1. An in depth investigation of Aedes aegypti and Aedes
albopictus in regards to dengue transmission and
prevention
Vitalia Vargo and Stacy Jacobs
December 16, 2013
“I affirm that my work upholds the highest standards of honesty and academic integrity at
Wittenberg, and that I have neither given nor received any unauthorized assistance.”
___________________________________
___________________________________

2. 1
Introduction
Over 40% of the world population is at risk from dengue infection, which is commonly
transmitted by the mosquito vector, Aedes aegypti (and less commonly transmitted by Aedes
albopictus). The A. aegypti vector is found globally and needs tropical conditions to be able to
reproduce and be a viable vector. There are ultimately many different factors that come into play
with dengue infection; however it becomes difficult since such a large population base is
affected. Up until recently, conventional prevention techniques, such as removal of positive
containers, were being implemented. However, the uses of genetically modified mosquitoes have
opened many different possibilities for dengue prevention and hopefully eradication. Finding
such a technique is imperative because 500 million people suffer from dengue infection every
year and of those 25,000 people die from dengue hemorrhagic fever. Due to the nature of dengue
infection and the regions where the infection primarily takes place, there are not any cures for
dengue, making targeting the vector crucial.
Group 3
Mosquitoes are among the best known groups of insects because of their importance to
man, as pests and as vectors, of some of the most distressing human diseases. These small, two
winged insects belong to the family Culicidae of the order Diptera (two winged flies). Nearly
three quarters of all mosquito species are found living in the humid tropics and subtropics
(Malar, 2006).
Culicidae are the major vector of arboviruses and filarias. In the subfamily Culicidae, the
Aedes are found among other vectors. There are more than 2500 species of Culicidae of which
the main genera are Aedes, with over 900 species. Aedes are best known as a vector of yellow
fever and dengue fever (Malar, 2006).

3. 1
Located in mainly tropical urban areas the A. aegypti mosquito is the leading vector of
dengue and other flaviviruses across the globe. Vector transmission begins with an infected
blood meal being taken in by the female mosquito, and then through a second blood meal, the
mosquito will infect the host (Bian et al., 2010). The genus of viruses that the mosquito is going
to be a vector of is the flavivirus; this includes the more commonly known yellow fever, West
Nile, dengue virus. Dengue, one of these flaviviruses, is of greater interest due to the lack of
prevention mechanisms (Maciel-de-Freitas et al., 2013).
Aedes aegypti is the primary vector of dengue and other flaviviruses, making it important
to implement prevention strategies. Two variables of prevention are water and temperature,
which come into play in the life cycle. The life cycle of A. aegypti is around 30 days under ideal
conditions. For the mosquito’s eggs to move into the larval stage, these variables must be
present: water and a temperature of around 32C (Christophers, 1960). Ovipostion is triggered by
the second blood meal taken in by the female mosquito. When the blood meal is taken in, the
female can lay up to 100-200 eggs; she will not lay all of these eggs at one time but over a period
of hours to a few days. They will be laid on the walls of water containers or any water surface.
This is crucial for the developmental process since the eggs will need water to enter the egg case
to move into the next stage of development (Christophers, 1960). The A. aegypti eggs can live in
desiccation or without water for up to two years before they move into the next stage. Once
water has entered the eggs, the larval stage has begun. In this stage, they will feed on
microorganisms and organic matter. The male larvae will develop faster than the females as they
shed their skin three times growing from the first to the fourth instar. The instar is the periods
between molting in an insect larva (Christophers, 1960). At the fourth instar the larva is about
eight millimeters long and metamorphous is triggered, changing the larva to pupa. In the pupal

4. 1
stage, they do not feed; they just change body form until they will emerge as an adult. However,
they are able to respond to stimuli. This process takes about two days, if conditions are ideal.
For the pupa to emerge and form into an adult, it ingests air into the abdomen; thus splitting open
the pupal case, emerging headfirst (Christophers, 1960). After two days the new mosquito will
mate; four to five days later, they will consume the first blood meal and then the cycle will repeat
(Malar, 2006). Adult A. aegypti can easily be
differentiated from other mosquitoes by the patterns
of white scales on the dorsal side of the thorax (Figure
1; Malar, 2006). In A. aegypti, the pattern consists of
two straight lines surrounded by curved lyre-shaped
lines on the side. In contrast, A. albopictus has only a
single broad line of white scales situated in the middle
of the thorax (Malar, 2006). A. aegypti and A.
albopictus have a different shape of comb scales and a
different shape of pecten teeth on the siphon (Figure
2: Malar, 2006). In A. aegypti, the larvae’s comb teeth have visible lateral dentricles, but they
have less defined dentricles on the pecten teeth.
Alternatively, in the A. albopictus larvae, the comb
teeth do not have lateral dentricles, but they have
three pointed dentricles on the pecten teeth (Malar,
2006).
The adult A. aegypti mosquito has several
morphological characteristics that distinguish it
Figure 1: The morphological patterns that aid
in identifying adult Aedes aegypti (left) and
Aedes albopictus (right) (Malar, 2006)
Figure 2: Morphological identification features of
Aedes albopictus and Aedes aegypti larvae (Malar
2006)

