This document provides an overview of insect pathogenic fungi and their molecular interactions with host insects. It discusses the major phyla of entomopathogenic fungi, important fungal genera like Beauveria, Metarhizium, and their infection processes. It also summarizes the molecular mechanisms of fungal pathogenesis, including cuticle degradation, secretion of antifungal compounds by insects, and fungal production of secondary metabolites to invade insects and suppress their immune responses. Finally, it touches on using comparative genomics and genetic engineering to improve fungal virulence and environmental tolerance.

1. WELCO
ME

2. PRESENTED BY:
P.KRANTHI
RAM/18-42
DEPT. OF ENTOMOLOGY
INSECT PATHOGENIC FUNGI : MOLECULAR
INTERACTION AND GENETIC IMPROVEMENT
CREDIT SEMINAR ON
CHAIRPERSON:R.SUNITHA DEVI
COURSE IN-CHARGE:T.UMA MAHESWARI

3. CONTENTS
• Introduction
• Entomopathogens of different phyla
• Important entomopathogenic fungi
• Fungal infection process
• Molecular mechanisms of fungal pathogenesis
• Comparative genomics of fungal entomopathogenicity
• Genetic improvement-virulence
 Using the pathogen’s own genes to improve virulence
 insect’s proteins for genetically engineering entomopathogenic fungi
 genes from insect predators and other insect pathogens for genetically engineering
entomopathogenic fungi
Invented proteins for genetically engineering entomopathogenic fungi
• Genetic engineering to improve fungal environmental stability
1. Improve tolerance to UV radiation
2. Improve tolerance to heat stress
• Concluding remarks and future perspectives

4. INTRODUCTION:
• Fungi are common pathogens of insects and therefore critical regulators of insect
populations in nature.
• Unlike bacteria and viruses entomopathogenic fungi do not require ingestion by
the host, so they can target sucking insects, such as mosquitoes and aphids
• There are more than 1,000 species distributed in the phyla
Entomophthoromycota
Blastocladiomycota
Microsporidia that are known to infect and kill insects
Basidiomycota
Ascomycota
• The phylum Entomophthoromycota is newly established from the former phylum
Zygomycota.

5. • Coelomomyces spp. were previously classified as chytrids and have
now been reallocated to the recently established phylum
Blastocladiomycota .
• Microsporidian parasites of insects are intracellular obligates, and
those that are pathogens of mosquitos and locusts have been
successfully used for the biological control of these insect pests .
• The genus Septobasidium contains more than 170 described species
• Of these five phyla, ascomycete insect pathogens, mainly species in
Cordyceps and Sensulato, include more than 600 species

6. • Insect pathogenic fungi have evolved highly diversified lifestyles in
nature .
• For example, the sexual stages of Metarhizium and Beauveria have been
identified as Metacordyceps and Cordyceps species; however, their
sexual stages rarely occur in the field and can hardly be induced in the lab
• Cordyceps species, such as C. militaris, can readily produce sexual
fruiting bodies ,which have been used for centuries in the traditional
medicine of Asian countries
• Fungal infections such as Ophiocordyceps unilateralis and
Entomophaga grylli, can alter host behaviour, inducing the host to climb
to an elevated position prior to its death to benefit the transmission of
fungal spores .

7. • Fungal pathogens currently have a small market share due to low
virulence compared to the chemical insecticides with which they
compete, and due to inconsistencies in their performance.
• Low virulence may be inbuilt as an evolutionary balance may have
developed between microorganisms and their hosts so that quick kill,
even at high doses, is not adaptive for the pathogen, in which case cost-
effective biocontrol will require genetic modification of the fungi
• The inconsistencies in performance in the field mainly result from the
sensitivity of entomopathogenic fungi to environmental stresses
• Genetic engineering has been proved to be an efficient tool to improve
the efficacy of mycoinsecticides by improving their tolerance to
environmental stresses and their virulence.

