Chapter 3. Microbial diseases of insects

▲ Microbial (infectious) disease

- Pathogenic microorganisms generally invade and multiply in an insect and

spread to infect other insects by contact or ingestion

- Amicrobial (noninfectious) diseases caused by

● mechanical agents

; wounds or injuries (damage to the tissues, loss of hemolymph, and

exposure to pathogens at the site of injury)

● physical agents

; high / low temperature, lack / abundance of moisture, oxygen

● chemical agents

; chemical insecticides, toxins, poisons

● biological agents

; parasites, predators

● genetic factors

; deformed body, no bristles or scales, vestigial wings

● nutritional deficiencies

; reduces growth, development, and reproduction

● deranged metabolism (malfunction of neuroendocrine system)

; hormonal disruption (failure in metamorphosis)

- Major pathogenic microorganisms are

; Bacteria, Virus, Fungi, Protozoa, and Nematodes

1. Potential / facultative / obligate pathogens

- not all microorganisms cause infectious diseases even after they gain entrance

into the insect's hemocoel

- this might be due to the resistant characteristics of the host insects or to the

inability of the microorganisms to survive and replicate in the host's

environment

1) Potential pathogens

- microorganisms that are incapable of invading host, either thru the body wall or

digestive tract, without the assistance of external factors (e.g., injury) that

lower the insect's resistance or enhance the ability of the microorganism to

invade the insect

; many opportunistic bacteria

; can be cultured easily outside of host

; can be found in multiple insect hosts

2) Facultative pathogens

- microorganisms that do not require an external factors (e.g., insect weakened

by injury or starvation) to cause infection

; can live and reproduce independent of hosts

; can be cultured easily in artificial medium

3) Obligate pathogens

- microorganisms that require live insect hosts for survival and replication

; specialized for invasion of insect host

; may occur outside of the host insect in a dormant stage (=environmentally

resistant stage, such as spore, cyst, viral occlusion body)

; cannot be easily cultured in artificial medium

; hard to mass produce

2. Infection process - Portals of Entry

- points or sites thru which a pathogen invades or gains entrance into an insect

- most common portals of entry: integument and mouth

1) Integument

- integument comprises the cuticle and the epidermal cells

- insect cuticle is very effective barrier for invading organisms

- fungi and nematodes generally invade thru the integument

2) Digestive tract

- pathogens enter the midgut because foregut and hindgut are lined with cuticle

- bacteria, microsporidia, virus, nematodes, protozoa

; alkaline (pH 8 or higher) gut required to dissolve proteins around virus

particles or dissolve crystal to activate the bacterial toxins

3) Congenital

- parent to offspring via egg or sperm

- majority are passed by female via the egg (transovum transmission)

- most are virus, protozoa (including microsporidia)

4) Vectors

- parasitoids

; contaminated ovipositor

- nematodes

; vector bacteria (deliver symbiotic bacteria to insect hemocoel)

5) Respiratory system

- cuticle serves as a barrier

- gain entrance thru spiracles

; nematodes

3. Barriers to entry of pathogens

1) Cuticle

- epicuticle: does not contain chitin

▷ lipids

; wax layer

; hydrophobic environment

; unsuitable for colonization by microorganisms

; able to suppress toxicity of microorganisms

; anti-microbial activity

☞ fatty acids

☞ aldehydes

☞ ketones (e.g.,acetone)

☞ alcohols

▷ cuticulin: thin layer of protein

; heavily sclerotized

▷ cuticular protein

; produce cecropin peptides that are anti-microbial

- exocuticle: containing both chitin and protein

▷ chitin

; heavily sclerotized

; extremely stable (hard and rigid)

; resistant to enzymes

; activation of phenoloxidase cascade

☞ melanization: binds toxins and blocks invasion

2) Alimentary tract (digestive tract)

- foregut

; lined with cuticle (impermeable)

- midgut

; certain plants that insects use as food contain toxic substances (i.e., tannin)

that can kill microorganism

; commensals and mutualists can produce anti-microbial materials

- hindgut

; lined with cuticle

; usually harboring various symbionts

< Larval black fly, Simulium innoxium >

4. Peritrophic matrix of midgut

- Lyonet (1762) first described it from caterpillars

- Balbiani (1890) found it from different insects and named it "peritrophic

membrane"

- Ramos (1994) proposed to substitute the term "membrane" with "matrix" since

PM is not a phospholipid bilayer

※ PMs are noncellular, semipermeable cellophane-like matrices composed of a

meshwork of chitin microfibrils attached to gel-like mixture of protein, glycoprotein,

and proteoglycans

1) Functions of the PM

- protection of the delicate microvilli on the surface of midgut cells from contact

with rough, coarse food particles

- a barrier against entry of viruses, bacteria, or other pathogens that would be

too large to pass thru the unbroken PM

- an aid in preventing the rapid excretion of digestive enzymes

- compartmentalization of digestion within the midgut

< PM of larval black flies >

< PM and foregut armature of larva of Simulium congareenarum >

2) PM in herbivorous insects

▶herbivorous insects avoid the potentially toxic effects of ingested plant

allelochemicals by...

