Author's personal copy

Provided for non-commercial research and educational use only.

Not for reproduction, distribution or commercial use.

This chapter was originally published in the book Genetics and Molecular Biology of
Entomopathogenic Fungi, Volume 94. The copy attached is provided by Elsevier for the
author's benefit and for the benefit of the author's institution, for non-commercial
research, and educational use. This includes without limitation use in instruction at
your institution, distribution to specific colleagues, and providing a copy to your
institution's administrator.

All other uses, reproduction and distribution, including

without limitation commercial reprints, selling or
licensing copies or access, or posting on open
internet sites, your personal or institution’s website or
repository, are prohibited. For exceptions, permission
may be sought for such use through Elsevier’s
permissions site at:
http://www.elsevier.com/locate/permissionusematerial

From Wang, J. B., St. Leger, R. J., & Wang, C. (2016). Advances in Genomics of
Entomopathogenic Fungi. In B. Lovett&R. J. St Leger (Eds.), Genetics and Molecular
Biology of Entomopathogenic Fungi (pp. 67–105).
ISBN: 9780128046944
Copyright © 2016 Elsevier Inc. All rights reserved.
Academic Press
 Author's personal copy

CHAPTER THREE

Advances in Genomics of
Entomopathogenic Fungi
J.B. Wang*, R.J. St. Leger*, 1 and C. Wangx, 1
*University of Maryland, College Park, MD, United States
x
Chinese Academy of Sciences, Shanghai, China
1
Corresponding authors: E-mail: [email protected]; [email protected]

Contents
1. Introduction 68
2. Evolutionary Relationships of Entomopathogenic Fungi 70
3. Evolution of Sex in Entomopathogenic Fungi 72
4. Evolution of Fungal Host Specificity 77
5. Protein Family Expansions and Contractions 80
5.1 Signal Transduction 85
5.2 Carbohydrate-Active Enzymes 87
5.3 Secondary Metabolites and Host Interaction 89
5.4 Protein Families Involved in Detoxification and Stress Responses 92
6. Horizontal Gene Transfer 94
7. Conclusions and Future Perspectives 96
Acknowledgments 97
References 97

Abstract
Fungi are the commonest pathogens of insects and crucial regulators of insect popula-
tions. The rapid advance of genome technologies has revolutionized our understanding
of entomopathogenic fungi with multiple Metarhizium spp. sequenced, as well as
Beauveria bassiana, Cordyceps militaris, and Ophiocordyceps sinensis among others. Phylo-
genomic analysis suggests that the ancestors of many of these fungi were plant endo-
phytes or pathogens, with entomopathogenicity being an acquired characteristic.
These fungi now occupy a wide range of habitats and hosts, and their genomes have
provided a wealth of information on the evolution of virulence-related characteristics,
as well as the protein families and genomic structure associated with ecological and eco-
nutritional heterogeneity, genome evolution, and host range diversification. In particular,
their evolutionary transition from plant pathogens or endophytes to insect pathogens
provides a novel perspective on how new functional mechanisms important for host
switching and virulence are acquired. Importantly, genomic resources have helped
make entomopathogenic fungi ideal model systems for answering basic questions in
parasitology, entomology, and speciation. At the same time, identifying the selective
Advances in Genetics, Volume 94
© 2016 Elsevier Inc.
j
ISSN 0065-2660
http://dx.doi.org/10.1016/bs.adgen.2016.01.002 All rights reserved. 67

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
68 J.B. Wang et al.

forces that act upon entomopathogen fitness traits could underpin both the develop-
ment of new mycoinsecticides and further our understanding of the natural roles of these
fungi in nature. These roles frequently include mutualistic relationships with plants. Ge-
nomics has also facilitated the rapid identification of genes encoding biologically useful
molecules, with implications for the development of pharmaceuticals and the use of
these fungi as bioreactors.

1. INTRODUCTION
Entomopathogenic fungi are particularly well suited for development
as biopesticides because unlike bacteria and viruses that have to be ingested
to cause diseases, fungi typically infect insects by direct penetration of the
cuticle followed by multiplication in the hemocoel (St. Leger, Wang, &
Fang, 2011). However, entomopathogenic fungi are very heterogeneous;
both they and their hosts have short generation times, and they occupy a
wide range of habitats, with near ubiquity in the soil and on plants. The
interactions between fungi, hosts, and the environment are therefore diverse
and dynamic, which complicates comparisons between different fungi
infecting different insects since their interactions may be necessarily dispa-
rate. The commonly accepted solution to this quandary was to pick a couple
of related fungal species for a thorough study of hostepathogen interactions,
and subsequently make comparisons with other distantly related species.
Consequently, most of what we know about the biochemical and molecular
basis of interactions between fungi and insects has been determined with the
experimentally tractable hypocrealean ascomycete genera Metarhizium
(family Clavicipitaceae) and Beauveria (family Cordycipitaceae). These fungi
are able to degrade, penetrate, and assimilate the insect cuticle using a com-
bination of cuticle-degrading enzymes and mechanical pressure while over-
coming any stresses encountered along the way (Ortiz-Urquiza & Keyhani,
2013). Upon reaching the hemocoel, the fungi quickly multiply by success-
fully competing for nutrients and avoiding host immunity.
Compared with the usual fungal model systems such as the yeast Saccha-
romyces cerevisiae, Metarhizium and Beauveria are extraordinarily versatile.
Metarhizium, for example, contains species that are narrow host range (eg,
Metarhizium album, Metarhizium acridum) or broad host range (eg, Meta-
rhizium robertsii, Metarhizium anisopliae) pathogens of arthropods, as well as
being saprophytes, and colonizers of the rhizosphere and plant root. Consis-
tent with their broad lifestyle options, most Metarhizium spp. exhibit an
extremely flexible metabolism. This metabolism enables them to grow

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 69

under various environmental conditions, with sparse nutrients (Rangel,

Alston, & Roberts, 2008) and in the presence of compounds lethal to other
fungi (Ortiz-Urquiza & Keyhani, 2015; Roberts & St. Leger, 2004).
Because of their mode of infection through the cuticle, fungi function as
contact insecticides (Thomas & Read, 2007). Biocontrol researchers have
therefore made a tremendous effort to find naturally occurring fungal
pathogens capable of controlling mosquitoes and other pest insects. This
typically involved the selection of strains pathogenic to target insects without
considering the mechanisms involved or the role of these fungi in their
natural habitats. Only recently has progress been made in determining the
factors that influence the distribution, population structure, and econutri-
tional characteristics of any entomopathogenic fungi, even Metarhizium
and Beauveria. These fungi are known to employ a vast array of metabolites
to aid in infection and to outcompete other microbes, and recent genetic
studies have revealed the mechanisms and importance of a portion of these
metabolites for virulence, but much still remains unknown (see Donzelli and
Krasnoff, 2016). These deficiencies have hindered realization of the poten-
tial of these fungi as classical biocontrol agents that persist in the environ-
ment and recycle through pest populations (Hajek, McManus, &
Delalibera, 2007; Hajek & Tobin, 2011; Wang & Feng, 2014).
As well as their direct benefit to agriculture and vector control as insect
pathogens, Cordyceps/Ophiocordyceps spp. are medicinally valued and insect
pathogens in general are prolific producers of enzymes and diverse secondary
metabolites with activities against insects, fungi, bacteria, viruses, and cancer
cells (Hu et al., 2013; Isaka, Kittakoop, Kirtikara, Hywel-Jones, &
Thebtaranonth, 2005; Kim et al., 2010; Liu et al., 2015; Zheng et al.,
2011). Enzymes from Metarhizium and Beauveria spp. are frequently
exploited as industrial catalysts (Pereira, Noronha, Miller, & Franco, 2007;
Silva et al., 2009), even though the responsible genes for these products
are rarely identified.
Comparative genomics offers a way forward by disentangling common
themes of fungal biology from specific components involved in insect
pathology and allowing broad host range pathogens to be studied in the
context of narrow host range pathogens (Wang & St. Leger, 2014). It is
also extremely valuable for assessing poorly characterized species such as
Ophiocordyceps sinensis. Comparative genomics has facilitated identifying
fungal fitness traits and the selective forces that act upon them to improve
our understanding of how and why entomopathogenic fungi interact
with insects and other components of their environments. Thus, sequence

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
70 J.B. Wang et al.

data can provide crucial information on the poorly understood ways that
these organisms reproduce and persist in different environments. Alongside
the recent availability of genomic resources, the wide array of experiments that
can be performed with entomopathogenic fungi make them ideal models for
answering basic questions on the genetic and genomic processes behind adap-
tive phenotypes (a “Holy Grail” in biology). Key challenges for fungi as
models for other eukaryotes include identifying the genes involved in ecolog-
ically relevant traits and understanding the nature, timing, and architecture of
the genomic changes governing the origin and processes of local adaptation
(Gladieux et al., 2014). These outstanding evolutionary questions are partic-
ularly important for biocontrol agents and address fundamental, yet poorly
understood, issues by asking: what roles do different kinds of mutations
play in adaptation? When organisms adapt to new environments, do they
do so because of changes in few genes or many? Are the same genes or net-
works involved in independent cases of adaptation to the same environment?
What is the timescale at which evolutionary processes happen?

2. EVOLUTIONARY RELATIONSHIPS OF
ENTOMOPATHOGENIC FUNGI
Fungal species of different phyla like Microsporidia, Chytridiomycota,
Entomophthoromycota, Basidiomycota, and Ascomycota are known to
infect and kill insects (Shang, Feng, & Wang, 2015; Sung et al., 2007).
The two best-studied groups are the ascomycete entomopathogens and
the Entomophthoromycota. Researchers studying Entomophthoromycota,
which are not easy to mass-produce, have focused on the ecology of these
organisms and their role as causative agents of mass epizootics. As of
February 2016, one published Entomophthoromycota genome (Conidiobo-
lus coronatus (Chang et al., 2015)) and nine incomplete Entomophthoromy-
cota genome sequencing projects were listed in the Genomes OnLine
Database (GOLD) (these are discussed in De Fine Licht, Hajek, Eilenberg
& Jensen, 2016). Ascomycete entomopathogens are usually developed as
inundative control agents which are applied en masse to a pest population
(Wang, Fan, Li, & Butt, 2004). To date, there is much more genomic infor-
mation on ascomycete insect pathogens, as sequences are available from nine
Metarhizium strains (Gao et al., 2011; Hu et al., 2014; Pattemore et al., 2014;
Staats et al., 2014), Beauveria bassiana (Xiao et al., 2012), Cordyceps militaris
(Zheng et al., 2011), Ophiocordyceps sinensis (anamorph, Hirsutella sinensis)
(Hu et al., 2013), Ophiocordyceps unilateralis (de Bekker et al., 2015),

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 71

Tolypocladium inflatum (Bushley et al., 2013), and Hirsutella thompsonii

(Agrawal, Khatri, Subramanian, & Shenoy, 2015). Ophiocordyceps unilateralis
is a specialized fungus responsible for zombie ant behavior and is discussed in
detail by Hughes et al., (2016).
The most well-studied insect ascomycete pathogens fall into three
families within the order Hypocreales: Cordycipitaceae, Clavicipitaceae,
and Ophiocordycipitaceae. From the reconstructed phylogeny (Fig. 1), it
is evident that entomopathogenicity evolved independently in these families
and that genera of hypocrealean entomopathogens cluster among closely
related phytopathogens, endophytes, and mycoparasites. These ancestral
associations are consistent with repeated transitions (host switching) between
plant, fungi, and insect hosts, as suggested by Suh, Noda, and Blackwell
(2001) in their study on Cordyceps spp. A comparative genome analysis of

Figure 1 Phylogenomic relationships of insect pathogenic fungi with other fungi. Note:
Some Metarhizium and Beauveria spp. have recently been recognized to be endophytes
(reviewed by Moonjely, Barelli & Bidochka, 2016).

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
72 J.B. Wang et al.

seven Metarhizium (Clavicipitaceae) genomes confirmed the genus as a

monophyletic lineage that diverged from clavicipetacean plant pathogens
and endophytes about 231 million years ago (MYA), and placed the hemip-
teran-specific M. album as basal to the Metarhizium clade with an estimated
divergence time about 117 MYA (Hu et al., 2014). It was suggested that
the close physical proximity of the plant-associated ancestor of M. album
to plant-sap sucking hemipteran bugs may have facilitated this particular
host switch to entomopathogenicity (Hu et al., 2014).
Several Metarhizium spp. maintain complicated relationships with plants
and colonize plant roots and the rhizosphere (the layer of soil influenced by
root metabolism) (Hu & St. Leger, 2002; Pava-Ripoll et al., 2011). Different
species/strains of Metarhizium show differing abilities to form associations
with different plant species (Bidochka, Kamp, Lavender, Dekoning, &
Amritha De Croos, 2001; Steinwender et al., 2015), but the genetic under-
pinning of this specificity is unknown. However, M. robertsii has mechanisms
for rapidly adapting to new soil habitats and plants which involves changes in
expression of cell wall and stress response genes (Wang, O’Brien, Pava-
Ripoll, & St. Leger, 2011). As shown by their antagonism to plant patho-
genic fungi (Kang, Goo, Gyu, & Heon, 1996), ability to survive exposure
to lead and other heavy metals (Rhee, Hillier, & Gadd, 2012), and pathoge-
nicity to soil amoebae (Bidochka, Clark, Lewis, & Keyhani, 2010), at least
some Metarhizium isolates have additional unpredicted flexibility in their tro-
phic capabilities. Beauveria bassiana (Cordycipitaceae) also forms intimate
endophytic relationships with a broad range of plant species, although it usu-
ally colonizes aerial parts of the plant (Biswas, Dey, Satpathy, & Satya, 2012;
Brownbridge, Reay, Nelson, & Glare, 2012; Ownley, Gwinn, & Vega,
2010; Vega et al., 2008; Wagner & Lewis, 2000), suggesting that flexibility
of lifestyle is a theme in hypocrealean insect pathogens.

