Insect+pathology+and+entomopathogens.pdf

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Insect pathology and

entomopathogens

Eustachio Tarasco

Università degli studi di Bari

[email protected]

The Use of Pathogens in Biological Control

� Insects, like most other groups of animals, are susceptible to

diseases.

� Disease is the impairment of normal physiological function.

� Pathogens are transmissible agents of a disease.

� Pathogens enter the insect body either passively, during feeding,

or actively, via natural orifices or by penetrating directly through

the cuticle.

� Once inside the insect, pathogen multiplies rapidly, eventually

killing the host by the production of toxic substances or by

depletion of its nutrients.

� Most pathogens exhibit high host specificity and some,

especially viruses, may infect only a single genus or species of

host.

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Insect Pathology and Microbial Control

• Agostino Bassi: "Insect Pathology" becomes an experimental science

– In 1835 he showed that the "bad sign" of silkworms was caused by a microorganism, fungus Beauveria bassiana

• Louis Pasteur in 1870

– He studied two silkworm diseases (one viral and the other caused by a Protozoan)

• 1878: the first significant experience of Microbiological (or Microbial) Control by the Russian Metchnikov

• fungus Metarhizium anisopliae was used to control a wheat pest, Anisopliaaustriaca.

• Krassilstschik organized the first mass production system of the fungus in Smela. Bio-factory prototype.

• Maestri e Cornalia (1856)

– indicate the presence of reflective particles (Virus) in the hemolymph of the silkworm larvae infected with "yellow vein“

– 1893: first application of Virus against Lymantria dispar in Ungharia collecting

infected larvae, grindingt them and using them for treatment

• Bacteria applications

– D’Herelle (1910): Coccobacillus acridorium against grasshoppers

– Berliner (1911): Bacillus thuringiensis

• Glaser (in 1930)

– First filed experiments with entomopathogenic nematodes, Neoaplectana glaseri, against the scarab Popillia japonica

• By 1965 the Insect Pathology is an integral part of the International Organization for Biological Control

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Comparative data on the biology of the major groups of insect pathogens

entomopathogens

1-5 daysChronic rather

than lethal4-7 days30 min - 1 day

3-10 days;

considerably

longer for

Oryctes virus

Speed of kill

Via natural openings

or cuticleOralVia cuticleOralOralMode of entry

Very broad

Broad

specificity at

family level

Very broad.

Strain

specificity

Lepidoptera,

Coleoptera and

Diptera. Strain

specificity

Lepidoptera and

Hymenoptera;

often genus or

species specific

Host range

NematodaProtozoaFungiBacteriaViruses

VIRUSES

Major families of insect pathogenic viruses

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VirusesCPVCPV

NPVNPV

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Viruses

Viruses

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Viruses - Baculoviruses

Viruses - Baculoviruses

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Viruses – Baculoviruses: Nuclear polyhedrosis viruses (NPV)

Viruses – Baculoviruses: Nuclear polyhedrosis viruses (NPV)

NPV (Baculoviridae) infecting Spodoptera exempta larvae (Lep.: Noctuidae) showing flaccid body in

an inverted V.

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Viruses – Baculoviruses: Granulosis viruses (GV)

Viruses – Baculoviruses: Group C Baculoviruses

Oryctes rhinocerus: Adult beetle (top)

and Infected (left) and healthy (right) larvae (bottom)

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Viruses – Baculoviruses: Entomopox Viruses

NPV NPV GV GV

Main bacterial control agents of insects

BACTERIA

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� Bacteria are microscopic prokaryotes, i.e., organisms

without membrane-limited nuclei and exhibiting mitosis.

� They lack a well-defined nucleus and organelles, but posses a structurally distinct cell wall.

� Principal bacterial biocontrol agents are species of the genus Bacillus.

� Bacillus spp. are aerobic, unicellular, usually rod-shaped

(bacilliform), spore-forming bacteria, most of which can be readily cultured.

