Other Biting Flies

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IMPORTANT ANNOUNCEMENT Tuesday April 22nd class
for cbio 4500 is at a different location Room
101 Environmental Health Sciences Building (same
building as the Creamery).
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CONTROL MEASURES Vector control
Medical Parasitology CBIO4500 April 17,
2008 Silvia N J Moreno
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PARASITE CONTROL
Control methods should be integrated with the
parasite life cycle Direct life cycles with no
IH (monoxenous) Only the DH and the environments
to be considered. For example, safe sewage
disposal will give satisfactory control of
fecally transmitted monoxenous parasites like
Ascaris. One or more Intermediate hosts
(heteroxenous) parasites with an intermediate
host often undergo asexual reproduction in the
intermediate host. This increase in biotic
potential can make control more difficult. For
the control of digeneans such as schistosomes,
prevention of fecal contamination of snail
habitats has to be almost perfect, since a
single infected snail can shed thousands of
cercariae. Protozoa multiply within the DH,
helminths do not.Low level of helminth can be
tolerated but low level of a protozoan can build
up to high numbers.

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Typical transmission cycle of a vector-borne
parasite or pathogen between a human host and an
arthropod vector, and potential steps for
intervention.
Examples of novel control strategies developed
based on arthropod genome resources (red shaded
text boxes) and the parasite or human host genome
resources (yellow text box) are shown. Nature
reviews Microbiology 3262 (2005).
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PARASITE CONTROL
Prevention of Environmental Contamination
Antiparasite drugs. Treated hosts should be
protected from re-infection. Sanitation.
Destruction of Free-Living Stages Protozoan
cysts, helminth eggs and infective larvae are
often extremely resistant to toxic
chemicals. Destruction of Intermediate Hosts and
Vectors chemicals to kill snails, insecticides.
Altering the environmental conditions so the the
target species do not find the suitable habitat
for its survival. Does not rely on public
cooperation. Destruction of Reservoir Hosts dogs
in leishmaniasis, game animals in
trypanosomiasis Prevention of Infection Many
infective stages gain entry in drinking water,
Cryptosporidium, Giardia, Dracunculus etc. These
can be controlled by safe water supplies. Meat
inspection to prevent Taenia. Bednets prevents
mosquito bites. Shoes stops hookworm larvae
burrowing through the skin. Prevention of
Parasite Maturation chemoprophylaxis and
vaccination Integrated Control