3. EVOLUTION OF SEX IN ENTOMOPATHOGENIC

FUNGI
As robust and diverse genetic models, fungi provide invaluable insights
into the evolution and mechanism of eukaryote sexuality (Dyer &
O’Gorman, 2012; Ni, Feretzaki, Sun, Wang, & Heitman, 2011). Fungi
exhibit diverse reproductive modes that often determine the rates and
patterns of genome evolution (Ni et al., 2011; Whittle, Nygren, &
Johannesson, 2011; Zheng, Xia, Zhang, & Wang, 2013), and, as exemplified
by entomopathogens, are linked as cause or effect with pathogenic strategies.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 73

There are three modes of sexual reproduction in ascomycetous fungi, ie,

heterothallic, homothallic, and pseudohomothallic, which are governed by
the mating-type (MAT) locus, a miniature fungal version of a sex chromo-
some coding for transcription factors (TFs) that induce the production of
pheromones and pheromone receptors (Fraser et al., 2004; Idnurm, 2011;
Kronstad & Staben, 1997; Whittle et al., 2011). Two opposite loci are
named as MAT 1-1 and MAT 1e2, respectively (or MATA and MATa,
in different fungi) (Alby, Schaefer, & Bennett, 2009; Whittle et al., 2011;
Zheng et al., 2013). The haploid genome of heterothallic species carries
only one of the MAT loci, thus they are self-sterile, requiring a haploid part-
ner with a compatible MAT locus to complete the sexual cycle. Homothal-
lic species (self-fertile) have both loci in their haploid genomes, while the
pseudohomothallic (also called secondary homothallic) species are similarly
self-fertile, but they contain two compatible haploid nuclei within their
sexual spores (Ni et al., 2011; Zheng et al., 2013). Respective genomes
of entomopathogenic fungi show single mating type loci in T. inflatum
(Ophiocordycipitaceae) (Bushley et al., 2013), Metarhizium spp. (Gao
et al., 2011), C. militaris (Zheng et al., 2011), and B. bassiana (Xiao et al.,
2012), suggesting that most members of the three hypocrealean entomo-
pathogen families (Clavicipitaceae, Cordycipitaceae, and Ophiocordycipi-
taceae) are heterothallic and are potentially therefore outcrossing fungi.
Syntenic analysis of these fungi showed that, except for the idiomorphic
regions, the genes flanking the mating-type locus are highly conserved,
especially between B. bassiana and C. militaris (Xiao et al., 2012). However,
unlike B. bassiana and many Metarhizium species (Kepler et al., 2012; Li, Li,
Huang, & Fan, 2001; Liu, Liang, Whalley, Yao, & Liu, 2001), C. militaris
commonly performs sexual reproduction (Zheng et al., 2011, 2013).
There are reproductive mode switches in the evolution of fungal
sexuality, eg, analysis of 43 species of Neurospora identified at least six in-
dependent switches from heterothallism to homothallism (Nygren et al.,
2011), while within the genus Aspergillus, Aspergillus nidulans is homothallic
while its relations Aspergillus fumigatus and Aspergillus oryzae are heterothal-
lic (Galagan et al., 2005; Peterson, 2008). It also seems that most asexual
Aspergillus lineages arose from sexual lineages (Geiser, Timberlake, &
Arnold, 1996). Signs of sex in supposedly asexual species include footprints
of repeat-induced point (RIP) mutations, an irreversible fungal genome
defense mechanism specific to fungi, occurring only during the sexual
stages on repeated sequences (Galagan et al., 2003). The consequences of
RIP are that repeated DNA segments, such as would result from the

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
74 J.B. Wang et al.

transposition of a retrotransposon, or the duplication of a gene, are inacti-

vated by mutations. Calculations of RIP indices indicated that RIP occurs
in narrow host range M. album and M. acridum, but not in the broad host
range species such as M. robertsii (Hu et al., 2014). RIP functions only dur-
ing meiosis, which suggests retention of sexuality in specialists, although
their sexual stages have not been verified (Hu et al., 2014). Intriguingly,
therefore, asexuality is associated with broad host ranges and sexuality
with narrow host ranges. Consistent with this, broad host range generalists
have many more heterokaryon incompatibility proteins (HETs) than spe-
cialists; this is indicative of tighter controls for achieving reproductive isola-
tion (Hu et al., 2014). HET gene function may have been dispensable in
specialists, as their life histories expose them to fewer alternative genotypes
than generalists, but, conversely, this suggests selection for retention of
reproductive isolation in nonspecialists. Reproductive isolation prevents
genetic homogenization and is crucial for speciation: in which case HET
genes may have played a key role in the evolution and diversification of
Metarhizium spp.
Unlike C. militaris, which is specific to lepidopteran pupae, B. bassiana
has a wide host range. Comparative genomics confirmed that asexual
B. bassiana is closely related to sexual Cordyceps spp. (Fig. 1), and a teleo-
morph (a fungus with a sexual reproductive stage) of B. bassiana has been
identified as Cordyceps bassiana (Li et al., 2001), but it is rarely observed in
the field. Beauveria bassiana, like generalist Metarhizium spp., lacks the RIP
mechanism, consistent with the sexual cycle being rare (Zheng et al.,
2013). Instead, B. bassiana reproduces clonally in the environment like broad
host range Metarhizium spp. (Meyling, L€ ubeck, Buckley, Eilenberg, &
Rehner, 2009; S.B. Wang, Fang, Wang, & St. Leger, 2011). Individual
isolates of B. bassiana carry either the MAT1-1 or MAT1-2 mating-type
locus (Yokoyama, Arakawa, Yamagishi, & Hara, 2006; Zheng et al.,
2013), indicating they retain the potential at least for heterothallic sexual
activity. Furthermore, many genes functioning in mating processes, karyog-
amy, meiosis, and fruiting-body development in other fungi are present in
B. bassiana (Xiao et al., 2012). However, B. bassiana lacks a meiosis-specific
topoisomerase, SPO11 which may contribute to its infrequent sexual cycle,
as SPO11 is crucial for initiating meiotic recombination by generating DNA
double-strand breaks (Xiao et al., 2012).
Despite their close phylogenetic relationship, there is an unexpectedly
high degree of genome structure divergence between B. bassiana and C. mil-
itaris. Transposable elements (TEs) are a major force driving genetic variation

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 75

and genome evolution (Cordaux & Batzer, 2009; Daboussi & Capy, 2003).
Beauveria bassiana has many more TEs than C. militaris (88 and 4, respec-
tively) (Xiao et al., 2012). RIP is incompatible with gene duplication events,
so its absence is consistent with expanded gene families and more TEs in the
B. bassiana and M. robertsii genomes, relative to C. militaris and M. acridum.
However, the genomes of Metarhizium species are highly syntenic in spite
of large differences in the number of TEs (148 TEs in M. robertsii vs 20
TEs in M. acridum) (Gao et al., 2011). Cordyceps militaris often reproduces
sexually (Zheng et al., 2011), whereas the sexual cycle in M. acridum is pre-
sumably less common, as it has not been observed in nature despite detection
of RIP signatures. Sexuality facilitates genome structure reorganization due
to frequent genetic and/or chromosomal recombination. These differences
in life cycle may have led to the considerable genome structure disparities
between B. bassiana and C. militaris (Xiao et al., 2012). In fact, syntenic re-
lationships between B. bassiana and Metarhizium spp. are closer than to
C. militaris (Xiao et al., 2012). This implies that genome reorganization,
and presumably therefore sexuality, in C. militaris has accelerated since it
shared a common ancestor with B. bassiana.
An extreme case of specialization is provided by the caterpillar fungus
O. sinensis, which mummifies ghost moth larvae (Thitarodes spp.) exclusively
in Tibetan Plateau alpine ecosystems. Touted as “Himalayan Viagra”, the
fungus’ sexual fruiting-body is highly prized due to its medical benefits
and dwindling supply (Hu et al., 2013). Attempts to culture the fruiting-
body have failed, and the huge market demand has led to severe devastation
of local ecosystems and has driven the fungus toward extinction. The route
of infection is unknown but probably occurs at the first instar larval stage.
The host caterpillars live underground for 4e5 years and have seven to
nine instars. During most of this time, the fungus is believed to remain
dormant and is only observed in the insect in later instars, just preceding
the host’s death (Cannon et al., 2009). The fungus then fully colonizes
the cadaver and produces a sexual structure. Before sequencing the
O. sinensis genome, the molecular basis for this lifestyle was entirely
unknown, as was the sex mode of O. sinensis.
In contrast to the other sequenced insect pathogens, which are all het-
erothallic, the O. sinensis genome contains two compatible MAT loci and
is sexually self-fertile, ie, homothallic (Hu et al., 2013). It is likely that
inbreeding is an adaptation by O. sinensis to its small population size resulting
from a very specialized lifestyle and the extreme environmental conditions in
its small geographical range. Consistent with this, O. sinensis has fewer HETs

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
76 J.B. Wang et al.

(5 genes) than other insect pathogens (15 genes) suggesting that, like
specialist Metarhizium spp., it encounters fewer genetically distinct individ-
uals than its more opportunistic relatives and, therefore, does not need bar-
riers to vegetative fusions. It was also determined that O. sinensis resembles
biotrophic plant pathogens (obligate pathogens that colonize living plant tis-
sue and obtain nutrients from living host cells) in having a genome shaped by
retrotransposon-driven expansions. This contrasts sharply with the genome
of the related T. inflatum (Ophiocordycipitaceae), with a lower proportion
(1.24%) of repeat sequences compared to other ascomycetes (Bushley
et al., 2013). Due to its repeat-driven expansion, the O. sinensis genome
size is approximately three times larger (w120 Mb) than the median of other
ascomycete insect pathogens but contains only 6972 protein coding genes as
compared to more than 9500 genes in other insect pathogens (Hu et al.,
2013) (9998 in T. inflatum (Bushley et al., 2013)). The RIP mechanism is
dysfunctional in O. sinensis, which has probably contributed to the massive
proliferation of retrotransposable elements, and thus genome size inflation.
Related TEs were clustered together in gene-poor or gene-free regions of
the O. sinensis genome indicative of repeated rounds of retrotransposition,
and the large number of retrotransposed and fragmented pseudogenes in
the genome implicates retrotransposition in most of the gene losses in
O. sinensis. The categories of pseudogenized genes are consistent with a
loss of capacity to adapt to heterogenous environments. For example, the
single O. sinensis nitrate reductase gene was pseudogenized, and the fungus
also lacks nitric oxide reductase, suggesting it cannot assimilate nitrate. An
inability to assimilate nitrate is also a feature of obligate plant pathogens
(Hu et al., 2013).
The data reported by Hu et al. (2013) are consistent with O. sinensis having
a biphasic pathogenic mechanism beginning with stealth pathogenesis in early
host instars and a lethal stage in late instars. It has been proposed that plant
pathogenic fungal lineages with large and flexible genomes are likely to adapt
faster during coevolution with hosts (Raffaele & Kamoun, 2012). It is reason-
able to assume that for the inbreeding O. sinensis, the massive proliferation of
TEs provides a trade off between advantages of increased genetic variation in-
dependent of sexual recombination and deletion of genes dispensable for its
specialized pathogenic lifestyle. As O. sinensis has lost many genes for oppor-
tunism, future transitions away from its current lifestyle seem unlikely, indi-
cating that while retrotransposition may facilitate rapid adaptation, it may
also contribute to stabile host interactions.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 77

4. EVOLUTION OF FUNGAL HOST SPECIFICITY

Comparative genomics utilizing the O. sinensis genome has provided
an unparalleled opportunity to develop a deeper understanding of how this
unique pathogen interacts with insects within its ecosystem. It is clear that
hostepathogen interactions are a major driving force for diversification,
but the genomic basis for speciation and host shifting remains unclear.
A major reason much remains unknown regarding speciation is because
there are few applicable model systems. The genus Metarhizium has been
subdivided into 12 different species according to the sequences of several
genes (Bischoff, Rehner, & Humber, 2009). Some of these species have a
wide host range, whereas others show specificity for certain insect families
and can be used to test hypotheses regarding speciation and host specificity.
Comparative genomic analyses of seven species revealed a directional speci-
ation continuum from specialists with narrow host ranges (ie, M. album and
M. acridum specific to hemipterans and acridids, respectively), to transitional
species with intermediate host ranges (Metarhizium majus and Metarhizium
guizhouense both have host ranges limited to two insect orders), and then
to generalists (ie, M. anisopliae, M. robertsii, and Metarhizium brunneum)
(Hu et al., 2014). Besides host range, generalist and specialist Metarhizium
species differ in the way they colonize hosts (Kershaw, Moorhouse,
Bateman, Reynolds, & Charnley, 1999). Generalists, like M. robertsii, typi-
cally kill hosts quickly via toxins and grow saprophytically in the cadaver.
In contrast, the specialist M. acridum causes a systemic infection of host tissues
before the host dies. This may reflect greater adaptation by the specialists to
subverting or evading the immune systems of their particular hosts so they
do not need to kill quickly. Generalists also have mechanisms for evading
host immunity, but perhaps by being “jack of all trades” they are less able
to subvert immune responses specific to certain insects. Lack of specific ad-
aptations could have selected for rapid killing of hosts before the host can
mount an enfeebling immune response. As described later in this chapter,
the gain and loss of the insecticidal cyclopeptide destruxin gene cluster is
correlated with host specificity in Metarhizium spp. (Hu et al., 2014;
Wang, Kang, Lu, Bai, & Wang, 2012).
Specialization in Metarhizium is associated with retention of sexuality and
rapid evolution of existing protein sequences, whereas generalization is asso-
ciated with protein family expansion, loss of genome-defense mechanisms,
genome restructuring, horizontal gene transfer, and loss of sexuality

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
78 J.B. Wang et al.

