Microbial Insecticides

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• Microbial Insecticides
• Ziad W Jaradat

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• Microbial Insecticides
•  
• Insects are the most abundant group of organisms
  on Earth, and they negatively affect humans in a
  variety of ways
• They cause massive crop damage,
• They act as vectors of both human and animal
  diseases.

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• History of insecticides
• During the 1940s, a number of chemical
  insecticides were developed as a means of
  controlling the proliferation of noxious insect
  populations.
• One of these was the chlorinated hydrocarbon DDT
  (dichlorodiphenyltrichloroethane). DDT proved
  to be exceptionally effective in killing and
  controlling many species of pests by attacking
  the nervous system and muscle tissue of insects.
• Other chlorinated hydrocarbons such as dieldrin,
  aldrin, chlordane, lindane, and toxophene have
  since been synthesized and applied on a massive
  scale.

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• Another class of chemical insecticides is called
  organophosphates and includes malathion,
  parathion, and diazinon.
• The first generation of organophosphates were
  developed as chemical warfare agents. Now they
  are used to control insect populations by
  inhibiting the enzyme acetylcholinesterase, which
  hydrolyzes the nerve transmitter acetylcholine.
  These insecticides disrupt the functioning of
  motor and brain neurons of the insect.

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• By early 1960s, over 100 million acres of U.S.
  agricultural land were being treated annually
  with chemical insecticides. However, at a latter
  time, researchers realized that chlorinated
  hydrocarbon insecticide and organophosphate
  insecticides had dramatic effects on animals,
  ecosystems, and humans.
• DDT, was found to persist in the environment for
  15 to 20 years and accumulates in increasing
  concentrations through food chains.
• This bioaccumulation in fatty tissues was having
  a significant biological impact on many
  organisms.
• For example, in North America, many species of
  birds including peregrine falcons, sparrow hawks,
  bald eagles, brown pelicans, and double-crested
  cormorants were severely depopulated.

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• Drawbacks of chemical insecticides
• Targeted insect pest populations be increasingly
  resistant to treatment with many chemical
  insecticides
• Chemical insecticides were found to lack
  specificity consequently, beneficial insects
  were being killed along with those that were
  considered to be pests.
• Some times the natural enemies of the insect pest
  species were killed more efficiently than the
  target organisms.
• Given all the drawbacks associated with the use
  of chemical inseciticides, alternative means of
  controlling harmful insects have been sought over
  the past 20 years.

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• Using insecticides that are produced naturally by
  either microorganisms or plants was an obvious
  choice, Why?
• Highly specific for a target insect species
  Biodegradable, Slow to select for resistance.
• Researchers can manipulate genes that encode
  insect pathogenic agents and introduce them in
  target microbes that can infect these insects.

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• Insecticidal Toxin of Bacillus thuringiensis
• A microbial insecticide can be an organism that
  either produces a toxic substance that kills an
  insect species or has the capability of fatally
  infecting a specific target insect.
• The most studied, most effective, most often
  utilized microbial insecticides are the toxins
  synthesized by Bacillus thuringiensis.
• This bacterium comprises a number of different
  strains each of which produces a different toxin
  that can kill certain specific insects, for
  example

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• B. thuringiensis kurstaki is toxic to
  lepidopteran larvae including moths, butterflies,
  skippers, cabbage worm, and spruce budworm.
• B. thuringiensis israelensis kills diptera such
  as mosquitoes and black flies.
• B. thuringiensis tenebrionis is effective
  against coleoptera (beetles) such as the potato
  beetle and the boll weevil.
• Other B. thuringiensis strains with different
  toxins that are specific toward certain insects.

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•  
• Mode of action
• To kill an insect pest, B. thuringiensis must be
  ingested as the contact of the bacterium or the
  toxin with the surface of an insect has no effect
  on the target organism.
• B. thuringiensis is generally applied by
  spraying, so it is usually formulated with insect
  attractants to increase the probability that the
  target insect will ingest the toxin.

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• Advantage
• Because for the toxin to be effective it has to
  be ingested, this limits the susceptibility of
  none target insects and other animals to this
  insecticide.
•  
• Drawbacks
• Insects that attack plant roots are less likely
  to ingest a B. thuringiensis toxin that has been
  sprayed on the surface of a host plant.
• B. thuringiensis toxin can only kill a
  susceptible insect during a specific
  developmental stage.
• It costs 1.5-3 times as much as chemical
  insecticides
• Resistance of insects to the toxins produced by
  these bacteria might occur.

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• Example of using Bacterial insecticides
• Bacillus thuringiensis sub specs. kurstaki was
  used as major means of controlling spruce budworm
  in Canada.
• Its use was increased from 1 in 1979 to around
  74 in 1986 for treating spruce budworm in
  Canada
• In other countries, B. thuringiensis kurstaki
  has been used against tent caterpillars, gypsy
  moths, cabbage worms, cabbage loopers, and
  tobacco hornworms.