5. 1
from other Aedes mosquitoes. These characteristics will aid in the transmission of flaviviruses.
The two identifying characteristics of this species are found on the clypeus and scutum. The
clypeus, which makes up the face of the mosquito, is found below the antennal sockets
(Christophers, 1960). On the clypeus, there will be two silver parallel markings, used for
identification. Paired with the clypeus is the scutum; in insect anatomy, this is another name for
the anterior portion of the mesonotum. The markings on the scutum can be identified by the
white lyre shapes that are always paired with the clypeus (Christophers, 1960).
There are two specific morphological structures that aid in the transmission of
flaviviruses: the proboscis and wing size. The proboscis of the mosquito has been adapted to
suck and puncture for the two blood meals that the mosquito will take up as part of vector
transmission. Inside of the proboscis there are two tubes: one that draws the blood and the other,
which injects saliva containing anti-coagulant and a mild painkiller (Andrew and Bar, 2013). In
dengue transmission, the saliva of the mosquito acts as a carrier of the virus through the
proboscis to ultimately infect the host. For this process to take place, the saliva must be
injected through the tube, carrying the anti-coagulant and mild-painkiller. For the mosquito
to become a vector, it will take up an infected blood meal through the proboscis traveling
through the salivary gland to the midgut until the mosquito takes its next blood meal (Andrew
and Bar, 2013).
The wingspan of the mosquito is also important for dengue transmission. Not commonly
thought to be of significance since they do not play a role in virus transmission. They do,
however, determine the fitness of the female mosquito and how far she will travel to take up a
blood meal (Andrew and Bar, 2013). This is crucial to disease dynamics since a larger area could
be infected if she has a larger wingspan. Typically, a female A. aegypti mosquito can fly about

6. 1
eight miles. Therefore, if her wings are altered by a variety of prevention strategies, such as
Wolbachia this will change the different niches the mosquito will inhabit (Andrew and Bar,
2013).
One way researchers have found to inhibit the spread of dengue is through the gram-
negative bacteria Wolbachia. This bacterium is not naturally occurring in the A. aegypti
population; however it can live in symbiosis with the mosquito. The bacterium is artificially
trans-infected in the laboratory with a virulent, life-shorting strain of Wolbachia (Turley et al.,
2009). The eggs are introduced to Wolbachia artificially in the laboratory setting. There are
many different biological implications that are of interest for dengue control. Wolbachia spreads
rapidly through populations by inducing a range of manipulations of host replication that benefits
the females (Ye et al., 2013). Three of these implications on mosquitoes infected with the
wMelPop strain are cytoplasmic incompatibility (CI), life shortening, and the “bendy proboscis”.
The phenomenon of CI causes embryotic mortality in the Wolbachia infected mosquito
eggs. This will occur when a Wolbachia infected male mates with either an unaffected female
(unidirectional) or a female that is infected with another strain of Wolbachia (bidirectional). Both
unidirectional and bidirectional mating will cause embryonic death. In the case of both the male
and female being infected with Wolbachia or just the female being infected, those embryos will
have other prevention implications since they are now carriers, such as “bendy proboscis” and
female life shortening.
Aedes aegypti eggs that are carriers of Wolbachia but do not die from CI suffer from the
other effects of Wolbachia. wMelPop is thought to be the life shortening strain native to
Drospohilia melanogaster, because it causes the over-replication and rupturing of host cells in
the mosquito (Turely et al., 2009). The aim of this novel biocontrol strategy is to reduce the