(Hu et al., 2014). As mentioned previously, the close physical proximity of

the plant-associated ancestor of M. album to plant-sap sucking hemipteran
bugs likely facilitated the switch from plant endophyte to entomopathoge-
nicity (Hu et al., 2014). The acridid-specific M. acridum split from the other
lineages 48 MYA (Fig. 2) within the mid-Eocene when recently evolved
grasses were growing along riverbanks and grass-feeding acridids first
appeared (Stidham & Stidham, 2000). Cocladogenesis such as this implies
host-driven divergence (Jackson, 2004; Schulze-Lefert & Panstruga,
2011). The transitional M. majus and M. guizhouense split 15 MYA
(Fig. 2) followed by the generalists M. robertsii and M. anisopliae that diverged
from each other only 7 MYA (Fig. 2), implying accelerated speciation asso-
ciated with increased phenotypic plasticity (Hu et al., 2014). Their radiations
coincided with climate change that was critical for massive diversification of
flowering plants, trees, and associated insects (Hay, Soeding, DeConto, &
Wold, 2002). Metarhizium majus and M. guizhouense genome sizes and
encoding capacities are similar to those of generalist species, and larger
than the specialists.

Figure 2 Reconstructed phylogeny of Metarhizium showing their insect host ranges

and divergence time. MAA, Metarhizium robertsii; MAN, Metarhizium anisopliae; MBR,
Metarhizium brunneum; MGU, Metarhizium guizhouense; MAJ, Metarhizium majus; MAC,
Metarhizium acridum; MAM, Metarhizium album; SAC, Saccharomyces cerevisiae. Rebuilt
from Hu, X., Xiao, G., Zheng, P., Shang, Y., Su, Y., Zhang, X., . Wang, C. S. (2014). Trajectory
and genomic determinants of fungal-pathogen speciation and host adaptation. Pro-
ceedings of the National Academy of Sciences of the United States of America, 111,
16796e16801. (See color plate)

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 79

Overall then, Metarhizium speciation provides a model for the evolution

of host preference, specificity, and virulence in other pathogens. An anal-
ysis of three Fusarium species also suggested a transition from specialist to
generalist coupled with genome and protein family expansions (Cuomo
et al., 2007; Ma et al., 2010). However, a few oomycete plant pathogenic
Phytophthora spp. appear to have transitioned from generalist to specialist
(Blair, Coffey, Park, Geiser, & Kang, 2008), so the general applicability
of the data reported by Hu et al. (2014) to other pathogens remains to
be determined.
Clearly, there are factors missing from specialists that limit their ability to
cause disease in multiple insects, as demonstrated by an increase in host range
following transfer of genes from generalist strains to the specialist M. acridum
(S.B. Wang et al., 2011). Nevertheless, M. acridum has a large number of
rapidly evolving genes (those with a high abundance of nonsynonymous
mutations), compared to generalists showing that it has not remained func-
tionally static; rather, specialization has involved rapid evolution of existing
protein sequences rather than the extensive gene duplication present in gen-
eralists (Hu et al., 2014). A likely explanation is that these are genes involved
in specific locust to M. acridum pathogen interactions and by evolving under
pairwise coevolution they are often subject to strong balancing or directional
selection. By contrast, generalist Metarhizium spp. interact with a wide range
of hosts in multiple environmental conditions and should therefore be
considered as evolving under “diffuse” interactions (Juenger & Bergelson,
1998). Diffuse coevolution to many insect hosts potentially explains why
signatures of positive selection were observed less frequently in the genome
of M. robertsii and other generalists.
Classical theory predicts specialists will be more likely than generalists to
lose sexuality in order to prevent shuffling of well-adapted gene combinations
(Sun & Heitman, 2011), but lack of RIP in generalist Metarhizium species sug-
gests that specialists are more likely to have a recent sexual history. It is
possible that an increase in gene family size and protein-coding composition
following loss of RIP in generalists facilitated their opportunistic lifestyles for
different insect hosts. Similarly, the broad host range plant pathogen Fusarium
oxysporum lacks RIP whereas the cereal-specialists Fusarium graminearum and
Fusarium verticillioides have strong RIP effects (Ma et al., 2010), suggesting
this pattern may be common in related fungal species with a wide range of
host preferences. The vast majority (c. 95%) of cataloged strains of Meta-
rhizium species in the ARSEF culture collection belong to generalist species
(http://www.ars.usda.gov/News/docs.htm?docid¼12125), suggesting that

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
80 J.B. Wang et al.

this is where most biodiversity within the Metarhizium genus resides and that a
broad host range is linked to ecological fitness.

5. PROTEIN FAMILY EXPANSIONS AND

CONTRACTIONS
Comparative genomics is crucial to understanding how fungi have
evolved to occupy diverse ecological niches. The secretome, ie, the entirety
of all proteins secreted by an organism, is of particular importance, as by
these proteins fungi acquire nutrients and communicate with their sur-
roundings. Secreted proteins are therefore on the front line of hostefungal
interactions. With the exception of O. sinensis, insect pathogenic ascomy-
cetes have a two- to threefold higher proportion of their genome
(w17%) devoted to secreted products than other ascomycetes, including
plant pathogens and the mycoparasitic Trichoderma spp. (Xiao et al., 2012).
The large secretomes of insect pathogens probably reflect the many habitats
they must adapt to in insecta, including the cuticle and the hemolymph, as
well as additional environmental habitats in the soil and with plants. These
complex lifestyles are reflected in the transcriptomes of insect pathogens;
many different genes are induced during adaptation to host cuticle, hemo-
lymph, or root exudate (Fang, Pava-Ripoll, Wang, & St. Leger, 2009; Feng,
Shang, Cen, & Wang, 2015; Freimoser, Hu, & St. Leger, 2005; C. Wang,
Butt, & St. Leger, 2005; C.S. Wang, Butt, & St. Leger, 2005; Wang & St.
Leger, 2005; Xiao et al., 2012). Some of these genes encode regulators
such as the protein kinase A that controls expression of many secreted
virulence factors (Fang et al., 2009), an osmosensor that signals to penetrant
hyphae that they have reached the hemocoel (Wang, Duan, & St. Leger,
2008), and perilipin, glycerol-3-phosphate acyltransferase, and cell auto-
phagy-related proteins that regulate lipolysis, turgor pressure, and formation
of infection structures (Duan et al., 2013; Gao, Shang, Huang, & Wang,
2013; C. Wang & St. Leger, 2007). The interactions between multiple re-
ceptors and their signal transduction pathways finely tune gene transcription
to adapt to different environmental niches and sites in the host. The insect
epicuticle or waxy layer comprises a heterogeneous mixture of long-chain
alkanes, wax esters, and fatty acids and represents the first barrier against
fungal attack. Metarhizium robertsii and B. bassiana highly transcribe cyto-
chrome P450 (CYP) subfamily CYP52 enzymes (for metabolism of insect
epicuticular lipids) and lipase genes as they germinate on the cuticle surface
(Lin et al., 2011; Zhang et al., 2012). Chitin constitutes up to 40% of the

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 81

procuticle but is absent from the epicuticular layer. Neither Metarhizium spp.
nor B. bassiana significantly upregulate chitinase genes on the epicuticle (Gao
et al., 2011; Xiao et al., 2012). Some genes are highly adapted to the specific
needs of Metarhizium, eg, Mcl1 (involved in immune evasion) with its
collagen domain is so far unique to Metarhizium and is only expressed in
the hemolymph (Wang & St. Leger, 2006). Metarhizium robertsii upregulates
a specific plant adhesin in the presence of plants (Mad2) and a specific insect
adhesin (Mad1) in the presence of insect cuticle, demonstrating that it has
specialist genes for a bifunctional lifestyle (C.S. Wang & St. Leger, 2007).
Other specifically regulated genes required to colonize the rhizosphere
and root include a novel oligosaccharide transporter for root-derived nutri-
ents (Fang & St. Leger, 2010a), an RNA-binding protein that has important
roles in both saprotrophy and pathogenicity (Fang & St. Leger, 2010b), and
an invertase that aids in the regulation of hydrolytic enzymes and provides a
plant-derived signal restricting fungal growth (Liao, Fang, Lin, Lu, & St.
Leger, 2013). Transcription of these genes is in part controlled by bZIP-
or C2H2-type TFs (Huang, Shang, Chen, Cen, & Wang, 2015; Huang,
Shang, Chen, Gao, & Wang, 2015).
Within the secretome there are different classes of proteins. Pathogenic
microbes evolved multiple enzymes to access nutrients from hosts (Schaible
& Kaufmann, 2005). Expansion and/or contraction of protein family size
have occurred in different fungal species in association with evolutionary ad-
aptations for different hosts and lifestyles (Table 1). A theme emerges of
niche-specific traits, ie, traits shared by fungi that occupy the same niche
irrespective of their phylogenetic position (Hu & St. Leger, 2004). For
instance, plant pathogens have expanded families of glycoside hydrolases
(GHs), carbohydrate esterases, cutinases, and pectin lyases in order to
degrade plant materials (Xu, Peng, Dickman, & Sharon, 2006), while
mammalian pathogens are enriched for aspartyl proteases and phospholipases
(van Asbeck, Clemons, & Stevens, 2009), and mycoparasitic fungi have
expanded numbers of chitinases to degrade fungal cell walls (Kubicek
et al., 2011). Typically, insect-pathogenic fungi infect insects by breaching
the cuticle using a combination of mechanical pressure, exerted by infection
structures (appressoria and penetration pegs), and cuticle-degrading
enzymes. The cuticle consists mainly of chitin microfibrils embedded in a
matrix of proteins and covered in lipids. Beauveria bassiana, Metarhizium
spp., and C. militaris, all have greatly expanded families of proteases, chiti-
nases, lipases, fatty acid hydroxylases, and acyl-CoA dehydrogenases (for
b-oxidation of fatty acids), illustrating an enhanced capacity to degrade

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy

82
Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105

Table 1 Comparison of selected protein families among different insect pathogens

Ophiocordyceps Cordyceps Beauveria Metarhizium Metarhizium Metarhizium
Protein family sinensis militaris bassiana roberstii acridum album

Protein kinases 116 167 159 161 192 127

Dehydrogenases 103 271 252 313 279 191
Cytochrome P450 57 57 83 123 100 74
G proteinecoupled receptors 22 29 32 63 37 32
Heterokaryon incompatibility 5 15 21 34 22 10
proteins
Horizontally transferred genes 25 45 42 79 56 27
Trypsins 2 12 23 32 17 12
Subtilisins 17 35 43 55 43 28
Aspartic proteases 12 21 21 33 25 22
Chitinases 9 20 20 21 14 12
Concanavalin A-like lectins 6 25 33 20 18 23
Glycoside hydrolases 66 135 145 159 130 108
Secondary metabolite 28 30 42 61 40 34
biosynthesis backbone
enzymes
Nonribosomal peptide 6 7 14 11 10 10
synthetase (NRPS)

J.B. Wang et al.

Polyketide synthase (PKS) 7 7 10 17 10 9
NPRS-PKS hybrid 1 6 6 8 5 3
Terpene synthase 10 4 6 9 6 5
 Author's personal copy

Advances in Genomics of Entomopathogenic Fungi

Major facilitator superfamily 55 132 139 134 107 81
Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105

ATP-binding 35 64 75 55 55 42
cassette superfamily
Sugar/inositol transporters 10 3 6 5 5 13
Appressorium differentiation 2 4 4 6 6 4
protein (MAS-like protein)
Adhesins 0 2 2 2 2 2
Chloroperoxidases 7 0 1 4 2 2
Small secreted cysteine-rich 138 255 351 322 240 209
protein
Compiled from Gao, Q., Jin, K., Ying, S. H., Zhang, Y., Xiao, G., Shang, Y., . Wang, C. (2011). Genome sequencing and comparative transcriptomics of the model
entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genetics, 7, e1001264; Zheng, P., Xia, Y., Xiao, G., Xiong, C., Hu, X., Zhang, S., . Wang,
C.S. (2011). Genome sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese medicine. Genome Biology, 12, R116; Xiao, G. H.,
Ying, S-H., Zheng, Z., Wang, Z. L., Zhang, S., Xie, X. Q. . Feng, M.G. (2012). Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria
bassiana. Scientific Reports, 2, 483; Hu, X., Zhang, Y. J., Xiao, G. H., Zheng, P., Xia, Y. L., Zhang, X. Y. . Wang, C.S. (2013). Genome survey uncovers the secrets of
sex and lifestyle in caterpillar fungus. Chinese Science Bulletin, 58, 2846e2854 and Hu, X., Xiao, G., Zheng, P., Shang, Y., Su, Y., Zhang, X., . Wang, C.S. (2014).
Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation. Proceedings of the National Academy of Sciences of the United States of America, 111,
16796e16801.