� Infection occurs only after ingestion of bacterial cells or

spores and mainly affects phytophagous or aquatic larval

stages

Bacteria

Bacteria

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� The most important

microbial control agent

� 1901: in Japan Ishiwata

isolated a bacterium from

silkworm (Bacillus sotto)

� 1911: Berliner isolated in

Thuringia (Germany) a

similar batterium from

Anagasta kuehniella

(Bacillus thuringiensis)

� Sporeine® (1938): first

commercial product with

Bt, in France

� Years ’50 -’60: with

Steinhaus researches great

impulse to Bt use

� 1957: Thuricide®, still on

the market

� During sporulation Bacillus thuringiensis-cells produce a

large proteinaceous crystal, characteristically bipyramidal in shape, in addition to a thick-walled endospore.

� The crystal is an inert toxin (endotoxin), but once inside the

susceptible host it dissolves in the alkaline gut fluids,

releasing toxic polypeptides which interact with the gut

lining causing paralysis of the muscles of the alimentary tract and the mouthparts.

� Feeding stops, and infected insects may develop

symptoms such as regurgitation and diarrhoea. Death

occurs rapidly, usually within a day.

Bacteria - Bacillus thuringiensis (Bt)

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� Bt invade and reproduce in the

insect haemocoel, inducing a

lethal septicaemia, although in

some hosts death is due entirely to starvation.

� many subspecies have been

distinguished so far, depending

in host association and biochemical properties.

� The pH of the host gut is a

critical factor in determining

susceptibility


� The vegetative cell contain endospores

(phase bright) and crystals of an

insecticidal protein toxin (delta

endotoxin).

� Most cells have lysed and released the

spores and toxin crystals (the structures

with a bipyramidal shape)


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�� BtBt subspeciessubspecies are are identifiedidentified byby

serologicalserological teststests……..

�� More than 40 recognized More than 40 recognized

serotypes on the basis of H serotypes on the basis of H

AntigenAntigen

�� Vegetative cells of Bt have at Vegetative cells of Bt have at

least 2 antigens on their surface: least 2 antigens on their surface:

flagellareflagellare (H) and somatic (or)(H) and somatic (or)

�� …… parasporalparasporal inclusionsinclusions

morphologymorphology ((toxintoxin shapeshape))

�� …….and classification of .and classification of δδ--

endotoxinsendotoxins according to their according to their

insecticidal propertiesinsecticidal properties

�� CRY I: CRY I: LepidopteraLepidoptera

�� CRY II: CRY II: LepidopteraLepidoptera and and

DipteraDiptera

�� CRY III: ColeopteraCRY III: Coleoptera

�� CRY IV: Diptera CRY IV: Diptera NematoceraNematocera

�� CytCyt: : BtiBti cytolisincytolisin

�� More More thanthan 60,000 60,000 BtBt strainsstrains isolatedisolated in the world, more in the world, more thanthan 60 60

subspeciessubspecies identifiedidentified, 25 , 25 differentdifferent crystalcrystal proteinsproteins, more , more thanthan 200 200

toxinstoxins isolatedisolated

�� The more The more importantimportant subspeciessubspecies::

�� BtBt kurstakikurstaki ((BtkBtk, strain HD, strain HD--1): 1): isolatedisolated in 1971 (in 1971 (LabLab. Abbott), . Abbott),

endospore endospore withwith 1 or more 1 or more crystalcrystal proteinsproteins, , activeactive againstagainst

LepidopteraLepidoptera larvaelarvae

�� BtBt tenebrionistenebrionis:: isolatedisolated nel 1982 nel 1982 fromfrom a a T. T. molitormolitor pupapupa , , activeactive

on Coleopteraon Coleoptera

� Bt israeliensis (Bti, serotype H14): isolated in 1976, spheric

crystal protein, with 4 toxins, active against Diptera

� Other subspecies: canadensis, galleriae, morrisoni, aizawai, alesti, kenyae, thompsoni, etc.

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� Bt is easily biodegradable in field conditions

� Temperature, water, pH, sun radiations (UV especially)

� After more than 30 years of use on millions of acres and several biotopes

there was no reporting of adverse effects on the environment as a result

of the use of Bt

� Several toxicity tests have repeatedly confirmed that toxins are harmless

to humans and animals (the low pH of mammalian intestine solubilizes

and denatures protein crystals). Bt is harmless to birds, fish,

invertebrates and vertebrates, aquatic and terrestrial, including insects

(parasites, predators and pollinators)

� One exception: the strains which produce β-exotoxin. Less selective δ-

endotoxins, harmful for 55 species of 10 different orders (i.e. Pieris

brassicae, Musca domestica, Locusta migratoria, Apis mellifera)

moreover nematods (Meloydogine) and vertebrates (mouses).

