Wolbachia is a powerful technology for controlling various insect pest.

2. WOLBACHIA-BASED STRATEGIES TO
CONTROL INSECT PESTS AND DISEASE
VECTORS

3. • Insect-borne diseases
impose an immense
burden on global health,
and insect crop pests
greatly influence
economic and agricultural
productivity.
• For example, malaria
alone is responsible for
over a million deaths
every year (Snow et al.,
2005).
• The conventional control
measures, such as using
insecticides for long-term
periods will be effective.
Introduction
•Furthermore, continued use of insecticides has led to concerns of negative
environmental effects.
•Thus, the need for novel environmentally friendly control strategies has been suggested
to complement current insect control measures.

4. • Recently, Wolbachia has received
attention as a potential bio-control
agent that may yield novel insect
control strategies.
• Wolbachia are maternally inherited
intracellular rickettsiae, like
bacteria belonging to the α-
Proteobacteria.
• Wolbachia was first reported in
reproductive tissues of Culex pipiens
by Hertig & Wolbach in 1924.
• Since then, Wolbachia has been found
worldwide in numerous arthropod
species, including: insects, mites,
spiders, terrestrial isopods, as well
as mutualistic association between
filarial nematodes.
• Approx. >65% of insect species
harbor Wolbachia (Hilgenboecker et
al., 2008).
• In invertebrates, Wolbachia has been shown to
manipulate cellular and reproductive processes.

5. • However, in arthropods, Wolbachia behaves more like a
reproductive parasite by inducing:
Feminization of genetic males,
Parthenogenesis,
Male-killing, and
Cytoplasmic incompatibility (CI).

6. • Two common methods
with which to
accomplish this are:
 Introgression and
Microinjection
Wolbachia inoculation in insects

7. Wolbachia Based Applied Strategies
• One applied strategy using CI is analogous to the sterile insect
technique (SIT).
• In these strategy, female sterility is artificially sustained by
repeated releases of cytoplasmically incompatible males.
• Wolbachia is not paternally transmitted, the infection type
present in the release strain does not become established in the
field.
1. Incompatible insect technique (IIT)
•As the size of the field population
decreases due to incompatible
matings, the proportion of males of
the release strain increases.
•Similar to conventional SIT, the
increasing ratio of incompatible
matings over time can lead to
population suppression and
possibly population elimination.

8. (1) Wolbachia infections in the release
strain should display high rates of
CI (i.e., egg hatch resulting from
incompatible matings low or absent);
(2) The release strain should show
high rates of maternal inheritance,
so that infections are passed on to
subsequent generations of release
strains and release males will
continuously harbor incompatible
infections;
(3) Release strain fitness and male
mating competitiveness should be
tested in natural systems and be
comparable to wild type males;
Criteria that should be met before the implementation of an IIT
strategy, include:

9. (4) There should be little
risk of unwanted side
effects in the
ecosystem caused by
releases;
(5)Proposals for
releases should be
presented to the public
and be met with
public acceptance
before release;
(6) A crucial criterion is
that only males can
be released.

10. Mechanisms of Action in CI
• Exact mechanisms are still unclear, incompatibility apparently
involves a two component system;
 bacterial “modification” of sperm and
 A bacterial “rescue” in the fertilized egg.
• According to this model, bacteria present in the testes modify the
developing sperm (possibly via chromatin binding proteins).
• The same bacterial strain must then be present in the egg to rescue
this modification.
• If rescue does not occur, then incompatibility between the egg and
sperm results.

11. Two general biochemical models have been proposed, either :
a) Wolbachia in the male produce a product that disrupts
sperm processing in the egg (unless rescued) or
b) Bacteria in the male act as a “sink” to bind away a product
necessary for normal processing of the sperm in the egg.
•The biochemical mechanisms of CI remain unknown, and this
clearly is a major area for research.

12. Reasons to disruption of sperm processing in eggs

13. Fig. Examples of cytoplasmic
incompatibility
a
c

14. 2. Population replacement strategies

15. Two strategies used in Population replacement
a) Gene drive strategies
• Two Wolbachia-based gene drive strategies have been
suggested based upon this hypothesis:
The first is based upon genetic transformation of the
Wolbachia (i.e., paratransgenesis).
• This strategy would involve the transformation of
Wolbachia to express a particular anti-pathogenic
product in its host.
A second strategy is that Wolbachia genes responsible
for CI could be inserted into a host chromosome and
driven into a population.
• Anti-pathogenic effector genes could be linked with the
CI genes to disrupt pathogen transmission by the host
insect.

17. b. Replacement of populations with natural Wolbachia
infections
• Min and Benzer, 1997 reported several Wolbachia strains are
associated with a shortened adult lifespan of their hosts.
• They suggest the life-shortening Wolbachia infections could be
used to reduce disease transmission by insect vectors.
• The time required for development and/or replication of an insect-
vectored pathogen is known as the extrinsic incubation period.
• For example, this time period is approximately two weeks for both
dengue and malaria.
• Therefore, life-shortening Wolbachia infections shortened the
vector insect life span before they develop/replicate the pathogen.
• Recently, a life-shortening infection (i.e., wMelPop) was
introduced into the dengue vector Aedes aegypti.
• The infected A. aegypti strains showed high rates of maternal
inheritance, complete CI, and a reduction of lifespan by half
compared to uninfected controls.

18. life-shortening Wolbachia infections in mosquito

19. 3. Parthenogenesis strategies
• There is widespread interest in possible
uses of PI Wolbachia strains in biological
control.
• The parasitoid wasps are uses in both
classical and augmentative biological
control programs.
• Parthenogenesis-inducing Wolbachia may
provide a benefit to biological control
strategies that use parasitic wasps.
Parthenogenesis-Inducing Wolbachia Strains
• PI Wolbachia strains have now been found in a wide range
of parasitic wasp genera, including Trichograma, Aphytis,
Encarsia, Leptopilina, and Muscidifurax.
• If females reproduced parthenogenetically all offspring
would be female (by thelytoky).