83
 Author's personal copy
84 J.B. Wang et al.

cuticles and other potential targets in insect hosts (Gao et al., 2011; Xiao
et al., 2012; Zheng et al., 2011). The B. bassiana/C. militaris lineage evolved
into insect pathogens independently of the Metarhizium lineage, so similar
expansion of these gene families is likely associated with functions necessary
for insect pathogenesis and reflects convergent evolution.
Analysis of entomopathogen protein families shows a dynamic loss and
gain of genes, but overall the generalists have more of these enzymes than
do the narrow host range species (Table 1). Thus, compared to M. album
and M. acridum, M. robertsii has evolved many expanded gene families of
proteases, chitinases, cytochrome P450s, polyketide synthases (PKSs), and
nonribosomal peptide synthetases (NRPSs) for cuticle-degradation, detoxi-
fication, and toxin biosynthesis, which may facilitate its ability to adapt to
heterogenous environments (Gao et al., 2011). The basal M. album genome
in particular highlights the early expansion of genes involved in cuticle
degradation, as it has greater than threefold more trypsin genes than related
plant endophytes and phytopathogens (Hu et al., 2014). However,
compared with M. album (87 proteases) and M. acridum (116 proteases), there
has been additional expansion of proteolytic capacity in other Metarhizium
species (average 165 proteases) (Table 1). The dramatic expansion of
proteases in B. bassiana resembles that of M. robertsii, suggesting that this is
also an adaptation to a broad host range. Thus, B. bassiana has more
subtilisins (43 vs 35) and trypsins (23 vs 12) than C. militaris and has four pro-
teolytic subfamilies that C. militaris lacks (Table 1, Hu et al., 2013).
Most of the secreted effector-type proteins of plant pathogens are small
(<300 amino acids) and contain four or more cysteine residues (Martin et al.,
2008). Plant pathogens usually have more of these small, secreted cysteine-
rich proteins (SSCPs) than saprophytes (Ohm et al., 2012). Therefore, the
identification and analysis of SSCPs has been highlighted in genomic studies
assessing many plant pathogenic and symbiotic fungi (Cheng et al., 2014). A
survey of SSCPs in 11 fungal species including entomopathogens revealed
396 clusters of which 12 contained SSCPs shared by insect pathogens and
the plant endophyte Epichloë festucae, and 26 are found in insect and plant
pathogens (Xiao et al., 2012). Ninety-one of the 396 SSCP clusters are
shared exclusively by the insect pathogens, suggesting many shared strategies
for interacting with plants and insects are currently unknown. Of these 91
clusters, 52 contain genes from B. bassiana. Relative to other insect patho-
gens (average 307), the B. bassiana genome encodes more SSCPs (373),
and many of them are species specific (154 vs an average of 95). As with
other classes of secreted proteins, generalist insect pathogens have more

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 85

SSCPs than specialists (Table 1), indicating that SSCPs play roles in adapta-
tion to diverse lifestyle options.
However, aside chitinases and hydrophobins (cell wall proteins that
facilitate adhesion and morphological changes (Bidochka, St. Leger, &
Roberts, 1995)), most of the entomopathogen SSCPs are of unknown
function. Of 373 B. bassiana SSCPs, only 130 contained conserved
domains. Of these, six B. bassiana SSCPs were identified as concanavalin
A-like lectins, which could potentially function in interactions with both
insects and plants. Beauveria bassiana also has four genes encoding proteins
containing eight cysteine-containing extracellular membrane (CFEM)
domains resembling pathogenicity determinants in plant pathogens (Xiao
et al., 2012). As for the rest, aside unknown roles in entomopathogenicity,
the functions identified for SSCPs in other fungi suggest they could play a
mutualistic role in interactions with plants but also may be involved in
antagonization of other fungi, bacteria, and higher soil organisms.
Ophiocordyceps sinensis, which probably infects insects through their
spiracles (breathing holes) or orally, provides an illuminating exception to
insect pathogens having large secretomes: gene families encoding epicutic-
ular degrading CYP52 enzymes, cuticle-degrading proteases, and chitinases
were greatly reduced in size (Table 1). In addition, protein families involved
in adhesion to cuticles and formation of appressoria were absent or reduced
in O. sinensis (Hu et al., 2013, Table 1). These gene losses are consistent with
the inability of O. sinensis to breach intact cuticle. Hydrolytic enzymes,
particularly proteases, can elicit host immune defenses (St. Leger, Joshi,
Bidochka, & Roberts, 1996). In which case, the reduced number of
cuticle-degrading enzymes in O. sinensis might also be an adaptation to
avoid detection by the host during its extended latent phase. Copy number
reduction was also evident for genes encoding known pathogen-associated
molecular patterns such as lectins (6 in O. sinensis vs average 24 in other in-
sect pathogens), further suggesting that selection for “stealth” (avoidance of
host defenses) was a major force driving O. sinensis evolution. Evidently, as
with plant pathogens (Ma et al., 2010; Stukenbrock et al., 2011), the differ-
ences found among the insect pathogens in protein family size are related to
their modus operandi and host range.

5.1 Signal Transduction

To recognize and adapt to invertebrate environments, such as the insect
cuticle, hemolymph, and cadaver, insect pathogens need to rapidly respond
to changes in nutrient availability, osmolarity, and the host immune system

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
86 J.B. Wang et al.

(Luo et al., 2012; Wang & St. Leger, 2006; Wang et al., 2008; Zhang et al.,
2009). BLASTing insect pathogen genomes against the pathogenehost
interaction gene database (a collection of experimentally verified pathoge-
nicity, virulence, and effector genes from fungi, oomycetes, and bacteria)
reveal large numbers of G proteinecoupled receptors (GPCRs), protein
kinases (PKs), and TFs that share similar sequences in the entomopathogen
genomes. Fungal GPCRs sense extracellular cues and transmit the signals to
distinct trimeric G protein subunits (Xue, Hsueh, & Heitman, 2008). Most
of the insect pathogen GPCRs resemble Pth11-like proteins of the rice-blast
fungus Magnaporthe oryzae (Xiao et al., 2012). Compared with M. album and
M. acridum, there was a major expansion of GPCR-related proteins in the
generalist Metarhizium spp., consistent with being able to recognize and
respond to many more environmental triggers. In particular, the generalists
showed a more than twofold expansion of Pth11-like receptors (average 51
vs 23 in specialists), with particular expansion in the subfamilies that are
developmentally upregulated during early Metarhizium infection processes
(germination and formation of infection structures on host cuticles) (Gao
et al., 2011; Xiao et al., 2012). Consistent with early host recognition events
being key to establishing specificity, M. acridum, but not M. robertsii, tran-
scribed different Pth11-like GPCR genes on locust and cockroach cuticles
indicating a role in recognizing specific hosts, or at least that these genes
have a function that varies between strains with different host ranges.
Comparatively analyzing the host-invading transcriptomes of M. acridum
and M. robertsii showed recognition by regulatory controls that exclusively
limit expression of genes for pathogenicity-related developmental processes
to individual hosts (Gao et al., 2011).
Relative to Metarhizium spp., B. bassiana and C. militaris have fewer
Pth11-like receptors (average 21). In particular, they lack a GPR1-like
GPCR, which in yeast is activated during nitrogen starvation (Xue, Batlle,
& Hirsch, 1998) and is upregulated by M. robertsii on the cuticle surface in
response to the nitrogen poor conditions that are an important trigger for
inducing production of infection structures (Gao et al., 2011; St. Leger,
Bidochka, & Roberts, 1994; St. Leger et al., 1992). Thus, B. bassiana and
Metarhizium have evolved different mechanisms for nutrient sensing.
Functional kinome analysis of the plant pathogen F. graminearum
indicated that many PKs are involved in fungal growth, conidiation,
pathogenesis, stress responses, toxin production, and/or sexual reproduction
(C. Wang et al., 2011). Excluding O. sinensis (116 kinases), the insect path-
ogens average 170 PKs as compared to an average of 145 in the plant

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 87

pathogens (Xiao et al., 2012). In contrast to GPCRs, the specialist M. acridum

has more PKs (192) than the generalist M. robertsii (161) (Table 1). Based on
just the M. robertsii and M. acridum genomes, Gao et al. (2011) suggested that
the additional kinases in M. acridum may allow a more stringent discrimina-
tion between potential insect hosts and subsequent control of cell differen-
tiation; consistent with this, B. bassiana has fewer PKs (159) than C. militaris
(167) (Xiao et al., 2012). However, the additional data from the M. album
(127 kinases) and O. sinensis genomes rule out a simple relationship between
evolution of narrow host range and/or sexuality and proliferation of PKs.
Like other pathogenic fungi, insect pathogens have large numbers of
TFs. However, B. bassiana has more (10) GATA-type TFs than C. militaris
(5) and Metarhizium species (4e5) (Gao et al., 2011; Xiao et al., 2012).
Fungal GATA-type TFs are involved in multiple functions, including nitro-
gen metabolism, light induction, siderophore biosynthesis, mating-type
switching, and chromatin rearrangement (Scazzocchio, 2000). Metarhizium
robertsii contains an array of 24 bZIP domainecontaining TFs; gene deletion
of one of these (MBZ1) revealed that it contributes to negative regulation of
subtilisin proteases, but positive control of adhesin MAD1 (Huang, Shang,
Chen, Cen, et al., 2015), consistent with transcriptomic data showing little
subtilisin production while spores are in the process of adhering to cuticle
(Gao et al., 2011). Functional study of an M. robertsii C2H2-type TF
(MrPacC) indicated that it was highly activated in alkaline conditions and
positively controlled chitinase genes (Huang, Shang, Chen, Gao, et al.,
2015). Cultures of M. robertsii always show a rapid increase in pH during
production of chitinases (St. Leger, Cooper, & Charnley, 1986a), and
M. robertsii alkalinizes a proteinaceous microenvironment, such as the insect
cuticle, by producing ammonia (St. Leger, Nelson, & Screen, 1999).
Digestion of cuticle proteins exposes the underlying chitin to enzymolysis
(St. Leger, Cooper, & Charnley, 1986b), so linking alkalinization with
chitinase production is adaptive for the fungus. It is likely that continued
dissection of the roles of individual TFs in the manner of MBZ1 and MrPacC
will untangle the complexity and illuminate the interactions between what
seem currently disconnected strands of biochemical and molecular data.

5.2 Carbohydrate-Active Enzymes

Chitin is the second most abundant polymer in insect cuticle. The necessity
to degrade it is reflected in an abundance of GH18 family chitinases in
B. bassiana, C. militaris, and Metarhizium spp. (on average 19) compared to
plant pathogens (on average 11). Fungal chitinases are subdivided into three

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
88 J.B. Wang et al.

subgroups (Seidl, 2008). Xiao et al. (2012) found that eight of the 20 B. bassi-
ana chitinases belong to subgroup A (without a chitin-binding domain,
CBM), four belong to subgroup B (one CBM at the C-terminal), and eight
belong to subgroup C chitinases (possessing LysM chitinebinding modules).
Insect and plant pathogens have similar numbers of subgroup A chitinases,
but the entomopathogens have many more chitinases with CBMs (average
11 vs 2). Phylogenetic analyses of subgroup B and C chitinases revealed that
most of the gene duplication events have occurred since B. bassiania/
C. militaris, Metarhizium spp., and Trichoderma spp. diverged from a common
ancestor, suggesting their abundance in each clade is due to convergent evo-
lution (Xiao et al., 2012).
Many plant pathogens need GHs, pectate lyases, and cutinases to degrade
the plant cuticle (waxy layer) and cell wall (Xu et al., 2006). Whole genome
analyses (Fig. 1) indicate that ascomycete insect pathogens evolved from
fungi adapted to grow on plants even though they now infect insects.
This inference is supported by the consistent existence of genes for plant-
degrading enzymes within their genomes (Gao et al., 2011; Hu et al.,
2014). The number of GHs possessed by M. robertsii (159), B. bassiana
(145), and M. acridum (130) is more than the endophytic close relation of
Metarhizium, E. festucae (98) but is fewer than plant pathogens (average
199) (Xiao et al., 2012). This is because overall, insect pathogens have fewer
genes associated with plant utilization than plant pathogens. This includes
fewer cellulases (average of 10 in insect pathogens vs 25 in plant pathogens),
oxidative lignin enzymes (29 vs 40), carbohydrate esterases (9 vs 33),
cutinases (4 vs 12), and pectin lyases (8 vs 20) (Xiao et al., 2012). Two
well-known virulence factors for plant pathogens are cutinases and pectin
lyases (Hugouvieux-Cotte-Pattat, Condemine, Nasser, & Reverchon,
1996). Metarhizium spp., particularly M. robertsii, showed overall profiles of
carbohydrate-active enzymes (CAZymes) more similar to plant pathogens
than to other insect pathogens (Bushley et al., 2013). In part, this is because,
compared to Metarhizium spp., B. bassiana and C. militaris have fewer
enzymes for degrading xylan; although unlike C. militaris (Zheng et al.,
2011), B. bassiana has a phosphoketolase required for xylose metabolism
and full virulence in Metarhizium (Duan, Shang, Gao, Zheng, & Wang,
2009). Thus, in contrast to C. militaris, B. bassiana grows on xylose, albeit
very weakly when compared to M. robertsii (Xiao et al., 2012). Interestingly,
E. festucae lacks the same xylanases as B. bassiana (the GH11 family) (Xiao
et al., 2012). As an alternate endophyte, B. bassiana presumably possesses
mechanisms to avoid stimulating plant defenses. Fungal endoxylanases are

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 89

known to trigger plant immune responses and induce necrosis of infected

plant tissue (Noda, Brito, & Gonzalez, 2010). Therefore, lack of GH11 in
B. bassiana and E. festucae could be an adaptation limiting induction of
necrosis by these endophytes, and thereby facilitating immune evasion.
To put the plant-degrading genes of entomopathogens in context,
fungal pathogens of humans that are seldom recovered from soil, such as
Coccidioides, exhibit few of these enzymes or none (Sharpton et al., 2009).
Even necrophytes such as Trichoderma reesei lack many families of plant cell
walledegrading enzymes (Martinez et al., 2008), and the existence of
such families in insect pathogens that are associated with plants implies
that these species are able to utilize living plant tissues. Potentially, these
enzymes could also facilitate colonization of plant surfaces, but this must
remain speculative because the genetic basis for rhizosphere competence is
largely obscure in fungi in general (St. Leger, 2008). Identification of the
full repertoire of Metarhizium and Beauveria genes will help to identify those
responsible for life on plant roots and shoots.
As in many other aspects of its biology, O. sinensis represents a special case
with respect to CAZymes, with many fewer GHs (66 vs average 139 in
other insect fungi) (Hu et al., 2013, Table 1). Most of the missing enzymes
are devoted to degradation of plant materials indicating that, unlike related
insect pathogens, O. sinensis may be exclusively insect parasitic.

