Chapter 6. Fungal infections

※ Mycology: study of fungi

※ The first microorganisms found to cause diseases in insects were fungi because

of their conspicuous macroscopic growth on the surfaces of their hosts

< Beauveria bassiana and Cordyceps spp. >

1. Characteristics of fungus

- somatic structure

‣ hypha (pl. hyphae): branching filamentous cell of a fungus

➜ collectively called "mycelium"

< Septate hyphae of Sordaria fimicola >

a) non-septate; b) unicellular; c) septate;

d) multinucleate cell; e) uninucleate cell

- heterotrophs (no chlorophyll)

‣ saprophytes (consuming dead or decaying organic matters)

‣ parasites (many are entomopathogenic)

‣ absorptive nutrition (release of digestive enzymes into the external

environment ➜ need some free water)

- eukaryotic

- cell wall contains chitin / cellulose / glucans

‣ "true fungi" does not contain cellulose in cell wall

‣ oomycota - contain cellulose (no chitin in cell wall)

➜ Laminarin: storage glucan (a polysaccharide of glucose) which is made up of

β (1,3) glucan with β (1,6) glucan linkages.

- reproduction by means of spores (sexual and asexsual)

2. Classification of fungus

- classification schemes vary among researchers

- for the Entomopathogenic fungus, we follow two kingdom system

● Kingdom Straminipila (=Stramenopila, Straminopila, Chromista): mostly are

algae

- Division Oomycota

● Kingdom Eumycota (true fungi)

- Division Chytridiomycota

- Division Zygomycota

- Division Ascomycota

- Division Basidiomycota

3. Entomopathogenic fungus

- fungi are unique because most do not need to be eaten to be infective since

they can penetrate directly thru the cuticles of insect hosts

- downside to this strategy

‣ fungal spores are highly susceptible to damage by solar radiation

‣ spores require some moisture (over 90% RH) to germinate

☞ exception: Metarhizium anisopliae var. acridum is capable of infecting

the desert locust, Schistocerca gregaria, at RH as low as 13%

1) Generalized mode of action

- Adhesion: fungal spore must be land on the surface of a susceptible host

- Germination: the spore germinates and germ tube and appressorium

(penetration structure) are produced

- Penetration: the fungus penetrates the host's cuticle, using both mechanical

pressure and enzymes to digest the cuticle

- Development: once within the hemocoel the fungus grows by vegetative growth

which results in the death of an insect (mycosis)

- some invade the host organs and tissues shortly before the host dies (may or

may not use toxins)

< Development cycle of entomopathogenic fungi >

☞ fungi can infect insects thru the buccal cavity (moth), spiracles, and other

external openings of an insect

ex) • Culicinomyces sp. invades the mosquito larvae thru the fore- and

hindguts.

• Metarhizium anisopliae occasionally infects elaterid (click beetle) larvae

thru the spiracles

• Beauveria bassiana infects several mosquito species thru the posterior

siphon (respiratory organ)

2) Pathogenicity

- Fungi usually cause insect mortality by one or more of the following:

• nutritional deficiency (starvation)

• invasion and destruction of tissues

• release of toxins

3) Spore dispersal

- in most entomopathogenic fungi, spore dispersal is passive, relying principally

on wind and water

- in the Entomophthorales, spores are forcibly discharged and can land many

centimeters from the host or be carried longer distances on air currents

- some insects infected with certain fungus climb to the top of plants just prior

to death, where they die firmly clasping the plant

4) Nature of relationships

- some fungi are obligate pathogens (e.g., Coelomomyces, some Entomophthora

spp.)

➜ their complete life cycles have not been cultured outside of a living insect

host

- most entomopathogenic fungi are facultative pathogens capable of growing

without an insect host

- some are virulent pathogens and kill the insect within a few days (e.g., in

Entomophthora spp.)