400,000 long-lasting insecticide-treated bed nets
start the last leg of their journey from a Red
Cross warehouse in Mozambique into the hands of
families who need them
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VECTOR CONTROL
Major vector-borne diseases of humans, and
associated aetiological agents and arthropod
vectors
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Global estimates of human mortality caused by
vector-borne diseases
The total numbers of human deaths that are
attributed to specific vector-borne diseases are
shown and can be compared with the numbers of
human deaths caused by two non-vector-borne
diseases HIV/AIDS and tuberculosis. The
percentages of human deaths attributed to
specific diseases as a percentage of the total
number of deaths attributed to all vector-borne
diseases are shown in the pie chart. Mortality
estimates are based on data collected from 112
countries by the World Health Organization.
Yellow fever estimate is based on data published
by the Centers for Disease Control and
Prevention. Nature reviews Microbiology 3262
(2005).
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The global distribution and burden of major
vector-borne diseases
The burden of vector-borne disease in
disability-adjusted life years (DALYs) for all
major vector-borne diseases in WHO regions (in
thousands). Disease burden is calculated based on
DALY statistics, one DALY is defined as one lost
year of 'healthy' life. The burden of disease is
a measurement of the gap between the current
health of a population and an ideal situation
where everyone in the population lives into old
age in full health. Morbidity and mortality
estimates are based on data published by the WHO.
Nature reviews Microbiology 3262 (2005).
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VECTOR CONTROL
Vector control has proven successful for disease
control Malaria eradication of malaria from
most of the temperate-climate countries in the
northern hemisphere Onchocerca the insecticide
phase of the Onchocerciasis Control Programme
(OCP) in West Africa has almost eliminated
onchocerciasis from11 countries
(www.who.int/ocp/index.htm) However there is
paucity of effective vector-control programmes
past neglect in this area of research, potential
environmental impact of existing agents,
reduction of their effectiveness because of
resistance and other biological complexities of
vector populations.
Abandoned village near the Volta river high
blindness rates resulted in the depopulation of
entire villages.
DDT (Dichloro-Diphenyl-Trichloroethane) spraying
does reduce disease transmission but
environmentalists have opposed to their use on
large scale. Bednets impregnated with
pyrethroids offer a simple and effective
approach to reduce vector-human contact and
transmission. However, limited adoption of this
approach, improper use and the emergence of
vector populations withresistance to
pyrethroids limit their effectiveness.
The result has been increased activity and
productivity in many of these areas. New villages
in areas formerly uninhabitable because of river
blindness
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VECTOR BIOLOGY
Advances in vector genomics an important
advance is the sequencing of the genome of the A.
gambiae mosquito (Science, 2002 298129). The
genome of Aedes aegypti (Science (2007)
3161718-23) (yellow fever) was also sequenced,
and a project is in place for the genome of
Glossina morsitans (Tsetse fly vector). This
information will provide opportunities for
understanding better vectors and devising new
methods for their control.
Vector biology is poised to explore new
strategies for vector-based disease
control Mosquitos refractory to pathogen
infection Novel insecticides targets Understanding
the molecular basis for vector behavior, ecology
and host-parasite-vector interactions
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Manipulation of insect vector by their parasite

• Parasites are bound to alter their host in some
  way. Does this alteration affect transmission?
  (increase would be favorable for a successful
  parasite)
• Parasite undergo a period of growth, development
  and sometimes reproduction within their vector.
• Parasite fitness is linked to vector fitness.
• Vector longevity
• Fast Development
• Host manipulation by parasite
• Hematophagy Infection results in altered vector
  feeding behavior. Most-studied examples in
  parasite affecting vector feeding behavior
  Leishmania and plasmodium.
• Fecundity, blood feeding, and infection Blood is
  important for the vector and the parasite.
  Parasite-induced fecundity reduction. Allows
  vector survival until transmission can occur?
• Infection and survivorship Do infected
  mosquitoes live longer? High infectivity also
  results in increase in mortality. Studies are not
  conclusive.

Lutzomyia longipalpis feeding
Anopheles albimanus mosquito feeding on a human
arm. This mosquito is a vector of malaria and
mosquito control is a very effective way of
reducing the incidence of malaria.
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Manipulation of insect vector by their parasite
Hematophagy Malaria-infected mosquitoes
Infected mosquitoes makes many attempts to feed,
each time depositing parasites at the feeding
site. The apyrase enzyme (degrades ADP) from the
mosquito saliva inhibits the stimulatory
activity of ADP upon platelet recruitment at a
wound site. This counters host hemostasis and
promotes easier and longer blood feeding.
Apyrase activity is reduced to one fourth in the
salivary glands of P. gallinaceum- infected
Aedes Aegypti resulting in an increase in three
times of the median blood location time. It is
not known how sporozoites are able to reduce the
levels of Apyrase in the SG of the mosquito. One
study showed that P. falciparum infected An.
gambiae bite more hosts and also that infected
mosquitoes were most likely to take multiple
meals and become fully engorged. This would
increase their chances for transmission. Is
infection increasing the threshold blood volume
at which blood-seeking behavior is
inhibited? More studies are needed in the natural
infection scenario.
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Leishmania manipulates sandfly feeding to enhance
its transmission

