Controlling Disease Transmission in Aedes aegypti Mosquitoes

Alyssa Forget

November 21, 2016

Montclair State University

Abstract:

Aedes aegypti mosquitoes are a major vector of many viral diseases including dengue, zika, and chikungunya. There are several ways to stop these diseases from spreading including Sterile Insect Technique (SIT), Releasing Insects with a Dominant Lethal (RIDL), introducing Wolbachia, and by eliciting an RNAi response. Although strategies that induce an RNAi response are still being researched, SIT, RIDL, and Wolbachia methods have been tested in both the laboratory and the field. RIDL field studies have shown that they are effective at reducing population size of Aedes aegypti mosquitoes in a target area, and therefore can reduce disease transmission. Meanwhile, Wolbachia has been observed to infect native populations and block viral infection via natural methods. All of these strategies have their positives as well as their drawbacks. Since each strategy has different techniques as well as positive and negative attributes, the discussion of this topic would make for an effective classroom debate. The techniques, advantages, and disadvantages to each topic are reviewed here, as is a plan for classroom use.

Introduction

With the recent increase in observed Zika virus cases, increased media attention

and discussion has been given to the importance of controlling disease transmission.

One of the main vectors of the Zika virus, the Aedes aegypti mosquito, is an especially

problematic vector because it can also carry dengue virus and chikungunya virus as well

(Center for Disease Control, 2016). Each of these viruses can cause severe flu-like

symptoms that may lead to fatalities if left untreated, while Zika can lead to

microcephaly in the newborn babies of mothers that contract the virus (European CDC,

2016; Center for Disease Control, 2016).

The increased interest in these viruses due to the increased prevalence of the

Zika virus and the subsequent media attention that it has received, has increased the

discussion on how to control the transmission of these diseases in both adults and

teenagers alike. Since students are innately interested in this real-world problem, how

to control the transmission of these diseases is a perfect topic for a class debate.

There are several potential strategies that can be used to decrease disease

transmission including the use of sterile insect technique (Knipling, 1955), release of

insects with a dominant lethal (Thomas et al., 2000), RNAi inducing strategies (Franz et

al., 2006), and Wolbachia infection (Zabalou et al., 2004). Each of these strategies uses

different techniques to control the population size of the vector mosquito, Aedes

aegypti, however each strategy also has its advantages and drawbacks. These strategies

as well as their potential advantages and disadvantages are discussed here, as is a plan

for using this topic to hold an effective debate in a classroom setting.

Literature Review

Sterile Insect Technique

Sterile Insect Technique (SIT) involves the release of large numbers of sterilized

males that cannot produce fertile offspring, which will thus decrease the size of the

target population (Knipling, 1955). This has been shown to induce sterility in

mosquitoes, since their gametes are not viable (Olivia et al., 2014). In order for this

technique to work effectively, Aedes aegypti mosquitoes must be mass-reared and

sterilized using irradiation to produce enough sterile male mosquitoes to compete with

the native population (Olivia et al., 2014).

After the sterilization process, the males must be separated from the females,

because the females must consume a blood meal in order to reproduce, while males do

not (Olivia et al., 2014). Therefore, males can be released into the environment without

increasing the likelihood that the various diseases carried by Aedes aegypti will be

spread through the human population (Olivia et al., 2014). Sex separation is typically

done manually by comparing the size of male and female pupae (Lee et al., 2013). Once

the sexes are separated, the sterile male mosquitoes are released into the wild in large

numbers in hopes that the males will be able to mate with the wild-type females and

reduce the likelihood that viable offspring are produced (Lacroix et al., 2012).

Effect of SIT on Disease Transmission

Open field studies have been carried out by Bellini et al. (2013) to show the

effectiveness of SIT. Sterile males were released into different areas in subsequent years

and the mean number of eggs observed in ovitraps per week was observed (Bellini et

al., 2013). The mean number of eggs observed varied greatly, but two trials showed a

significant reduction in egg density, which shows that SIT can be an effective means of

controlling vector population size despite some inconsistencies that were observed

(Bellini et al., 2013). However, in a similar study where sterile Aedes aegypti males were

released in Kenya, only a 50% reduction in sterility was observed (McDonald et al.,

1977).

This observed variety of sterilization is contributed to issues with mating

competitiveness observed in sterilized males due to the amount of radiation used to

sterilize them. A study completed by Bellini et al. (Jan. 2013) compared how mating

competiveness in sterilized males varied with varying levels of gamma radiation. They

observed that higher levels of radiation caused a decrease in mating competitiveness,

which can hinder the effectiveness of SIT (Bellini et al., Jan. 2013).

Despite using what has been determined to be optimal levels of radiation, a

reduction in mating competitiveness can still be observed when SIT is used. In a study

carried out by Munhenga et al. (2016) male mosquito pupae were irradiated with

optimal irradiation levels and tested for mating vigor and competiveness. There was no

difference observed in mating vigor, as irradiated males were just as capable of

insemination as wild-type males, however the irradiated males were not as competitive

as wild-type males in both laboratory and semi-field conditions (Munhenga et al.,

2016). In laboratory conditions, the irradiated males were only one tenth as

competitive as the wild-type males and only slightly higher in semi-field conditions

(Munhenga et al., 2016).

This same difference in mating competiveness caused by irradiation has also

been observed in other species such as Culex tarsalis (Milby et al., 1983). When

released into open field condition this difference in mating competiveness resulted in

sterility levels that only reached 11% in the population, which is not a high enough

number to cause any decrease in the population (Milby et al., 1983). This shows that

the process of being developed in a laboratory and irradiation has a negative impact on

the mating competitiveness of irradiated males.

This reduction in mating competitiveness occurs because over time, the captive

mosquitoes used for disease transmission control get used to reproducing in the

confined spaces within the laboratory and they lose their ability to mate under natural

conditions (Munhenga et al., 2016). Introducing newly sterilized males into the

laboratory population frequently can rectify this by stimulating natural mating

behaviors.