- others produce chronic and prolonged infections

5) Signs and symptoms

- at an early stage of fungal infection, the infected insect shows little or no signs

and symptoms except a few necrotic (=dead) spots at the invasion sites

- at a late stage, the insects generally become

• less active

• reduced appetite

• lose coordination

- infected insects often move to high places (summit disease)

6) Commercial use for insect pest management

- Lagenidium giganteum: mosquito control

- Beauveria bassiana: thrips, whiteflies, aphids, diamondback moth

- Metarhizium anisopliae: vine weevils, wheat cockchafer

- Metarhizium anisopliae var. acridum: locusts and grasshoppers

- Nomuraea rileyi: lepidopteran pest

4. Kingdom Straminipila - includes diatoms and brown algae

◆ Stramen (Latin: straw) + pilous (Latin: hair): describe the structure of the

flagellar hairs produced by these organisms

◆ most straminipiles are algae with chlorophyll

4-1. Class Oomycetes (Division Oomycota) - watermold

1) Characteristics

- aquatic

- possess aseptated (without septum), coenocytic hyphae (multinucleate)

- no chitin in cell wall (cellulose + glucan)

- biflagellata (zoospores): asexual reproduction

- oospores: sexual reproduction

< SEM of biflagellate zoospore of Phytophthora infestans and infected mayfly

nymph >

tinsel flagellawhiplash flagella

anterior movement direction posterior

4-2. Order Saprolegniales

1) Genus Leptolegnia- only two species Leptolegnia caudata and Leptolegnia chapmanii have been

isolated from mosquito larvae (no other insect order has been infected)

- highly pathogenic to first and second instar larvae (100% mortality within 24hrs

after exposure)

- it is easy to culture in vitro, but tend to loose their larvicidal activity after

prolonged culture

4-3. Order Lagenidiales (some classified as Pythiales)

1) Genus Lagenidium- only one species, Lagenidium giganteum, is known to be a facultative parasite

of mosquito larvae

- can grow either as a saprophyte in the aquatic environment or as a mosquito

pathogen

< Life cycle of Lagenidium giganteum >

- infection of a larval host is initiated by motile biflagellate zoospores that

selectively recognize chemical signals on the epicuticle of larval mosquitoes

(A)

- the zoospores form cysts producing germ tubes that penetrate thru the

integument and develop a branched mycelium that produces oval-shaped

sporangia (B)

- depending upon the temperature and zoospore density, the larva dies of

starvation within 1-4 days. At that time each individual cell (=sporangia) can

form an exit tube and release 10-50 zoospores, which in turn seek out a new

host (C) - asexual cycle

- alternately, two cells can fuse (D), ultimately resulting in the formation of a

thick-walled dormant oospore within the host (E) - sexual cycle

- this sexual stage of L. giganteum can remain viable in a dehydrated state for

at least 7 years

- under appropriate environmental conditions, oospores will germinate, resulting in

the production of infective biflagellate zoospores similar to those produced

during asexual reproduction

2) Lagenidium giganteum as a pesticide

- Lagenidium giganteum was registered (approved for sale) as a pesticide active

ingredient in 1991. As of November 2000, there were three registered

pesticide end products containing this fungus as the active ingredient

- similar to Leptolegnia spp., Lagenidium giganteum can grow in vitro, but

pathogenicity is lost after several generation on agar

5. Kingdom Eumycota (true fungi)

◆ Entomopathogenic fungi in the Zygomycota, Ascomycota, and Basidiomycota do

not form motile spores and are primarily terrestrial fungi

◆ cell wall contains chitin and glucans (no cellulose)

5-1. Class Blastocladiomycetes

1) Characteristics

- microscopic fungi that occur as saprobes on a wide variety of dead plants and

animals, and as pathogens of aquatic and terrestrial plants and animals

- produce motile zoospores with single, posteriorly directed whiplash flagellum (do

not produce tinsel flagella, as in Oomycetes)

- mostly inhabit fresh water (some are found in soil, which is transformed into

an aquatic habitat when flooded)

2) Genus Coelomycidium- infect black fly larvae thru the cuticle as well as thru the intestinal wall

- one of the most widespread parasites of larval black flies, but patent infections

typically are found in less than 4% of any larval population

- patently infected larvae, recognized by the small spherical sporangia that fill the

body cavity, assume a C shape, where upon the cuticle ruptures, releasing

the fungal zoospores

- Coelomycidium simulii has been found in 95 species (37%) of the North

American black flies

- C. simulii enters the host larval black flies through the intestinal track, but

probably also through the cuticle from outside

3) Genus Coelomomyces- specialized obligate parasites of mosquitoes and other aquatic Diptera

- require crustacean alternate hosts to complete life cycle ➜ HETEROECISM

- highly pathogenic to many larval mosquito species

‣ Life cycle

‣ Sporophytic generation (asexual - mosquito larva)

‣ Gametophytic generation (sexual - copepod)

Zygote

Gamete fusion

Gamete Meiospores

Sporangia

Gametophytes

< Generalized life cycle for species of Coelomomyces >

(A) Biflagellate zygote penetrates the larval cuticle by means of penetration tube

(B) Enters the hemocoel of mosquito larvae forming hyphae

(C) Hyphae grow in the hemocoel and form resistant sporangia which virtually fill

the hemocoel.