� In this Group also B. mycoides and B. anthracis

� B. cereus has been recognized for many eye infections and intoxications

These 3 Bacillus

belong to the

same group

These 3 Bacillus

belong to the

same group

� Bt and B. cereus, are

genetically and

phenotypically

indistinguishable,

except for the plasmid encoding the

production in Bt

parasporale bodyBacteriological

weapon?

Bacteriological

weapon?

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Toxin structure of B. thuringiensis

showing Domain I, Domain II and

Domain III.


bipyramidal crystal protein

of Bt spp. kurstaki

Bt

Crystals

alkaline pH,

enzymes

protoxin (130 kDa)

proteinase

toxin (65 kDa)

receptor binding and

formation of pores

in the insect intestines


Mode of action

Bt infection of silkworm larva

(Lep.: Bombycidae).

Healthy larva below for comparison.

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Bacteria – other biocontrol agents

- Very active towards larvae of mosquitoes (Culex, Anopheles,

and not on Aedes)

- Stable protein crystals (4°C and pH 7)

- Produces 2 toxins, binary (Btx) and zanzaricida (Mtx). The

most virulent strains produce both.

- It is most persistent than Bti

Bacteria – other biocontrol agents

� Enterobacteriaceae

� Serratia entomophila

� “Ambra desease” on Costelytra zealandica(Scarabeidae)

� Serratia marcescens

� Septicemia in Orthoptera

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� Saccharopolyspora spinosa � New species of Actinomicete (bacteria

close to fungi), isolated in the Caribbean

� Active ingredient: Spinosad

� Active metabolyte: Spinosine

(mainly A e D, more than 30)

� Naturalyte: New class of control

agents (from Natural e metabol-yte)

� Registred in 60 Countries on 150

crops.

� Broad spectrum of action

� Tisanotteri (Frankliniella), Lepidotteri

(Lobesia, Spodoptera, Ostrinia, Plutella), Coleotteri (Leptinotarsa),

Ditteri (Lyriomiza, Ceratitis, Bactrocera, Anopheles)

� Action for ingestion and contact

Non-toxic for auxiliaries

(except for Encarsia

and Orius)

Low environmental impact

… …. some doubt….

Natural Product

Microbial insecticide

(micro-organism-derived)

� Protozoa classified in several phyla: Ciliophora, Sacromastigophora, Apicomplexa and Microspora.

� Disease caused by protozoa are chronic in nature, rather

than lethal, and generally kills only when very high levels of

organisms have built up, destroying the normal function of organs and debilitating the host.

� Infections occurs by ingestion of spores and subsequent

penetration of the digestive tract.

� For biocontrol most important phylum is Microspora, a

group of obligate parasites of arthropods.

� Microspora are not highly pathogenic, but significantly

reduce the rate of development and fecundity of the host.

PROTOZOA

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� Spores of Microspora have characteristic polar capsule

which, after ingestion and subsequent germination,

develops into a tube capable of penetrating the wall of the gut cells.

� Gut or fat body cells provide the main foci of infection and

spore production.

� Nosema and Vairimorpha are two of the genera which

contain species that are used in biocontrol. N. locustae

infects a wide range of grasshopper, whilst V. necatrix is

broad-spectrum disease agent in may Lepidoptera.

Protozoa

Protozoa

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A wet mount preparation of spores

viewed with phase contrast microscopy

Protozoa

� A transmission electron micrograph through a

microsporidian spore.

� The arrows point to cross sections through the

polar filament, which is used to inject the

infectious sporoplasm into the host tissue.

� Typically, several million to as many as several

billion spores are produced per host. � The usual mode of infection is by ingestion, after

which the spores extrude the polar filament,

injecting the microsporidian sporoplasm into mid

gut epithelial cells, or directly into tissues such as

the fat body.