Drawbacks to Sterile Insect Technique

Even though SIT is species specific and environmentally friendly, there are some

drawbacks to this technique. Mass-rearing and sterilization are both major components

of SIT procedures, both of which can have major environmental risks if any accidents

happen at the site where they are taking place (Nangle & Peveling, 2005). Another

major concern with mass-rearing procedures is that unintentional release of female

insects can occur (Legros et al., 2012). If this were to occur, there would be excess

disease vectors released into the area where the mass rearing is taking place, which

could unintentionally increase the risk of disease transmission if the area around the

facility is within the range of the species being reared there (Legros et al., 2012).

Radiation also poses a potential threat to the environment around the facility if the

radioactive isotopes used in the sterilization of the pupae are ever accidently released

(Nangle & Peveling, 2005). Therefore, it is imperative that every precaution is taken in

order to ensure that these accidents do not occur.

Aedes aegypti sex-separation is also a major concern when discussing SIT

implementation in the field since it can lead to the accidental release of female

mosquitoes. The manual method typically used for sex separation is labor intensive and

can lead to high levels of females accidently being released along with the sterile males

(Lee et al., 2013). Because manually separating sexes is unreliable, some other

mechanical methods have been developed such as the use of a sieve, which uses a nylon

grid placed in water to separate pupae based on size, so the smaller male pupae float to

the top, while the larger females remain under the grid (Sharma et al., 1972).

Besides mechanical separation, other methods for separating the sexes have

been developed. Genetic sexing strains (GSS) use irradiation and classic genetics to kill

the females in the embryonic or pupae stage, prior to the release of any mosquitoes

into the environment (Franz, 2005). The GSS contain a selectable marker necessary for

sex separation that should kill the females at an early stage through either physical or

chemical means (Franz, 2005). These GSS are made by using radiation-induced

translocations to the sex-determining regions of a chromosome as dominant selectable

markers, which can be selected for under laboratory conditions (Thomas et al., 2000).

One way to use this method is by genetically modifying the pupae to be two

different colors depending on the gender, with male pupae expressing a white color,

while female pupae express a brown color (Franz et al., 1994). The pupae can also be

genetically modified to express temperature-sensitive lethality, so when the colors are

separated the female pupae can be killed by exposing them to high temperatures (Franz

et al., 1994). The physical color traits caused by these genetic modifications make sex

separation easier than previous manual techniques that relied on size alone.

Genetic modification using fluorescent proteins is another method of genetically

sexing strains. In order to make the males express fluorescence, the fluorescent marker

can be added to the male sex-determining region of a chromosome (Condon et al.,

2007). The expressed fluorescent marker appears weaker when it is transposed into the

sex-determining region, than when it is transposed into an autosome (Condon et al.,

2007). However adding the fluorescence into the sex-determining region in necessary,

despite the reduction in observed strength, since the fluorescence is being used to

determine males from females.

Release of Insects Carrying a Dominant Lethal

Since traditional Sterile Insect Technique has many drawbacks, scientists have

been trying to improve upon this technique. In a study completed by Thomas et al.

(2000), major modifications were made to traditional insect technique by genetically

modifying mosquitoes to express a homozygous dominant gene that is lethal to female

mosquitoes. The addition of transgenes can be used to genetically separate the sexes,

but a more beneficial use of transgenes is to use them to sterilize the insects as well

(Thomas et al., 2000).

Thomas et al. (2000) coined this variation of SIT as the Release of Insects

Carrying a Dominant Lethal (RIDL). In order for RIDL to work correctly, the target

population must carry a conditional, dominant, sex-specific lethal, so that the condition

that allows the females to survive can only encountered under laboratory conditions

(Thomas et al., 2000). The simplest type of conditional laboratory setting would be to

genetically modify the pupae to grow on media containing a chemical additive, such as

tetracycline, that when consumed will allow for female survival, but when absent, as

would be observed in nature, would stop any female progeny from surviving (Thomas et

al., 2000).

In order for this to work, the dominant lethal gene must be added to a female-

specific promoter so it is only specific to females and will not affect the males in the

population (Alphey, 2002). An example of this is the tetracycline-repressible system in

which the tetracycline-repressible transactivator protein (tTA) is placed under the

control of a female specific promoter, typically Act4 in Aedes aegypti female mosquitoes

(Alphey, 2002). When this promoter is expressed in the presence of tetracycline it will

ultimately cause cell death (Alphey, 2002). This technique has since been used to create

several lines of Aedes aegypti mosquitoes such as OX513A, which have been

consistently observed to pass on the female lethality trait to their offspring based on a

simple Mendelian pattern (Lacroix et al., 2012).

The progeny of the transgenic OX513A males and wild-type females would result

in females that die during the larval or pupae stage, while the male progeny would be

heterozygous and be able to pass on the dominant lethal to another generation (Lacroix

et al., 2012). However, another strain of genetically modified Aedes aegypti mosquitoes,

OX3604C, contains a different tetracycline regulated transgene that causes the female

mosquitoes to be flightless (Facchinelli et al., 2001). Since the females are flightless they

cannot feed or reproduce, which leads to their death (Facchinelli et al., 2001). Although

the OX513A mosquitoes contain an early acting female killing transgene and the OX3604

mosquitoes contain a late acting female killing transgene, both strategies can pass the

gene from one generation to the next and therefore can theoretically be used to control

disease transmission.

Using RIDL to Control Disease Transmission

In order to compare the effectiveness of RIDL in comparison to SIT a study

carried out by Lee et al. (2013) used a temporal model of mosquito population dynamics

to compare mosquito dispersal rate and how large of an area the release of modified

mosquitoes can control disease transmission. After running several models comparing

minimal release region size required for eradication, extinction time and strategy cost,

and the dependence of extinction time on release rate ratio, Lee et al. (2013)

determined that RIDL strategies are superior to SIT strategies. This is because SITs

reduce larval populations, which actually enhance the survival of wild-type offspring,

which counteract the reduction in population size caused by the larval death (Lee et al.

