spores from the small size, robustness, excellent reproductive performance and
hemolymph (or midgut) to various tissues types (Yu et al., 2021). rapid development render it easy and cost-effective for laboratory
The silkworm has been established for studying bacterial infections maintenance and experimentation (Spence et al., 2008). With
as well as number of medical relevant fungal species like Candida detailed genomic information, well-established tools for genetic

manipulation, transparency of the larvae, allowing observation cases of mucormycosis in immunocompetent patients (Hammami
through live microscopy as well as its closer resemblance to et al., 2021; He et al., 2021). Earlier work, examining the influence of a
humans compared to invertebrate models make it a strong model different endosymbiont of R. microsporus, namely Burkholderia spp.,
for the study of infectious diseases (Howe et al., 2013; Rosowski showed contradictory results, as no link between the presence of
et al., 2018). Both larvae and adult fish are employed for these Burkholderia spp. as an endosymbiont and fungal virulence could be
investigations, with larvae being particularly suitable for exploring established. These studies highlight the species specific interactions of
the innate immune response due to the absence of an adaptive bacterial-fungal symbiosis in virulence (Ibrahim et al., 2008).
immune system (Rosowski et al., 2018). Regardless of differences in Kousser et al. revealed that co-culturing of R. microsporus and
anatomy, the zebrafish can be used to study infections occurring in Pseudomonas aeruginosa lead to inhibition of fungal growth
humans by injecting analogous organs with defined inocula (Gomes through the bacteria’s secretion of siderophores, specifically
and Mostowy, 2020). One major disadvantage of this model, pyoverdine (Kousser et al., 2019). They further demonstrated that
compared to invertebrates, is the need for specialized equipment administering this siderophore would reduce the virulence of R.
for laboratory maintenance (Jacobsen, 2019). Moreover, even microsporus through iron starvation in the zebrafish larvae model
though wild zebrafish are able to tolerate temperature ranges compared to a siderophore that the fungus is able to to take up.
from 10 to 38°C, domestication of zebrafish led to a reduced Expanded up on previous findings related to phagocyte
physiological plasticity due to their constant maintenance at their recruitment, Ló pez-Muñoz et al. showed that an infection with
optimal temperature around 28°C (Morgan et al., 2022). sporangiospores of M. lusitanicus lead to a decrease in myeloid cells
in the hematopoietic organ of the adult zebrafish, indicating their
4.1.2 The use of Danio rerio for studying the mobilization to the site of infection (Ló pez-Muñoz et al., 2018).
interplay of immune responses and Additionally, they demonstrated that the fungus could evade
virulence factors immunity though inducing cell-death in the macrophages but not
For the first time employing the zebrafish model for the study of in neutrophils, emphasizing the importance of neutrophils in
mucormycosis, Voelz et al. conducted experiments using the larval protection from fungal diseases and the vulnerability of patients
model to analyze the real-time-interaction between innate immune with neutropenia (Ghuman and Voelz, 2017). Furthermore, the
cells and M. lusitanicus (formerly M. circinelloides f. lusitanicus) authors demonstrated that a larger size of sporangiospores
sporangiospores in-vivo (Voelz et al., 2015). They could show that constitute a factor leading to higher virulence of the fungus.
severity of the infection depended on infection site and was more Employing the adult zebrafish as a model to examine the effects
pronounced and caused death sooner under dexamethasone-induced of an infection by Rhizopus oryzae, with and without
immunosuppression. In a follow-up study that combined zebrafish immunosuppression, Tatara et al. did not observe a statistical
larvae with mathematical modelling, Inglesfield et al. could show that significant difference between the two trial groups (Tatara et al.,
commencement of the disease depended on the number of recruited 2018). These results contradict earlier observations made by Voelz
phagocytes in-silico (Inglesfield et al., 2018). These results were et al. for the zebrafish larvae, yet no specific hypothesis could be
backed by the absence of spore killing and reactive oxygen burst found for this phenomenon (Voelz et al., 2015).
in-vivo. Furthermore, they demonstrated that formation of
granulomas is crucial for the control of fungal growth, which could
recrudesce through immunosuppression. Building up on these 4.2 Embryonated chicken (Gallus
previous studies, Itabangi et al. investigated the impact of domesticus) egg
endosymbiotic bacteria, specifically Ralstonia picketti, in R.
microsporus on virulence and immune evasion (Itabangi et al., 4.2.1 General aspects, advantages
2022). The authors demonstrated that bacteria free spores were and disadvantages
avirulent compared to bacteria harboring wild-type spores in A little more than 10 years ago, fertilized chicken eggs have been
zebrafish larvae. For symbiont-free spores, the effect was employed as one alternative vertebrate model to study fungal
independent of inoculum size and their morphological state (pre- pathogenicity. The fertilized eggs provide the expected advantages
swollen or resting). In contrast, endosymbiont-harboring spores like low costs, possibility of high throughput screening, facile
showed higher virulence when pre-swollen. Additionally, the maintenance and, importantly, are incubated at 37°C.
authors employed transgenic zebrafish larvae with fluorescently Furthermore, administration of the pathogenic agent is possible
labeled phagocytes to examine macrophage and neutrophil via different routes (Jacobsen et al., 2012).
recruitment. They demonstrated that the recruitment of both cell
types was significantly lower in zebrafish larvae infected with 4.2.2 The use of embryonated chicken egg for
endosymbiont-containing spores. The authors regarded the lower studying virulence factors
recruitment of immune cells as a form of immune evasion possibly So far, the model has only been used in two works involving
mediated through secreted metabolites by the bacteria or their clinically relevant mucormycetes. They have shown to be a valuable
influence on the biological traits of the fungus, such as cell wall tool for comparing the virulence potential of a high number of
composition. Furthermore, they hypothesized that endosymbiont strains. Evaluating the virulence potential in Rhizopus and its
supported immunevasion could provide a possible explanation for correlation to temperature, stress resistance and morphological