5.3 Secondary Metabolites and Host Interaction

Most hypocrealean insect pathogenic fungi produce large numbers of
secondary metabolites that seem to have no role in normal fungal
metabolism but are highly active in insect tissues (Xu, Luo, Gao, Shang,
& Wang, 2015). These are assumed to be part of an ongoing evolutionary
arms race between fungi and insects. As pointed out by Donzelli and
Krasnoff (2016), there are a lot of Metarhizium secondary metabolites that
cannot currently be connected to genes and lots of genes in entomopatho-
gen genomes that cannot be connected to known chemistry. The key fungal
secondary metabolite-producing enzymes are NRPSs, PKSs, and terpene
cyclases (TCs). Hypocrealean fungi are known to synthesize a number of
products with activity against insects via both NRPSs and PKSs, such as des-
truxins (M. robertsii) (Pal, St. Leger, & Wu, 2007; Wang et al., 2012), efra-
peptins (Tolypocladium spp.; Krasnoff, Gupta, St. Leger, Renwick, &
Roberts, 1991), and ergot alkaloids (clavicipitalean endophytes of grasses
including Claviceps and Epichloë spp.; Torres, Singh, Vorsa, & White,
2008). Many of these compounds also have pharmaceutical applications

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
90 J.B. Wang et al.

and/or roles in antibiosis, pathogenesis, and competitive interactions

between organisms (Pal et al., 2007).
In terms of PKS genes and putative polyketides, generalist Metarhizium
spp., such as M. robertsii, appear to possess a greater potential for the produc-
tion of secondary metabolites than specialist strains and other sequenced
ascomycetes, even Fusarium species (Chen, Feng, Shang, Xu, & Wang,
2015; Gao et al., 2011; Hu et al., 2014). Donzelli and Krasnoff (2016) report
that the lowest number of secondary metabolite genes is found in M. album
(48 in total) and the highest is in M. guizhouense (93 in total). Acting collec-
tively with the secretome, the number and diversity of these effectors may
contribute to the ability of generalists to infect and kill a much wider variety
of insects than a specialist, such as M. acridum. This also relates neatly to path-
ogenic strategies because, as described previously, M. robertsii kills hosts
quickly via toxins and grows saprophytically in the cadaver. In contrast,
like many specialists, M. acridum causes a systemic infection of host tissues
before the host dies which suggests a much smaller role for toxins (Kulkarni,
Thon, Pan, & Dean, 2005). The best-studied Metarhizium toxins are the des-
truxins, which suppress insect cellular and humoral immune responses (Pal
et al., 2007; Wang et al., 2012). The gene cluster responsible, dtxS1-dtxS4
(Wang et al., 2012), is present in all surveyed Metarhizium species except
for M. acridum and M. album. Intriguingly, minor differences in virulence
were found between applications (Donzelli, Krasnoff, Sun-Moon, Church-
ill, & Gibson, 2012; Wang et al., 2012) of M. robertsii DTX mutants and
wild-type strains. When the removal of a redundant toxin cluster fails to
markedly reduce virulence, this points to a complexity of host-range deter-
mination far beyond individual toxic metabolites (Donzelli and Krasnoff,
2016).
As reviewed by Donzelli and Krasnoff (2016), B. bassiana and C. militaris
(Cordycipitaceae) share very few secondary metabolite pathways with
Metarhizium (Clavicipitaceae); more often, sequenced species from Ophio-
cordicipitaceae have more pathways in common with Metarhizium. Beauveria
is well known for producing a large array of biologically active secondary
metabolites (eg, oosporein, bassianin, tenellin, beauvericin, bassianolides,
and beauveriolides) and secreted metabolites involved in pathogenesis and
virulence (eg, oxalic acid) that have potential or realized industrial, pharma-
ceutical, and agricultural uses (Molnar, Gibson, & Krasnoff, 2010). As
reviewed by Ortiz-Urquiza and Keyhani (2016), the pathways for a number
of these B. bassiana secondary metabolites have been examined, including
oosporein, bassianolide, and beauvericin. In most cases, targeted gene

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 91

inactivation of key genes in these pathways resulted in a significant reduction

in virulence that, in the case of oosporein, may be accounted for by an
impaired suppression of insect host immunity (Feng et al., 2015). Silkworm
larvae infected by B. bassiana (batryticated silkworms, also called Bombycis
corpus or white-stiff silkworm) have for centuries been a traditional Chinese
medicine, and their potential as a medicine has been validated by modern
technologies, eg, water extract of batryticated silkworms protect against
a-amyloid-induced neurotoxicity (Koo et al., 2003). Cordyceps militaris pro-
duces cordycepin and cordycepic acid, which have been linked to a variety
of benefits from antiaging to sleep-regulating effects (Paterson, 2008). Cor-
dycepin has been investigated for use against cancers (Liao et al., 2015). Like-
wise, the pyridine alkaloid fumosorinone identified from Isaria fumosorosea
has potential to treat type II diabetes and other associated metabolic syn-
dromes (Liu et al., 2015).
In this chapter, we have seen that there are genomic similarities between
O. sinensis and obligate plant pathogens. However, O. sinensis differs from
the plant pathogens, which have lost secondary metabolites, in having
multiple PKSs, modular NRPSs, and TCs for production of an array of sec-
ondary metabolites (Table 1). These most likely play roles after the latent
period when the fungus is colonizing and killing the host, and they are
also likely candidates for production of the pharmacologically active com-
pounds that this fungus is famous for. Ophiocordyceps sinensis encodes four
terpenoid synthases and one terpenoid cyclase which are absent in other
fungi, indicating that this fungus, likened to “natural Viagra,” produces
novel terpenoids. However, many putative secondary metabolism clusters
were conserved between O. sinensis and other insect pathogens, providing
singular exceptions to the restructuring of the O. sinensis genome by repeat
elements and suggesting that the physical linkage of secondary metabolite
biosynthetic genes has strong adaptive significance for entomopathogenicity.
Other members of the family Ophiocordycipitaceae host a variety of sec-
ondary metabolites involved in pathogenesis with potential usefulness to
humans. Hirsutella thompsonii produces Hirsutellin A, which has been tested
against honey bee parasitic Varroa mites (https://www.google.com/patents/
US6277371). Tolypocladium inflatum produces a range of insecticidal
compounds: most notably cyclosporine, an immunosuppressant in humans,
as well as in insects, which is exploited in that role to prevent transplant
rejection. Phylogenomic analyses revealed complex patterns of homology
between the NRPS that encodes for cyclosporin synthetase and those of
other secondary metabolites with activities against insects (eg, beauvericin,

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
92 J.B. Wang et al.

destruxins, etc.) and demonstrated the roles of module duplication and gene
fusion of distantly related NRPS modules in diversification of NRPSs
(Bushley et al., 2013). Famously, O. unilateralis s.l. alters infected ant
behavior (reviewed by Hughes et al., 2016), instigating them to climb and
bite down on elevated vegetation to assist the fungus in spore transmission
(Shang et al., 2015). Recent genomic and transcriptomic data revealed
that O. unilateralis secretes a plethora of putative compounds that have an
effect on behavior, including ergot, enterotoxins, alkaloids, polyketides,
and nonribosomal peptides (de Bekker et al., 2015). Ergot famously causes
serotonergic overstimulation of the central nervous system in humans and
livestock leading to ergotism (Eadie, 2003), and polyketides, nonribosomal
peptides, and alkaloids have been suggested to be neuromodulatory agents
(Molnar et al., 2010).

5.4 Protein Families Involved in Detoxification and Stress

Responses
As a plant symbiont and an entomopathogen, Metarhizium encounters many
different selective pressures. During these two lifestyles, the fungus is
confronted with a wide range of abiotic (eg, UV, pH, temperature, etc.)
and biotic stresses (eg, insect host defenses), which the fungus has adapted
to overcome. The specific genes involved in these processes are discussed
in Lovett and St. Leger (2015).
Relative to plant pathogens, amidohydrolases, glyoxalases, and monoox-
ygenases are all expanded in insect pathogens implying that consistent with
their broad lifestyle options, the latter are better able to detoxify correspond-
ing compounds. Dehydrogenases and CYPs are also abundant in insect path-
ogens and are reflective of the lifestyle of the organism. They are involved in
multiple essential physiological processes, including degradation/detoxifica-
tion of xenobiotics, biotransformations, and the biosynthesis of bioactive
metabolites (Cresnar & Petric, 2011). The profile of dehydrogenases can
be used to highlight metabolic pathways and metabolic flexibility, ie, the
ability to switch between energy sources. By this measure, M. robertsii has
the greatest flexibility (313 dehydrogenases), and O. sinensis the least (103
dehydrogenases; Table 1), which is consistent with their known lifestyles.
Beauveria bassiana has a comparatively modest 252 dehydrogenases,
suggesting fewer lifestyle options than M. robertsii. Entomopathogen
dehydrogenases with known functions are widely distributed. Thus,
mannitol-1-phosphate dehydrogenase and mannitol dehydrogenase con-
tribute to B. bassiana’s tolerance to H2O2, ultraviolet radiation, and heat

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 93

stresses by regulating mannitol accumulation (Zhang, Xia, & Keyhani, 2011).

Homologs of these genes are present in Metarhizium spp. genomes. In fact,
M. robertsii is particularly enriched in dehydrogenases (17 in M. roberstii; 7
in M. acridum) required for the biosynthesis of mannitol (Gao et al., 2011).
The genome of M. robertsii encodes 123 highly divergent CYP genes
versus an average of 118 in ascomycete plant pathogens (Xiao et al.,
2012), 100 in M. acridum, 83 in B. bassiana, and 57 in both C. militaris and
O. sinensis (Table 1). CYP genes are involved in oxygenation steps during
alkane assimilation and the biosynthesis of secondary metabolites, as well
as with detoxification. Twenty-four CYP families present in Metarhizium
spp. are absent in B. bassiana and C. militaris, including nitric oxide reductases
(CYP family 55) used for anaerobic denitrification (Xiao et al., 2012). Thus,
unlike Metarhizium spp., B. bassiana and C. militaris may not be able to
respond to hypoxic conditions in a host by fermenting nitrate or ammonia.
Ophiocordyceps sinensis also lacks CYP55 (Hu et al., 2013), unlike the closely
related H. thompsonii (Agrawal et al., 2015), confirming that this enzyme has
been lost repeatedly in different lineages of insect pathogens.
Outer membrane transporters serve as the means by which nutrients are
absorbed by the cell and as a mechanism for the export of toxic molecules.
Most transporters belong to the major facilitator superfamily (MFS) and the
ATP-binding cassette (ABC) superfamily. In fungi, many ABC transporters
have been implicated in drug resistance and could, therefore, serve to defend
the fungus from host-produced secondary metabolites, whereas MFS pro-
teins are typically involved in the transport of a wide range of substrates
and may function as nutrient sensors (Morschhauser, 2010). Interestingly,
entomopathogen species in general have many more amino acid and peptide
transporters than other fungi, consistent with their ability to access a range of
protein degradation products from insect sources (Gao et al., 2011; Xiao
et al., 2012). Otherwise, as with other categories of genes involved in stress
responses and detoxification, generalist genomes are particularly enriched in
transporters (Table 1). Metarhizium robertsii and B. bassiana have a similar
repertoire of MFS transporters, but B. bassiana has a clear advantage in
ABC transporters compared to M. robertsii (75 versus 55, respectively).
Ortiz-Urquizza and Keyhani review the impacts on virulence and stress
resistance imparted to B. bassiana by ABC transporters in this volume.
Among insect pathogens, B bassiana has the highest numbers of epoxide
hydrolases (7 vs 4e5 in others), nitrilases (11 vs 7e10 in others), and mono-
oxygenases (36 vs 16e30 in others) (Xiao et al., 2012). Along with its
enhanced profile of ABC transporters and comparative paucity of CYP

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
94 J.B. Wang et al.

genes, this indicates that B. bassiana may have evolved unique solutions to
overcoming environmental challenges in plants and insects. The monooxy-
genases in particular might be involved in rapid elimination of insect
polyphenolics by ortho-hydroxylation of phenols to catechols. Beauveria bassi-
ana is a well-known whole cell catalyst in industrial applications, and some of
these enzymes have the potential to be involved in bioconversions. Epoxide
hydrolases are highly versatile biocatalysts for the hydrolysis of epoxide, and
nitrilases are valuable alternatives to chemical catalysts for biotransformation
of various organic nitriles (Xiao et al., 2012).

6. HORIZONTAL GENE TRANSFER

Horizontal gene transfer (HGT) is the transfer of genetic material
directly from the genome of one organism to that of another, rather than
between parent and offspring. HGT between distantly related bacteria is a
commonplace process that allows them to quickly share adaptive genes,
ie, an antibiotic-resistance set of genes to adapt to an antibiotic. It also
contributes significantly to the emergence of new bacterial pathogens, but
HGT is usually thought to play a minor role in eukaryotes. However,
data from multiple genomic sequences suggest that HGT has also occurred
from prokaryotes to eukaryotes and between eukaryotes (Crisp, Boschetti,
Perry, Tunnacliffe, & Micklem, 2015). Among the eukaryotes, the chitin
cell wall of fungi is considered a structural barrier, likely reducing the
frequency of HGT, but even so, there are numerous instances of HGT in
fungi. The amount of transferred DNA ranges from single genes, to second-
ary metabolite gene clusters and even to whole chromosomes (Fitzpatrick,
2012). Richards, Leonard, Soanes, and Talbot (2011) analyzed the functions
of 323 genes probably originating from HGT into fungi and concluded that
HGT played a role in expanding the nutrient acquisition and environmental
colonization capacities of many fungi.
Detection of horizontally transferred genes traditionally depends on in-
congruences in phylogenies: the phylogeny of a given gene does not match
a known species phylogeny. However, discrepancies in phylogeny can
have other explanations besides HGT, including gene loss, eg, a gene that
existed in a distant ancestor could have simply been lost in many relatives.
A phylogenomic survey of fungal gene family evolution suggested that indi-
vidual protease genes have been lost many times independently in different
lineages, and that flux of genes is an ongoing process (Hu & St. Leger, 2004).