• Hematophagy
• Metacyclic promastigotes of L. mexicana are
  regurgitated from the midgut of the sand fly
  vector accompanied by a viscous gel-like material
  of parasite origin. The major component of this
  secretory gel (PSG) gel is filamentous
  proteophosphoglycan (fPPG) which is also thought
  to block the gut preventing flushing of
  promastigotes back into the abdominal midgut with
  the blood meal.
• The resulting blockage of sand flies interferes
  with feeding and limits the volume of blood a fly
  can obtain and this could explain why infected
  flies have been noted to probe the skin more
  frequently and spend more time feeding.
• Experiments with Phlebotomus chinensis showed
  that flies were more likely to transmit L.
  donovani to hamsters when they probed and took no
  blood than those infected flies that took a blood
  meal.
• Feeding persistance correlates with the infection
  level
• This demonstrated that Leishmania could
  manipulate the feeding ability of the sand fly to
  promote its own transmission success.

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Transmission from the Vertebrate host

• Host attractiveness is enhanced possibly due to
  modified host odor and vectors such as tsetse
  flies feed more frequently on infected hosts.
• Host defensive behaviors may be reduced making
  feeding on infected hosts less risky.
• Pathology associated with the infection could
  assist the blood-feeding efforts of vectors
• Thrombocytopenia induced by malaria, African
  trypanosomiasis and babesiosis decrease
  hemostasis.

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Manipulation of insect vector by their parasite
FECUNDITY, BLOOD FEEDING, AND INFECTION

• Fecundity (ability to reproduce) was reduced
  when mosquitoes developed oocyst infections.
• Loss of fecundity has been attributed to reduced
  blood intake or intake of impoverished blood when
  mosquitoes fed on infected hosts.
• However, experiments show no correlation with
  the the blood meal size or quality.
• Some aspect of the infection in the mosquito
  reduces reproductive output in a manner separate
  to any effect caused by feeding on an infected
  host.
• Since increased reproductive effort decreases
  lifespan if vector survival results from
  fecundity reduction then the parasite increases
  its chances of successful transmission.

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Recommendations for vector control
Genetic manipulation of vectors Transgenic
mosquitos with impaired ability to transmit the
parasite. This is attractive because is likely to
be economically viable and relatively low
technology. Vector immunity and
vector-parasite interactions Genome projects as
the A. gambiae one would help identify novel
targets in the mosquito gut and salivary glands
that are involved in digestion of the blood meal
and host-parasite-vector interactions could be
used to develop vaccines that block the
transmission of parasites and mosquito immune
regulators or smart sprays that disrupt the
development of the parasite in the
mosquito. Vector behavior and other approaches
to vector control Elucidating the molecular
basis of many mosquito behavior may be an
expensive research investment, but the simple
traps and repellant devises anticipated from this
research could be easily adopted in
malaria-endemic countries
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Genetic manipulation of vectors

• Two broad categories
• Genetic modification of mosquito populations.
  Could be achieved by releasing transgenic
  mosquitoes carrying genes whose products impair
  pathogen development.
• Population suppression use of sterile insect
  technique (SIT) in conjunction with release of
  insects carrying a dominant lethal (RIDL).
  Insects are transformed with a transgene whose
  product suppresses offspring production, leading
  to a decrease of the vector population.

A transgenic mosquito carrying a gene that
confers resistance to the malaria parasite. The
mosquito can be recognized as transgenic by the
green fluorescence of the eye facets.
http//news.mongabay.com/2007/0319-mosquitoes.html
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Genetic manipulation of vectors

• Controversial but attractive and potentially
  self-propagating. Many questions need to be
  addressed first about the feasibility and
  consequences of this approach. Serious issues
  are reduced fitness of modified vectors, the
  ecological impact of transgenic arthropods and
  the evolutionary consequences of their release.
• In addition scientists need
• Methodologies to introduce foreign genes into
  vectors mosquitoes (e.g. transposable elements)
• Promotores that can drive the expression of
  foreign genes in the correct tissues and at the
  appropriate times need to be characterized
• Find blocking gene products capable of
  interfering with parasite development in the
  mosquito. Deleterious effects of these gene
  products on the mosquito should be considered and
  avoided