characteristics Kaerger et al. showed that adaptation to host effects of the toxin onto the amoeba, ultimately providing
temperature is not the single factor determining pathogenicity in protection to the fungus (Richter et al., 2022). Whether virulence
comparison to species with less tolerability to 37°C (Kaerger et al., factors of other mucorales, such as those inducing apoptosis in
2015). Similarly, different virulence potential was determined for macrophages as observed by Ló pez-Muñoz et al. for M.
species of the genus Lichtheimia which reflect the species isolated circinelloides, originated from interactions between amoeba and
from patient material (Schwartze et al., 2012). To our best this Mucor species requires verification in an amoeba model
knowledge, no other studies including embryonated chicken eggs (Ló pez-Muñoz et al., 2018).
to study mucormycosis relevant mechanisms have been published.
Even though, this model could also be used for testing antifungal
efficacy and toxicity of novel drugs (Ribeiro et al., 2022). 6 Summary and outlook
As described in this review, the different well-established
5 Other alternative models greatly facilitate compiling data to enhance our
understanding of mucormycosis. The types of experiments
According to the ‘Amoeboid Predator-Fungal Animal performed and their significance for potential clinical applications
Virulence’ Hypothesis, amoeba predation on fungi leads to are summarized in Table 1. All models comprise advantages and
evolutionary selection of fungal traits being advantageous for disadvantages (summarized in Table 2), which mostly hold true for
infecting animal hosts. These traits not only protect fungi from investigating other fungal pathogens as well. The lack of
the host immune system but also influence interactions with standardized procedures and protocols and the lack of mimicking
phagocytizing amoeba (Casadevall et al., 2019). The evasion of other forms of disease than the disseminated one are the most
host phagocytosis by certain fungal species like Cryptococcus prominent. Besides those limitations, the beneficial nature of the
neoformans to evade mediated host immunity has been presented models for pre-screening and evaluation of large strain
investigated using amoeba, such as Acanthamoeba castellani or sets or high number of antifungal agents, is especially important to
Dictyostelium discoideum (Steenbergen et al., 2001). Using the meet the great variability seen in mucormycosis and,on long track,
Dictyostelium model for phagocytosis, Itabangi et al. helps to minimize mammalians needed in follow-up experiments.
demonstrated that R. microsporus, in symbiosis with a metabolite To incorporate translational aspects, like the potential of diagnosing
secreting bacterial endosymbiont, suppressed the amoeba growth pulmonary mucormycosis via a breath test, or the efficacy of novel
and inhibited their predation on other microbes (Itabangi et al., antifungal drugs, data for all clinically relevant Mucorales species
2022). These findings aligned with results from human macrophage will be needed, an aim that can easier be met with alternative model
assays, where endosymbiont-containing spores protected the systems. Enhancing transcriptomics and proteomics studies of the
fungus from phagocytosis. Similarly, Richter et al. demonstrated individual model challenged with Mucorales, will greatly advance
that co-culturing the amoeba Prostelium aurantium with Rhizopus the suitability and comparability of the respective system. Following
spores, hosting rhizoxin producing bacteria, resulted in detrimental the principle of the 3 Rs (replacement, reduction, and refinement),
TABLE 1 Type of experiments performed in mucormycosis and their potential translational application.
Model organism Tests performed in mucormycosis studies Translational aspects