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• How Does it Work
• The insecticidal activity of B. thuringiensis
  kurstaki and other strains is contained within a
  very large structure called the parasporal
  crystal, which is synthesized during bacterial
  sporulation.
• The crystal is an aggregate of one kind of
  protein that can be dissociated by mild alkali
  treatment to yield two subunits of 130 kDa each
  (Fig. 12.1).
• The parasporal crystal is not the active form of
  the insecticide rather, it is a protoxin, a
  precursor of the active toxin.

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• When the parasporal crystal is ingested by a
  target insect, the protoxin is activated within
  its gut by the combination of alkaline pH (7.5 to
  8.0) and specific digestive proteases, which
  converts the protoxin into an active form with
  68 kDa (Fig. 12.1).
• When the toxin changes to its active form, it
  inserts itself into the membrane of the gut
  epithelial cells of the insect and creates an ion
  channel through which excessive loss of cellular
  ATP occurs (Fig. 12.2). About 15 min after the
  channels forms cellular metabolism ceases, the
  insect stops feeding, becomes dehydrated, and
  eventually dies.

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• Two things make this process very specific
• The need for an alkaline media
• The need for specific proteases
• B. thurigienis kurstaki is applied by spraying
  approximately
• 1 .3 to 2.6 X 108 spores per square foot of the
  target area at the peak of the larval population
  of the target organism.
• The crystal is short lived as it breaks down
  after exposure to sunlight so it is appropriate
  to spray it in cloudy days.

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• Toxin Gene Isolation
• Isolate and characterize the protoxin gene(s).
• determine whether the toxin genes are located on
  a plasmid or on the chromosomal DNA
• Test for plasmid-borne toxin genes a toxin
  producing strain can be conjugated to a strain
  that lack the insecticidal activity. If the
  latter strain acquires the ability to synthesize
  the insecticidal toxin, then the toxin gene(s) is
  most likely present on a plasmid because the
  transfer of chromosomal DNA during conjugation is
  a rare event.

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• Isolation of the protoxin encoding DNA sequence
• Total cellular DNA is isolated and separated into
  plasmid and chromosomal DNA using cesium chloride
  gradient centrifugation.
• A clone bank is constructed from the chromosomal
  DNA
• When toxin gene is plasmid encoded, the plasmid
  can be further fractionated by sucrose gradient
  to separate the plasmids to different sizes.
  Figure 12.3
• Bacillus thuringiensis kurstaki contains an
  insecticidal protoxin gene on one of 7 different
  plasmids (2, 7.4, 8.2, 14.4, 45 and 71 kb in
  length.

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• To determine which B. thuringiensis kurstaki
  plasmid carries the protoxin gene, following
  sucrose gradient centrifugation, the plasmid DNA
  sample is divided into three fractions
• Small (2.0 kb)
• Medium (7.4, 7.8, 8.2, and 14.4 kb)
• Large (45 and 71 kb)
• The fraction with the small plasmid (2.0 kb) is
  discarded, because this plasmid is too small to
  encode a protein equivalent to the 130-kDa
  protoxin.

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• The medium and large plasmid fractions are each
  partially digested with the restriction enzyme
  Sau3AI and then ligated into the BamHI site of
  plasmid pBR322.
• These clone banks were transformed into E. coli
  and then screened immunologically using the
  following procedure
• Colonies are transferred from agar plates to a
  nitrocellulose membrane.
• The transferred colonies are partially lysed
  with organic solvents.

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• All available sites on the membrane to which
  primary and secondary antibodies could bind are
  blocked by treating the membrane with bovine
  serum albumin.
• The bovine serum albumin-treated membranes are
  treated with rabbit antiserum that contains
  antibodies against the insecticidal toxin.
• The membranes are washed to remove unbound
  antibodies and then treated with 251-labeled S.
  aureus protein A, which binds to the Fc portion
  of the bound antibodies.
• Spots on the membrane corresponding to colonies
  that actively synthesize the insecticidal toxin
  are visualized by autoradiography.

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• Using the protoxin gene, the 71-kb plasmid of B.
  thuringiensis. kurstaki was found by DNA
  hybridization to encode the toxin gene.

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• Genetic Engineering of B. thuringiensis Strains
• First step is to isolate and sequence the toxin
  gene, then to obtain the amino acid sequence of
  toxin.
• When the amino acid sequence was compared for
  other toxins they all showed a common toxin
  domain.
• It was found that the whole gene is not necessary
  for the toxin to have its insecticidal activity,
  rather a portion, a chemically synthesized
  coding sequence or of course the whole gene can
  be used for further genetic manipulation

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• The B. thuringiensis protoxin protein is only
  synthesized during the sporulation phase of
  growth therefore,
• It might therefore be advantageous to have the
  toxin gene transcribed and translated during
  vegetative growth.
• This would permit the toxin to be synthesized by
  a continues fermentation process which decreases
  the cost of toxin production
•  

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• However, when a continuously active
  (constitutive) promoter form tetracycline
  resistant gene was introduced into B.
  thuringiensis the active toxin protein was
  produced continuously through out the whole
  growth cycle ( spore and vegetative phases)
• Figure 12.4, and even the toxin synthesis
  occurred in B. thuringiensis defective in
  sporulation gene.