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 95

Issues distinguishing between HGT and gene loss can be potentially cir-
cumvented using fully sequenced genomes and filters based on known
phylogenetic relationships to look for genes that have been horizontally
transferred since speciation.
To evaluate the extent of gene transfer into insect pathogenic fungi, Hu
et al. (2014) analyzed seven Metarhizium genomes to quantitate how closely
a gene aligned to a nonfungal sequence compared to a bacterial or metazoan
sequence. Based on this score, they determined that the broad host range
species have obtained more bacterial genes than the specialists (on average
63 vs 27), indicating considerable variability in the rates of gain and/or
loss of bacterial genes within the estimated 117 million year divergence of
the Metarhizium species. Orthology analysis revealed that only eight HGT
genes are present in all Metarhizium species, whereas the remaining genes
were probably acquired clade- or species specifically because they are absent
from multiple species. In a few cases the importance of the transferred genes
has been functionally demonstrated with gene knockouts. For example,
experimentally verified, functionally important, bacterial-like proteins in
M. robertsii encode a chymotrypsin (Screen & St. Leger, 2000), a pentose
metabolizing phosphoketolase (Duan et al., 2009), a cold shock protein
(Fang & St. Leger, 2010b), and a putative chitinase that are present in all
seven Metarhizium species, indicating that these genes were acquired before
the Metarhizium lineages radiated. These genes are all most similar to se-
quences in soil- and insect-dwelling bacteria, providing an ecological niche
overlap between the bacteria and the fungus. Not surprisingly, therefore,
hosts are also a source of adaptive traits. A virulence factor (Mr-NPC2a)
of M. robertsii was horizontally acquired from an insect and allows Meta-
rhizium to compete with insect hosts for the sterols necessary to maintain
cell membrane integrity (Zhao et al., 2014). This sequence was also present
in all seven Metarhizium species, suggesting it was acquired by a common
ancestor and that HGT from hosts, as well as associated bacteria, has played
a role in shaping functional diversity of the gene repertoire in entomopath-
ogens. More recent acquisitions may include a putative insect-like trypsin
that is absent only in M. album and a putative TcdB-like insecticidal toxin
protein that is unique to M. robertsii (Hu et al., 2014). Likewise, around
half of the gene clusters encoding secondary metabolites in M. album
(11/19) and M. acridum (11/25) are species-specific (vs an average of 7 in
the other species), although most are also present in other fungal genera.
This implies that they were laterally acquired from other fungi during Meta-
rhizium speciation (Hu et al., 2014). For example, Hu et al. (2014) suggest

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
96 J.B. Wang et al.

that the destruxin gene cluster may have been acquired 15 MYA by
Metarhizium lineages that were broadening their host range.
These results suggest that Metarhizium genomes have all been subject to
low levels of HGT; some of the adaptive HGT events are ancient
(>100 MYA), but generalist species have been more susceptible than
specialists to HGT events in the last 30 MY, consistent with their being
exposed to a greater variety of potential donor DNA. Thus, throughout
their evolution, Metarhizium spp. may have acquired new virulence mecha-
nisms by HGT and presumably increases in pathogen virulence through this
process evolved in a stepwise manner.

7. CONCLUSIONS AND FUTURE PERSPECTIVES

Sequencing related entomopathogen species that have evolved
specialist or generalist lifestyles has increased their utility as models and pro-
vided insights into the evolution of pathogenicity. Thus, cross-species
comparative analysis has identified novel and specialized virulence mecha-
nisms and, compared to experimental methods, has allowed for more rapid
identification of genes encoding biologically active molecules and genes
responsible for interactions between fungi, plants, and insects. These analyses
also show how much we still need to do to comprehensively understand these
interactions. For example, most of the abundant SSCPs produced by entomo-
pathogens are of unknown function, highly species-specific, and lack similar-
ity to known proteins. Undoubtedly, the information from comparative
genomics will benefit future functional studies of insectefungus interactions.
Entomopathogenic fungi present a vast reservoir of biopolymer-degrading
enzymes adapted to a wide range of temperatures and environments. The
new information on these abundant enzymes will also facilitate more exten-
sive work to determine mechanisms of the biotransformation reactions that
make these fungi such useful industrial catalysts, as well as reveal potentially
useful compounds for medicinal use. Overall, entomopathogen genome se-
quences will help realize the still undeveloped potential possessed by these
fungi both as insect pathogens and as microbial biocatalysts, as well as illumi-
nate their, as yet, poorly understood role as endophytes and plant symbionts
(reviewed by Moonjely et al., 2016).
Given that specialization has occurred many times in Metarhizium, it pro-
vides an opportunity to study a genus with species containing a large number
of independently evolved models of adaptation and response. Multispecies

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 97

exploration of genome evolution of Metarhizium spp. has already led to

better understanding of mechanisms by which novel pathogens emerge
with either wide or narrow host ranges (Hu et al., 2014). However, the
thousands of different entomopathogen species display extraordinary diver-
sity, especially given the number of different products that can be studied
(Isaka et al., 2005). In this context, we can look forward to the completion
of the current Entomophthoromycota sequencing projects. These are very
virulent pathogens that are highly adapted to their hosts and often devastate
pest populations in spectacular epizootics. In order to increase the power and
accuracy of comparative analyses, we need to sequence a much more exten-
sive sampling of entomopathogenic fungi.

ACKNOWLEDGMENTS
This work was supported by the US National Science Foundation Grant IOS-1257685 and
the NIAID of the National Institutes of Health under award number RO1 AI106998 to
Raymond St. Leger and by the National Nature Science Foundation of China Grants
31530001 and 31225023 to Chengshu Wang.

REFERENCES
Agrawal, Y., Khatri, I., Subramanian, S., & Shenoy, B. D. (2015). Genome sequence,
comparative analysis, and evolutionary insights into chitinases of entomopathogenic fun-
gus Hirsutella thompsonii. Genome Biology and Evolution, 7, 916e930.
Alby, K., Schaefer, D., & Bennett, R. J. (2009). Homothallic and heterothallic mating in the
opportunistic pathogen Candida albicans. Nature, 480, 890e893.
van Asbeck, E. C., Clemons, K. V., & Stevens, D. A. (2009). Candida parapsilosis: a review of
its epidemiology, pathogenesis, clinical aspects, typing and antimicrobial susceptibility.
Critical Reviews in Microbiology, 35, 283e309.
de Bekker, C., Ohm, R. A., Loreto, R. G., Sebastian, A., Albert, I., Merrow, M. …
Hughes, D. P. (2015). Gene expression during zombie ant biting behavior reflects the
complexity underlying fungal parasitic behavioral manipulation. BMC Genomics, 16,
620.
Bidochka, M. J., Clark, D. C., Lewis, M. W., & Keyhani, N. O. (2010). Could insect phago-
cytic avoidance by entomogenous fungi have evolved via selection against soil amoeboid
predators? Microbiology, 156, 2164e2171.
Bidochka, M. J., Kamp, A. M., Lavender, T. M., Dekoning, J., & Amritha De Croos, J. N.
(2001). Habitat association in two genetic groups of the insect-pathogenic fungus Meta-
rhizium anisopliae: uncovering cryptic species? Applied and Environmental Microbiology, 67,
1335e1342.
Bidochka, M. J., St. Leger, R. J., & Roberts, D. W. (1995). The rodlet layer from aerial and
submerged conidia of the entomopathogenic fungus Beauveria bassiana contains
hydrophobin. Mycological Research, 99, 403e406.
Bischoff, J. F., Rehner, S. A., & Humber, R. A. (2009). A multilocus phylogeny of the Meta-
rhizium anisopliae lineage. Mycologia, 101, 512e530.
Biswas, C., Dey, P., Satpathy, S., & Satya, P. (2012). Establishment of the fungal entomo-
pathogen Beauveria bassiana as a season long endophyte in jute (Corchorus olitorius) and
its rapid detection using SCAR marker. BioControl, 57, 565e571.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
98 J.B. Wang et al.

Blair, J. E., Coffey, M. D., Park, S. Y., Geiser, D. M., & Kang, S. (2008). A multi-locus phy-
logeny for Phytophthora utilizing markers derived from complete genome sequences.
Fungal Genetics and Biology, 45, 266e277.
Brownbridge, M., Reay, S. D., Nelson, T. L., & Glare, T. R. (2012). Persistence of Beauveria
bassiana (Ascomycota: Hypocreales) as an endophyte following inoculation of radiata
pine seed and seedlings. Biological Control, 61, 194e200.
Bushley, K. E., Raja, R., Jaiswal, P., Cumbie, J. S., Nonogaki, M., Boyd, A. E. …
Spatafora, J. W. (2013). The genome of Tolypocladium inflatum: evolution, organization,
and expression of the cyclosporin biosynthetic gene cluster. PLoS Genetics, 9, e1003496.
Cannon, P. F., Hywel-Jones, N. L., Maczey, N., Norbu, L., Samdup, T. T., & Lhendup, P.
(2009). Steps towards sustainable harvest of Ophiocordyceps sinensis in Bhutan. Biodiversity
and Conservation, 18, 2263e2281.
Chang, Y., Wang, S., Sekimoto, S., Aerts, A. L., Choi, C., Clum, A. … Berbee, M. L. (2015).
Phylogenomic analyses indicate that early fungi evolved digesting cell walls of algal
ancestors of land plants. Genome Biology and Evolution, 7, 1590e1601.
Chen, Y. X., Feng, P., Shang, Y. F., Xu, Y.-J., & Wang, C. S. (2015). Biosynthesis of non-
melanin pigment by a divergent polyketide synthase in Metarhizium robertsii. Fungal
Genetics and Biology, 81, 142e149.
Cheng, Q., Wang, H., Xu, B., Zhu, S., Hu, L., & Huang, M. (2014). Discovery of a novel
small secreted protein family with conserved N-terminal IGY motif in Dikarya fungi.
BMC Genomics, 15, 1151.
Cordaux, R., & Batzer, M. A. (2009). The impact of retrotransposons on human genome
evolution. Nature Reviews Genetics, 10, 691e703.
Cresnar, B., & Petric, S. (2011). Cytochrome P450 enzymes in the fungal kingdom. Bio-
chimica et Biophysica Acta, 1814, 29e35.
Crisp, A., Boschetti, C., Perry, M., Tunnacliffe, A., & Micklem, G. (2015). Expression of
multiple horizontally acquired genes is a hallmark of both vertebrate and invertebrate
genomes. Genome Biology, 16, 50.
Cuomo, C. A., G€ uldener, U., Xu, J. R., Trail, F., Turgeon, B. G., Di Pietro, A. …
Kistler, H. C. (2007). The Fusarium graminearum genome reveals a link between localized
polymorphism and pathogen specialization. Science, 317, 1400e1402.
Daboussi, M. J., & Capy, P. (2003). Transposable elements in filamentous fungi. Annual
Review of Microbiology, 57, 275e299.
De Fine Licht, H. H., Hajek, A. E., Eilenberg, J., & Jensen, A. B. (2016). Utilizing Genomics
to Study Entomopathogenicity in the Fungal Phylum Entomophthoromycota: A
Review of Current Genetic Resources. Advances in Genetics, 94, 41e65.
Donzelli, B. G. G., Krasnoff, S. B., Sun-Moon, Y., Churchill, A. C. L., & Gibson, D. M.
(2012). Genetic basis of destruxin production in the entomopathogen Metarhizium
robertsii. Current Genetics, 58, 105e116.
Donzelli, B. G. G., & Krasnoff, S. B. (2016). Molecular Genetics of Secondary Chemistry in
Metarhizium Fungi. Advances in Genetics, 94, 365e436.
Duan, Z. B., Chen, Y. X., Huang, W., Shang, Y. F., Chen, P. L., & Wang, C. S. (2013).
Linkage of autophagy to fungal development, lipid storage and virulence in Metarhizium
robertsii. Autophagy, 9, 538e549.
Duan, Z. B., Shang, Y. F., Gao, Q., Zheng, P., & Wang, C. S. (2009). A phosphoketolase
Mpk1 of bacterial origin is adaptively required for full virulence in the insect-pathogenic
fungus Metarhizium anisopliae. Environmental Microbiology, 11, 2351e2360.
Dyer, P. S., & O’Gorman, C. M. (2012). Sexual development and cryptic sexuality in fungi:
insights from Aspergillus species. FEMS Microbiology Review, 36, 165e192.
Eadie, M. J. (2003). Convulsive ergotism: epidemics of the serotonin syndrome? Lancet
Neruology, 2, 429e434.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 99

