Wolbachia infections in Aedes aegypti: The ‘Bigfoot’ of endosymbionts

Words: Perran Ross

Wolbachia are endosymbiotic bacteria found within the cells of many insects, from butterflies and bees to cockroaches and dung beetles. Wolbachia are so common because they often provide their insect hosts with an advantage, aiding their spread through populations.

Whether an insect carries Wolbachia is an important question, especially if they’re a pest or vector. Wolbachia can affect host traits like fertility, thermal tolerance, susceptibility to predators and virus protection. By harnessing these properties, Wolbachia can be used to suppress insect populations or the pathogens they transmit.

Insects that don’t naturally carry Wolbachia can often be infected in the laboratory. Wolbachia have been introduced into Aedes aegypti mosquitoes, the main vector of dengue. Mosquitoes carrying Wolbachia are now being released around the world. This is an effective way to control dengue fever transmission, because Wolbachia can spread through mosquito populations and block the dengue virus as well as other viruses like Zika.

Until recently, Wolbachia have not been found in this species naturally. The absence of natural Wolbachia makes Aedes aegypti a great target species for these interventions because it’s a blank slate; release enough lab-reared mosquitoes carrying Wolbachia and the Wolbachia infection can spread naturally throughout the local population.

Mosquitoes with dengue-blocking Wolbachia rely on a mechanism called ‘cytoplasmic incompatibility’ to spread through local mosquito populations. Cytoplasmic incompatibility results in no viable offspring when a Wolbachia-infected male mates with an uninfected female. On the other hand, females infected with Wolbachia are fertile regardless of what type of male they mate with. This provides females carrying Wolbachia with an advantage. As the Wolbachia infection is passed from mother to offspring, the infection can spread rapidly through a population.

Cytoplasmic incompatibility occurs when males carrying Wolbachia mate with uninfected females. These females do not produce viable offspring.

But this all changes if the local population already has Wolbachia. Different strains of Wolbachia can be compatible with each other, interfering with the mechanism of cytoplasmic incompatibility. The strains can alternatively be incompatible with each other, in either direction. For populations already carrying Wolbachia, introducing a new strain of Wolbachia into the population could be challenging, if not impossible.

The Aedes albopictus mosquito is also a dengue vector, but unlike Aedes aegypti it carries two strains of Wolbachia. Introducing a new Wolbachia strain into Aedes albopictus populations would require a triple infection that includes the two natural strains. Without them, the new Wolbachia strain would have little hope of establishing in the population.

Pilot releases of experimentally-generated Wolbachia infections have been recommended by the World Health Organization and programs are now operating in over 10 countries. But the presence of natural Wolbachia infections in Aedes aegypti could be problematic. So when a 2016 study by Coon et al. detected Wolbachia in this species, it led to the possibility that these natural infections could interfere with releases.

This study was followed by seven more detections of Wolbachia in Aedes aegypti from laboratories across the world. However, in a new paper, published this week in Ecology and Evolution, we show that the evidence isn’t compelling.

To convince a skeptic that a mythical creature like the Loch Ness monster or Bigfoot exists, you need multiple lines of evidence. A photo won’t cut it, because Nessie could just be a sock puppeteer going for a swim. The same is true for the existence of Wolbachia in Aedes aegypti. What if Wolbachia were just big, hairy Anaplasma (a relative of Wolbachia)? In a situation where the presence or absence of Wolbachia can affect disease suppression programmes, it’s crucial to get an accurate diagnosis.

The evidence for natural Wolbachia infections in Ae. aegypti (left) is not much more compelling than the evidence for the Loch Ness monster (right). The transmission electron microscopy image on the left is from Balaji et al. (2019).

We scoured the literature and compiled the evidence for Wolbachia in Aedes aegypti to date. Most studies report only molecular evidence from field-collected mosquitoes. This is a good first step because it shows that there is Wolbachia DNA somewhere in the sample, but it can’t tell us if the Wolbachia are alive and kicking inside the mosquitoes. To confirm a genuine infection requires three main lines of evidence:

1. Visualizing Wolbachia in different host tissues
2. Demonstrating that the infection is passed on through the mother
3. Showing that Wolbachia can be removed from mosquitoes by antibiotic or heat treatment

Molecular evidence is also prone to false positives such as contamination or misidentification of the host species.

More recent studies have gone one step further and brought these putatively-infected mosquitoes back to the laboratory. We contacted the authors of these studies to ask if they were willing to share their Wolbachia-infected mosquito colonies, but from the one population we obtained we couldn’t find any evidence for Wolbachia infection. Other studies provide additional evidence like electron microscope images and Wolbachia removal through antibiotic treatment, but these results have not been independently verified.

In collaboration with the Institute for Medical Research Malaysia we surveyed natural populations in Kuala Lumpur, where Wolbachia were putatively detected in Aedes aegypti. We found no evidence for Wolbachia here either. Our results are consistent with a comprehensive survey of Aedes aegypti across the world that also found no evidence for Wolbachia in this species.

So Wolbachia probably don’t occur naturally in Aedes aegypti after all. However, since releases of Wolbachia-infected mosquitoes are expanding around the world, it’s important to continue to monitor populations in case Wolbachia infections are geographically restricted. Our study highlights the need for more robust evidence when claiming that Wolbachia infections occur naturally, because the results could be a matter of life or death for people living in dengue endemic areas.