- biochemical detoxification (in midgut)

- rapid excretion

- storage excretion

- non-absorption (limit permeability)

3) Types of PM

- type I (in most insects)

; from the entire length of midgut

; envelops the food along the whole midgut

: synthesized in direct response to blood feeding in hematophagus insects

(e.g., mosquito, black fly) as well as other flies

; continuously synthesized (e.g., coleopteran larvae, caterpillars)

- type II

; from the specialized cells at the anterior portion of midgut (=cardia)

; larvae of higher Diptera including hematophagus insects

; earwig (Dermaptera)

- in Hemiptera: nearly absent

5. Course of infection

▶ After a pathogen invades an insect...

- incubation period

- beginning of disease

- hight of disease

- termination of disease

No of pathogens in host or pathogen activity Time

Healthy

Pathogen introduced

Beginning of disease

Period of lethal infection

Incubation period

Height of disease

DeathRecovery

Chronic infection

First sign of symptom

1) Incubation period

; the period from the entrance or introduction of the pathogen into an insect's

body until the development of signs and symptoms

2) Beginning of the disease

; the first appearance of signs or symptoms until the disease is fully

developed

3) Height of the disease

; the period when the signs and symptoms attain its maximum and may

begin to fall or reach a steady state

; pathogens are most active with severe signs and symptoms

; many pathogens, esp. bacteria and fungi, continue to grow and reproduce

after the death of an insect

4) Termination of the disease

; the recovery of an insect due to immune response

; the death of an insect in the absence of immune response

; an insect may have a chronic infection and survive to reproduce if the

pathogen has low pathogenicity

※ Signs, symptoms and syndromes

- during the infection, the insect exhibits characteristic aberrations or dysfunctions

☞ Signs: physical or structural abnormality

; abnormalities in the structure

; changes in color

; malformation of appendages, integument

< Greater wax moth, Galleria mellonella larvae. A live, healthy versus infected by

Heterorhabditis bacteriophora nematode >

< Black fly larvae, Simulium pertinax infected with microsporidia >

< Popillia japonica larvae, healthy versus infected by

Paenibacillus popilliae >

☞ Symptoms: functional or behavioral disturbance

; abnormal movement

; abnormal response to stimuli

; digestive disturbance

; inability to mate

- NPV (nucleopolyhedrosis virus> infected larvae

; become sluggish

; climb to the top of trees

; inverted "V" shape

☞ Syndrome: a particular combination, set, or sequence of signs and symptoms

; when a silkworm larva ingests the spore and endotoxin of Bt

- it stops feeding in a few minutes

- become sluggish in about 10 min

- the pH of the blood becomes higher

- the larva collapses and dies within an hour

< Bacillus thuringiensis >

< Bt toxin crystals and spores >

6. Interactions among microorganisms

- different pathogenic and non-pathogenic microorganisms are often found within

the same insect

- the interactions may result in...

; independent coexistence

; complementation (synergism)

; interference (antagonism)

- these interactions are affected by factors, such as...

; larval stage

; dosages of the pathogens

; sequence and time of feeding of each pathogen

; environmental condition (i.e., temperature)

1) Complementation (synergism)

- the pathogens are associated with each other in such a way as to

enhance pathogenicity by the combined pathogens

- Baculovirus (BV)

; all are known from insects and related invertebrates

; NPV (nucleopolyhedrovirus), GV (granulovirus)

< NPV and armyworm, Pseudaletia unipuncta >

; GV is the synergist and enhances the infection of the NVP

(GV contains an enhancing factor, a lipoprotein)

- Entomopathogenic nematode (EPN)

; Steinernema carpocapsae nematode

; Xenorhabdus nematophila, symbiotic bacteria

; bacteria, by itself, cannot enter the insect host

; bacteria is carried in the intestinal vesicle of the infective juvenile

(IJ) and released thru the anus when the nematode invades the

host insect's hemocoel

; axenically (without symbiotic bacteria) cultured nematode can kill the

insect host, but it is much less efficient

; in fact, many of other nematode species cannot kill the insect host

without symbiotic bacteria

2) Interference (antagonism)

- interference occurs commonly when microorganisms present in the midgut

Enterococcus sp.

Xenorhabdus bovienii

Acinetobacter sp.

< Growth curves for three bacterial species from G. mellonella larvae, infected

with Steinernema feltiae >

7. Koch's postulates

▶ Nobel Prize winner in 1905 in physiology and medicine for his work on

tuberculosis

▶ He made brilliant discoveries on the causal agents of tuberculosis,

anthrax, and cholera thru the application of the postulates

- Observe: a specific pathogenic organism must be seen in all cases of a

disease

- Isolate: this organism must be attained in pure culture

- Re-infect: the organism from the pure culture must reproduce the disease

in experimental animals

- Re-isolate: the same organism must be recoverable from the experimental

animal

※ It is not possible to apply the postulates in all circumstances

- some microorganisms are hard or apparently impossible to detect

- certain pathogens (i.e., many obligate pathogens) are not possible to culture

in artificial medium or even in tissue culture

- different pathogens may produce similar signs, symptoms, and syndromes

- the causal agent may produce symptoms in the laboratory that differ from

those in nature