2013)

Once RIDL was observed to be a more effective strategy than SIT, Legros et al.

(2012) used a model to determine whether a homogeneous or a point source release of

transgenic female killing mosquitoes would be more effective. After running the models,

it was observed that homogenous release conditions would be more effective in

diminishing population size (Legros et al., 2012). However the model also suggests that

complete elimination of Aedes aegypti mosquitoes from a large heavily populated area,

such as a city, is highly unlikely due to reintroduction and limited community

participation (Legros et al., 2012). Since complete extinction of a mosquito species in a

given area is unlikely, the focus should be placed on reducing the number of mosquitoes

to a level where disease transmission is sufficiently reduced rather than complete

extinction (Legros et al., 2012).

There have been several laboratory and field studies where the reduction in

Aedes aegypti mosquito population size has been observed. The first trials using

transgenic mosquitos were carried out in a laboratory setting, where transgenic males

were introduced into large laboratory cages at a ratio of 10 transgenic males to 1 wild-

type Aedes aegypti mosquito (Facchinelli et al., 2001). In this study, the wild-type

population was eliminated in all experimental cages in less than 20 weeks, which shows

that this strain of transgenic mosquito can successfully reduce vector population size

and therefore reduce disease transmission under laboratory settings (Facchinelli et al.,

2001).

Transgenic males were subsequently released into field cages containing a wild-

type Aedes aegpyti population at ratios of 10:1 or greater. Similar to the model

predictions of Legros et al. (2012), there was a decline in the population size of the wild-

type mosquitoes, but complete extinction was not observed (Facchinelli et al., 2001).

This lack of extinction was thought to be due to the difference between mating habits

between captive mosquitoes kept in the laboratory and those in field settings

(Facchinelli et al., 2001).

Due to the successful decline in population size observed by Facchinelli et al.

(2001) open field release experiments were warranted, but first dispersal patterns and

appropriate recapture techniques had to be determined (Winskill et al., 2015). Mark-

release-recapture studies were carried out under both urban (Winskill et al., 2015) and

rural conditions (Lacroix et al., 2012). Although the environments were very different

both studies determined that the recapture rate quickly decreased over time, with the

large majority of the released mosquitoes being recaptured within the first five days of

the study (Winskill et al., 2015; Lacroix et al., 2012). The average distance traveled by

the released transgenic mosquitoes was also very similar at approximately 50 meters,

with very few mosquitoes traveling more than 100 meters (Winskill et al., 2015; Lacroix

et al., 2012).

However, the mean distance traveled for the wild-type males was significantly

further at 99.8m (Lacroix et al., 2012). This may indicate that transgenic males do not

have the same flight capacity as wild-type males do (Lacroix et al., 2012). This is

important to know when designing disease transmission control strategies, because it

means that transgenic males have to be released in closer proximity to one another

than wild-type males would in order to ensure that the population in the targeted area

is controlled.

The information gathered from the aforementioned studies about dispersal

patterns and recapture rates was then used to determine effective techniques for a

large-scale field studies that test the effects of transgenic mosquito release on

population size. There have been two open release studies that have shown a decline in

population density as a result of releasing transgenic males (Harris et al., 2012; Carvalho

et al., 2015) In both cases, OX513A transgenic males were released into the targeted

area, Grand Cayman and Brazil respectively, and ovitraps were used to determine

population size (Harris et al., 2012; Carvalho et al., 2015). In the study carried out in

Grand Cayman, an 80% decline in population density was observed over the course of

the experiment in the targeted areas (Harris et al., 2012). The study carried out in Brazil

showed a 78% reduction in population size as determined by ovitrap data, which is very

similar to the results found in the Grand Cayman experiment (Carvalho et al., 2015).

Even though this study did not cause extinction of all Aedes aegypti mosquitoes in the

targeted area, it did reduce the population size to a level that would reduce the rate of

disease transmission in the targeted area, which shows that this is an effective strategy

(Harris et al., 2012; Carvalho et al., 2015).

Advantages and Drawbacks Associated with RIDL

Besides being observed to be an effective way of controlling population size, late

acting female killing strategies commonly used with RIDL are also commonly thought to

be more effective than traditional SIT because they allow for larval competition to occur

between the wild-type and genetically modified larvae before the females are killed (Lee

et al., 2013). In a study completed by Gentile et al. (2015) a model was used to compare

the effectiveness of various population reduction strategies including both early acting

and late acting female killing strategies. In every scenario ran the late acting female

killing strategy, commonly used in RIDL, decreased the wild-type population size more

efficiently than the early acting female killing strategy, which is the method used in SIT

(Gentile et al., 2015). This occurs because late-acting genes allow for larval competition

to occur between the wild-type and transgenic mosquitoes, which will increase the

suppression of the wild-type population (Gentile et al., 2015).

There are many positive attributes of using RIDL such as effective separation of

the sexes, which eliminates the threat of accidental female release, observed equality in

mating competiveness, and most importantly, a reduction in the rate of disease

transmission due to Aedes aegypti population control. However, there are also some

potential drawbacks to using RIDL methods for large-scale disease transmission control.

RIDL works by genetically modifying genes to kill the females later on in their lifecycle,

so females will go through the early stages of their lifecycle, but they will never survive

long enough to reproduce due to the specific transgene incorporated into their genome

(Alphey, 2002). However, the male progeny that come from the reproduction of a

transgenic male and a wild-type female will live and will all be heterozygous (Gentile et

al., 2015). When the heterozygous males mate with wild-type females, the only females

found in the population, their offspring will only have a 50% chance of inheriting the

female killing transgene (Gentile et al., 2015) Therefore, half of the offspring will not

have the transgene and will be considered wild-type, which will help to preserve the

wild-type population (Gentile et al., 2015). This will make it very difficult for the

transgenic males to eradicate or even reduce the wild-type population to a level that

will stop disease transmission in all cases.