8. DIVERSIFICATION

10. M.robertsi M.acridum B.bassiana
Cardyceps militaris Cardyceps cicadae Ophiocardyceps unilateralis

11. Entomopathogens of Different Phyla
S.NO PHYLUM EXAMPLE
1 Oomycota Lagenidium giganteum
2 Chytridiomycota Coelomomyces (Blastocladiales) infect Hemipterans and Dipterans.
Myriophagus (Chytridiales) infects the pupae of Diptera
3 Zygomycota Entomophthora, Massospora
4 Ascomycota Cordyceps
5 Dueteromycota Aspergillus, Metarhizium, Hirsutella, Beauveria, Aschersonia,
Culicinomyces, Lecanicillium, Paecilomyces, Tolypocladium, etc.
6 Basidiomycota Uredinella and Septobasidium

12. Life Cycle of Entomopathogens
• Entomopathogens are found in the environment as spores or resting
spores
• The life cycle of insect pathogenic fungi begins with the spore
germination on the host cuticle
• Spores infect the insects by penetrating through the insect cuticle,
often joints or creases
• The fungus grows in the insect haemocoel
• After the insects die, the fungus grows out through the exoskeleton
produce spores

14. IMPORTANT ENTOMOPATHOGENIC
FUNGI

15. Beauveria spp.
pathogenic on
• Termites
• Thrips
• Whiteflies
• Aphids
• Grasshoppers
• Beetles
• Caterpillars
• Silkworms-white muscardine
Grasshoppers killed by B. bassiana

16. Metarhizium spp.
pathogenic on
• Grubs of Coconut rhinoceros beetle
• Grasshopper
• Rice BPH
• Sugarcane Pyrilla
• Bollworm
• Green Muscardine

17. Lecanicillium spp.
• L. lecanii and L. chlamydosporium are the important species of this
genus.
• It is an effective biocontrol agent against Trialeurodes vaporariorum
in greenhouses. This fungus attacks nymphs and adults and sticks to the
leaf underside by means of filamentous mycelium.
• L. lecanii is considered to control whitefly and several aphid species.

18. Nomuraea spp.
• Few important species of this genus
are
• Nomuraea rileyi
• Nomuraea atypicola
• Have been reported for several insects
hosts such as
• Trichoplusia sp.,
• Heliothis zea,
• Bombyx mori, etc

19. Isaria spp.
pathogenic on
• Trichoplusia sp.
• Heliothis zea
• Bagrada cruciferarum
• Bombyx mori
• Two important species, I.fumosoroseus and I.lilacinus.
• I.fumosoroseus is one of the most important natural enemies of whiteflies worldwide
and causes the sickness called “yellow muscardine.”

20. Hirsutella spp.
Specific to the eriophyid mites
• Coconut mite
• Citrus rust mite
• Genus Hirsutella includes three
important species,
• H.thompsonii, H. gigantea, and
H. citriformis
• Hirsutella thompsonii is used for
the control of citrus rust mite.
• This fungus is also pathogenic to
Acarida, Lepidoptera, and
Hemiptera groups of insects

21. MOLECULAR INTERACTION BETWEEN
ENTOMOPATHOGENIC FUNGI AND HOST

22. SCHEMATIC OF THE INFECTION PROCESS OF
ENTOMOPATHOGENIC FUNGI –M.robertsii

23. • By using M.robertsii, M.acridum, B.bassiana as model species,
different genes have been functionally characterized for their
contributions to fungal infection processes, such as
Spore adhesion
Infection structure differentiation
detoxification
Insect hemocoel adaptation
Nutrient deprivation
Immune evasion

24. Insect cuticle degradation by entomopathogenic fungi
1. Compositional compounds
 Hydrocarbons
 Protein and chitin
2. Secreted compounds that are deposited on the cuticle
Antifungal aldehydes and ketones
Insect cavity invasion by entomopathogenic fungi
1.Fungal secondary metabolite production
Insect response to fungal infection and fungal strategies to evade the
insect's immune system

25. Hydrocarbons :
• The most external surface of the insect cuticle, the epicuticle, is formed by a
thin lipid layer making it hydrophobic, which facilitates the attachment of
the hydrophobic conidia
• Major component are mix of straight-chain and methyl-branched, saturated
and unsaturated hydrocarbons.
• insects with saturated straight and branched chains are more susceptible
to entomopathogenic fungi than insects with predominantly unsaturated
(alkenes and/or alkadienes) chains .
• This might represent a first attempt of insects to stop fungal infection, i.e.
the possibility of covering its surface with hydrocarbons of different
chemical nature that provide a potential advantage to defend against the
entry of fungi.
• Entomopathogenic fungi have evolved to produce a variety of
hydroxylating enzymes that can successful assimilate hydrocarbons and
thus help degrade the lipid cuticular layer