7. 1
female population so they die before the second blood meal. This ultimately prevents the spread
of dengue back to the host as part of the vector transmission, as well as reducing the overall A.
aegypti population. In addition, the mosquitos that lived long enough to take up a second blood
meal had developed the mutation called “bendy proboscis” (Turley et al., 2009). The change in
morphology of the female mosquitoes’ proboscis prevented them from being able to take up a
complete blood meal and/or they were not able to successfully pierce the skin to begin blood
feeding. The “bendy proboscis” defect in wMelPop-infected mosquitoes points to a model of
infection-induced virulence, or the degree of infection in the host. It also points to disruption of
host activity of damage at the level of tissue or cell that could lead to consequences for host
physiological function and hence complex behaviors like feeding (Turely et al., 2009). These
behaviors were seen to increase as mosquito’s age in a laboratory setting.
Group 2
Mosquitoes are simple organisms with an open circulatory
system; this will give the virus the ability to proliferate throughout
each internal organ structure. This is extremely important since virus
transmission begins with the proboscis and propagates throughout the
entire organism’s tissues once infected. When the mosquito takes in a
blood meal, it goes through the salivary glands to the midgut and
through the midgut epithelium into the hemolymph (Figure 3;
Zieler et al., 2000). The hemolymph is the fluid in the
circulatory system that makes up the blood and interstitial
fluids of the mosquito. Once the hemolymph is infected, the
open circulatory system allows the virus to spread to other
Figure 3: The midgut epithelium of Aedes
aegypti after a blood meal scanned at low
magnification to show the hemolymph
(bottom), lumen side (top), and the
basement membrane. (Zieler et al., 2000)

8. 1
organs rapidly, including the ovaries, fat body, trachea, and central nervous chain as well as the
salivary glands (Tchankouo-Nguetcheu et al., 2010). The infection in the salivary glands is
essential to the spread of dengue virus to humans.
The midgut is the first insect tissue that the virus will come in contact with during its
migration. It is also the first cellular barrier that the virus must overcome to be transmitted to a
new vertebrate host (Figure 4; Zieler et al., 2000).
A. aegypti’s midgut is composed of a single layer of
epithelial tissue surrounded by a basement
membrane, muscle fibers, nerve fibers, and
tracheoles. The epithelial cells found in the midgut
are mostly columnar and heavily microvillated
(Zieler et al., 2000). The function of
these cells is secretory and absorptive;
they secrete digestive enzymes and
absorb the nutrients from the blood meal.
It is likely that midgut cells retain some proliferative potential since a damaged midgut can
rapidly be repaired at the site of damage (Zieler et al., 2000). This is important since dengue does
not show symptoms in the mosquito, only the host. When the midgut is vulnerable to attack by
phagocytes or other cells in the blood meal, the function of the microvilli-associated network
(MN) is to prevent contact between the midgut cells and the blood meal following feeding, The
MN strands are capable of close associations with other membranes, as demonstrated by the fact
that they attach closely to the surface of the virus when in contact (Zieler et al., 2000). Thus, the
MN may also function to trap viruses inside the lumen of the midgut and prevent their contact
Figure 4: An electron micrograph of microvillated midgut cell
from an unfed mosquito. The arrows indicate the MN, which is
visible above and between the microvilli. Scale, .05m. (Zieler et
al., 2000)

9. 1
with the midgut surface.
Bare cells (BC; Figure 5), which are
distinguished by the lack of microvilli
compared to other midgut cells, are found
singly or in small clusters throughout the
midgut except at the extreme posterior end.
The term ‘bare cells’ was chosen not to
imply a specific cell type but to refer to a set
of midgut cells that have few or no microvilli it is possible that they include several distinct cell
types (Zieler et al., 2000). Some of the bare cells may correspond to a division of cells that are
invaded by dengue (Zieler et al., 2000).
The salivary gland of the mosquito performs two main roles: facilitating blood feeding
and transmitting the virus. During a mosquito bite, the salivary glands release components that
include antihistamines, vasodilators, and anticoagulants, such as thrombin and
immuomodulators, in order to facilitate entry of the virus in the host (Dhar and Kumar, 2003).
Saliva also exhibits immunomodulatory activities by suppressing or enhancing the host immune
response. Specifically, the mosquito salivary glands produce tachykinin, a peptide that acts as a
vasodilator by increasing the diameter of the blood vessels and allows for greater blood flow
(Dhar and Kumar, 2003). Not only does saliva increase blood flow but it also suppresses or
enhances the hosts’ immune response (Dhar and Kumar, 2003).
Each salivary gland in the mosquito is made up of three lobes: two lateral lobes and one
median lobe. Each lobe has a central duct constituted by a layer of epithelial cells, bound
externally by basal lamina (Dhar and Kumar, 2003). The extracellular apical cavities of the
Figure 5: An example of a bare cell (BC) on the luminal
surface of the midgut, from a blood-fed mosquito. (Zieler
et al., 2000)