RNA Interference

In both SIT and RIDL the reproductive capabilities of the mosquito are targeted,

however there are drawbacks to each of these approaches, so a novel form of

population control that enables RNA interference (RNAi) is being developed that

specifically targets the ability of the Aedes aegypti mosquito to transmit a particular

virus. Since dengue virus (DENV) is the most serious of the diseases transmitted by

Aedes aegypti, most of the research being done with RNAi involves genetically

modifying the mosquito to stop the ability of the dengue virus to spread through it

(Franz et al., 2006).

In order to do that an anti-dengue virus gene must be added to the mosquito

genome to block the replication and transmission of the virus (Travanty et al., 2004).

The anti-dengue virus gene that triggers RNAi must be expressed in the whole mosquito

or at least in the midgut, which would prevent replication, or the salivary glands, which

would prevent the transmission (Travanty et al., 2004). The RNAi pathway is an anti-viral

pathway found in many organisms including humans and mosquitoes, which causes the

destruction of the virus’ mRNA sequence as soon as the virus’ double-stranded RNA is

recognized (Travanty et al., 2004). By destroying the virus’ mRNA sequence the RNAi

mechanism is effectively blocking the viral replication system, so it won’t be able to

replicate (Travanty et al., 2004).

There are two studies that have used different methods to induce RNAi in the

target mosquito. A study by Travanty et al. (2004) made an anti-DENV effector made

from a hairpin turn derived from the premembrane (prM) protein-coding region of

DENV-2. Meanwhile another study completed by Franz et al. (2006) did this by

modifying the premembrane protein-coding region of the DENV-2 RNA genome to

express an inverted-repeat (IR) RNA instead of a hairpin turn. The effector in both cases

was then added to a mosquito embryo by co-injecting two plasmids, one containing the

gene and a fluorescent marker and one containing transposase (Travanty et al., 2004;

Franz et al., 2006).

As soon as the dsRNA is formed when the virus tries to replicate in the midgut of

the mosquito after a blood meal, the mosquito will activate the RNAi against it, which

will break down the viral RNA into small interfereing RNAs (siRNAs) (Travanty et al.,

2004; Franz et al., 2006). These siRNAs, when observed, as they were in both of these

studies, are an indicator that RNAi has taken place (Travanty et al., 2004; Franz et al.,

2006).

In order to test that this process works in a live mosquito, a family of transgenic

mosquitoes called Carb77, which contain midgut epithelial cells that express IR-RNA

after a blood meal, were developed (Franz et al., 2006). They were given a blood meal

containing DENV-2 and their susceptibility to the virus was tested (Franz et al., 2006).

After a week only one out of the fifteen Carb77 female mosquitoes infected was

detected to contain the virus and all of them reduced viral replication because less sites

of viral replication were detected, thus the virus’ ability to replicate was reduced (Franz

et al., 2006).

Although this methodology did produce the intended RNAi response in the

midgut of the mosquito as intended, the Carb77 mosquitoes did have a drawback. After

only seventeen generations grown in culture, the Carb77 mosquitoes stopped

expressing the IR-RNA and therefore were not able to effectively stop the replication of

DENV-2 long term (Franz et al., 2009). However, the research continued and Franz et al.

(2014) developed a new line of transgenic mosquitoes, Carb109M that works using the

same IR-RNA technique. This line of mosquitoes has remained stable for over thirty-

three generations and the IR-RNA continues to be passed on as a heritable trait (Franz et

al., 2014). This shows that the IR-RNA and the RNAi response that it elicits can be

maintained in a population.

Additionally, the use of IR-RNA on a gene in the salivary glands of the female

mosquitoes has been observed to reduce the infection rate of DENV-2 in this region

(Mathur et al., 2010). The anti-DENV2 gene used in this study uses an inverted repeat

similar to the Carb109M line, except it targets the 30K genes in the mosquito salivary

glands (Mathur et al., 2010). Out of the fifteen lines of transgenic mosquitoes tested,

none of them transmitted the virus in detectable numbers, showing that using IR-RNA to

induce RNAi can also be as effective if expressed in the salivary gland as it has been

when incorporated into the midgut.

Drawbacks to RNAi

There are two main drawbacks to using RNAi to reduce disease transmission. The

first drawback is that in the three experiments mentioned here this method has not

been observed to be 100% capable of stopping DENV-2 from replicating within the

mosquito. In the study by Travanty et al. (2004) RNAi was not induced in every tissue in

the mosquito, most importantly, it did not occur in the midgut, so viral replication was

not blocked and these mosquitoes could still carry and transmit the virus. In both

studies by Franz et al. (2006 & 2014) the IR-RNA did not elicit an RNAi response in at

least one of the Carb77 or Carb109M mosquitoes. Since this method is not yet 100%

effective at stopping the DENV-2 virus from replicating, the virus may still be

transmitted.

Another main drawback of using transgenics to elicit an RNAi response is that

the resulting mosquito has been observed to not be as fit as the wild-type mosquito,

which caused it to be lost in the Carb77 population after a few generations (Franz et al.,

2009) This loss of the trait shows that it is not being selected for in the population,

which suggests that it has a high fitness cost associated with having it that needs to be

studied further (Franz et al., 2014)

Wolbachia

Wolbachia are maternally inherited endosymbiotic bacteria that live within many

species of insects and manipulate the reproductive strategies of their hosts by inducing

cytoplasmic incompatibility (CI) in the larvae that result from an unmatched mate

pairing (Zabalou et al., 2004). Males infected with Wolbachia cannot mate with

uninfected females, however infected females can mate with uninfected males (Zabalou

et al., 2004). Additionally if both males and females are infected with Wolbachia, they

must be infected by the same strain to produce viable offspring (Zabalou et al., 2004).

Although the Wolbachia bacteria are found naturally in many species, they have

been observed to be transferable into species that don’t typically carry them (Zabalou et

al., 2004). Aedes aegypti are not naturally infected with Wolbachia, however Xi et al.