Protozoa

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the fat body.

Protozoa











the fat body.

Protozoa

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the fat body.

Protozoa

Amblyospora sp. (Protozoa: Microspora) infection of

mosquito larva (centre and right) (Dipt.; Culicidae).

Protozoa

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Nosema sp. (Protozoa: Microspora) infection of silkworm larva,

showing smaller size and dark spots

Protozoa

� Fungi are Eukaryotes, i.e. organisms having membrane-limited nuclei which divide by mitosis.

� Fungi have a well-defined nucleus and organelles,

characterised by chitinised cells.

� These are typically formed into filaments or strands

(hyphae) which collectively constitute a mycelium .

� Reproduction is predominantly by spores, which may be formed asexually or sexually.

� True entomopathogenic fungi are to be found in the

subdivisions Mastigomycotina, Zygomycotina and

Deuteromycotina.

Entomopathogenic Fungi

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� Entomopathogenic fungi penetrate and infect the insect

host directly through the cuticle, using enzymes liberated during sporulation.

� Spores frequently have morphological or biochemical

adaptations which enable them to attach securely to the insect cuticle.

� Inside the host haemocoel, fungus multiplies rapidly by

budding or hyphal fission, and the resultant yeast-like cells are disseminated throughout the insect body.

� In primitive fungal pathogens (Mastigomycotina,

Zygomycotina) the host usually dies only after extensive

mycelial colonisation, death being due to asphyxiation or

starvation.


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� In more advanced fungal pathogens (Ascomycotina,

Deuteromycotina), mortality is the result of toxin release by

the yeast phase and the true mycelium then develops saprophytically within the cadaver.

� Uptake of host nutrients and water by the rapidly growing

hyphae results in desiccation (mummification) of the insect.

� In majority of entomopathogenic fungi, the hyphae break

through the host cuticle only after death.

� Spore-forming structures develop from the external

mycelium and spores are liberated passively or violently to continue the cycle.

� Fungi have broad host range within the Insecta, and

Arthropoda in general, and display varying degrees of

specificity, susceptibility probably depending on the initial fungal spore-insect exosceleton interaction.


Typical life cycle of an

entomopathogenic fungus


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Metarhizium anisopliae

(Deuteromycotina: Hyphomycetes)

infecting adults of

Aenolamia varia (Hemiptera: Cercopidae)

Beauveria bassiana


infecting Schistocerca gregaria

(Orthoptera: Acrididae)


Hirsutella citriformis


infecting adult of Nilaparvata lugens

(Hem.; Delphacidae)

Aschersonia cubensis

(Deuteromycotina: Coelomycetes)

infecting scale insects (Hem.; Diaspididae) on citrus leaves


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Entomophthora muscae

(Zygomycotina: Zygomycetes)

infecting Delia sp. (Dip.; Anthomyiidae)

Erynia delphacis

(Zygomycotina: Zygomycetes) infecting

Cofana spectra (Hem.; Cicadellidae)


• Metarhizium anisopliae on Red Palm Weeviel

Rhynchophorusferrugineus

• Beauveria bassiana

on Red Palm

Weeviel

Rhynchophorus

ferrugineus

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� Nematodes are a large and diverse group of simple

multicellular eukaryotic organisms belonging to the phylumNematoda (roundworms).

� In general, nematodes are bilaterally symmetrical,

elongate, and vermiform; taper at both ends; and are

covered by a cuticle that they must molt to progress in development.

� Most species have a stylet plus specialised feeding glands

and alimentary tract. Life stages include an egg, several

juvenile stages (larvae), and adults. The latter, depending on the species, can be sexually dimorphic as well as

hermaphroditic.

Entomopathogenic Nematodes (EPNs)

� Two species used for mosquito control, i.e. Romanomermis culicivorax and R. iyengari.

� Mermethids are obligately parasitic nematodes.

� Females are found in wet soil near aquatic habitats.

� The L2 swims to the surface of the pond, looks for hosts, and with the help of the stylet invades early larval instars of mosquitoes.

� The L2 feeds for 7-10 days in the host.