10. 1
posterior regions of female salivary glands are highly dilated with salivary secretions. The
median lobe is full of large secretory cells separated from the rest of the gland by the neck region
(James and Rossignol, 1991). The neck region contains nonsecretory cells and aids in fluid
transport, not secretion. The lateral lobes produce enzymes that are involved in sugar feeding,
which is morphologically and functionally identical to males (James and Rossignol, 1991). The
median and distal lateral lobes consist of secretory cells that are female specific; these are
necessary for blood feeding
Wolbachia, one prevention mechanism for inhibiting dengue transmission, works by
proliferating every tissue in the mosquito. In particular, it can be found in high concentrations in
the midgut, this is essential since this is the first tissue that the virus will come in contact with
before dispersing throughout the organism (Hughes et al., 2011). Also, there are a high number
of immune system genes that are expressed in the midgut, so when Wolbachia is present it
initiates an antimicrobial cascade to prevent dengue transmission (Hughes et al., 2011)
Group 1
Viruses are composed mainly of RNA or DNA enclosed in a protein shell, which is often
highly symmetric (Grandi, 2007). They use this symmetry to compact the RNA or DNA to fit the
limited space for their genome. Viruses inject their genetic material into host cells and utilize
their cellular machinery (Grandi, 2007). Structural rearrangement of the viral coat protein is
critical in the infection process because it allows for replication of the virus. The dengue virus is
an enveloped, positive strand RNA virus (Rodenhuis-Zybert et al., 2010). It is made of three
structural proteins: the Capsid or C protein, the Membrane or M protein, and the Envelope or E
protein (Rodenhuis-Zybert et al., 2010). The C protein synthesizes a protein shell to protect the
nucleic acids called the nucleocapsid. It is surrounded by the viral envelope, which is made by

11. 1
the E protein. The M protein helps the virus bind to the cell surface receptors. The E and M
proteins attach to the viral envelope (Rodenhuis-Zybert et al., 2010).
There are four serotypes of dengue: DENV-1, DENV-2, DENV-3, and DENV-4. A
serotype is a distinct variation in the type of antigens it binds to on the Fc receptor, which is a
protein in the plasma membrane of human immune cells (Rodenhuis-Zybert et al., 2010). Each
individual serotype provides specific lifetime immunity and short-term cross immunity to other
serotypes; some genetic variants within each serotype create a greater epidemic potential
(Rodenhuis-Zybert et al., 2010). For example, a meta-analysis performed between 1994 and
2006 in Bangkok, Thailand, found that the most common serotype of dengue in humans was
DENV-1, but the serotype most likely to cause dengue hemorrhagic fever was DENV-2 (Fried et
al., 2010).
The dengue E protein dimer is an
integral glycosylated protein in binding
to a target cell in either a mosquito or
human host. It is divided into 3 domains,
as seen in Figure 6 (Rodenhuis-Zybert et
al., 2010). The acidic pH from the
eukaryotic host cell triggers the dengue E protein dimer to dissociate so domain II can project
outward. This exposes the hydrophobic fusion peptides of the virus to the host membrane.
Domain III folds toward the fusion peptides, forcing the target cell membrane and viral
membrane to bend toward each other and fuse (Rodenhuis-Zybert et al., 2010).
Dengue enters the cell through clatherin-mediated endocytosis, which is the process
where host vesicles internalize the virus with the receptor sites (Rodenhuis-Zybert et al., 2010).
Figure 6: Dengue virus’ E Protein dimer with three domains:
domain I (red), domain II (yellow), and domain III (blue). It
functions in binding to the target cell (Rodenhuis-Zybert et al.,
2010).

