Dengue Vector Control: A Review for Wolbachia-based Strategies

Mosquito-borne diseases continue to pose a major health problem globally and have had a significant impact on human life and economy. Consequently, many countries have implemented national vector control programs in an effort to suppress/eradicate mosquitoes contributing to spread of diseases including Malaria, Dengue, Yellow fever, Rift valley fever, West Nile fever, Zika, Chikungunya etc. Of these endemic diseases, Dengue fever is an arbovirus and transmitted primarily by Aedes aegypti mosquito that has become a rapidly emerging infection, especially in the tropical countries. Insecticides spraying remains the main method to control the transmition of dengue virus. However, the overuse and misuse of insecticides can result in negative consequences such as the development of insecticides resistance. This, in part, has led to the development of a more eco-friendly measures to suppress mosquitoes e.g. genedrive based controls and Wolbachia-based approaches. The latter approach has the ability to block the dengue virus transmission by inhibiting virus intracellular replication in mosquito. In addition, Wolbachia decreases adult mosquito lifespan and can be naturally passed from one generation to the next. In recent years, Aedes aegypti mosquitos infected with Wolbachia released and tested in the field in several countries and have achieved very promising results. In this review, we focus and discuss the emerging Wolbachia-based biocontrol approaches that are already being deployed, evaluated and tested in the field.

Global incidences of mosquito-borne diseases are growing up due to people travel, fast urbanization and ineffective of control programs measures 1 . Dengue fever (DENV) is the most serious arboviral epidemic threatening humanity and it is responsible of death cases in the tropical and subtropical regions. About 50 % of the inhabitants across the world are now at dengue risk. Official reports estimated around 390 million person are infected annually, half million of them are critical situations and require hospital treatment. About of 2.5 % of those infected cases die. Although several countries used vaccine against DENV in humans ranged between 9 and 45 years of age inhabiting in endemic areas, but the mosquito control is still the main approach to stop the dengue disease 2 .A. aegypti mosquito is the main vector of Dengue, Zika,chikungunya and yellow fever diseases, also A. albopictus is a possible transmitter. A. aegypti mosquito is well adapted to mankind. The mosquito females obtain blood meals by biting and digestive it inside their bodies to produce their eggs. Unfortunately, the elimination of A. aegypti is not easy task due to their ability to lay the eggs in many sites included those of a little amount of water. Alsothe eggs able to stay alive months in the absence of water and hatch as soon as water is available. Moreover they have resistance to common insecticides 3,4 . All these conditions make the eradication of A. aegypti by traditional techniques useless 5 . The use of insecticides can be effective on mosquito control program, but it is often prohibitively, highly cost, harmful on non-target organisms, has negative environmentally effects.Furthermore, the long term use of insecticides led mosquitoes to develop resistance against insecticides 6,7 . Alternative methods were used to mosquitoes management such as the elimination of eggs laying sites, the using of animals that naturally preys on mosquitoes like copepods and fish 8 and avoid mosquito bites byprotection tools. These strategies are considered beneficial way in some cases but it can be difficult and high cost to apply in urban areas.Accordingly, Novel arbovirus vector control tools are needed. Currently, two novel techniques revealed promise in reducing the dengue transmission 9 . The first one depends on is a genetic management by spread mosquitoes that are treated with lethal or flightless trait 10 and the second technique is establishment of mosquitoes carrying Wolbachia bacterium. Wolbachia block and prevent the growth of the dengue virus inside A. agypti mosquitoes 11,12 . In this technique, the Wolbachia-infected mosquitoes are released into the wild. Because of cytoplasmic incompatibility (CI), the wolbachia are passed on through generations of mosquitoes and the ratio of Wolbachia-infected mosquitoesis growing upuntil it become high and predominance without any additional releases. This strategy is applied in several countries 13,14,15 .

What is Wolbachia?
Wolbachia is considered type of Gram-negative bacterium fall under the order Rickettsiales and the family Anaplasmataceaet (Table 1). This type of bacterium are naturally availablein invertebrates and infect about60% of insect community 11 . However, Wolbachia is naturally absent from A.aegypti mosquito,the main transmitter responsible for the spread of human diseases including dengue and other diseases of RNA-virus.Wolbachia has the ability to prevent the growth of several of RNA-viruses in mosquitoes and Drosophila. If infected or uninfected males fertilize wolbachia-possitive females, the resulting generations will be healthy and carrying Wolbachia and are expanded in the wild population. Otherwise if infected males fertilize uninfected females, the resulting offspring could not developed. This event is named by the term of cytoplasmic incompatibility (CI). Meanwhile, Wolbachia-positive mosquitoes produce less eggs and reduce mosquito lifespan.
In general, the Wolbachia species are named based on the source where they first discovered. For example, Wolbachia pipientis (wPip) strain was isolated for the first time from Culex pipiens mosquito. Also, wMel speciesfrom the fruit fly Drosophila melanogaster, while wAlb species isolated fromthe mosquito Aedes albopictus. Scientists have revealed that Wolbachia stimulate the resistance of arthropods against viruses and inhibit their reproductive ability inside the host. Recently, Australian researchers of the control program showed of dengue have showed that the expand of wolbachia into wild mosquitoes A. aegypti populations is considered promising technique to overcome the dengue virus transmission. This led WHO and health authoritiesto encourageuse Wolbachia approachas a way to overcome the transmission of dengue and arboviral diseases 12 .
Wolbachia strategy provides eco-friendly and a safe alternative to insecticide use. Although Wolbachia-infected A. aegypti were originally developed for biocontrol of dengue, it may able toreduce the transmission of other mosquito-borne diseases including chikungunya and yellow fever 16 , potentially malaria 17,18 and Zika 19 .

