The vector-borne-diseases, particularly those transmitted by mosquitoes such as malaria and dengue fever cause an enormous health burden to people living in tropical and subtropical regions of the world. Despite years of intense effort to control them, many of these diseases are increasing in prevalence, geographical distribution and severity, and options to control them are limited.
Most control methods are failing to prevent the global increase in the incidence of these diseases . Even if, the use of insecticides to target mosquitoes as a means of disease control can be effective, it is often prohibitively expensive, unsustainable and environmentally undesirable.
Therefore, new approaches are clearly needed if these trends are to be reversed.
Since about 2011, the use of the maternally inherited endosymbiotic bacteria named Wolbachia is showing as a promising biocontrol approach.
Wolbachia is a widespread bacterial endosymbiont found in a large number of arthropods, including more than 60% of all insect species, encompassing all orders .
Insects infected with Wolbachia often exhibit major disruptions in reproductive biology, including sterility, production of one sex, or parthenogenesis .
Traditionally, Wolbachia. spp detected in arthropods are divided into two groups (A and B) based on the sequences of their 16S rRNA*, ftsZ and wsp genes , .
Both groups contain Wolbachia. spp that have been detected in several genera of sandflies (vectors of Leishmaniasis) and mosquitoes. Group A includes the Wolbachia. spp detected in sandflies belonging to Sergentomyia and Phlebotomus genera as well as detected in Aedes mosquitoes belonging to Finlaya genus, while Group B contains Wolbachia. spp detected in sandflies belonging to the Phlebotomus and Lutzomyia genera as well as detected in Culex mosquitoes from Culex genus –.
These bacteria have been detected using molecular tools such as the polymerase chain reaction (PCR)** and reported as “ Wolbachia species” from arthropod and only a single species has thus far been properly isolated and bears a valid name, Wolbachia. pipientis .
Thanks to the research program Eliminate Dengue, the transinfection of Aedes. aegypti mosquitoes with a strain of this bacteria isolated from Drosophila. melanogaster showed spectacular results about the manipulation of the mosquitoes reproduction by Wolbachia allowing to limit their capacity to transmit different diseases such as dengue, chikungunya and zika viruses but also yellow fever –.
Indeed, Wolbachia can be only transmitted among mosquitoes inside the female’s eggs.
When a Wolbachia-infected male mates with an uninfected female, it prevents those eggs from hatching. If a female mosquito is infected, it will produce offspring normally, gradually increasing the numbers of Wolbachia-infected mosquitoes. These mosquitoes may still bite people but won’t transmit infections.
Likewise, it has been recently shown that Wolbachia transinfections in Anopheles mosquitoes, the human malaria vectors, induced refractoriness to the human malaria parasite Plasmodium falciparum –.
Therefore, this has led to disease control programs using Wolbachia-infected-mosquitoes releases in hopes of replacing the target Wolbachia-free mosquito population, thus reducing the risk of disease transmission by the vector.
Until 2014, Wolbachia was known to be present naturally in a lot of arthropods but not in mosquito populations transmitting dengue (Aedes. aegypti) and malaria (Anopheles. spp). However, since 2014, different studies have found the presence of natural Wolbachia infections at low prevalence in the field populations of Anopheles. gambiae in a limited geographic range in Burkina Faso  and in Mali .
Moreover, these natural Wolbachia infections were negatively correlated with the Plasmodium development and may reduce the prevalence of the human malaria disease, suggesting that Wolbachia may be an important player in malaria transmission dynamics , .
While natural Wolbachia infections were found in the malaria vector, studies exploring the existence of these natural infections in the dengue vector Ae. aegypti have been limited  and were negative.
However, one study reports the presence of Wolbachia sequences in three Ae. aegypti individuals from Florida .
Recently, a research team from the University of Yale (USA) in collaboration with the Connecticut Agricultural Experiment Station (CAES, USA) studied this question at large-scale using a wider sample of Ae. aegypti and tried to find natural Wolbachia infections in this mosquito vector by PCR.