12. 1
Then, the nucleocapsid is uncoated so RNA can be translated into a polyprotein and folded with
in the host cell. The new RNA is packaged into a nucleocapsid. After replication is completed,
the nucleocapsid enters the endoplasmic reticulum, where translocation and budding occurs
(Rodenhuis-Zybert et al., 2010). This allows the nucleocapsid to get its envelope coating back.
Then, the nucleocapsid enters the Golgi, where furin cleavage occurs. Furin is an endoprotease
that cleaves precursor proteins at their paired basic amino acid processing sites (Rodenhuis-
Zybert et al., 2010). This allows the precursor proteins to bind to a new cell to continue the
infection process. The Golgi has a slightly acidic pH between 5.8 and 6.0, which triggers the
release of the virus (Rodenhuis-Zybert et al., 2010). Finally, the virus matures and exits the cell
through the process of exocytosis (Rodenhuis-Zybert et al., 2010).
Antibody-Dependent Enhancement, also known as ADE, is a process where non-
neutralizing antiviral proteins, or antibodies, facilitate the virus’ entry into host cells (Rodenhuis-
Zybert et al., 2010). Antibodies direct the virus to the Fc receptors, which are located on the
plasma membrane. The virus binds to the antigen site of the receptor (Rodenhuis-Zybert et al.,
2010). It can infect macrophages, monocytes, dendritic cells, and other types of immune cells.
The viral replication process exhausts the cell of its ATP, or energy source, resulting in a higher
rate of virus infection and more severe symptoms (Rodenhuis-Zybert et al., 2010). If dengue
infects macrophages and other cells important in defense, these immune cells are too energy
depleted to fight infection. ADE may occur when a person who has previously been infected
with one serotype of dengue becomes infected with a different serotype later (Rodenhuis-Zybert
et al., 2010). This can happen months or even years apart. People in their secondary infection
have a higher viremia compared with those in whom ADE has not occurred. Viremia is the
period of time when the dengue virus is at its highest levels of concentration in the blood of the

13. 1
host, and they are most likely to be able to transmit dengue to the mosquito (Rodenhuis-Zybert et
al., 2010). This explains why primary infections cause mostly minor disease, and secondary
infections are more likely to be associated with severe disease (Rodenhuis-Zybert et al., 2010).
Aedes aegypti has had their entire genome mapped, a total of 1,376 Mb; this is the largest
mosquito genome (Timoshevskiy et al., 2013). An extensive linkage map of morphological and
molecular markers was localized on quantitative trait loci (QTLs). Four QTLs related to the
transmission of dengue were physically mapped on four chromosomal bands, which
encompassed approximately 11% of chromosome 2 (Timoshevskiy et al., 2013). Therefore, the
susceptibility of A. aegypti to diverse pathogens is controlled by very few genomic loci
(Timoshevskiy et al., 2013).
There are two categories of genes: differentially up-regulated genes (DURGs) and
differentially down-regulated genes (DDRGs). DURGs occur when a cell is deficient in a
hormone or neurotransmitter so more receptor protein is synthesized (Colpitts et al., 2011).
DDRGs occur when a cell is overstimulated by a hormone or neurotransmitter so the expression
of the receptor protein is decreased. These mechanisms bring the cell back to homeostasis, and
they demonstrate the significance of how the expression of genes can be altered based on
manipulation (Colpitts et al., 2011). The AAEL011045 gene, a DDRG, codes for the Pupal
Cuticle Protein, which is synthesized by the imaginal disk epithelium (Colpitts et al., 2011). This
makes the pupal epithelium during pupation of the mosquito. The AAEL003012 gene codes for
the Matrix Metalloprotease (MMP), which is a zinc-dependent endopeptidase that functions in
cell metabolism, cell migration, cell proliferation, and immune response (Colpitts et al., 2011).
Overexpression of the PC protein and MMP causes flaviviruses to be inhibited one million fold
in mosquitoes (Colpitts et al., 2011). When the PC protein from the mosquito binds onto the E

14. 1
protein of a virus, whether it is dengue, yellow fever, or West Nile Virus, the flavivirus infection
was inhibited in mosquitoes and mice (Colpitts et al., 2011). On the other hand, the
overexpression of MMP inhibits infection in mosquitoes but not in mice. Therefore, MMP may
not inhibit infection in humans (Colpitts et al., 2011).
The AAEL014440 gene codes for
the Juvenile Hormone Inducible Protein
and the AAEL003685 gene codes for the
Core Histone H3 Protein. Both were
found to be up-regulated at every time
point for dengue, West Nile, and yellow
fever (Colpitts et al., 2011). However,
the Core Histone H3 Protein was found to
have a four-fold up-regulation, compared to the Juvenile Hormone Inducible Protein (Colpitts et
al., 2011). Importantly, the Juvenile Hormone Inducible Protein regulates other genes that allow
the mosquito to properly develop, and the Core Histone H3 Protein helps tightly package DNA
in chromatin (Colpitts et al., 2011). Therefore, these two proteins could have large roles in
dengue transmission; although, their exact significance is unknown due to this research being so
novel. There are two primary pathways in Aedes aegypti: the Immune Deficiency (Imd) Pathway
and the Toll Pathway. The Imd Pathway provides defense against gram-negative bacteria
(Zhiyong et al., 2008). Activation of this pathway causes degradation of the negative regulator,
the Caspar gene, translocation of Relish (a transcription factor) to the nucleus, and production of
antimicrobial compounds (Zhiyong et al., 2008). The Toll Pathway causes a similar anti-
microbial cascade, which functions in the immune system. As seen in Figure 7, Pattern
Figure 7: Diagram of the Toll pathway in Aedes aegypti,
showing dengue virus inhibition. ROS stands for Reactive
Oxygen Species. http://www.pnas.org