(D) Sporangia are released from larvae and meiospores are formed by meiosis

(E) Meiospores infect a copepod and produce gametophytes

(F) Gametophytes are cleaved and formed gametangia which are forming

uniflagellate gametes (G)

(H) Gametes are released from copepod and fuse to form biflagellate zygote that

completes the cycle by infecting another mosquito larva

- Occasionally, however, the infected larva pupates and ecloses to produce

infected adults

- the infection is mostly localized in the ovaries. The hyphae in the ovaries will

mature to resting sporangia in response to changes in host ecdysone levels

following a blood meal. Even though Coelomomyces-infected females will

mate, no eggs will develop in the ovaries. Females do, however, attempt to

oviposit, but instead of eggs, resting spores are laid. Infected adult female

mosquitoes therefore play a role in the transmission of the fungus to new

habitats, which is especially useful for those species of Coelomomyces that

infect mosquitoes occupying small habitats such as tree holes or water

containers

5-2. Class Zygomycetes (Division Zygomycota)

1) Characteristics

- form a sexual spore, termed a zygospore (as resting spores)

- form a asexual spore (sporangiospore)

- coenocytic mycelium

- no motile spores (flagellate spores)

- most entomopathogens within the Zygomycota occur within the order

Entomophthorales ("insect destroyer")

2) Genus Entomophthora (plus Entomophaga)

- produce coenocytic hyphae (occasionally some septation occurs, particularly in

older hyphae)

- most are obligate parasites possessing restricted host ranges

- characterized by the production of forcibly discharged spores at maturity

■ Host, signs, and symptoms

• infect over 32 families in the order Hemiptera,

Diptera, Lepidoptera, Coleoptera, Orthoptera, and

Hymenoptera

• larva, pupa, and adult are infected but adult is

the most common stage infected

• infected insect does not display any obvious

signs and symptoms at the early stage of infection

• at a late stage of infection, the insect loses its

power of locomotion and settles down on the

underside of a brach or leaf

• it may climb to the tops of plants where it dies clasping the stem with its

head upwards as in grasshoppers (called summit disease)

< Entomophthora muscae > < Entomophaga grylli > < Adult grasshoppers,

Melanoplus differentialis, that have been infected with either "summit disease,"

Entomophaga grylli or an unidentified bacterial pathogen >

< Life cycle of Neozygites fresenii >

3) Other entomopathogenic genera

- Erynia aquatica: infects aquatic stages of invertebrates (can be found in adults)

- Zoophthora radicans: found on aphids and diamondback moth

- Entomophaga grylli: infect only grasshoppers and cause summit disease

- Neozygites floridanum: found on two spotted spider mite

- Neozygites fresenii: found on a variety of aphid species; valuable biocontrol

agent in cotton aphid population

- Neozygites parvispora: found on a variety of thrips spp.

5-3. Class Trichomycetes (Division Zygomycota)

※ Trichomycete fungi are common, widespread group of filamentous fungi

that, as obligate symbionts, inhabit the digestive tracts of many terrestrial

and aquatic insects (flies, mayflies, stoneflies, caddisflies)

※ previously 4 orders had been recognized

‣ Asellariales: colonize isopods, springtails

‣ Harpellales: true trichomycete fungi

‣ Eccrinales: now included in Ichthyosporean protist

‣ Amoebidiales: now included in Ichthyosporean protist

1) Characteristics

- mostly commensals

- must be ingested to colonize the guts

- midgut (Harpellaceae) vs hindgut (Legeriomycetaceae) inhabitant

- at least one species is pathogenic to larval mosquitoes (Smittium morbosum)

- asexual spores (trichospores), sexual spores (zygospores)