(unique for mucormycosis in bold)
C. elegans • Study of virulence factors (Arf and Arf-like proteins, • Identification of potential new drug targets
fermentative metabolism, siderophore production) • Control of serum iron levels in patients
• Impact of growth medium on virulence • Targeted antibiotic use against endosymbiont-carrying strains
• Impact of endosymbionts/their products on virulence
D. melanogaster • Study of virulence factors (CotH-genes, iron-metabolism, • Identification of potential new drug targets
tornadic-sheer stress, VOCs) • Control of serum iron levels in patients
• Use of calcineurin inhibitors in traumatic wounds
• Use of VOCs in patients’ breath for diagnosis
G. mellonella • Study of virulence factors (CotH-genes, growth speed, • Identification of potential new drug targets
myosin genes, oxidative stress tolerance, siderophore • Control of serum iron levels in patients
production, spore size, spore surface composition) • Synergetic effects of drugs in antifungal therapy
• Testing of (new) antifungals and combination therapies • Identification of new virulence traits as putative novel drug
• Impact of growth medium on virulence (iron targets
concentration) • Awareness of changes in epidemiology
• Impact of incubation temperature
• Screening of virulence potential of different strains
B. mori • Testing of new antifungals • Potential new antifungals substances

• Screening of virulence potential of environmental • Identification of potentially pathogenic species (awareness
derived strains in diagnostic)
(Continued)

TABLE 1 Continued
Model organism Tests performed in mucormycosis studies Translational aspects

(unique for mucormycosis in bold)
D. rerio • Study of virulence factors (spore size) • Identification of potential new drug targets
• Study of different infections sites • Identification of strains with clinical relevance
• Study of phagocyte recruitment • Manifestation of diseases depending on tissue type
• Impact of endosymbionts • Antifungal prophylaxis in patients with neutropenia
• Impact of microbial interactions with P. aeruginosa in • Targeted antibiotic use against endosymbiont-carrying strains
traumatic wounds • Cautious use of antibiotics in traumatic wounds with
potential fungal infections
Chicken egg • Screening of virulence potential of different strains and • Identification of potential new drug targets
their virulence traits • Identification of strains with clinical relevance and
correlation to origin
Amoeba • Impact of endosymbionts in evasion of phagocytosis • Targeted antibiotic use against endosymbiont-carrying strains
TABLE 2 Comparison of the alternative model organisms with focus on mucormycosis.
Caenorha- Drosophila Galleria Bombyx Danio Chicken Amoeba

bditis melanog- mellonella mori rerio egg
elegans aster
General characteristics
- Breeding Yes Yes Yes Yes Yes Yes Yes
- costs Very low Reasonable Very low Very low Reasonable Low Very low
- commercial availability No Yes Yes Yes Yes Yes No