15. 1
recognition receptors (PRRs) activate the reactive-oxygen species, depicted as ROS, which
initiates the cascade. Reactive oxygen species are chemically reactive molecules containing
oxygen (Zhiyong et al., 2008). They are by-products of normal metabolism and are important in
cell signaling and homeostasis. Then, the negative regulator, the Cactus gene, is degraded,
transcription factors are translocated to the nucleus, and the antimicrobial proteins, such as
defensin and cecropin, are produced, as demonstrated in Figure 6 (Zhiyong et al., 2008). This
results in suppression of viral infection. Wolbachia can activate the reactive-oxygen species.
However, antioxidants can stop the Toll Pathway by removing free radicals. If this response is
stopped, the virus proliferates in the tissues (Zhiyong et al., 2008).
The Toll Pathway uses a large number of down-regulated genes and up-regulated genes
that function in immune response. Roughly 34.5% of these genes are found to be expressed in
the midgut, while 27.5% are expressed in the carcass (Zhiyong et al., 2008). The Myeloid
Differentiation Primary Response Gene 88, also known as MYD88, codes for cytoplasmic
adaptor protein, which functions to bind to a receptor (Zhiyong et al., 2008). This activates the
Toll Pathway. Therefore, the Toll Pathway is suppressed when MYD88 is silenced (Zhiyong et
al., 2008). When this occurs, dengue has a higher infection rate. However, when the negative
regulator, Cactus gene, is activated the Toll Pathway is also repressed from viral proliferation,
essentially stopping the Toll Pathway from protecting the mosquito (Zhiyong et al., 2008).
Group 4
Aedes aegypti and Aedes albopictus are the two primary vectors of dengue. A. albopictus
is a semidomestic mosquito; whereas A. aegypti is a domestic mosquito found in urban areas. A.
aegypti is also more widespread geographically, causing more dengue cases than A. albopictus.
A. aegypti is an effective vector of dengue because its ability to breed in artificial containers in

16. 1
and around the homes, close to human beings (Barrera et al., 2006). Before implementing either
chemical or genetically modified organisms, the ecological implications and the mechanism of
disease transmission must be understood, especially with regard to the vector’s life cycle, the
population dynamics, and the environmental conditions. A keystone species is one where there is
a disproportionate impact on the ecosystem in relation to their biomass and productivity; A.
aegypti is not a keystone species. Therefore, removing it, will have little effect on the trophic
levels of the ecosystem since no organism directly feeds off of it. This is important for future
prevention techniques that are being researched, in particular modified mosquitoes, since they
have the potential to wipe out the population. A. aegypti is usually found between latitudes 35°N
and 35°S. During the warm season, A. aegypti are found expanding their geographic distribution
to more northern and southern latitudes. The ideal habitat is an average temperature range of
26°C (78.8°F) to 36°C (96.8°F) with average rainfall of 85 cm annually. These conditions that
the mosquitoes are found in aid in the transmission of dengue (Barrera et al., 2006).
The number of emerging adults is regulated by abiotic (rainfall, temperature, and
evaporation) and biotic factors (predation, parasitism, competition, and food) interacting in
diverse aquatic container habitats, which have varying internal properties (organic matter,
microbial communities, and other aquatic insects) depending on their size and shape, location
(under tree canopy, exposed to the sun), and season (tree leaf-shedding). Rainfall and
temperature are the two factors that determine potential breeding sites and there success. The
eggs need the presence of water to move to the larval stage and temperature is important since
the female mosquitoes will not lay their eggs if is not warm enough. Moreover, eggs that have
already been laid will die in cooler temperatures (Barrera et al., 2006). Water temperature is a
determinant of A. aegypti’s development and survival as well as its seasonal occurrence in