- some invade the adult female's ovary and replace the eggs with cysts and

female oviposits fungal cysts instead of eggs

2) Genera Harpella and Stachylina- attach to the midgut peritrophic matrix of larval flies

< Holdfast, trichospores, and conjugations of Harpella mellusinae>

3) Genera Pennella, Genistellospora, and Simuliomyces- attach to the hindgut cuticle of larvae of aquatic insects

< Genistellospora homothallica holdfast and zygospore >

< Pennella simulii, Simuliomyces microsporus >

5-4. Class Sordariomycetes (Division Ascomycota) [Asco, G.=bag]

➜ sac fungi

1) Characteristics

- produce an ascospores (sexual stage, usually 8) within an ascus

- sexual reproduction (teleomorph = ascospore stage)

- asexual reproduction (anamorph = conidial stage)

2) Genus Ascosphaera - affect honey bee larvae (Ascosphaera apis) ➜ chalkbrood

- infection occurs when the larvae ingests fungal spores or may occur thru the

external body surface

- dead larvae are chalky white and usually covered with filaments (mycelia) that

have a fluffy, cotton-like appearance

- chalkbrood usually disappears or declines as the air temperature rises in the

summer (high temperature inhibits the development of the fungus)

< Chalkbrood infected larvae of honey bee, Aphis mellifera >

3) Genus Cordyceps- all Cordyceps spp. are endoparasitoids, mainly on insects and other arthropods

- Cordyceps are precious ingredient in oriental medicines

- most common entomopathogenic fungi in tropical forests

- mainly found in Hemiptera and Lepidoptera (other insects orders are also

susceptible)

- because of its unique and striking fungal body, it was referred as "vegetable

wasp" and "plant worm"

- some Cordyceps spp. are obligate parasite on ants

< Camponotus ant with Cordyceps lloydii and bullet ant>Ant infected with Cordyceps

- some Cordyceps spp. are able to affect the behavior of their insect host;

Cordyceps unilateralis causes ants to climb a plant and attach there before

they die, assuring maximal distribution of the spores from the fruiting body

that sprouts out of the dead insect's body

- once infected ants are spotted by healthy workers, they are readily removed

from the nest

■ Fungi traditionally classified as Hyphomycetes (Division Deuteromycota - Fungi

Imperfecti, since no sexual stage was known)

➜ cause muscardine diseases in insects

muscardine: derived from Italian, muscardino, meaning a comfit, grape.

4) Genus Beauveria - white muscardine fungus

- one of the most frequently isolated entomopathogenic fungi

- worldwide distribution (naturally occurs in soil as a ubiquitous saprophyte)

➜ so they are not obligate pathogens

- Beauveria bassiana: has been used for biological control of pest insect for

more than 100 years

- white spores form a complete covering over the mummified insect

- white muscardine was the first disease in animals shown to be caused by a

fungus

< Weevil infected by Beauveria bassiana >

- infection takes place thru the integument

- produce mycotoxins (not directly related to the pathogenicity)

- spores are almost completely inactivated by exposure to 60min of direct

sunlight

5) Genus Metarhizium - green muscardine fungus

- Like Beauveria, one of the most common, soil-dwelling entomopathogenic fungi

- Metarhizum anisopliae: discovered by Metchnikoff from wheat cockchafer

(Anisoplia austrica), infects over 200 insect host species

- highly virulent (killed 70-90% of treated locust within 2-3weeks)

- produce mycotoxins (destruxins - toxic to insects only by ingestion)

- the colony of M. anisopliae appears white when young, but as the conidia

mature, the color turns to dark green

- commercially available for the control of pests such as white grub (wheat

cockchafer), mole crickets, caterpillars, fire ants, ticks, and termites

6) Genus Aspergillus - yellow muscardine fungus

- mainly saprophytic but may infect a wide range of insect species

- common contaminants of starchy foods (bread, potatoes)

- Aspergillus oryzae: to ferment soybeans to produce soy sauce and miso as

well as sake and soju

- infect honey bee brood and cause "stonebrood" since the cadaver becomes

hard, stone-like mummies (rarely observed)