Fang, W. G., Pava-Ripoll, M., Wang, S. B., & St. Leger, R. J. (2009). Protein kinase A
regulates production of virulence determinants by the entomopathogenic fungus,
Metarhizium anisopliae. Fungal Genetic and Biology, 46, 277e285.
Fang, W. G., & St. Leger, R. J. (2010a). Mrt, a gene unique to fungi, encodes an oligosac-
charide transporter and facilitates rhizosphere competency in Metarhizium robertsii. Plant
Physiology, 154, 1549e1557.
Fang, W. G., & St. Leger, R. J. (2010b). RNA binding proteins mediate the ability of a fun-
gus to adapt to the cold. Environmental Microbiology, 12, 810e820.
Feng, P., Shang, Y. F., Cen, K., & Wang, C. S. (2015). Fungal biosynthesis of the bibenzoqui-
none oosporein to evade insect immunity. Proceedings of the National Academy of Sciences of
the United States of America, 112, 11365e11370.
Fitzpatrick, D. A. (2012). Horizontal gene transfer in fungi. FEMS Microbiology Letters, 329,
1e8.
Fraser, J. A., Diezmann, S., Subaran, R. L., Allen, A., Lengeler, K. B., Dietrich, F. S. …
Heitman, J. (2004). Convergent evolution of chromosomal sex-determining regions
in the animal and fungal kingdoms. PLoS Biology, 2, e384.
Freimoser, F. M., Hu, G., & St. Leger, R. J. (2005). Variation in gene expression patterns as
the insect pathogen Metarhizium anisopliae adapts to different host cuticles or nutrient
deprivation in vitro. Microbiology, 151, 361e371.
Galagan, J. E., Calvo, S. E., Borkovich, K. A., Selker, E. U., Read, N. D., Jaffe, D. …
Birren, B. (2003). The genome sequence of the filamentous fungus Neurospora crassa.
Nature, 422, 859e868.
Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L. J., Wortman, J. R., Batzoglou, S. …
Birren, B. W. (2005). Sequencing of Aspergillus nidulans and comparative analysis with
A. fumigatus and A. oryzae. Nature, 438, 1105e1115.
Gao, Q., Jin, K., Ying, S. H., Zhang, Y., Xiao, G., Shang, Y. … Wang, C. (2011). Genome
sequencing and comparative transcriptomics of the model entomopathogenic fungi
Metarhizium anisopliae and M. acridum. PLoS Genetics, 7, e1001264.
Gao, Q., Shang, Y. F., Huang, W., & Wang, C. S. (2013). Glycerol-3-phosphate acyltransfer-
ase contributes to triacylglycerol biosynthesis, lipid droplet formation and host invasion in
Metarhizium robertsii. Applied and Environmental Microbiology, 79, 7646e7653.
Geiser, D. M., Timberlake, W. E., & Arnold, M. L. (1996). Loss of meiosis in Aspergillus.
Molecular Biology and Evolution, 13, 809e817.
Gladieux, P., Ropars, J., Badouin, H., Branca, A., Aguileta, G., de Vienne, D. M. … Giraud, T.
(2014). Fungal evolutionary genomics provides insight into the mechanisms of adaptive
divergence in eukaryotes. Molecular Ecology, 23, 753e773.
Hajek, A. E., McManus, M. L., & Delalibera, I., Jr. (2007). A review of introductions of
pathogens and nematodes for classical biological control of insects and mites. Biological
Control, 41, 1e13.
Hajek, A. E., & Tobin, P. C. (2011). Introduced pathogens follow the invasion front of a
spreading alien host. Journal of Animal Ecology, 80, 1217e1226.
Hay, W. W., Soeding, E., DeConto, R. M., & Wold, C. N. (2002). The late cenozoic uplift-
climate change paradox. International Journal of Earth Science, 91, 746e774.
Hu, G., & St. Leger, R. J. (2002). Field studies using a recombinant mycoinsecticide (Meta-
rhizium anisopliae) reveal that it is rhizosphere competent. Applied and Environmental
Microbiology, 68, 6383e6387.
Hu, G., & St. Leger, R. J. (2004). A phylogenomic approach to reconstructing the diversi-
fication of serine proteases in fungi. Journal of Evolutionary Biology, 17, 1204e1214.
Hu, X., Xiao, G., Zheng, P., Shang, Y., Su, Y., Zhang, X. … Wang, C. S. (2014). Trajectory
and genomic determinants of fungal-pathogen speciation and host adaptation. Proceedings
of the National Academy of Sciences of the United States of America, 111, 16796e16801.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
100 J.B. Wang et al.

Hu, X., Zhang, Y. J., Xiao, G. H., Zheng, P., Xia, Y. L., Zhang, X. Y. … Wang, C. S. (2013).
Genome survey uncovers the secrets of sex and lifestyle in caterpillar fungus. Chinese Science
Bulletin, 58, 2846e2854.
Huang, W., Shang, Y. F., Chen, P. L., Cen, K., & Wang, C. S. (2015). Basic leucine zipper
(bZIP) domain transcription factor MBZ1 regulates cell wall integrity, spore adher-
ence, and virulence in Metarhizium robertsii. Journal of Biological Chemistry, 290,
8218e8231.
Huang, W., Shang, Y. F., Chen, P. L., Gao, Q., & Wang, C. S. (2015). MrpacC regulates
sporulation, insect cuticle penetration and immune evasion in Metarhizium robertsii. Envi-
ronmental Microbiology, 17, 994e1008.
Hughes, D. P., Araujo, J. P. M., Loreto, R. G., Quevillon, L., de Bekker, C., & Evans, H. C.
(2016). From So Simple a Beginning: The Evolution of Behavioral Manipulation by
Fungi. Advances in Genetics, 94, 437e469.
Hugouvieux-Cotte-Pattat, N., Condemine, G., Nasser, W., & Reverchon, S. (1996). Regu-
lation of pectinolysis in Erwinia chrysanthemi. Annunal Review and Microbiology, 50, 213e257.
Idnurm, A. (2011). Sex and speciation: the paradox that non-recombining DNA promotes
recombination. Fungal Biology Reviews, 25, 121e127.
Isaka, M., Kittakoop, P., Kirtikara, K., Hywel-Jones, N. L., & Thebtaranonth, Y. (2005). Bioac-
tive substances from insect pathogenic fungi. Accounts of Chemical Research, 38, 813e823.
Jackson, A. P. (2004). A reconciliation analysis of host switching in plant-fungal symbioses.
Evolution, 58, 1909e1923.
Juenger, T., & Bergelson, J. (1998). Pairwise versus diffuse natural selection and the multiple
herbivores of scarlet gilia, Ipomopsis aggregata. Evolution, 52, 1583e1592.
Kang, C. S., Goo, B. Y., Gyu, L. D., & Heon, Y. (1996). Antifungal activities of Metarhizium
anisopliae against Fusarium oxysporum, Botrytis cinerea and Alternaria solani. Korean Journal of
Mycology, 24, 49e55.
Kepler, R. M., Sung, G. H., Ban, S., Nakagiri, A., Chen, M. J., Huang, B. … Spatafora, J. W.
(2012). New teleomorph combinations in the entomopathogenic genus Metacordyceps.
Mycologia, 104, 182e197.
Kershaw, M. J., Moorhouse, E. R., Bateman, R., Reynolds, S. E., & Charnley, A. K. (1999).
The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of
insects. Journal of Invertebrate Pathology, 74, 213e223.
Kim, H. G., Song, H., Yoon, D. H., Song, B. W., Park, S. M., Sung, G. H. … Kim, T. W.
(2010). Cordyceps pruinosa extracts induce apoptosis of HeLa cells by a caspase dependent
pathway. Journal of Ethnopharmacology, 128, 342e351.
Koo, B. S., An, H. G., Moon, S. K., Lee, Y. C., Kim, H. M., Ko, J. H., & Kim, C. H. (2003).
Bombycis corpus extract (BCE) protects hippocampal neurons against excitatory amino
acid-induced neurotoxicity. Immunopharmacology and Immunotoxicology, 25, 191e201.
Krasnoff, S. B., Gupta, S., St. Leger, R. J., Renwick, J. A. A., & Roberts, D. W. (1991).
Antifungal and insecticidal properties of the efrapeptins: metabolites of the fungus
Tolypocladium niveum. Journal of Invertebrate Pathology, 58, 180e188.
Kronstad, J. W., & Staben, C. (1997). Mating type in filamentous fungi. Annual Review of
Genetics, 31, 245e276.
Kubicek, C. P., Herrera-Estrella, A., Seidl-Seiboth, V., Martinez, D. A., Druzhinina, I. S.,
Thon, M. … Grigoriev, I. V. (2011). Comparative genome sequence analysis under-
scores mycoparasitism as the ancestral life style of Trichoderma. Genome Biology, 12, R40.
Kulkarni, R. D., Thon, M. R., Pan, H., & Dean, R. A. (2005). Novel G-protein-coupled
receptor-like proteins in the plant pathogenic fungus Magnaporthe grisea. Genome Biology,
6, R24.
Li, Z., Li, C., Huang, B., & Fan, M. (2001). Discovery and demonstration of the teleomorph
of Beauveria bassiana (Bals.) Vuill., an important entomogenous fungus. Chinese Science
Bulletin, 46, 751e753.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 101

Liao, X., Fang, W., Lin, L., Lu, H. L., & St. Leger, R. J. (2013). Metarhizium robertsii produces
an extracellular invertase (MrINV) that plays a pivotal role in rhizospheric interactions
and root colonization. PLoS One, 8, e78118.
Liao, Y., Ling, J., Zhang, G., Liu, F., Tao, S., Han, Z. … Le, H. (2015). Cordycepin induces
cell cycle arrest and apoptosis by inducing DNA damage and up-regulation of p53 in
Leukemia cells. Cell Cycle, 14, 761e771.
Lin, L. C., Fang, W. G., Liao, X. G., Wang, F. Q., Wei, D. Z., & St. Leger, R. J. (2011). The
MrCYP52 cytochrome P450 monoxygenase gene of Metarhizium robertsii is important
for utilizing insect epicuticular hydrocarbons. PLoS One, 6, e28984.
Liu, L. S., Zhang, J., Chen, C., Teng, J. T., Wang, C. S., & Luo, D. Q. (2015). Structure and
biosynthesis of fumosorinone, a new protein phosphatase 1B inhibitor firstly isolated
from the entomogenous fungus Isaria fumosorosea. Fungal Genetics and Biology, 81,
191e200.
Liu, Z. Y., Liang, Z. Q., Whalley, A. J., Yao, Y. J., & Liu, A. Y. (2001). Cordyceps brittleban-
kisoides, a new pathogen of grubs and its anamorph, Metarhizium anisopliae var. majus.
Journal of Invertebrate Pathology, 78, 178e182.
Lovett, B., & St. Leger, R. J. (2015). Stress is the rule rather than the exception for
Metarhizium. Current Genetics, 61, 253e261.
Luo, X., Keyhani, N. O., Yu, X., He, Z., Luo, Z., Pei, T., & Zhang, Y. (2012). The MAP
kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect
pathogenic fungus Beauveria bassiana. Fungal Genetics and Biology, 49, 544e545.
Ma, L. J., van der Does, H. C., Borkovich, K. A., Coleman, J. J., Daboussi, M.-J., &
Pietro, A. D. (2010). Comparative genomics reveals mobile pathogenicity chromosomes
in Fusarium. Nature, 464, 367e373.
Martin, F., Aerts, A., Ahren, D., Brun, A., Danchin, E. G. J., Duchaussoy, F. …
Grigoriev, I. V. (2008). The genome of Laccaria bicolor provides insights into mycorrhizal
symbiosis. Nature, 452, 88e92.
Martinez, D., Berka, R. M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S. E. …
Brettin, T. S. (2008). Genome sequencing and analysis of the biomass-degrading fungus
Trichoderma reesei (syn. Hypocrea jecorina). Nature Biotechnology, 26, 553e560.
Meyling, N. V., L€ ubeck, M., Buckley, E. P., Eilenberg, J., & Rehner, S. A. (2009). Com-
munity composition, host range and genetic structure of the fungal entomopathogen
Beauveria in adjoining agricultural and seminatural habitats. Molecular Ecology, 18,
1282e1293.
Moonjely, S., Barrelli, R., & Bidochka, M. J. (2016). Insect Pathogenic Fungi as Endophytes.
Advances in Genetics, 94, 107e136.
Molnar, I., Gibson, D. M., & Krasnoff, S. B. (2010). Secondary metabolites from entomo-
pathogenic Hypocrealean fungi. Natural Product Reports, 27, 1241e1275.
Morschhauser, J. (2010). Regulation of multidrug resistance in pathogenic fungi. Fungal
Genetics and Biology, 47, 94e106.
Ni, M., Feretzaki, M., Sun, S., Wang, X., & Heitman, J. (2011). Sex in fungi. Annual Review
of Genetics, 45, 405e430.
Noda, J., Brito, N., & Gonzalez, C. (2010). The Botrytis cinerea xylanase Xyn11A contributes
to virulence with its necrotizing activity, not with its catalytic activity. BMC Plant
Biology, 10, 38.
Nygren, K., Strandberg, R., Wallberg, A., Nabholz, B., Gustafsson, T., García, D. …
Johannesson, H. (2011). A comprehensive phylogeny of Neurospora reveals a link be-
tween reproductive mode and molecular evolution in fungi. Molecular Phylogenetics and
Evolution, 59, 649e663.
Ohm, R., Feau, N., Henrissat, B., Schoch, C. L., Horwitz, B. A., Barry, K. W. … Grigoriev, I.
(2012). Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of
eighteen Dothideomycetes fungi. PLoS Pathogens, 8, e1003037.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
102 J.B. Wang et al.