1 2
- Ethics approval necessary No No No No Yes Yes No
- Temperature range 15-25°C 12-30°C 20-42°C up to 37°C 28-28.5°C 37°C 22-28°C3
- Genetic manipulation Possible Possible Not possible Possible Possible Not possible Possible
- Gene homology to humans 65% 60% n. d. 58% 70% 75% n. d.
Experimental handling
- Ease of handling Easy Special training Very easy Very easy Special training Very easy Very easy
and and
facilities needed facilities needed
- Injection of defined inoculum Not possible Not possible Possible Possible Possible1 Possible Not possible
- Multiple injections No injection Not possible Possible Possible Possible1 Possible No injection
without harm
- Dissection of organs Not possible Not possible Possible Possible Possible1 Possible Not possible
- Different infection routes Not possible Not possible Not possible Possible Possible1 Possible Not possible
- Transcriptomics/proteomics Yes Yes Yes Yes Yes n. d. Yes
Evaluation of antifungal agents Yes Yes Yes Yes Yes Yes Yes
- Tested against Mucorales No Yes Yes Yes No No No
Correlation of virulence potential Yes Yes Yes Yes Yes Yes Yes
to mammals
Immunology
- Similarities in innate immunity Yes Yes Yes Yes Yes Yes Yes
1 2
- adaptive immunity & cytokines No No No No Yes Yes No
- Ex vivo studies with Not possible Possible Possible Possible Possible Not possible Not possible
immune cells
(Continued)

TABLE 2 Continued
Caenorha- Drosophila Galleria Bombyx Danio Chicken Amoeba

bditis melanog- mellonella mori rerio egg
elegans aster
- Induction Possible Possible n. d. Possible Possible n. d. Not possible
of immunosuppression
1
for adult zebrafish.
2
by the 18th day.
3
depending on species.
replacing animal models for studying mucormycosis with more Conflict of interest
advanced cell-culture based methods has not yet been exhausted
fully, which holds promising applications for advancing The authors declare that the research was conducted in the
mucormycosis research in the future. absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Author contributions
JS: Writing – original draft, Writing – review & editing. UB:
Conceptualization, Writing – original draft, Writing – review
Publisher’s note
& editing.
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
Funding reviewers. Any product that may be evaluated in this article, or
The author(s) declare that no financial support was received for claim that may be made by its manufacturer, is not guaranteed or
the research, authorship, and/or publication of this article. endorsed by the publisher.
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TYPE Review

PUBLISHED 01 February 2024

Alternative in-vivo models

Frontiers in Cellular and Infection Microbiology 01 frontiersin.org

Frontiers in Cellular and Infection Microbiology 02 frontiersin.org

Frontiers in Cellular and Infection Microbiology 03 frontiersin.org

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Frontiers in Cellular and Infection Microbiology 05 frontiersin.org

Frontiers in Cellular and Infection Microbiology 06 frontiersin.org

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Model organism Tests performed in mucormycosis studies Translational aspects

B. mori • Testing of new antifungals • Potential new antifungals substances

Frontiers in Cellular and Infection Microbiology 09 frontiersin.org

Model organism Tests performed in mucormycosis studies Translational aspects

TABLE 2 Comparison of the alternative model organisms with focus on mucormycosis.

Caenorha- Drosophila Galleria Bombyx Danio Chicken Amoeba

- Breeding Yes Yes Yes Yes Yes Yes Yes

- commercial availability No Yes Yes Yes Yes Yes No

- Temperature range 15-25°C 12-30°C 20-42°C up to 37°C 28-28.5°C 37°C 22-28°C3

- Gene homology to humans 65% 60% n. d. 58% 70% 75% n. d.

- Transcriptomics/proteomics Yes Yes Yes Yes Yes n. d. Yes

- Tested against Mucorales No Yes Yes Yes No No No

Frontiers in Cellular and Infection Microbiology 10 frontiersin.org

Caenorha- Drosophila Galleria Bombyx Danio Chicken Amoeba

Frontiers in Cellular and Infection Microbiology 11 frontiersin.org

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Frontiers in Cellular and Infection Microbiology 14 frontiersin.org

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