26. Protein and chitin :
• The next cuticle layer that fungi must go through is the procuticle,
which contains the major bulk cuticle components, protein and chitin
• St. Leger et al. characterized a variety of hydrolytic enzymes
implicated in procuticle degradation, i.e., proteases, peptidases and
chitinases
• This information has subsequently facilitated obtaining fungal strains
that overexpress a subtilisin-like protease (Pr1) a chitinase and a fusion
protein with both activities , all of which improved virulence against
insect hosts.
• Pr1 expression and activity is currently used as a virulence marker in
Metarhizium species

27. 2. Secreted compounds that are deposited on
the cuticle
 Antifungal aldehydes and ketones
• Endocrine glands-thorax ,abdomen and legs: repel the predator
• Most of them are volatiles, these substances embed into the cuticle after
released and also confer protection against invasive microorganisms.
• In secretion one carbonyl group-resistant to entomopathogenic fungi
• Pentatomidae - saturated and α,β-unsaturated aldehydes
• Tenebrionidae - aromatic ketones (quinones)
• Stink bug- (E)-2-decenal (Aldehyde) was selectively fungistatic to
entomopathogenic fungi

28. • Mix of (E)-2- hexenal, (E)-2-octenal, and (E)-2-decenal released by
the rice stalk stink bug is responsible for the fungal growth inhibition
detected in Metarhizium anisopliae
• Both methyl- and ethyl-benzoquinone were the only compounds of
the volatile blend secreted by the red flour beetle T. castaneum able to
inhibit both germination and growth of B. bassiana
• By overexpression of a fungal 1,4-benzoquinone reductase,
B. bassiana became more virulent against the red flour beetle
T. castaneum, but not against other non-quinone secreting beetles

29.  Insect cavity invasion by entomopathogenic
fungi
 Fungal secondary metabolite production
• Filamentous fungi produce them mainly for use as antimicrobial.
• Insect pathogenic fungi also use them to facilitate fungal invasion of the
insect cavity and act as an immunosuppressant.
• B. Bassiana synthesize
Beauvericin
 Bassianolide (cyclooligomer non ribosomal peptides)
 Variety of beauverolides (cyclic peptides)
Oosporein (dibenzoquinone)
Bassiatin (diketomorpholine)
Enellin (2-pyridone)

30. • Metarhizium spp. produce mainly destruxins, a family of cyclic
hexadepsipeptides composed of α-hydroxy acid and 5 amino acid
residue
• Toxic compounds has been frequently linked with virulence
• Secondary metabolites are known to synthesize by gene clusters,
including non-ribosomal peptides synthetases (NRPS), polyketides
synthetases (PKS), and hybrid NRPS-PKS genes
• Oosporein- act by evading insect immunity and thus facilitating fungal
multiplication inside hosts
• Destruxin-detoxification of host immune compounds

31.  Insect response to fungal infection and fungal
strategies to evade the insect's immune system
• Two types of innate immune reactions
• The cellular response involves phagocytosis, hemocyte aggregation,
and pathogen encapsulation.
• The humoral response includes the induction of several antimicrobial
peptides (AMPs), lectins, and the prophenoloxidase cascade
• some differences in the recognition of microbial cells inside body
cavity specifically for entomopathogenic fungi
• Blastospores and hyphal bodies are weaker inductors of the immune
system than conidia and hyphae due to epitope profile

32. • Entomopathogenic fungi evolved more specific mechanisms to avoid the
immune response of the insect host including changing these epitopes to
escape from hemocyte encapsulation, as well as down-regulating protease
activities.
• Proteases are used for cuticle degradation, and once inside an insect they can
also degrade host defense molecules and thus help in fungal invasion
• The up-regulation of AMPs is regulated mainly by the Toll signal transduction
pathway
• The resulting peptides are then secreted into the hemocoel to prevent
microbial proliferation
• Fungi counterattack by secreting secondary metabolites
• Destruxins in Metarhizium spp.
• Myriocin in Isaria sinclairii
• Oosporein in B. bassiana

33. (Lobo et al.2015)