Laser scanner image of transgenic mosquito
larvae. The white areas of high fluorescent
intensity indicate a high level of transgene
expression - particularly in the gut of the larvae
The transformation technique involves the
microinjection of the recombinant DNA into the
posterior end of mosquito embryos (fresh laid
eggs) prior to pole cell formation.
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Effector strategies that could inactivate malaria
parasites within the mosquito
Parasite ligands can be good targets for effector
molecules Single chain antibody fragments (scFv)
have been produced to target proteins as the
Pbs21, a surface protein on the zygote and
ookinete stages of Plasmodium berghei. CSP from
P. gallinaceum sporozoites. Pfs25 is a surface
protein expressed on the surface of
ookinetes. Mosquito tissue recognition Lectins
inhibit P. gallinaceum sporozoite invasion of
salivary glands. The SM1 peptide which binds the
midgut and salivary gland of A. stephensi. Snake
venom phospholipase A2 (PL2) inhibit oocyst
formation in A. aegypti, A. stephensi and A.
gambiae. Parasite-secreted proteins are
excellent targets for transmission-blocking
strategies. Ookinetes secrete chitinases that
facilitate their crossing of the peritrophic
matrix. Parasite strains carrying mutations or
gene knockouts of chitinase are markedly
impaired in their ability to form oocysts.
Feeding anti-chitinase antibodies to mosquitoes
also impaired the development of oocysts.
Involvement of other proteins in parasite
development has been implicated in a study where
monoclonal antibodies made to parasite proteins
secreted by ookinetes in culture bind P.
gallinaceum zygotes and ookinetes in diverse
patterns of spatial localization and temporal
expression. Immune response effectors use of the
lytic activity of synthetic derivatives of
immune peptides Antiparasite toxins expression
of toxins in mosquitos that kill parasites.
Scorpine inhibits ookinete formation in cultures
of P. berghei.
Chitin
Ookinete (O) penetration of the chitin-containing
peritrophic matrix (PM) of the mosquito midgut.
The parasite is exiting the bloodmeal on the
left, producing chitinase focally to disrupt the
peritrophic matrix, en route to the microvilli of
the midgut epithelial surface at the right.
Trends in parasitology, (2001) 17269
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Genetic manipulation of vectors

• Transgenic A genetically modified organism
  (GMO) an organism whose genetic material has
  been altered using genetic engineering
  techniques. DNA molecules from different sources
  are combined in vitro into one molecule to create
  a new gene. This DNA is transferred into an
  organism and causes the expression of modified or
  novel traits. In transgenic mosquitoes the idea
  is to alter the transmission of the malaria
  parasite.
• The main issue was to find the appropriate tools
  for the transfer of genetic material. A Minus
  transposon with exogenous DNA can be integrated
  by a transposase-mediated mechanism into the
  genome of A. spephansi. pMinEGFP was created with
  a Drosophila promoter, the gene for Green
  Fluorescent Protein (EGFP), and a marker for
  selection of the mutants Hyg, amp. Nature 405959
  (2000).
• Pre-blastoderm embryos were injected with the
  constructs and an average of 29 of the injected
  ones survived. From these, 50 were fluorescent.

Map of the transformation vector pMinEGFP.
actinP, D. melanogaster actin5c promoter hspP,
D. melanogaster hsp70 promoter. hspT, D.
melanogaster hsp70 terminator sequence. The EGFP,
ampicillin-resistance (AmpR) and
hygromycin-resistance (HygR) genes are indicated
by black arrows and the left (ML) and right (MR)
arms of Minos are in green, with the inverted
repeats represented by black triangles.
Confocal fluorescence and transmission
microphotographs of putative heterozygous (left)
and homozygous (middle) transgenic larvae
expressing EGFP compared with a wild-type
mosquito (right).
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Genetic manipulation of vectors