(2005) did successfully create a line of infected Aedes aegypti by injecting their embryos

with Wolbachia. When the females infected with Aedes aegypti were allowed to

reproduce under laboratory conditions high rates of cytoplasmic incompatibility was

observed, which resulted in a decrease in population size (Xi et al., 2005). Moreover,

there have been minimal fitness costs observed and the line has been maintained under

laboratory conditions for at least six years (Xi et al. 2005).

In addition to cytoplasmic incompatibility reducing the number of offspring

produced, mosquitoes infected with Wolbachia have also exhibited resistance to several

RNA viruses including dengue (Bain et al., 2010). When the Aedes aegypti becomes

infected with Wolbachia, it increases the immune response within the mosquito (Bain et

al, 2010). Bain et al. (2010) observed this in a study, where the number of copies of the

DENV2 RNA was significantly lower in the mosquitoes infected with Wolbachia than in

those that did not contain Wolbachia. Additionally, when the amount of plaque forming

units transferred from the proboscis were measured after a simulated feeding, the

mosquitoes inflected with Wolbachia only transferred 45 plaque forming units (pfu)/ml,

compared to 550 pfu/ml in the control group (Bain et al, 2010).

Moreover, Wolbachia has also been observed to reduce the lifespan of Aedes

ageypti mosquitoes under laboratory conditions, which can help reduce the spread of

viral infection, because viruses such as dengue, take longer to develop within the

mosquito before they can be transmitted (McMeniman et al., 2009). Therefore, by

reducing the lifespan of the mosquito, the virus is less likely to be able to fully develop

and be transmissible before the mosquito dies (McMeniman et al., 2009). Together

these results show that infection with Wolbachia increases resistance to viral infection,

which can be an effective tool for controlling disease transmission.

The ability for Wolbachia to invade native populations of Aedes aegypti has been

tested in both semi-field an open field conditions (Walker et al., 2011; Hoffman et al.,

2011). When tested under semi-field conditions, a strain of Aedes aegypti mosquitoes

infected with Wolbachia (wMel) were released into a population of wild-type Aedes

aegypti (Walker et al., 2011). The wMel strain reached fixation within both of the test

cages in eighty days or less, showing that it can successfully be spread throughout a

wild-type population (Walker et al., 2011). Similar results were seen under open field

conditions where the frequency that Wolbachia was detected in the population

increased throughout the experiment even after new wMel mosquitoes stopped being

released (Hoffman et al., 2011). These studies demonstrated that Wolbachia infection

can be passed through a population, which can cause a reduction in population in those

not infected with Wolbachia, driving the population to have a greater frequency of

infection and therefore a greater rate of natural resistance against viral infection.

Drawbacks to Using Wolbachia to Control Disease Transmission

Although using Wolbachia infection to reduce disease transmission, there are

some fitness costs associated with using this strategy long term. Several strains of

Wolbachia infected strains of Aedes aegypti, including the wMel strain used in the

studies above, have been observed to have low fitness in the field (Yeap et al., 2014). A

lower rate of oviposition success was observed in infected mosquitoes in comparison to

uninfected mosquitoes because female mosquitoes infected with Wolbachia exhibit

lower performance on blood-feeding, mating success, and oviposiiton site seeking (Yeap

et al., 2014).

Additionally, larvae infected with Wolbachia also exhibited a lowered ability to

survive under starvation conditions in comparison to uninfected mosquitoes (Ross et al.,

2016). The mosquitoes that do reach adulthood are often smaller in body size, which

can inhibit their ability to feed and mate as effectively as their uninfected counterparts

(Yeap et al., 2014). The metabolic rate of adults infected with Wolbachia is also

increased because of the Wolbachia infection (Ross et al., 2016). These reductions in

fitness can have major implications for long term sustainability of Wolbachia infected

individuals within a population, which may be a deterrent for using Wolbachia infection

for a long term strategy to control disease transmission.

Two-Step Strategies Used to Control Disease Transmission

In order to effectively control disease transmission of the several viruses

transmitted by Aedes aegypti mosquitoes a two-step strategy using a combination of

the methods discussed above may be beneficial (Carvalho et al., 2014). Since these

mosquitoes live in a wide range of areas, Carvalho et al. (2014) suggested that disease

transmission could be reduced more effectively by first releasing a set of transgenic

mosquitoes modified using RIDL methods to reduce the population size followed by a

second release of RNAi transgenic mosquitoes that would replace the previous

population.

The first release would have to be the one that suppresses the population to the

lowest number possible, so the wild-type mosquitoes would not be able to compete

with the later released RNAi inducing mosquitoes (Carvalho et al. 2014). Using RIDL to

genetically modify the females to die prior to adulthood would be one effective method

of achieving this (Carvalho et al. 2014). There have been multiple studies that show late

female killing strategies to be effective in reducing the population size by as much as

80% in the Grand Cayman (Carvalho et al., 2014; Carvalho et al., 2015; Harris et al.,

2012). However, late acting bisex strategies, in which released males mating with wild-

type females produces offspring that only survive through the larval stage and then both

sexes die, has been shown to be more effective at reducing population size (Gentile et

al., 2015). This strategy allows for larval competition to occur prior to the death of all

the offspring of a wild-type female and a transgenic male, which maximizes the number

of mosquito deaths with the least amount of males that have to be released (Gentile et

al., 2015).

After either a late acting female killing or bisex strategy is used the population of

the wild-type Aedes aegypti will be reduced drastically, which will allow for them to be

replaced by a new line of Aedes aegypti mosquitoes that have been modified to stop the

transmission of viruses such as dengue virus (Carvalho et al., 2014). By having a whole

population of mosquitoes that have been modified so that the RNAi response is

triggered whenever the virus is present in the mosquito, disease transmission of that

virus would be significantly reduced (Carvalho et al. 2014).