35. Toxins Produced by Different Entomopathogenic Fungi
Toxin Fungi Function
Efrapeptins Tolypocladium niveum Inhibitors of mitochondrial oxidative
phosphorylation and ATPase activity
Destruxins Metarhizium anisopliae Immuno depressant activity in insect
and cytotoxic effect
Beauvericin Beauveria bassiana Cytotoxic effect and insecticidal
properties
Bassianolide B. bassiana,
Verticillium lecanii
Acts as ionophore, toxic effect on
insects
Leucinostatins Isaria lilacinus
I.marquandii
Insecticidal activity by interfering
with oxidative phosphorylation

36. COMPARATIVE GENOMICS OF
FUNGAL ENTOMOPATHOGENICITY
• Genomes of 26 species of insect pathogens, which include 23
ascomycete species and 3 species of Entomophthoromycota
• Most of them are Metarhizium species, which include 13 sequenced
strains that belong to 9 species.
• In addition to understanding the fungal tree of life , obtaining genome
information will advance our knowledge of the genomic traits that
determine fungal entomopathogenicity and will further promote
molecular biology studies of fungus-environment interactions that will
benefit agriculture, the environment, and human health.

38. GENETIC IMPROVEMENTS OF
FUNGAL BIOCONTROL EFFICACY:
• The ultimate goal of studying entomopathogenic fungi is to promote the
cost-effective applications of mycoinsecticides for the control of different
insect pests .
• Barriers to the large-scale application of fungal biocontrol agents still
exist due to their slow killing speed and environmental stability issues
• Apart from improving formulation techniques, genetic engineering
efforts have been spent on improving the efficacy of mycoinsecticides
• Genetic modifications to increase fungal virulence against targeted pests
and enhancing fungal environmental stabilities against stress factors, such
as UV irradiation and high temperature.

39. GENETIC IMPROVEMENT
VIRULENCE
• Based on the sources of genes for genetic engineering , four major
strategies are currently being exploited to improve virulence of
entomopathogenic fungi

40. Using the Pathogen’s Own Genes to Improve
Virulence
Aim Type Source
Pr1A Subtilisin-like protease Metarhizium robertsii
CDEP1 Subtilisin-like protease Beauveria bassiana
Bbchit1 Chitinase Beauveria bassiana
Mr-Npc2a Sterol carrier Metarhizium robertsii
ATM1 Trehalase M.acridum
Mr-Ste1 Esterase Metarhizium robertsii
BbBqrA Benzoquinone
oxidoreductase
Beauveria bassiana
(Zaho et al. 2016)

41. • Overexpressing the gene encoding the subtilisin-like protease Pr1A
increased the virulence of Metarhizium anisopliae to Manduca sexta, and
the recombinant strain showed a 25% mean reduction in survival time (ST50)
toward the insect as compared to the parent wild-type (WT) strain
• Overproduction of B. bassiana’s chitinase CHIT1 improved virulence by
23%
• Expression of Pr1A from M. anisopliae also increases the killing speed of
B.bassiana ,showing that pathogenicity-related genes from one
entomopathogenic fungus can be used to improve virulence of other
entomopathogenic fungi.
• Overexpression of the acid trehalase ATM1 accelerated the growth of
M. acridum in the hemocoel of locusts, reducing the number of conidia
causing 50% mortality (LC50) by 8.3-fold compared with the WT strain.
• Transfer of an esterase gene (Mest1) from the generalist
Metarhizium robertsii to the locust specialist M. acridum enabled the latter
strain to expand its range to infect caterpillar.

42. Insect’s Proteins for Genetically Engineering
Entomopathogenic Fungi
Aim Type Source
MSDH Diuretic hormone Manduca sexta
Serpin Serine proteinease
inhibitor
Drosophila melanogaster
TMOF Trypsin modulating
oostatic factor
Anopheles aegypti,
Sarcophaga bullata(grey
flesh fly)
β-NP Pyrokinin β-neuropeptide Solenopsis invicta
(Zaho et al. 2016)

43. • Diuretic hormones regulate insect water salt balance
• Expression of the M. sexta diuretic hormone (MSDH) significantly
increased the virulence of B. bassiana against various Lepidopteran
targets (eg. M. sexta and Galleria mellonella) as well as mosquitoes
(Anopheles aegypti)
• expression of the species-specific pyrokinin β-neuropeptide from fire ants
(Solenopsis invicta) increased the virulence of B. bassiana against fire
ants, but not against Lepidopteran hosts G. mellonella and M. sexta
• Expression of TMOF from A. aegypti significantly increases the
virulence of B. bassiana against adults and larvae of the mosquito.
• This TMOF expressing strain was also potent against the malaria
mosquito Anopheles gambiae.