• The ookinete cross the midgut epithelium and
  differentiate into oocyst which after 10-15 days
  liberate sporozoites into the hemocel. The
  development of the parasite in the mosquito is
  completed when sporozoites cross the salivary
  gland epithelium.
• Scientists identified a peptide (SM1 for salivary
  gland- and midgut-binding peptide 1) that binds
  specifically to the two epithelia that are
  traversed by the parasite the distal lobes of
  the salivary glands and the lumenal surface of
  the midgut. SM1 inhibits crossing of the two
  epithelia by the parasites.
• The idea is that if you can produce SM1 into the
  gut lumen with a blood meal the Plasmodium
  development would be blocked.
• They created a synthetic gene AgCPSM14. They
  used a promoter which is activated by a blood
  meal, and a signal sequence to drive secretion
  of the protein into themidgut lumen. The
  synthetic gene consisted of four SM1 units
  joined by 4-amino-acid linkers attached to the
  CP signal sequence and driven by the
  gut-specific and blood-inducible CP promoter.
  This gene was inserted into a piggyBac vector and
  transformed into the germ line of Anopheles
  stephensi.
• Nature (2002) 417452.

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Genetic manipulation of vectors
After selecting the positive larvae, they
analyzed if the expression of this gene (they
looked at the presence of SM1) was induced by a
blood meal. Timing was important since the
invasion of the ookinete occurs 24 hours after a
blood meal. They fed control and transgenic
mosquitos on the same infected mouse and measured
the number of oocysts formed. The results show a
reduction in the number of oocysts formed (69-95
inhibition) and also these transgenic mosquitos
had fewer sporozoites in their salivary glands
after feeding in infected mice. Transmission was
completely blocked in two out of three
experiments and in a third transmission was
greatly reduced. Other group of scientists
obtained similar results using the venom
phospholipase A2 gene instead of SM1. Other genes
used csp antibodies and others.
Detection of AgCPSM14transgenic mosquitoes by
transformation marker-mediated fluorescence. Top,
a wild-type (non-transgenic) larva (middle)
flanked by transgenic larvae viewed from the
dorsal (top) or ventral (bottom) sides. Note
green fluorescence of the ventral nerve cord in
the latter, which is similar to marker-mediated
fluorescence in Drosophila13. Bottom, the head of
a wild-type (left) and a transgenic (right)
mosquito. The entire eye expresses GFP but which
facets fluoresce depends on the angle of the
incident light.http//www.genomenewsnetwork.org/ar
ticles/05_02/transgenic_mosquitoes.shtml
Midguts were dissected 24 h after a blood meal,
opened into a sheet. Midgut from a female
heterozygous for the AgCPSM14 gene. d, Midgut
from a wild-type (non-transgenic) female. Nature
(2002) 417452.
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Population reduction SIT and RIDL
SIT Sterile Insect technique male insects are
mass-reared, sterilized by irradiation and then
released in large numbers in the infested areas
in order to contribute to sterile mating with
wild mosquitoes. SIT lacks efficient methods to
select for males and irradiation-caused lethality.
RIDL release of insects with a dominant lethal .
RIDL uses males carrying female dominant lethal
transgenes that can produce purely male
offspring. In the laboratory, this strain is
maintained by using a repressible system to
control transgene expression absence of the
repressor from the insect diet in the field
activates the lethal trait. A tetracycline-induci
ble and repressible expression system has been
recently developed for A. stephensi. This
approach does not appear suitable for malaria
control because of its subsceptibility to
immigration from outside the target area.
Sterilized male flies are mass released across
the target area by airplanes. The sexually
sterile males, which outnumber the wild-type
males, mate with wild-type females resulting in
infertile mating events. This results in a
decrease of the pest levels and, if continued
over several generations, the potential
eradication of the pest from the target area.
Nature Biotechnology 23433
SIT has been used successfully to eradicate
tsetse flies from Burkina Faso, Tanzania, Nigeria
and Zanzibar where it eradicated Glossina austeni
from the 1600 km2 Unguja Island.
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Population replacement
A transgenic mosquito (Left) with green
fluorescent eyes, and a non-transgenic mosquito
(Right), with no eye fluorescence. The transgenic
mosquito carries a gene that confers resistant to
the malaria parasite.
An important issue to consider before the release
of genetically manipulated organisms is the
fitness of the mosquitoes carrying the
transgene. The transgenic insect needs to compete
with the local populations to efficiently
introgress the effector genes into the wild gene
pool. Fitness is the relative success with which
a genotype transmits its genes to the next
generation. Survival and reproduction are the
important components.
Fitness costs Burden from the transgene product
Transgenic insects may express multiple genes (a
fluorescent marker and an anti-pathogen effector
protein. In addition constructs for RIDL contain
a repressible transactivator protein for tight
control of the system. Insertional mutagenesis
Disruption of native gene function. Fitness
reduction through this strategy is not frequent.
Viability is not changed because any random
insertion is likely to be deleterious.
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Transgenic mosquitoes have a fitness advantage
when feeding on Plasmodium-infected blood
Plasmodium decrease the fertility of their insect
hosts. The hypothesis was that when fed on
Plasmodium-infected blood, transgenic mosquitoes
expressing antiparasite genes would be more fit
than WT because of their reduced parasite
prevalence. To investigate this idea,
cage-invasion experiments were performed in which
a starting population of equal numbers of WT and
SM1-transgenic A. stephensi mosquitoes were
maintained on P. berghei-infected blood. They
find that when fed on Plasmodium-infected blood,
the transgenic malaria-resistant mosquitoes had a
significant fitness advantage over WT they had
higher fecundity and lower mortality when fed on
blood containing gametocyte-producing parasites.
The progression of the infection was an important
factor to see the difference. This was done
with a model system and it needs to be tested in
non-model organisms under field conditions. PNAS
(2007), 1045580.
Fecundity of transgenic and WT mosquitoes fed on
a parasite-containing blood meal. Hemizygous
transgenic males were crossed with virgin
nontransgenic females to yield a population of
50 hemizygous transgenic and 50 nontransgenic
sibling mosquitoes originating from the same
rearing pans. Mosquitoes were fed on mice
infected with gametocyte-producing (ANKA 2.34) or
gametocyte-deficient (ANKA 2.33) parasites after
which single engorged females were kept in
separate containers for egg collection.
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Other research initiatives