� Thereafter the L2 moults to L3 and this instar punctures the host,

thereby killing it, leaves the host, descends to the bottom of the

pond and matures without further feeding after 7-10 days to the

adult stage.


Mermithidae

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� Subsequently adults mate and the female lay eggs. However,

rates of parasitism did not exceed 85%, not sufficient to interrupt the vector potential of mosquitoes.

� In addition, other biocontrol agents like Bti were more successful in mosquito control.

� Therefore, there is little interest nowadays in further development

of Mermethids as biological control agents.


Mermithidae

Hexamermis sp.

� Small (less than 1-3 mm) terrestrial nematodes. Mostly parasites of soil-inhabiting insects.

� Life cycle include egg and 4 larval stages.

� Nematodes have mutualistic relationship with bacteria that they harbour in their alimentary tract.

� These bacteria kill the insect after the nematode has invaded the host body.

� Nematodes produce quasi-resistant larval stage, so-called ‘dauer

larva’, which is actually the 3rd larval instar surrounded by the

moulted cuticle of the L2.


Steinernematidae and Heterorhabitidae

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� The dauer larva infects host via the mouth, anus or spiracles. In the hemocoel, nematode begins to feed on hemolymph.

� When it defecates, the bacteria are released which kill the hostwithin 1-3 days.

� Nematodes feed on the bacteria and the tissue of the dead insecthost.

� Usually after 2-3 generations nematodes leave the host, i.e., after 1-2 weeks thousands of dauer larvae leave the cadaver.

� Dauer larvae can be easily mass-produced in liquid culture in

industrial fermenter.





Life cycle

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Steinernematidae and HeterorhabitidaeInfective juveniles (L3) or

dauer larvae

EndotokiaEndotokia

matricidamatricida

• Steinernema: anfigonia

• Heterorhabditis: Hermaphroditism (first generation)

HostHost searchingsearching

ActiveActive

H. bacteriophoraH. bacteriophora

H. megidisH. megidis

S. glaseriS. glaseri

notnot activeactive

S. carpocapsaeS. carpocapsae

S. S. scapterisciscapterisci

S. feltiaeS. feltiae

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Once they break into the host EPNs released bacteria; nematodes begin to grow, multiply and do within the host more generations (2-3), as long as there is availability of food. The lack of food induces the formation of the infective stages (III Infective juvenile – dauer juvenile) who leave the cadaver and are ready to infest a new host

The infective stage (dauer juvenile) of entomopathogenic nematodes, which is formed as a result of particular stress conditions (lack of food, extreme temperatures) has physiological and morphological adaptations to survive without feeding, waiting for a new host.

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�� SymbiosisSymbiosis

�� Bacteria are released into the Bacteria are released into the hemolymphhemolymph

where they multiply and produce toxins, where they multiply and produce toxins,

enzymes and antibioticsenzymes and antibiotics

�� Nematodes can kill the host even by Nematodes can kill the host even by

themselves, but without the bacteria cannot themselves, but without the bacteria cannot

breedbreed

�� The bacteria transform the insect tissues in a The bacteria transform the insect tissues in a

““nutrient brothnutrient broth””, ideal for the development of , ideal for the development of

nematodesnematodes

�� Nematodes are the means of transport of Nematodes are the means of transport of

bacteria and provide them protectionbacteria and provide them protection

�� Photorhabdus Photorhabdus –– HeterorhabditisHeterorhabditis�� very specific Symbiosis, 1 species of very specific Symbiosis, 1 species of

bacteria is associated with 1 single bacteria is associated with 1 single

species of nematodespecies of nematode

�� Xenorhabdus Xenorhabdus –– SteinernemaSteinernema�� less specific Symbiosis, 1 species of less specific Symbiosis, 1 species of

bacteria can be associated with more bacteria can be associated with more

species of nematodesspecies of nematodes

�� BacteriaBacteria metabolitesmetabolites ((xenorhabdinexenorhabdine, , xenocumacinexenocumacine))�� Antimicrobial activity, insecticide, Antimicrobial activity, insecticide, nematicidenematicide, antitumor and antiviral drugs,, antitumor and antiviral drugs,

�� Use of bacteria for the production of antibioticsUse of bacteria for the production of antibiotics

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