Manipulation of the manipulators

Wolbachia bacteria infect insects and can cause mating incompatibilities, an outcome that is used to fight insect-transmitted disease. The proposed genes responsible illuminate this process and the disease-control mechanisms. See Letter p.243

The papers in brief

• Wolbachia bacteria can infect the male and female germ line of insects.

• This bacterial infection can manipulate insect reproductive outcomes to benefit Wolbachia transmission through a phenomenon known as cytoplasmic incompatibility.

• Wolbachia has promise as a potent means of pest control and is being used to limit mosquito-transmitted human diseases such as dengue and Zika.

• Understanding the molecular basis of cytoplasmic incompatibility could aid efforts to improve disease-control strategies.

• On page 243 and in Nature Microbiology , respectively, LePage et al.¹ and Beckmann et al.² present evidence that they have identified bacterial genes responsible for cytoplasmic incompatibility.

Incompatibility pathway

William Sullivan

The ability of bacterial pathogens to usurp the core processes of host cells has long fascinated biologists. The bacterium Wolbachia can infect more than half³ of all insect species and manipulates the reproduction of its host⁴. Although Wolbachia infects the germ line of both sexes, only females can transmit it to their offspring.

Cytoplasmic incompatibility (CI) refers to the sterility or strikingly low hatch rate that can arise when a Wolbachia-infected male mates with an uninfected female (Fig. 1). Matings between insects that are both infected with Wolbachia (known as rescue matings) and matings between Wolbachia-infected females and uninfected males all result in normal hatch rates. Wolbachia-infected females have a selective advantage over uninfected females because they have a normal brood size, regardless of the Wolbachia-infection status of the males they mate with. CI can therefore help to spread a Wolbachia infection rapidly through an insect population.

Figure 1: Cytoplasmic incompatibility and Wolbachia in disease control.

a, Wolbachia bacteria can infect the germ lines of insects, including mosquitoes (bacterial infection shown in pink), and only females can transmit the bacteria to their offspring. In the phenomenon known as cytoplasmic incompatibility (CI), matings between Wolbachia-infected males and uninfected females results in embryo lethality or low hatch rates. This phenomenon is being used to suppress insect populations in some pest-control approaches. LePage et al.¹ and Beckmann et al.² present evidence that they have identified bacterial genes responsible for CI. b, CI can be prevented in rescue matings between Wolbachia-infected males and Wolbachia-infected females. Because the effect of Wolbachia infection on insect reproduction favours the survival of Wolbachia-infected females over uninfected females, Wolbachia can rapidly spread through an insect population. c, Wolbachia infection can prevent insects from being infected by some viruses that cause human disease such as dengue or Zika, and the spread of Wolbachia through an insect population is being used to control insect-transmitted human diseases.

Although CI was discovered more than 45 years ago⁵, its molecular basis has remained unknown until now. Previous work has provided descriptive observations of the cellular consequences of CI. When a Wolbachia-infected sperm nucleus enters the egg of an uninfected female, this nucleus encounters problems that include delays to DNA replication and cell-cycle progression⁶. These abnormalities result in embryonic lethality. The identification by LePage et al. and Beckmann et al. of genes in Wolbachia strains that seem to be required to induce CI should provide a major step forward in the efforts to understand this process.

Through a combination of genomic, bioinformatic and molecular approaches, LePage et al. identified potential CI-associated sequences in the Wolbachia wMel strain that contain the genes cifA and cifB. These sequences originate from viral DNA that has integrated into the Wolbachia genome. LePage and colleagues observed that cifA and cifB sequences are present in CI-inducing Wolbachia strains, but are absent, or present in a highly divergent form, in non-CI-inducing Wolbachia strains. The CI-effect strength, in terms of the level of embryonic lethality, correlates with the number of copies of cifA and cifB present in crosses between Wolbachia-infected males and uninfected females. This strongly suggests that this gene pair directly mediates CI.