Discussion

Even though none of the strategies discussed here are ready for use in real

world, it is clear that some form of disease transmission control is necessary and that

science is capable of figuring out the more efficient way to do it. Each of the

aforementioned methods of disease control, SIT, RIDL, RNAi and Wolbachia, are all

effective strategies that can theoretically be used to reduce disease transmission of a

specific virus in a target area. However, they both have some environmental, physical,

and ethical drawbacks that could be used as evidence for or against each topic when

debated in a high school classroom.

Many states including New Jersey have recently adopted a new set of state

standards called the Next Generation Science Standards that require students in all

levels of science to be able to think critically about science topics. According to these

standards, all teachers must be able to integrate disciplinary core ideas, science and

engineering practice, and crosscutting concepts into their lessons in order to develop

students that have impeccable critical thinking skills, so they can analyze a problem or

situation on their own and be able to determine how to find a solution (Quinn et al.,

2012).

By having the class debate which type of disease control should be used in a

given area, a number of the science practices, core ideas, and crosscutting concepts

would be met. The science practices that would be displayed would include analyzing

and interpreting data, constructing explanations, engaging in argument from evidence,

and obtaining, evaluation, and communication information (Quinn et al., 2012). First,

students would work in groups to develop a presentation based on primary resource

documents and articles about one of the assigned strategies. The presentations would

include an overview of the assigned strategy as well as the advantages and drawbacks to

each strategy. Each group would then present to the class in the format of their

choosing, so that the rest of the class was informed about each strategy. Once the

presentations were complete, the class would be able to have a class discussion to

debate which strategy would be the most effective form of disease transmission control

to use in the United States. Each student would then write a culminating essay

explaining which strategy he/she believes to be most effective based on specific

evidence from the presentations and the debate. (The full lesson plan for this activity

can be seen in the Appendix.)

By participating in this activity, students will not only be performing several

science practices in the classroom, but the topics being debated can help them to better

understand several disciplinary core ideas. One disciplinary core idea that would be

incorporated into this lesson is the inheritance of traits (Quinn et al., 2012). By

comparing the different methods of disease control, the importance of heritable genetic

traits is emphasized, as is the ability for those traits to be manipulated under laboratory

settings. In order to understand the different genetic principles used to manipulate the

genes within the mosquito, students must have a firm grasp on the basic genetics

concepts taught in high school.

Another disciplinary core idea that would be displayed in this activity is the

importance of interdependent relationships within ecosystems (Quinn et al., 2012). By

examining the role that mosquitoes play in disease control, students are learning about

the interactions between viruses and their vectors and their potential human hosts. The

relationships between these species are being discussed as well as how the population

dynamics in each species are affected based on the population size of the others. When

students are formulating explanations for why their form of disease control is superior,

they will come across several environmental effects of each strategy that can be used to

support their claims. Some of these environmental effects include the potential impacts

of radiation on the environment caused by SIT and the potential impacts on the food

web cause by significantly reducing the population size of a species.

In addition to the potential environmental effects that may be caused by each

strategy, there are several evolutionary effects that students should take into account

when formulating their arguments for each form of disease control. Some of the

evolutionary concepts taught in biology that should be considered when students

formulate their explanations include fitness costs of incorporating the transgenes or

Wolbachia into the mosquito and how a founder effect caused by population that is all

grown in a laboratory together, as is the case with the RNAi inducing strategies, will

affect the traits found in the offspring over time. These evolution concepts that would

be incorporated into this debate would also fit another disciplinary core idea, biological

evolution including unity and diversity (Quinn et al., 2012).

Lastly, debating the use of the abovementioned strategies to control disease

transmission in Aedes aegypti mosquitoes has real world implications that align with the

engineering practices, which are a part of the NGSS. In addition to teaching biology, high

school science teachers in states that have adopted the NGSS also have to teach

engineering practices. Each of the engineering disciplinary core ideas focuses on the

important of analyzing, evaluation, and designing solutions for complex real-world

problems (Quinn et al., 2012). The transmission of the various diseases caused by viral

infection of Aedes aegypti mosquitoes is a real problem facing the world today and in

this debate, students would be analyzing, evolution, and determining which of the three

strategies presented would be the most effective at controlling disease transmission.

Therefore, completing a debate in a high school classroom to determine which of the

three strategies presented is the most effective would be a practical method for

incorporating several of the NGSS disciplinary core ideas and science and engineering

practices into one lesson that displays how effectively the students can use their critical

thinking skills to analyze, evaluate, and communicate, while having them understand a

current real-world problem.

Conclusions

Since the recent increase in the prevalence of the Zika virus and the increased

media attention that it has received in the United States, there has been resurgence in

the discussion of possible ways to control disease transmission in Aedes aegypti

mosquitoes even among mainstream citizens. This increase in the discussion

surrounding control of disease transmission has made it a real-world problem that can

easily grab the attention of high school students. The various disease transmission

control strategies being researched each have their advantages and disadvantages,

which makes this topic perfect for debate in the high school classroom.

By presenting students with data from research-based articles that discuss the

advantages and drawbacks to each strategy, students can use their knowledge of

biology disciplinary core ideas and science practices to create an argument that

supports each of the disease transmission control strategies, SIT, RIDL, RNAi inducing

methods, and Wolbachia infection. These arguments can then be debated in the

classroom to show mastery of both biology content and science practices. Although

having a debate based on current scientific research would be very beneficial for

students, it should only be done with classes that have a highly proficient understanding

of the major biology concepts including ecology, genetics, and evolution. Without

substantial background knowledge of these concepts, it would be difficult for students

to analyze the data given to them and use it to make an effective argument for a

strategy.

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Appendix

Evaluating Strategies for Controlling Disease Transmission Lesson Plan Primary Subject Area and Grade Level: This lesson requires prior knowledge of genetics, ecology, and evolution, and therefore must take place at the end of the year in an honors level high school biology classroom or in AP Biology after these concepts have been covered.