- produce mycotoxins: aflatoxins - potent carcinogens and produce tumors in the

liver of vertebrates including humans

7) Genus Sorosporella - red muscardine fungus

- unlike other muscardines, the fruiting bodies do not cover the exterior of the

dead insect (instead the cadaver becomes filled with reddish spores)

- mainly infects caterpillars

- cadaver remains soft (not mummified)

8) Genus Culicinomyces- Culicinomyces clavisporus: unique in that it produces conidia (asexual spore)

underwater and infects mosquito larvae (and other insect's larvae) thru the

foregut and hindgut cuticles

- following invasion, the fungus colonizes the body cavity (hemocoel) and kills

the larvae within 2-7 days

- the cause of death is unknown, and it may result from toxic substances

produced by the massive invasion of the hyphae

5-5. Class Teliomycetes (Division Basidiomycota)

- form sexual spores, termed basidiospores

- only a small number are entomopathogens

1) Genus Septobasidium- obligate pathogens of armored scale insects

- mutualistic at the host population level, as the fungi provide shelter for the

scales while parasitizing only a small percentage of individuals, making them

a pathogen at the level of the individual scale

6. Microsporidia (=Microspora)

- obligate, spore-forming, intracellular parasites that invade vertebrates and

invertebrates

- have long been classified as protozoans, the microsporidia are now considered

by many workers to have affinities with the true fungi

- it is currently being classified under the Kingdom Fungi

• presence of chitin in the spore wall

- lack mitochondria (need host cell for chemical energy)

- many species are common pathogens of arthropods, few microsporidia have

been used in microbial control programs because

; complicated life cycles (some requires intermediate hosts)

⇨ Amblyospora spp. requires copepod intermediate host to complete their

life cycle

; obligate parasitism (reproduce only in living cells)

; chronic rather than acute effects on their hosts

- more than 250 species are known from insects, predominantly Lepidoptera and

Diptera

- microsporidia infection is generally classified as chronic, rarely acute

; sublethal effects such as reduced fertility, shortened longevity

- first identified as the cause of "pebrine" of silkworms in 1857

; pebrine is a collective name of a silkworm disease caused by microsporidia

parasites (mainly caused by Nosema bombycis)

; flacherie is a collective name of a silkworm disease caused by virus in

combination with bacteria

- produce environmentally resistant spores with coiled polar filament

- microsporidia species often grow only in specific host tissues, such as fat body

(most common), the midgut epithelium, or the reproductive tissues

- ingestion is the most common route

; thru cuticle by means of the ovipositor of a parasitoid

; transovum transmission (invade the egg or embryo within the females'

reproductive organ)

- insects infected with microsporidia generally have changes in color, size,

morphology and activity

- infected hosts often survive to adulthood but infected individuals are usually

much smaller than normal and their reproductive output is often reduced

< Spores of microsporidia >

< Generalized life cycle of microsporidia >

■ Life cycle

① the environmentally resistant, infectious spore is ingested (a)

② this stimulus activates the polar filament extrusion (acts like a syringe) and

punctures a host cell (b)

③ release their sporoplasm (containing one or two nuclei and other organelles)

into host cell thru the hollow polar filament (c)

④ the microsporidia multiply within the cytoplasm of the cell (d)

⑤ differentiate into mature spores (environmentally resistant) by having polar

filament and thick spore wall (e)

⑥ when spores completely fill the host cell, the plasma membrane is affected

and releases the spores to its surroundings. The spores may then infect other

surrounding cells, be transported to new sites within the host, or be excreted

in feces or urine to infect other hosts

6-1. Genus Nosema

- Nosema bombycis

• first named microsporidia species from the silkworm (pebrine disease)

- Nosema apis

• important parasite of the honeybee

- Paranosema locustae (=Nosema locustae)

• the only registered microsporidia insecticide to control grasshopper

< Grasshopper infected with Nosema locustae and product >

• P. locustae persisted to the next year and area with infected individuals

expanded

• grasshoppers stop feeding, become lethargic and die (few days or weeks)

• cannibalistic behavior ⇨ primary method of horizontal transmission

6-2. Human infected microsporidia

- of the 14 species of microsporidia currently known to infect humans,

Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most

common causes of human infections

- associated with diarrhea and systemic disease in AIDS patients, organ

transplant recipients, and the elderly

- microsporidiosis: an opportunistic intestinal infection that causes diarrhea in

immunocompromised individuals