LePage and colleagues expressed cifA and cifB sequences in Drosophila melanogaster fruit flies. When these genes were expressed in the germ line of males mated to females that did not express cifA and cifB, the offspring had reduced hatch rates and embryonic cell-division defects that were strikingly similar to those observed in CI. The hatch rate was restored when males flies expressing cifA and cifB were mated to Wolbachia-infected females.

The findings by LePage et al. are consistent with the work of Beckmann and colleagues. Previous studies⁷ by Beckmann et al. identified a protein called WPA0282 that is present in sperm from males infected with the Wolbachia strain wPip. In the operon sequence that encodes the WPA0282 protein (renamed CidA by Beckmann et al.), the authors identified a gene they called cidB. This gene encodes a deubiquitylating enzyme, an enzyme that can remove ubiquitin proteins attached to other proteins. Such bound ubiquitins often act as a tag that targets a protein for destruction. When D. melanogaster males expressing CidA and CidB proteins in their germ line were mated with female flies that were not infected with Wolbachia, the offspring had the type of nuclear abnormalities during early embryonic divisions that are characteristic of CI.

It seems likely that cifA and cifB identified by LePage and colleagues are the same as (or related to) cidA and cidB identified by Beckmann and colleagues. The relationship between these genes, which were identified in different Wolbachia strains, will require further investigation.

If a deubiquitylating enzyme is responsible for CI induction, where does it localize, and what are its targets? What pathway connects the action of the identified genes to the observed embryonic defects in CI, and how does rescue of CI occur? Researchers are now positioned to rapidly address many of these important questions.

Bacteria that fight disease

Scott L. O'Neill

There has been an upsurge in interest in the use of Wolbachia bacteria to control mosquito-transmitted viruses such as dengue and Zika — devastating diseases that threaten about half of the world's population⁸. Most of the current control measures for these diseases are proving to be ineffective.

Two main Wolbachia-based disease-control methods are being tested in the field. The first approach targets virus replication in mosquitoes. Wolbachia prevents a range of human viruses and parasites, such as dengue, chikungunya and Zika virus, from replicating in the Aedes aegypti mosquito. If Wolbachia can be successfully introduced into natural populations of these mosquitoes, it should greatly reduce the disease-transmission potential of the insect populations (Fig. 1). It is thought that the Wolbachia-mediated pathogen-interference mechanism involves a combination of upregulated mosquito immune pathways and competition for key lipid molecules such as cholesterol⁹. The second approach uses Wolbachia to directly suppress the abundance of mosquito populations and thereby reduce viral transmission. Both approaches rely on the CI phenomenon for their success.

For disease-control approaches that introduce Wolbachia into mosquito populations, such as the work being carried out by the Eliminate Dengue Program¹⁰, CI is the central mechanism that allows Wolbachia to become established and maintain itself sustainably in the mosquito population. In the alternative suppression approach, the embryo mortality that CI induces is being used to reduce the mosquito population over time. In this approach, male mosquitoes are released that are infected with a Wolbachia strain that will induce CI in matings with wild females.

A deeper understanding of the CI mechanism from the work by LePage et al. and Beckmann et al. opens the door for manipulating the system and potentially tuning Wolbachia-induced CI to enhance the ability of different Wolbachia strains to invade insect populations. Perhaps the CI mechanism can be separated from the pathogen-blocking effects of different Wolbachia strains so that better strains for insect treatment can be generated. It might be possible in future to genetically engineer Wolbachia strains to replace strains that have been previously used in control programmes as a way of managing resistance issues, if Wolbachia were to lose effectiveness over time. Understanding CI might enable this phenomenon to be used in other insect species that Wolbachia does not naturally infect, or that are resistant to Wolbachia infection.

Many of these potential approaches would require the release of genetically modified insects into the environment. Recent controversy in Florida about the release of genetically modified mosquitoes for control of mosquito-transmitted viruses indicates that technology alone is insufficient for programmes to be successfully implemented in the field, and public understanding and acceptance are required for such technologies to be effective. ¹