Lesson Duration:

This lesson will take place over the course of three eighty-minute block classes.

Overview: In this activity students will analyze articles and data related to four different strategies used to control viral disease transmission in Aedes aegypti mosquitoes, the primary vector for several diseases including Zika, Dengue, and Chikungunya. This lesson incorporates many different topics taught in biology into one lesson based on a real-world issue. By completing the activities included in this lesson, students will integrate their comprehensive knowledge of biology to analyze each of these strategies and develop a claim supported by evidence that predicts which strategy would be the most effective to use for large-scale control of disease transmission in the United States.

Objectives: - Students will be able to evaluate various strategies used to control viral disease transmission and

develop a claim supported by evidence to show which strategy is the most effective. - Students will be able to analyze data and effectively communicate their findings to their peers. - Students will be able to make and defend a claim using evidence to support their ideas.

Content Standards and Common Core Learning Standards:

Next Generation Science Standards: - Disciplinary Core Idea LS2.B - Ecosystems have carrying capacities resulting from biotic and abiotic

factors. The fundamental tension between resource availability and organism populations affects the abundance of species in any given ecosystem.

- Disciplinary Core Idea LS2.C – Ecosystem Dynamics, Functioning, and Resilience: If a biological or physical disturbance to an ecosystem occurs, including one induced by human activity, the ecosystem may return to its more or less original state or become a very different ecosystem, depending on the complex set of interactions within the ecosystem.

- Disciplinary Core Idea LS3.A – Inheritance of Traits: DNA carries instructions for forming species’ characteristics. Each cell in an organism has the same genetic content, but genes expressed by cells can differ

- Disciplinary Core Idea LS3.B - The variation and distribution of traits in a population depend on genetic and environmental factors. Genetic variation can result from mutations caused by environmental factors or errors in DNA replication, or from chromosomes swapping sections during meiosis.

- Disciplinary Core Idea LS4.B – Natural Selection: Natural selection occurs only if there is variation in the genes and traits between organisms in a population. Traits that positively affect survival can become more common in a population.

- Disciplinary Core Idea LS4.C – Adaptation: Evolution results primarily from genetic variation of individuals in a species, competition for resources, and proliferation of organisms better able to survive and reproduce. Adaptation means that the distribution of traits in a population, as well as species expansion, emergence or extinction, can change when conditions change.

- Disciplinary Core Idea ETS1.B – Developing Possible Solutions: When evaluating solutions it is important to take into account a range of constraints, including cost, safety, reliability, and aesthetics and to consider social, cultural, and environmental impacts.

English Language Arts Standards: - CCSS.ELA-LITERACY.SL.9-10.4 - Present information, findings, and supporting evidence clearly, concisely,

and logically such that listeners can follow the line of reasoning and the organization, development, substance, and style are appropriate to purpose, audience, and task.

- CCSS.ELA-LITERACY.SL.9-10.3 - Evaluate a speaker's point of view, reasoning, and use of evidence and rhetoric, identifying any fallacious reasoning or exaggerated or distorted evidence.

- CCSS.ELA-LITERACY.SL.9-10.1.D - Respond thoughtfully to diverse perspectives, summarize points of agreement and disagreement, and, when warranted, qualify or justify their own views and understanding and make new connections in light of the evidence and reasoning presented.

Lesson Procedures: Day One:

- Think-Pair-Share: What do you know about Zika virus? (10 minutes)o Students will think about the question and discuss their knowledge of Zika virus with a partner.

Then each group will share their knowledge with the class and a brief class discussion regarding Zika virus will occur, including what is it, symptoms, transmission, vectors, and treatment. If students are not knowledgeable about Zika virus, the teacher will lead the discussion to draw

connections between their prior knowledge of viruses and how it relates to Zika. - Asking Questions: Since Zika and many similar viruses such as dengue virus, are not curable, there is a

need to decrease the transmission of these diseases in order to stop their spread. How could humans reduce the transmission of these diseases? (20 minutes)

o This question will be posed to the students and they will be given time to develop an answer. Then a list will be made on the board of the possible ways of controlling disease transmission that they come up with.

o Some possible forms of controlling disease transmission that students may come up with include: Killing the vector that carries the virus Sterilizing the vector Stopping the vector from feeding on humans Stopping the vector from reproducing Stopping the virus from reproducing

o Once the list is made, the teacher should point out that many of these strategies could be viable options and that scientists are currently researching many of them. The teacher should then introduce the activity that is going to be carried out in class by briefly explaining each of the components of the lesson including the presentation, the class debate, and the individual reflective essay.

o After explaining the overview of the project, the teacher should explain the presentation component in detail and hand out the rubric being used to grade this component.

- Presentation Development in Groups (50 minutes)o Students will then be broken into eight groups of approximately two to four students. Two

groups will be assigned each of the following topics: Sterile Insect Technique Releasing Insects Carrying a Dominant Lethal Inducing an RNAi response Using Wolbachia to increase an immune response

o Groups will then be given the necessary materials including access to websites and articles containing general information about how each strategy works, data collected from research-based journal articles, and access to journal articles about each topic for further review. The amount of data given to the students and the amount they must retrieve on their own from selected primary sources can vary with class level. More advanced classes should be more responsible for gathering their own data than introductory level classes.

o Students will then analyze the data to summarize how the strategy works, the advantages and drawbacks of each strategy, as well as any potential risk factors associated with it. This information will then be used to create a presentation in the form of their choice, which will be presented to the class. Options for the format of student presentations include posters, PowerPoints, videos, brochures, etc.

Day Two:

- Presentation Expectations (5 minutes)o Prior to students working in their groups for the rest of the block, the teacher should clarify the

expectations for the presentation, including proper presentation techniques. Particular emphasis should be placed on voice volume, the importance of eye contact, and explaining the topic, not just reading off the slides.