• Understanding vector biology
• Mosquitoes show a remarkable preference for
  humans as hosts for blood-feeding
• They are highly susceptible to infection
• Olfaction plays a crucial role in shaping
  behaviors such as host seeking and feeding and
  determines their vectorial capacity.
• Research on the behavior of vectors
• modification of vector behavior for disease
  control
• basic research to understand the genetic and
  environmental components of vector behavior and
  reproductive biology
• mosquito genomics comparative genomics to
  provide information about lineage-specific
  adaptations, population biology, ecology and
  genetics, dynamics, regulation and variation of
  vector populations, and vector survival
  strategies.
• The gene families implicated in olfactory
  processes are regarded as promising novel targets
  for the design of mosquito attractants and/or
  repellents.

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Other research initiatives
Investigating mechanisms of insecticide resistance
At present, the control of malaria vectors relies
extensively on the use of indoor house spraying
with residual insecticides and the use of
insecticide-impregnated bednets. The problem is
the lack of available licensed insecticides and
the growing resistance. Novel targets and the
understanding of resistance are also important
areas of research. Scientists are trying to
identify specific members of the detoxification
enzymes whose expression are elevated in
insecticide-resistant populations
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SUMMARY
Remarkable progress has been made towards a
better understanding of vector biology and the
potential manipulation of its host for its own
benefit (transmission and metacyclogenesis). New
tools are available and are being produced for
the production of transgenic vectors. Fitness is
an issue still to be solved. Mosquito control
measures are often complicated by the presence of
multiple vectors in the same area. In Africa, as
many as five different anopheline species can
function as malaria vectors, either
simultaneously or seasonally. Genome information
can be used to find molecules that will inhibit
the development of the parasite in the vector.
It will also help to understand the molecular
basis for vector behavior, ecology and
host-parasite-vector interactions and to find
novel insecticides targets