44. Pyrokinin β-Neuropeptide Affects Necrophoretic Behaviour in Fire
Ants (S. invicta), and Expression of β-NP in a Mycoinsecticide
Increases Its Virulence
(Fan et .al. 2011)

45. (Fan et .al. 2011)

46. Genes from Insect Predators and Other Insect Pathogens
for Genetically Engineering Entomopathogenic Fungi
AIM TYPE SOURCE
AaIT1 Sodium channel blocker Androctonus australis (fat tailed
scorpion)
BmKit Sodium channel blocker Buthus martensi (Chinese
scorpion)
LqhIT2 Sodium channel blocker Leiurus quinquestriatus
hebraeus (death stalker)
BjαIT Sodium channel blocker Buthotus judaicus (black
scorpion)
ω-HXTX-Hv1a Calcium channel blocker Atrax robustus (web spider)
k-HXTX-Hv1c Ca+2-activated K+1 channel
blocker
Hadronyche versuta(web
spider)
Hybrid-toxin Ca+2-activated K+1 channel
blocker
Hadronyche versuta
(Zaho et al. 2016

47. Genes from other insect pathogens
AIM TYPE SOURCE
Vip3A protein Vegetative insecticidal Bacillus thuringiensis
(Zaho et al. 2016)

48. A scorpion neurotoxin increases the potency of
a fungal insecticide
(Wang et.al. 2007)

49. Construction of a Hypervirulent and Specific
Mycoinsecticide for Locust Control
(Fang et.al.2014)

50. (Fang et.al.2014)

51. • The virulence of M. acridum was improved by using the insect-specific
neurotoxin LqhIT2 from the Israeli yellow scorpion Leiurus quinquestriatus
and the neurotoxin BjαIT from the Judean black scorpion Buthotus judaicus
• Vip3A Bt toxins expressed by B. bassiana may be more environmentally
stable.
• Transgenic B. bassiana may be effective at lower concentrations than the WT.
• The toxin BmKit from Buthus martensi was expressed in L. lecanii, and the
mortality rates in cotton aphids at 7.1-fold lower conidial doses than the WT
strain, and the median survival time reduced by 26.5% compared with WT
• The scorpion Na+ channel blocker AaIT1 gene tested in M. anisopliae.
It gave mortality rates in tobacco hornworm (M. sexta) at 22-fold lower
conidial doses than the WT fungus, and survival times at some doses
were reduced by 40%.

52. Invented Proteins for Genetically Engineering
Entomopathogenic Fungi
AIM TYPE SOURCE
CDEP1:Bbchit1 Protease and chitinase
activity
Engineered
CDEP1:CBD Chitin-binding domain
fused to a protease
Engineered
Bbchit1:CBD Chitin-binding domain
fused to a chitinase
Engineered
(Zaho et al. 2016)

53. • Expression of the fusion protein CDEP1:Bbchit1 that contains the
Pr1A-like protease CDEP1 and the chitinase Bbchit1 accelerated
cuticular penetration by B. bassiana when compared to the WT
• The chitinase Bbchit1 identified from B. bassiana lacks chitin-binding
domains.
• Fan et al. (2007) constructed several B. bassiana hybrid chitinases
where Bbchit1 was fused to chitin-binding domains derived from plant,
bacterial, or insect sources.
• A hybrid chitinase containing the chitin-binding domain from the
silkworm Bombyx mori chitinase fused to the B. bassiana chitinase
showed the greatest ability to bind to chitin and the insect cuticle
compared to the WT Bbchit1 and other hybrid chitinases

54. Increased Insect Virulence in B. bassiana
Strains Overexpressing an Engineered
Chitinase
(Yanhua Fan et.al. 2006)

55. (Yanhua Fan et.al. 2006)