Potential risk of Wolbachia-infected mosquitoes on human
There is no evidence indicate that wolbachia transfer to human or to the mosquito predators such as geckos and spiders. No antigenic or immune response developed by mosquitoes bites 20 . The Australian Commonwealth Scientific Organization produced a risk assessment of releasing Wolbachia in the wild 21 before the official authorities granted the acceptance 13 .
It is worth noting that the Wolbachia does not horizontally transfer to other organisms. Potentially, there is horizontally transfer of Wolbachia DNA into mosquito genomes, but this situation of transferhappen rarely 22,23,24,25 .Such lateral transfer are unlikely to raise the risk related with the Wolbachia-positive mosquitoes release. Wolbachia-based biocontrol holds the promise of an environmentally and safe alternative that is not expensive to implement and has the chance to be effective on a global scale.

Biocontrol of Dengue Virus Using Wolbachia Strategy
Recently, the wolbachia has been studied by several researchers for its potential to useas a biocontrol strategy of Aedes mosquito 26 . Laven (1967) was the first researcher started the use of Wolbachia-infected Cx. pipiens mosquitoes to eliminate the the population of mosquito Culex pipiens through cytoplasmic incompatability (CI) 27 . CI is a phenomenon occurs when wolbachiainfected males are mating with uninfected females and the resulting offspring can not develop. In contrast,whenboth Wolbachia-positive male and femaleare mating, the offspring will hatch and develop normally 28 .
Wolachia release was done in Yorkeys Knob and Gordonvale, Australia, in early January 2011,asfirst trial sites, both wMel-positive females and males of A.aegypti were weekly released for a totally of ten weeks.
After five weeks-post finishing release, A. aegypti mosquitoes were Wolbachia positive with the percentage of 100 % and 90 % in Yorkeys Knob and Gordonvale, respectively.

Second release was in January 2012 by wMelPop-infected A.aegypti in both Machans
Beach and Babinda areas. A promising proportion of wMel Pop-positive A.aegypti was reported (with 49% and 75%, respectively) in the wild population during 2-3 weeks after the release start.However, one month post-finishing release, the proportions of wMelPop-infected A.aegypti decreased to less than 50 and 71 % in both Machans Beach and Babinda, respectively.This is may be attributed to inability of that Wolbachia strain to keep themselves for a long time in the field 29 .
Releases of wAlbB-infected Ae. Aegypti mosquitoes were done in greater Kuala Lumpur, Malaysia, including 6 diverse sites with high dengue cases. The wolbachia strain was established successfully with very high population frequency at some sites and fluctuations at other sites which were supported by additional releases. Based on the monitoring of the situation and compared to control sites, decrease in human dengue cases was observed in the release sites. The wAlbB strain of Wolbachia offers a promising strategy as a tool for dengue control, especially in very hot weather 30 .
Durovniet al., 2019 described study for evaluatingthe impact of wide-scale Wolbachia releases on the control of dengue, chikungunya and Zika in Brazil. The study is in progress and the monitoringand data analysis will continue until 2023. In case of success, the experiment will be expanded nationally and regionally. Releases programs of mosquito carrying Wolbachia are implemented or still in progress in 8 countries, fortunately no record of dengue, chikungunya and zika cases in areas where wide spread of Wolbachia-infected mosquitoes are established 31 .  This model shows clearly that the disadvantages of CI could outweigh the advantages and the Wolbachia may be lost. They set the optimal release strategy that determines the ability of Wolbachia for invasion and also, they deduced locally and globally stability of the equilibrium points 32 .
Mathematical modelling represents a significant tool to understand the effect of factors in infectious diseases dynamics and help in making decisions regarding the implementation of control programs 33 . These models simulate the invasion of Wolbachia-infectedA. aegypti into wild mosquito populations 34,35,36 . CI represents important factor on the replacement between Wolbachia-uninfected and Wolbachia-infected mosquitoes populations 37  OReilly et al. used different models to evaluate the negative consequences of dengue in Indonesia. They expect that Wolbachia technique can avoidup to75 % of disease consequences in the country.Area-wide interventions such as wolbachia can display an effective way to protect humans more than individually measures, such as vaccinations, in such huge population density 42 . Finally, all above mentioned models support the approach of ability of Wolbachia-infected mosquitos to replace the uninfected ones in wild populations