17. 1
subtropical zones. Rainfall and heat influence evaporation, thus determining when there are
containers with standing water. Temperature also influences feeding, impacting the rate of
immature development and the size of emerging adults (Barrera et al., 2006). There are several
regulatory factors for the maturation of A. aegypti, as they move from the egg to the larval stage.
For instance, heavily concentrated water containers may have larval competition due to limited
resources. The containers that are used for human consumption are thought to be relatively clean;
therefore, the prevalence of A. aegypti is most likely minimal. Due to the fact that the mosquito
larva feed off of microorganism that would be found in the water and if the water is going to be
used for human consumption, the presence of these organisms would not be enough to support A.
aegypti life (Barrera et al., 2006).
Current prevention methods that are being implemented include removing the positive
water containers, spraying insecticides, and using mosquito nets. It is extremely difficult to
remove all positive water containers because they include any container of still water in the
environment. In addition, the removal of these containers will have a significant impact on the
ecosystem since they provide water for humans, animals, as well as a niche for various other
species. When water containers are removed or during the dry season there is a fluctuation in the
mosquito population. Although, the prevalence of water containers in urban regions has been
reportedly linked to endemic dengue hemorrhagic fever. In these areas, it is difficult to control
the spread of dengue because there are so many water containers that complete removal is
unrealistic. Furthermore, dengue viruses often have two or more serotypes existing in one area,
causing more secondary infections. Insects have become genetically resistant to the insecticides
that have been used over the years, and it is extremely difficult to target just one organism when
spraying, such as birds, amphibians etc. Hence, such organisms are negatively affected by the

18. 1
spraying of these chemicals, causing lasting negative effects on the ecosystem. Lastly, mosquito
nets can only cover so much space so they are typically used at night to cover beds, but they do
not directly address the vector; they just provide protection for the host when being used.
Another method of prevention is genetically modifying the organism; these mosquitoes
have had their genetic make up manipulated in the lab to prevent dengue transmission. One way
to do this is through the overexpression of DDRGs, such as AAEL011045 (PC protein) and
AAEL003012 (MMP). An example of genetic manipulation to prevent the spread of dengue has
been carried out in one experiment called the “Cayman Island Trials”. These trials were
performed by the Oxitec Corporation, a leading company in GMO mosquito research. In the
trial, genetically modified mosquitoes (GMM) were males that carried a lethal gene, causing
death of the female progeny. Once manipulated, the organism was released back into the wild so
mating could occur. This is an example of one trial that used gene alteration for dengue
prevention. The risks of releasing an entirely new strain of organism into the environment, has
brought up a question of concern by environmental activists. These individuals fear that releasing
a transgenic organism into the wild type population might cause potential damage in the
ecosystem (Scott, 2002). Creating an 'empty niche', could potentially lead to other damaging
insects, but also affect organisms higher in the food chain that rely on mosquitoes as a dietary
source. However, these mosquitoes are not a keystone species so little effect would occur on the
ecosystem. There are, however, other reasons to question releasing mass amounts of GMM into
the environment without proper research (Scott, 2002). When these experiments are conducted,
they assume random mating with the population, where as the more natural occurrence is for
assortative mating, or the mating of similar phenotypes, to take place. Another factor is the
calculation of population size because these vector populations fluctuate with seasons due to

19. 1
rainfall (Scott, 2002). GMMs have a lot of potential for future dengue research and prevention,
unlike the removal of positive containers, since the research is still new the arguments against
GMM have not been thoroughly investigated, up to this point.
Wolbachia another method of dengue prevention targets the mosquito in a different way.
Since it is a bacterium that lives in symbiosis with many different insects, it can be artificially
introduced into the life cycle of the mosquito for prevention. These mosquitoes not only inhibit
dengue transmission but also malaria parasites, Chikungunya viruses, filarial nematodes, and
Erwinia bacteria. Unlike GMM, Wolbachia does not change the genetic make-up of the
mosquito; however it does impact reproduction and morphology. This can have a similar
argument as to why they should not be released into the environment without extensive research,
due to unknown implications. In 2011, thousands of these modified mosquitoes were released in
Queensland, Australia each week. Within a few months, the Wolbachia infected mosquitoes
overran the uninfected mosquito population. Future research is being conducted to see how
Wolbachia affects the vector when it has the highest viral counts for dengue transmission.
Integration
For dengue transmission to occur, there is an ideal period of time when the mosquito
vector and host are most likely to transmit the virus to each other. The period of viremia that
occurs in the human between day two and day six after the first blood meal is when the viral load
is highest in the blood of the host, leading to prime conditions for the mosquito to take up the
second blood meal as a vector. This is important because the second blood meal triggers
oviposition, and it is also at its highest viral load of dengue in the mosquito. At this time, it is
necessary to take note that temperature and water play a significant role in the mosquito’s life