56. IMPROVE TOLERANCE TO ABIOTIC
STRESSES
AIM TYPE SOURCE
Try Tryosinase Aspergillus fumigatus
BbSOD1 Superoxide dismutase Beauveria bassiana
DHN-melanin synthesis
pathway
Three genes Alternaria alternate
MrPhr1 CPD photolyase Metarhizium robertsii
HsPHR2 CPD photolyase Halobacterium salinarum
trxA Thioredoxin Escherichia coli
HSP25 Heat shock protein 25 Metarhizium robertsii
(Zaho et al. 2016)

57. IMPROVE TOLERANCE TO UV
RADIATION
• UV radiation causes not only DNA damage but also produces reactive
oxidative species (ROS) that elevate oxidative stress in cells
• Overexpression of a superoxide dismutase (SOD) increased the ability of
B. bassiana to detoxify ROS, enhancing UV tolerance
• Expression of thioredoxin (trxA) from the bacterium Escherichia coli also
increased the tolerance of B. bassiana to UV-B irradiation, oxidation, and
heat.
• Pigments such as melanin are effective absorbers of UV light and can
dissipate the absorbed UV radiation.

58. • Pigments on conidial cell surfaces usually act as a coat to protect fungal
cells from UV damage
• Expression of a tyrosinase from Aspergillus fumigatus activated the
production of pigments in B. bassiana and thus increased the tolerance
of this fungus to UV radiation .
• Tseng et al. (2011) transferred the DHN-synthesis pathway of
Alternaria alternata into M. anisopliae. Compared to the WT strain, the
transformant showed a 2-fold greater tolerance to UV radiation
• DHN-melanin also enhanced the virulence of M. anisopliae to insects
by promoting germination, appressorium formation, and increasing
the expression of virulence
• In M. robertsii, the laccase gene involved in synthesis of conidial
pigment is also related to cell wall rigidity of appressorium and thus is
involved in pathogenicity.

59. Integration of Escherichia coli thioredoxin (trxA) into B. bassiana
enhances the fungal tolerance to the stresses of oxidation, heat and
UV-B irradiation
(Sheng-Hua Ying et.al. 2011)

60. IMPROVE TOLERANCE TO HEAT
STRESS:
• Tolerance of entomopathogenic fungi to heat stress can be increased
through experimental evolution via continuous culture of fungal cells
under heat stress.
• In these conditions, thermotolerant variants of M. robertsii were
obtained, which displayed robust growth at 37 C
• Tolerance to heat stress by entomopathogenic fungi can also be
improved by transferring single genes
• Similar to UV radiation, heat stress produces ROS

61. • Expression of ROS scavengers, such as SOD and bacterial
thioredoxin, increases the heat tolerance in entomopathogenic fungi
• Small heat shock proteins have been shown to confer thermotolerance
in many organisms, and HSP25 expression was found to be
upregulated when M. robertsii was grown at extreme temperatures or in
the presence of oxidative or osmotic agents.
• Overexpressing HSP25 in M. robertsii increased fungal growth under
heat stress either in nutrient-rich medium

62. Enhancing the stress tolerance and virulence of an
entomopathogen by metabolic engineering of
dihydroxynaphthalene melanin biosynthesis genes
(Tseng et,al. 2011)

63. (Tseng et,al. 2011)

64. (Tseng et,al. 2011)

65. CONCLUSION AND FUTURE PERSPECTIVES
• Genomic analyses of entomopathogenic fungi have revealed the
evolutionary and protein family features associated with fungal adaptation to
insect hosts.
• Fungal entomopathogenicity evolved multiple times, and relative to non
insect pathogenic fungi, the formation of similar protein family size in
divergent species suggests the occurrence of convergent evolution during the
coevolutionary arms race between fungi and insects.
• Insect pathogenic fungi also evolved specificity for different ranges of host
species.
• Species with narrow host ranges usually have reduced protein-coding
capacities and protein family sizes.
• In addition, they frequently undergo sexual reproduction as compared with
generalist species

66. • Molecular biology studies have revealed the genes that function in
fungal interactions with insect hosts at different infection stages.
• Relative to our greater level of understanding regarding fungus-plant
and fungus-human interaction mechanisms, future efforts will be needed
to investigate the function of effector-like proteins in fungus-insect
interactions and to dissect the molecular mechanisms involved in
regulating the cell dimorphic switch during fungal colonization of the
insect hemocoel.
• The knowledge obtained will further improve the cost-effective
application of mycoinsecticides in the field.