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• Therefore, under these conditions the toxin will
  be produced in high quantities.
• Many crops might be attacked by different insect
  species, therefore, it would be advantageous if
  we could create microbial insecticides with broad
  spectrum of target insects. This could be done
  by
• Transferring a gene against one insect into a B.
  thuringiensis that already produced another toxin
  against another insect
• Fusing portions of two different species-specific
  toxin genes to one another so that a unique
  hybrid toxin is produced and is effective against
  these to different insects.

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• Testing whether the insect target can be widened
• Toxin genes were taken from B. thuringiensis
  subspecies aizawai and tenebrionis and used to
  make one construct (vector)
• The vector was introduced onto B. thuringiensis
  subspecies aizawai, tenebrioinis, kurstaki,
  israelensis
• All the transformed strains were tested for their
  toxicity toward three different larvae.
• Table 1 shows the results of this experiment.

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• Some times B. thuringiensis might not be the best
  bacteria to be sprayed to combat an insect, so
  the toxin genes must then be introduced into
  anther suitable vector such as Caulobacter
  crescentus or cyanobacteria.
• Another example is that some insects attack the
  roots of the plants which makes them
  inaccessible to the B. thuringiensis based
  insecticidal therefore, it is possible to
  introduce the toxin gene from the Bacillus to a
  bacteria that colonizes close to the roots.
• The engineered bacteria could be introduced into
  the soil so they

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• release the toxin close to the roots
• Since they colonize the soil, they keep producing
  the toxin continuously thus obviating the need
  for continuous spraying.
• Example the B. thuringiensis kurstaki
  insecticidal toxin was introduced into the
  chromosomal DNA of Pseudomonas fluorescens that
  colonize corn roots.
•  

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• 1. A transposon Tn5 element that had been cloned
  into a plasmid was genetically modified by
  altering portions of its left and right borders
  and deleting its transposase. Such an altered Tn5
  element cannot be excised from the plasmid, even
  by exogenous transposase.
•  
• Transposes is an enzyme that is encoded by
  transposon gene and facilitates the insertion or
  excision of the transposon form a chromosomal
  site.
•  
• 2. An isolated B. thuringiensis subsp. kurstaki
  insecticidal toxin gene was spliced into the
  middle of the altered Tn5 element on the plasmid
  and placed under the control of a constitutive
  promoter.

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• 3. A wild-type Tn5 element was transposed into
  the chromosome of the root-colonizing strain of
  P. fluorescens.
• 4. The plasmid carrying the altered Tn5 element
  with the insert toxin gene was introduced into
  the host bacterium that had the integrated
  wild-type Tn5 element.
•  
• 5. Homologous recombination by means of a double
  crossover between the nontransposable Tn5 element
  on the plasmid that carries the toxin gene and
  the chromosomally integrated wild-type Tn5 led
  the integration of the altered Tn5 with the toxin
  gene into the chromosomal DNA, with the
  concomitant loss of the wild-type Tn5 element.

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• This form of engineering controls two things
• toxin gene is unlikely to be lost either during
  the large-scale laboratory growth or after
  release of the engineered microorganism to the
  environment.
• Probability of transfer of the toxin gene to
  other microorganisms in the environment is very
  low.

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• Baculoviruses as Biocontrol Agents
•  
• Baculoviruses are rod-shaped, double-stranded DNA
  viruses can infect and kill a large number of
  different invertebrate organisms. Sub groups of
  this viral family are pathogenic to several
  classes of insects including Lepidoptera,
  Hymenoptera, Diptera, Neuroptera, Trichoptera
  Coleoptera, and Homoptera.
• Therefore, some of the baculoviruses are
  important for the control of certain pests and
  thus are registered as pesticides.
• Problems!!!
• These viruses kill insects slowly within days or
  weeks.

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• Solution !!!
• Enhance the virulence of the virus by introducing
  foreign genes that will severely impair or kill
  the target insect such using the gene that
  disrupts the cell cycle of the insect.
• During the insect development, larva juvenile
  hormone is needed for the metamorphosis to
  happen, now this hormone is degraded by the
  action of the juvenile hormone estrase which is
  an enzyme that inactivate the juvenile hormone.
  Therefore, an increase in the production of this
  hormone will interrupt the insect life cycle and
  leads to its death.

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•  The gene for the juvenile estrase was purified
  from the insect Heliothis virescens ( tobacco
  budworm) and the coding sequence was isolated
  from the cDNA library and inserted into the
  genome of a baculovirus under the control of the
  baculovirus transcription signals.
• When this virus was fed to target insects, the
  larva feeding and growth was severely limited
  relative to the control.

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• Problems of this method
• This method is specific for the control of the
  insects at this larval stage only so it has a
  limited effect. Therefore, the incorporation of
  a toxin gene in the genome of the virus that will
  be transcribed and translated during the viral
  normal cycle in the insect would help in killing
  the insect any time regardless of its developing
  stage.
• Example, the gene that encodes the insect
  specific neurotoxin produced by the North
  American scorpion was cloned into a baculovirus
  and was tested against target insects and found
  to decrease the damage to crops by 50 . The cost
  of propagating this virus is high

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