20. 1
cycle because the mosquito is found in warmer temperatures with higher rainfall so the eggs can
move into the larval stage.
Currently the prevention methods for dengue infection include removing positive water
containers, using insecticides, such as DDT, and community education. These methods are not
meeting the needs of prevention so the manipulation of the vector is a strong possibility to
eliminate dengue. There was a sterilization spray used in the 1940s to prevent the females from
reproducing and ultimately preventing the spread of dengue. However, the sterilization spray was
extremely toxic to the environment (Cattand et al., 2006). There are two types of modification
that can be done to prevent dengue in A. aegypti: the overexpression of AAEL001445 gene (PC
protein) and AAEL003012 gene (MMP) in their genome and the introduction of Wolbachia
bacteria. These modified mosquitoes, theoretically, will eliminate dengue by wiping out the A.
aegypti population. However, since the mosquito inhabits many different niches and is found
across the globe, modified mosquitoes would have to be introduced globally. Also, they would
have to be introduced over a two year time period since the eggs can live in desiccation. This
could theoretically cause global eradication of the virus.
Even though both are being released back into the wild in separate experimental
locations, researchers have not studied the effects of the population, if Wolbachia infected
mosquitoes and DDRG mosquitoes mated. Hypothetically, male Wolbachia infected mosquitoes
mated with wild type female mosquitoes through random mating, for four generations, and the
same process took place for the male manipulated DDRG mosquitoes. After four generations, the
F4 generations of the Wolbachia and DDRG progenies cross (See Figure 8, 9). In the Wolbachia
crosses unidirectional CI is observed, in 50% of the embryos. The F4 generation mates in
between niches creating a double prevention mechanism against dengue (See Figure 10). In the

21. 1
F4 punnet square (Figure 10), there will be an 8:8 phenotype observed, with 50% of the embryos
unable to transmit dengue to DDRGs and the 50% not being able to transmit dengue to due to
complication of Wolbachia; however they will be vectors. The mosquitoes that are carriers of
Wolbachia (100%) will die before the second blood meal or soon after due to the life shorting
effects of wMelPop Wolbachia. Plus, if they do live to take it a second blood meal, they will
suffer from the phenomenon of “bendy proboscis”, which was discussed above. Even though, CI
does not occur in the F4 generation punnet square, due to the cross that was chosen. It is
imperative to keep in mind that the original population will be decreased since CI occurred in
50% of the parent through the F3 generation already limiting the possible genetic crosses.
This is important on many levels because it provides the genetic variation over time and
through that it gives a multilevel approach to dengue prevention that cannot be achieved without
targeting the vector. By using two different modified mosquitoes, the likelihood of dengue
transmission is diminished considerably as is demonstrated by the punnet square (Figure 9).
Since mosquitoes are not keystone species, eradicating them through dengue prevention is not
going to alter the trophic levels in the ecosystem. Therefore, the mosquito’s predators would not
be significantly affected.
By implementing the use of modified mosquitoes into dengue prevention plans, the
likelihood of vector host transmission greatly diminishes over time. Either, the genetic
modification or the Wolbachia infection has the potential to be passed down through its progeny.
In addition, since these modifications decrease the population size, this also can be taken into
account for limiting dengue infections. However, for both of these methods to be effective, the
desiccation period must be taken in account, as well as each niche the mosquito may inhabit.

22. 1
Targeting the vector in dengue transmission is extremely crucial for prevention; two of
those ways are Wolbachia and DDRGs. Although research is fairly new for modifying the
mosquito in these ways, they hold promise for dengue eradication. If dispersed globally, these
prevention techniques have the potential to impact over half the world’s population.
Figure 8: The parent Wolbachia infected
mosquito. The red demonstrations CI, green are
recessive. W- Wolbachia infected, w- Wolbachia
recessive
Nn Nn nn nn
ww wwNn wwNn wwnn wwnn
ww wwNn wwNn wwnn wwnn
ww wwNn wwNn wwnn wwnn
ww wwNn wwNn wwnn wwnn
Figure 10: The F4 cross between Wolbachia
infected mosquito and a DDRG mosquito. Blue=
unable to transmit dengue due to DDRGs, orange=
carrier of Wolbachia.
W w
w Ww ww
w Ww ww
Figure 9: The parent DDRG infected mosquito
cross,the blue demonstrates those unable to
transmits dengue,green – uninfected. N-
DDRGs infected
n- uninfected
N n
n Nn nn
n Nn nn

23. 1
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