- Presentation Development in Groups (75 minutes) o Students will then analyze the data to summarize how the strategy works, the advantages and

drawbacks of each strategy, as well as any potential risk factors associated with it. This information will then be used to create a presentation in the form of their choice, which will be presented to the class. Options for the format of student presentations include posters, PowerPoints, videos, brochures, etc.

- If presentation is not finished in class, it should be finished for homework.

Day Three:

- Group Presentation Instructions (5 minutes)o The teacher will explain that the groups will present in order with the groups presenting on the

same topic presenting back to back. The teacher will also emphasize the importance of using proper presentation skills and showing respect while others are presenting.

o Prior to the presentations, the teacher should also discuss the role of the observer during the presentations, stressing the importance of asking questions to help with understanding. The students should be reminded that they will have to write an essay explaining which strategy they believe to be most effective and why, so taking notes on each presentation would also be helpful.

- Group Presentations (40 minutes)o Each group will present their strategy including an overview of how the strategy works, the

advantages of using the strategy, the drawbacks associated with it, and any potential risk factors. o While students are watching the presentations, they will ask questions relevant to the

presentation and take notes that will be used later on to write their individual response essays. o Student presentations will be graded using a presentation rubric.

- Open Forum Debate (25 minutes)o Once each group has presented, the class will have an open forum debate discussing which

strategy should be used to decrease Zika disease transmission in the United States. Students will be allowed to speak freely, but respectfully, to each other in order to discuss the advantages and disadvantages of each strategy.

o Prior to the debate, the teacher should review how students should talk to each other in a debate setting while still being respectful to one another.

o The teacher should allow students to take a leadership role in the discussion and only intervene when the debate is dwindling or off topic.

- Writing Task (10 minutes)o After the debate, each student will write an essay explaining which strategy would be the most

beneficial to use to control disease transmission in Aedes aegypti mosquitoes in the United States. Each student must make claim and then support their claim using evidence from the presentations. This writing task will be graded using the “Generate an Argument” rubric.

o This culminating writing task will be assigned in class and finished for homework. This will allow students to analyze the presentations and debate on their own and summarize their analysis into

a cohesive essay.

Assessment: Formative Assessment:- While students are working on their presentations in their groups, the teacher will circulate the room to

check on their progress, answer questions, and keep them on task. - While the students are debating which strategy is the most effective, the teacher will monitor the

progress of the debate, keep it on task, and help spawn discussion when appropriate.

Summative Assessment: - The presentation will be formally graded for both content and presentation skills. - The culminating essay will also be graded based on the “Generate an Argument” rubric.

Name: Date: Total Score = /22

Topic: _____________________________________________

SCORING RUBRIC: GENERATE AN ARGUMENT

Aspect of the ArgumentPoint Value Comments or Suggestions

0 1 2The Claim

The claim is sufficient

The claim is accurate

The EvidenceIncludes data

Includes an analysis of the data

Includes an interpretation of the analysis

The justification of the evidence

Explains why the evidence is important or why it is relevant

Links the evidence to an important concept or principle

Language of scienceAppropriate use of scientific

terms

Uses phrases that are consistent with the nature of science

MechanicsThe order and arrangement of

the sentences enhances the development of the main idea

Appropriate spelling and grammar is included

Name: ___________________________ Total: _____/60Date: _____________________

Disease Control Strategy Presentation Scoring Rubric

10 Points 8 Points 6 Points 4 Points 2 Points 0 PointsPresentation contains a detailed summary

of the assigned strategy and explains

the specific steps used to carry out the

strategy

Presentation contains a detailed

summary of the assigned strategy

and briefly explains the specific steps

used to carry out the strategy

Presentation contains a brief summary of the

assigned strategy and briefly explains the specific steps

used to carry out the strategy

Presentation contains a detailed

summary of the assigned strategy,

but does not explain the steps used to

carry out the strategy

Presentation contains a brief summary of the

assigned strategy, but does not explain

the steps used to carry out the

strategy

Presentation does not contain

a summary of the assigned

strategy

Presentation explains more than two

advantages of using the strategy and provides ample

evidence from the literature for support

Presentation explains two

advantages of using the strategy and provides ample

evidence from the literature for support

Presentation explains at least two advantages of using

the strategy, but provides minimal evidence from the

literature for support

Presentation explains one

advantage of using the strategy and provides some

evidence from the literature for support

Presentation explains one

advantage of using the strategy, but does not provide

evidence from the literature for support

Presentation does not explain any advantages

of using the strategy

Presentation explains more than two

drawbacks of using

Presentation explains two

drawbacks of using

Presentation explains at least two drawbacks of using

Presentation explains one

drawback of using

Presentation explains one

drawback of using

Presentation does not explain any drawbacks

the strategy and provides ample

evidence from the literature for support

the strategy and provides ample

evidence from the literature for support

the strategy, but provides minimal evidence from the

literature for support

the strategy and provides some

evidence from the literature for support

the strategy, but does not provide

evidence from the literature for support

of using the strategy

Presentation cites at least three sources in correct APA format

Presentation cites at least three sources with some errors in

APA format

Presentation cites at two sources in

correct APA format

Presentation cites at two sources with

some errors in APA format

Presentation lists sources, but not in

any acceptable format

Presentation does not list any

sources

Presentation is organized and has no

spelling or grammatical errors

Presentation is organized and has

less than five spelling or grammatical

errors

Presentation is organized, but it has

many spelling or grammatical errors

Presentation is disorganized and has less than five spelling

or grammatical errors

Presentation is disorganized and has

many spelling or grammatical errors

Presentation is not handed in

During the presentation all members of the group speak in a

clear, audible voice, make eye contact, and do not read to

the audience

During the presentation all members of the group speak but there are minor

errors in one of the following: audible voice, eye contact, and reading to the

audience

During the presentation all members of the group speak but there are minor

errors in two of the following: audible voice, eye contact, and reading to the

audience

During the presentation all group members

speak, but they read to the audience

During the presentation not all

group members speak and those that

do read to the audience

Group does not present