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa
Malaria is mosquito-borne disease and transmit to human by some Anopheles mosquito species. Historically, anopheles genus has been considered Wolbachia-free but has recently discovered in 5 Anopheles species in west Africa, Anopheles coluzzii, Anopheles gambiae, Anopheles arabiensis, Anopheles moucheti and Anopheles species A.These novel strains of Wolbachia have possibility to establish Wolbachia-infected anopheles mosquitoes, which could be used for control strategies to overcome the plasmodium parasite responsible for malaria incidence 43 .

Prevalence dynamic of Wolbachia and Cytoplasmic incompatibility (CI)
It is known that Wolbachia passed on from generation to the next generation by vertical transmission. Infected-males cause excitation of cytoplasmic incompatibility (CI) and modification of their sperm and lead to offspring death in early development stages ( Figure 1A). However, in females case, Wolbachia make encoding that keep the offspring to stay alive and successful development allowing the bacterium to dominate over the populations ( Figure A1) 44 . When Wolbachia spread with high rate then the reproductive advantage of infected females will be greatest. Otherwise, If Wolbachia is low, females rarely mate with Wolbachia-infected males, then low chance of compatibility with these males and Wolbachia could be lost from the population ( Figure 1B). Similar case happens when infected females don't carry Wolbachia to the whole offspring. Wolbachia release should be with high amount to avoid losing the infection after release stop 45,46,47 . Releasing of Wolbachia-positiveA. aegypti was implemented in 2011 in both isolated Yorkeys Knob and Gordonvale areas and the infection kept spread for 2 yearpost release stop 46 . Recently release was applied in Cairns area where the mosquitos' migration may occur from the release areasto surrounding areas and vice-versa, this may lead to decrease of Wolbachia infection level and ultimately the loss of Wolbachia. However, the infection was developed well showing that Wolbachia approach can be implemented in a wide-scale 48 .

Wolbachia and pathogen interference in Aedes aegypti
Creative strategy to control mosquito-born diseases started by using artificially Wolbachiainfected mosquitoes. Data from the field work has proved that Wolbachia represents promising technique to reduce natural populations of Aedes aegypti and control the diseases they transmit.
The mechanism of Wolbachia interference with the pathogens is complicated issue and need to be understood. Several scientists have attempted to explain the pathogen blocking by Wolbachia.
They have discussed the properties of mosquito's samples collected from the field and other related insects. They demonstrated the correlation between Wolbachia density and the ability to block pathogen by high load that destroy host tissue.Also the probability of induction the immune response system of the host which could resist the pathogens inside the insect. Furthermore, recent studies showed that Wolbachia play role in immune system modulation of the host and affecton the immunity system of A. aegypti and Culex quinquefasciatus to suppress Dengue andWest Nile virus replication 49 .
Other mode of action suggests modification of the cell membrane of the host, lead to preventing the vector to transmit the pathogens. Other explanation suggests competition development between Wolbachia and pathogens inside the host 50 .We mentioned above that wMelPop cause reducing in the lifespan of mosquito, this limits the pathogens spread because the lifespan of mosquito became shorter and not enough to complete the incubation interval for pathogens 51 57 .

Wolbachia strategy in Saudi Arabia
In the Kingdom of Saudi Arabia (KSA) dengue disease was recorded for the first time in 1994 58 and the number of cases is growing up as reported by researchers, Malaria cases also represent issue especially in Jazan region. Although the control activities during the past period, but people remain at risk as the epidemics transmission does not stop. Thus, new control strategies are needed to overcome these health problems. Accordingly, the strategy of Wolbachia-based biocontrol of dengue is started and still in the initial stages. Outcomes will be subject to evaluation and reported after finishing releases. If the experiment achieved success, it could be implemented on a large-scale.

CoNClUSIoN
Mosquitoes transmit Dengue and several diseases.We discussed here the current available informations about the relation between Wolbachia and moaquitoes. Insecticide-based approaches are currently the key tools in combat of major mosquito-borne diseases.However, the ability of mosquitoes to develop resistance against insecticides in addition to its harmful effects to ecosystem push to thinking to find alternative strategies. Wolbachia is a promising as a biocontrol technique in fighting mosquitoes-borne diseases. More research is urgently needed to find better understand about behavior of artificially Wolbachia-infected mosquitoes and the mechanisms of interference between Wolbachia, pathogens and hosts.