Their results are published in the Journal of Medical Entomology .
To explore this question, researchers assayed a worldwide collection of more than 200 samples of Ae. aegypti from different continents as well as field and colony specimens Ae. mascarensis, the closest relative to Ae. aegypti with which it can hybridize and produce fertile offspring , thus potentially transferring endosymbionts.
Fig. 1: World distribution of field sampling locations of Aedes aegypti and Aedes mascarensis screened for Wolbachia
Source: Lack of Evidence for Natural Wolbachia Infections in Aedes aegypti (Diptera: Culicidae): Gloria-Soria et al. 2018
In total, 2 663 Ae. aegypti mosquitoes from 63 populations representing 27 countries around the world, 230 Ae. aegypti from laboratory strains, and 32 field-collected Ae. mascarensis from Mauritius and 40 Ae.mascarensis from a colony established in 2014 (Figure 1) were screened for the presence of Wolbachia DNA by PCR targeting two genes, the Wolbachia outer surface protein gene wsp which is highly conserved in the different Wolbachia groups (A and B), and the GroE operon containing the highly conserved bacterial heat shock proteins, GroES and GroEL. The screening was performed using different primers whose their high efficacy and high sensitivity to detect the presence of Wolbachia DNA was demonstrated by positive amplification in a variety of Diptera such as Ae. albopictus, D. melanogaster, Culex. pipiens and C. quinquefasciatus…belonging to the group A and B.
Results showed that no Wolbachia DNA was detected in no one of Ae. aegypti and Ae. mascarensis mosquitoes screened for this endosymbiont.
This study is the first using a quite complete Ae. aegypti sampling from a wide range of geographic areas.
Although the used primers successfully detected natural infections in a variety of Diptera, belonging to the 2 groups of Wolbachia, it is possible that strain(s) of natural Wolbachia infect Ae. aegypti but their potential divergence prevented their detection in this experiment.
Moreover, researchers used different Dipterans (mosquitoes and sandflies) as well as non-Dipterans (ants and beetles) as a positive control of the PCR and their characterization suggested that the screen would have picked up a wide variety of Wolbachia strains if they were present.
However, researchers did not measure the limits of detection of this assay to the individual genome copies, so it is possible that a much lower abundance of Wolbachia, relative to their control species, may prevent detection by this method.
Maybe it would have been interesting to set up a protocol of nested PCR to increase the sensitivity and the detection of potential natural Wolbachia infections in these wide populations of Ae. aegypti.
Thanks for reading.
And don’t forget: Fight Malaria
*16S ribosomal RNA: Component of the 30S small subunit of a prokaryotic (bacterial) ribosome. The genes coding for it are referred to as 16S rRNA gene and are used in reconstructing phylogenies, due to the slow rates of evolution of this region of the gene
**Polymerase chain reaction (PCR): A molecular method allowing to make many copies of a sequence of DNA. This method needs an enzyme called DNA polymerase. It is called chain reaction because the result of one cycle is used immediately for the next cycle.
The method consists of repeated heating and cooling, causing “melting” (separation of the two strands) and replication of the original DNA, also called a template.
Short DNA fragments consisting of DNA sequences complementary to the ends of the template, called primers, and a DNA polymerase are key materials for selective and repetitive steps.
It proceeds in three steps:
- DNA denaturation: The template strands that are bound together cannot be replicated, so the first step of PCR is to separate them by heating up the sample, breaking the hydrogen bonds between them.
- Primers hybridization: The sample is cooled just enough to allow the primers to bind to the ends of each of the two template strands
- Primers extension: DNA polymerase attaches to the primers and makes a copy of each template strand.
After the first cycle, there are 4 DNA strands. The process repeats with the 4 DNA strands, which will go on to make 8 strands, then repeat itself again to make 16 strands. In this way, PCR doubles the amount of DNA in a sample after each cycle, making it possible to obtain millions of copies of a DNA strand overnight.
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