20. • Stouthamer et al. (1999) has
identified several potential
advantages:
(a) Parthenogens will have higher
population rates of increase and
higher stinging rates,
(b) Parthenogens are likely to be
better colonizers and more easily
established at low population
densities because the problem of
finding a mate is circumvented,
and
(c) Parthenogens will be less costly
to produce in mass-rearing
programs because production is
not “wasted” on the males.

21. Mechanism of parthenogenesis induction in insects by Wolbachia:
• The cytogenetic mechanisms of PI Wolbachia have
been studied most extensively in Trichogramma
spp..
• Meiosis is normal.
• In the first mitotic division, the chromosomes
condense properly in prophase but fail to segregate
in metaphase, resulting in diploidization of the
nucleus.
• This mechanism is known as gamete duplication and
results in homozygosity at all loci.
• Subsequent mitotic divisions appeared to be normal.

22. Reason to chromosome segregation fail in metaphase:
• Possible mechanisms
involve disruption of the
centrosome or spindle
formation, attachment of the
spindle to the
chromosomes, or spindle
kinetics.
• Consistent with possible
disruption of the mitotic
spindle is the observation
that CI Wolbachia are
associated with the spindle
apparatus and can serve as
foci for astral formation.

23. 4. Feminizing strategies
• In isopod crustaceans and
Lepidopteran, Wolbachia turns males
into female or infertile pseudofemales.
• Infected females produce twice as
many daughters as uninfected ones,
allowing their cytoplasm to be
transmitted to twice as many grand-
daughters.
• The best-studied example is the
woodlouse, Armadillidium vulgare.
• The feminizing bacterium in this species acts by
suppressing an androgenic gland, thus converting
males into reproductively competent females
(although intersexes are also produced).

24. • Populations of the pill woodlouse, Armadillidium
vulgare, which are exposed to the feminizing effects of
Wolbachia, have been known to lose their female-
determining chromosome.
• Because the standard genetic mechanism of sex
determination in the woodlouse is female heterogamety
(males ZZ and females ZW).
• However, presence of the feminizing bacterium and/or
other feminizing factors in appreciable frequencies
results in loss of the female-determining chromosome
(W), thus destabilizing the sex-determining mechanism.
• A secondary result is the accumulation of suppressing
genes that act as female-determining factors in some
populations.
Mechanism of Feminizing induction in insects by Wolbachia:

25. • In these cases, only the presence of Wolbachia can cause an
individual to develop into a female.
• The genome of Wolbachia bacterial endosymbionts was
horizontally transferred into a chromosome of the common
pill bug Armadillidium vulgare, which resulted in this
chromosome evolving as a new female (W) sex chromosome.
• This represents a remarkable mechanism underpinning the
birth of sex chromosomes.

26. Fig. Mechanism of Feminization induction in pill woodlouse by Wolbachia.

27. 5. Male killing strategies
• Male killing occurs when infected
males die during larval
development.
• Gregory et al., 2000 reported
Wolbachia strains that kill male
hosts during embryogenesis in
different host species, Ex: the
ladybird beetle, Adalia bipunctata
and the butterfly Acraea encedon,
Mediterranean flour moth Ephestia
kuehniella, Cadra cautella and
Drosophila bifasciata.
•They assessed the potential use of these microorganisms in the
control of insect populations.

28. • In Coleoptera, Lepidoptera and Diptera; Wolbachia kills the
sons of infected females’.
• It is advantageous if the hosts’ daughters benefit from their
brothers’ death.
• Benefits might include eating their brothers (which happens in
Ladybirds, and flour beetles), a reduced probability of
inbreeding or reduced intensity of antagonistic interactions
between siblings.
• Infected females produce daughters with a higher probability
of survival than uninfected ones, allowing their cytoplasm to
be more efficiently transmitted, and the infection to spread.

29. Mechanism of Male-Killing
2) After gastrulation, not associated with
the normal brown coloration of
necrotic embryos; rather, the embryo
blackens as a result of breakdown of
internal structures and pycnosis of
nuclei.
• Little is known about how male-killing is achieved.
• Studies of embryos from D. willistoni lines infected
with S. poulsonii show that death occurs at two stages:
1) Before gastrulation, associated with abnormal cleavage
patterns; in particular, achromatic spindles, with other
abnormalities of the mitotic process, which account for
most embryonic deaths in male-killed lines.

30. • The points of interaction between host and bacterium
have been investigated in D. willistoni lines transfected
with S. poulsonii.
• In Drosophila, sex is determined by the ratio of the X
chromosomes to autosomes.
• In females, which are 2X:2n, the peptide Sxl is
produced.
• Sxl induces female development of the soma and the
germ line.
• In males, which are X: 2n, Sxl is not produced.
• Absence of Sxl is associated with upregulation of genes
on the single X chromosome (dosage compensation),
male somatic development, and male germ line
development.
Reason of Male-Killing at before gastrulation & after
gastrulation

31. Success of Wolbachia in pest management programme
• The tremendous works done in Wolbachia based mosquito
management programmes in all around the world.
• A notable success was achieved in Wolbachia based pest management
in all around the world on mainly, mosquito Culex pipiens, Aedis
aegypti, Wuchereria bancrofti, tsetse fly (vector of African
trypanosomiasis) and kissing bugs (vector of Chagas’ disease),
Ceratitis capitata (med fly) was eliminated by releasing males
harboring an incompatible Wolbachia infection type (Laven, 1967).

32. CONCLUSION