Ortiz-Urquiza, A., & Keyhani, N. O. (2013). Action on the surface: entomopathogenic fungi
versus the insect cuticle. Insects, 4, 357e374.
Ortiz-Urquiza, A., & Keyhani, N. O. (2015). Stress response signaling and virulence: insights
from entomopathogenic fungi. Current Genetics, 61, 239e249.
Ortiz-Urquiza, A., & Keyhani, N. O. (2016). Molecular Genetics of Beauveria bassiana
Infection of Insects. Advances in Genetics, 94, 165e250.
Ownley, B. H., Gwinn, K. D., & Vega, F. E. (2010). Endophytic fungal entomopathogens
with activity against plant pathogens: ecology and evolution. BioControl, 55, 113e128.
Pal, S., St. Leger, R. J., & Wu, L. P. (2007). Fungal peptide Destruxin A plays a specific role
in suppressing the innate immune response in Drosophila Melanogaster. Journal of Biological
Chemistry, 292, 8969e8977.
Paterson, R. R. (2008). Cordyceps: a traditional Chinese medicine and another fungal thera-
peutic biofactory? Phytochemistry, 69, 1469e1495.
Pattemore, J. A., Hane, J. K., Williams, A. H., Wilson, B. A., Stodart, B. J., & Ash, G. J.
(2014). The genome sequence of the biocontrol fungus Metarhizium anisopliae and
comparative genomics of Metarhizium species. BMC Genomics, 15, 660.
Pava-Ripoll, M., Angelini, C., Fang, W. G., Wang, S., Posada, F. J., & St. Leger, R. J. (2011).
The rhizosphere-competent entomopathogen Metarhizium anisopliae expresses a specific
subset of genes in plant root exudate. Microbiology, 157, 47e55.
Pereira, J. L., Noronha, E. F., Miller, R. N., & Franco, O. L. (2007). Novel insights in the use
of hydrolytic enzymes secreted by fungi with biotechnological potential. Letters in Applied
Microbiology, 44, 573e581.
Peterson, S. W. (2008). Phylogenetic analysis of Aspergillus species using DNA sequences
from four loci. Mycologia, 100, 205e226.
Raffaele, S., & Kamoun, S. (2012). Genome evolution in filamentous plant pathogens: why
bigger can be better. Nature Reviews Microbiology, 10, 417e430.
Rangel, D. E., Alston, D. G., & Roberts, D. W. (2008). Effects of physical and nutritional
stress conditions during mycelial growth on conidial germination speed, adhesion to
host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus. Myco-
logical Research, 112, 1355e1361.
Rhee, Y. J., Hillier, S., & Gadd, G. M. (2012). Lead transformation to pyromorphite by
fungi. Current Biology, 22, 237e241.
Richards, T. A., Leonard, G., Soanes, M., & Talbot, N. J. (2011). Gene transfer into the
fungi. Fungal Biology Reviews, 25, 98e110.
Roberts, D. W., & St. Leger, R. J. (2004). Metarhizium spp., cosmopolitan insect-pathogenic
fungi: mycological aspects. Advances in Applied Microbiology, 54, 1e70.
Scazzocchio, C. (2000). The fungal GATA factors. Current Opinion in Microbiology, 3,
126e131.
Schaible, U. E., & Kaufmann, S. H. E. (2005). A nutritive view on the host-pathogen
interplay. Trends in Microbiology, 13, 373e380.
Schulze-Lefert, P., & Panstruga, R. (2011). A molecular evolutionary concept connecting
nonhost resistance, pathogen host range, and pathogen speciation. Trends in Plant Science,
16, 117e125.
Screen, S. E., & St. Leger, R. J. (2000). Cloning, expression and substrate specificity of a
fungal chymotrypsin. Journal of Biological Chemistry, 275, 6689e6694.
Seidl, V. (2008). Chitinases of filamentous fungi: a large group of diverse proteins with mul-
tiple physiological functions. Fungal Biology Reviews, 22, 36e42.
Shang, Y., Feng, P., & Wang, C. (2015). Fungi that infect insects: altering host behavior and
beyond. PLoS Pathogens, 11, e1005037.
Sharpton, T. J., Stajich, J. E., Rounsley, S. D., Gardner, M. J., Wortman, J. R.,
Jordar, V. S. … Taylor, J. W. (2009). Comparative genomic analyses of the human fungal
pathogens Coccidioides and their relatives. Genome Research, 19, 1722e1731.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 103

Silva, W. O. B., Santi, L., Berger, M., Pinto, A. F. M., Guimaraes, J. A., Schrank, A. …
Vainstein, M. H. (2009). Characterization of a spore surface lipase from the biocontrol
agent Metarhizium anisopliae. Processing Biochemistry, 44, 829e834.
St. Leger, R. J. (2008). Studies on adaptations of Metarhizium anisopliae to life in the soil. Jour-
nal of Invertebrate Pathology, 98, 271e276.
St. Leger, R. J., Bidochka, M. J., & Roberts, D. W. (1994). Germination triggers of Meta-
rhizium anisopliae conidia are related to host species. Microbiology, 140, 1651e1660.
St. Leger, R. J., Cooper, R. M., & Charnley, A. K. (1986a). Cuticle-degrading enzymes of
entomopathogenic fungi: regulation of production of chitinolytic enzymes. Journal of
General Microbiology, 132, 1509e1517.
St. Leger, R. J., Cooper, R. M., & Charnley, A. K. (1986b). Cuticle-degrading enzymes
of entomopathogenic fungi: cuticle degradation in vitro by enzymes from
entomopathogens. Journal of Invertebrate Pathology, 47, 167e177.
St. Leger, R. J., Joshi, L., Bidochka, M. J., & Roberts, D. W. (1996). Construction of an
improved mycoinsecticide overexpressing a toxic protease. Proceedings of the National
Academy of Sciences of the United States of America, 93, 6349e6354.
St. Leger, R. J., May, B., Allee, L. L., Frank, D. C., Staples, R. C., & Roberts, D. W. (1992).
Genetic differences in allozymes and in formation of infection structures among isolates
of the entomopathogenic fungus Metarhizium anisopliae. Journal of Invertebrate Pathology,
60, 89e101.
St. Leger, R. J., Nelson, J. O., & Screen, S. E. (1999). The entomopathogenic fungus Meta-
rhizium anisopliae alters ambient pH, allowing extracellular protease production and
activity. Microbiology, 145, 2691e2699.
St. Leger, R. J., Wang, C. S., & Fang, W. G. (2011). New perspectives on insect pathogens.
Fungal Biology Reviews, 25, 84e88.
Staats, C. C., Junges, A., Guedes, R. L., Thompson, C. E., de Morais, G. L., Boldo, J. T., &
Schrank, A. (2014). Comparative genome analysis of entomopathogenic fungi reveals a
complex set of secreted proteins. BMC Genomics, 15, 822.
Steinwender, B. M., Enkerli, J., Widmer, F., Eilenberg, J., Kristensen, H. L.,
Bidochka, M. J., & Meyling, N. V. (2015). Root isolations of Metarhizium spp. from
crops reflect diversity in the soil and indicate no plant specificity. Journal of Invertebrate
Pathology, 132, 142e148.
Stidham, T. A., & Stidham, J. A. (2000). A new Miocene band-winged grasshopper (Orthop-
tera: Acrididae) from Nevada. Annals of the Entomological Society of America, 93, 405e407.
Stukenbrock, E. H., Bataillon, T., Dutheil, J. Y., Hansen, T. T., Li, R., & Zala, M. (2011).
The making of a new pathogen: insights from comparative population genomics of the
domesticated wheat pathogen Mycosphaerella graminicola and its wild sister species. Genome
Research, 21, 2157e2166.
Suh, S. O., Noda, H., & Blackwell, M. (2001). Insect symbiosis: derivation of yeast-like en-
dosymbionts within an entomopathogenic filamentous lineage. Molecular Biology and
Evolution, 18, 995e1000.
Sun, S., & Heitman, J. (2011). Is sex necessary? BMC Biology, 9, 56e59.
Sung, G. H., Hywel-Jones, N. L., Sung, J. M., Jennifer Luangsa-ard, J., Shrestha, N., &
Spatafora, J. W. (2007). Phylogenetic classification of Cordyceps and the clavicipitaceous
fungi. Studies in Mycology, 57, 5e59.
Thomas, M. B., & Read, A. F. (2007). Can fungal biopesticides control malaria? Nature Re-
views Microbiology, 5, 377e383.
Torres, M. S., Singh, A. P., Vorsa, N., & White, J. F., Jr. (2008). An analysis of ergot alkaloids
in the Clavicipitaceae (Hypocreales, Ascomycota) and ecological implications. Symbiosis,
46, 11e19.
Vega, F. E., Posada, F., Aime, M. C., Pava-Ripoll, M., Infante, F., & Rehner, S. A. (2008).
Entomopathogenic fungal endophytes. Biological Control, 46, 72e82.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
104 J.B. Wang et al.

Wagner, B. L., & Lewis, L. C. (2000). Colonization of corn, Zea mays, by the entomopatho-
genic fungus Beauveria bassiana. Applied and Environmental Microbiology, 66, 3468e3473.
Wang, B., Kang, Q. J., Lu, Y. Z., Bai, L., & Wang, C. (2012). Unveiling the biosynthetic
puzzle of destruxins in Metarhizium species. Proceedings of the National Academy of Sciences
of the United States of America, 109, 1287e1292.
Wang, C., Butt, T. M., & St. Leger, R. J. (2005). Colony sectorization of Metarhizium ani-
sopliae is a sign of ageing. Microbiology, 151, 3223e3236.
Wang, C., Fan, M. Z., Li, Z. Z., & Butt, T. M. (2004). Molecular monitoring and evaluation
of the application of the insect-pathogenic fungus Beauveria bassiana in southeast China.
Journal of Applied Microbiology, 96, 861e870.
Wang, C., & Feng, M.-G. (2014). Advances in fundamental and applied studies in
China of fungal biocontrol agents for use against arthropod pests. Biological Control,
68, 129e135.
Wang, C., & St. Leger, R. J. (2006). A collagenous protective coat enables Metarhizium ani-
sopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of
the United States of America, 103, 6647e6652.
Wang, C., & St. Leger, R. J. (2007). The Metarhizium anisopliae perilipin homolog MPL1 reg-
ulates lipid metabolism, appressorial turgor pressure, and virulence. Journal of Biological
Chemistry, 282, 21110e21115.
Wang, C., Zhang, S., Hou, R., Zheng, Q., Xu, Q., Zheng, D. … Xu, J. (2011). Functional
analysis of the kinome of the wheat scab fungus Fusarium graminearum. PLoS Pathogens,
12, e1002460.
Wang, C. S., Duan, Z. B., & St. Leger, R. J. (2008). MOS1 osmosensor of Metarhizium
anisopliae is required for adaptation to insect host hemolymph. Eukaryotic Cell, 7,
302e309.
Wang, C. S., Hu, G., & St. Leger, R. J. (2005). Differential gene expression by Metarhizium
anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals
mechanisms of physiological adaptation. Fungal Genetics and Biology, 42, 704e718.
Wang, C. S., & St. Leger, R. J. (2005). Developmental and transcriptional responses to host
and non-host cuticles by the specific locust pathogen Metarhizium anisopliae sf. acridum.
Eukaryotic Cell, 4, 937e947.
Wang, C. S., & St. Leger, R. J. (2007). The MAD1 adhesin of Metarhizium anisopliae links
adhesion with blastospore production and virulence to insects, and the MAD2 adhesin
enables attachment to plants. Eukaryotic Cell, 6, 808e816.
Wang, C. S., & St. Leger, R. J. (2014). Genomics of entomopathogenic fungi. In F. Martin
(Ed.), The ecological genomics of fungi (pp. 243e260). John Wiley & Sons, Inc.
Wang, S. B., Fang, W. G., Wang, C. S., & St. Leger, R. J. (2011). Insertion of an esterase
gene into a specific locust pathogen (Metarhizium acridum) enables it to infect
caterpillars. PLoS Pathogens, 7, e1002097.
Wang, S., O’Brien, T. R., Pava-Ripoll, M., & St. Leger, R. J. (2011). Local adaptation of an
introduced transgenic insect fungal pathogen due to new beneficial mutations. Proceedings
of the National Academy of Sciences, 108, 20449e20454.
Whittle, C. A., Nygren, K., & Johannesson, H. (2011). Consequences of reproductive mode
on genome evolution in fungi. Fungal Genetics and Biology, 48, 661e667.
Xiao, G. H., Ying, S.-H., Zheng, Z., Wang, Z. L., Zhang, S., Xie, X. Q. … Feng, M. G.
(2012). Genomic perspectives on the evolution of fungal entomopathogenicity in Beau-
veria bassiana. Scientific Reports, 2, 483.
Xu, J. R., Peng, Y. L., Dickman, M. B., & Sharon, A. (2006). The dawn of fungal pathogen
genomics. Annual Reviews of Phytopathology, 44, 337e366.
Xu, Y. J., Luo, F. F., Gao, Q., Shang, Y. F., & Wang, C. S. (2015). Metabolomics reveals
insect metabolic responses associated with fungal infection. Analytical and Bioanalytical
Chemistry, 407, 4815e4821.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105
 Author's personal copy
Advances in Genomics of Entomopathogenic Fungi 105

Xue, C., Hsueh, Y. P., & Heitman, J. (2008). Magnificent seven: roles of G protein-coupled
receptors in extracellular sensing in fungi. FEMS Microbiology Review, 32, 1010e1032.
Xue, Y., Batlle, M., & Hirsch, J. P. (1998). GPR1 encodes a putative G protein-coupled
receptor that associates with the Gpa2p G-alpha subunit and functions in a Ras-
independent pathway. EMBO Journal, 17, 1996e2007.
Yokoyama, E., Arakawa, M., Yamagishi, K., & Hara, A. (2006). Phylogenetic and structural
analyses of the mating-type loci in Clavicipitaceae. FEMS Microbiology Letter, 264, 182e191.
Zhang, S., Widemann, E., Bernard, G., Lesot, A., Pinot, F., Pedrini, N., & Keyhani, N. O.
(2012). CYP52X1, representing new cytochrome P450 subfamily, displays fatty acid
hydroxylase activity and contributes to virulence and growth on insect cuticular
substrates in entomopathogenic fungus Beauveria bassiana. Journal of Biological Chemistry,
287, 13477e13486.
Zhang, S. Z., Xia, Y. X., & Keyhani, N. O. (2011). Contribution of the gas1 gene of the
entomopathogenic fungus Beauveria bassiana, encoding a putative glycosylphosphatidy-
linositol-anchored beta-1,3-glucanosyltransferase, to conidial thermotolerance and
virulence. Applied and Environmental Microbiology, 77, 2676e2684.
Zhang, Y., Zhao, J., Fang, W., Zhang, J., Luo, Z., Zhang, M. … Pei, Y. (2009). Mitogen-
activated protein kinase hog1 in the entomopathogenic fungus Beauveria bassiana
regulates environmental stress responses and virulence to insects. Applied and Environ-
mental Microbiology, 75, 3787e3795.
Zhao, H., Xu, C., Lu, H. L., Chen, X., St. Leger, R. J., & Fang, W. (2014). Host-to-
pathogen gene transfer facilitated infection of insects by a pathogenic fungus. PLoS
Pathogens, 10, e10040.
Zheng, P., Xia, Y., Xiao, G., Xiong, C., Hu, X., Zhang, S. … Wang, C. S. (2011). Genome
sequence of the insect pathogenic fungus Cordyceps militaris, a valued traditional Chinese
medicine. Genome Biology, 12, R116.
Zheng, P., Xia, Y., Zhang, S., & Wang, C. (2013). Genetics of Cordyceps and related fungi.
Applied Microbiology and Biotechnology, 97, 2797e2804.

Genetics and Molecular Biology of Entomopathogenic Fungi, First Edition, 2016, 67e105