I mentioned Wolbachia in my parthenogenesis post; here I will talk about it in more detail, because it’s a really cool parasite, as well as in the center of much research nowadays.
Wolbachia are Gram-negative ⍺-Proteobacteria (order Rickettsiales, family Anaplasmataceae; Dumler et al.,2001), systematically split into 6 supergroups, lettered from A to F (Lo et al., 2002). Those in supergroups C and D have coevolved with their nematodan hosts and are mutualistic (Bandi et al., 1998). Those in supergroups A and B are parasites in arthropods. Morphologically, they come in two groups: ~1µm large rods, and ~0.5µm diameter coccoids. They can be found in a membrane-bound vacuole in the host cell’s cytoplasm (Yen & Barr, 1971); in arthropods, this will be in egg and ovarian cells (see diagram above, from Weiss et al. (2009)), although it has also been found in other tissues, including the Malpighian tubules, and muscle and nervous tissue (Stouthamer et al., 1999). When found in sperm cell cytoplasm, it disappears during spermatogenesis (Clark et al., 2002).
Note that in this post and in the literature, what’s referred to as Wolbachia is Wolbachia pipientis. Before DNA sequencing, when bacteria were classified morphologically, W. persica and W. melaphagi were also thought to be related, but these have nothing to do with the famous Wolbachia (W. persica is a γ-proteobacterium, W. melaphagi is a rhizobacterium).
Being present endosymbiotically in 20+% of all known insect species (Werren & Windsor, 2000), and given that some estimate that over half of all insect species are infected by it (Hilgenboecker et al., 2008), it’s obviously extremely successful, although it should be noted that the virulence of each Wolbachia strain is different (Min & Benzer, 1997), and each species is has a different susceptibility. In Australian and Panamanian fig wasps, Wolbachia has been found to infect over 70% of all species (Haine & Cook, 2005). A host database can be found here.
The biggest reason for its success is its parasitic effect: it’s always transferred maternally in the cytoplasm of the eggs, and in order to ensure maximal transfer rates, it alters its host’s reproductive abilities to favour their own reproductive success (Charlat et al., 2003). It has four ways to do this, in contrast to the one or two ways present in other such parasites:
- Inducing thelytokous parthenogenesis in haplodiploid organisms, e.g. hymenopterans, thus making sure that only female offspring are produced (e.g. Stouthamer et al., 1993). It occurs by forcing the fusion of the two nuclei of the first mitotic division (Huigens et al., 2000).
- Feminizing males, i.e. males reproduce as females (Wilkinson, 1998). The mechanism for this varies; for example, in the pillbug, Wolbachia blocks the formation of the androgenic gland which produces the masculinising androgenic hormone (Martin et al., 1990).
- Killing males, either in the embryonic stages or later (Hurst, 1991). The mechanisms of action are still largely unknown, although recent pioneering research by Riparbelli et al. (2012) shows that it happens due to Wolbachia (purposely?) messing around with male chromosomes at various stages in development, leading to defective embryos and death.
- Inducing cytoplasmic incompatibility (CI) between infected males and uninfected females (Hurst & Werren, 2001), causing sterility (Bordenstein & Werren, 2007) and eventually reproductive isolation within a species.
The latter was the first of Wolbachia‘s effects to be discovered, by Yen & Barr (1971) in mosquitoes; Wolbachia itself was first spotted by Hertig & Wolbach (1924). What happens is that the sperm enters the cell as normal, but its chromosomes don’t decondense and fuse with maternal chromosomes due to a delay in the breakdown of the nuclear envelope in the male’s pronucleus (Tram & Sullivan (2002); see Landmann et al. (2009) for more molecular details), and so can’t enter the first mitosis, meaning they get discarded (Lassy & Karr, 1996). The embryo either becomes a haploid female (in haplodiploid organisms), or it dies. In evolutionary theory terms, in a population susceptible to Wolbachia-induced CI, uninfected females become more unfit, therefore giving a fitness advantage to infected females (Bourtzis et al., 2003), hence explaining how the phenotype persists.
Which manipulation happens cannot be predicted even within the same species, as demonstrated by Hornett et al. (2008), who found that in their North American Hypolimnas bolina butterflies, Wolbachia was a male killer while in Southeast Asian ones, it was a CI-inducer. The difference came from a dominant allele in the SE Asian butterfly genome suppressing the male-killing. Charlat et al. (2007) showed that such a mutation can become fixed in a population in under 10 generations due to the extreme selection pressure to maintain a decent sex ratio, a testament to the ecological power of Wolbachia.
However, in some cases, Wolbachia can be so prominent that the entire affected population gets a very female-biased sex ratio, which in the case of the butterfly Acarea encedon has led to females reversing their sexual roles and behaving like males (Jiggins et al., 2000). In other cases, Wolbachia is counteracted by other elements, for example the B chromosome in the parasitoid wasp Trichogramma kaykai which turns Wolbachia-feminised males back into regular males (Stouthamer et al., 2001).
It must be mentioned that part of their success is their ability to be transmitted horizontally across different species (Raychoudhury et al., 2009) – they aren’t host-specific. This has been shown as happening through parasitoids (Heath et al., 1999) or through the environment (e.g. by sharing a common food source (Huigens et al., 2000)). This also means that a single individual may have multiple Wolbachia species (or, better said, strains) co-existing and mingling inside it; the largest number I know of is eight, in the fire ant Solenopsis daguerrei (Dedeine et al., 2005). Note that this ability only comes in the arthropod-associated Wolbachia, whose genomes are more plastic, with recombination and phage-derived elements (Wu et al., 2004), none of which are characteristics present in their nematodan counterparts (Foster et al., 2005).
One study also reported the possibility of Wolbachia having transferred part or all of its genome to its hosts, albeit with only 2% of the genes able to be transcribed and none of them having any described effect (Hotopp et al., 2007).
The negative effects of Wolbachia are obviously of great interest for biocontrol of pests and disease vectors. For example, Alam et al. (2011) discuss the possibility of using Wolbachia to control the tsetse fly, a vector of trypanosomiasis; Atyame et al. (2011) do the same for mosquitoes. Such biocontrol would work by allowing a chosen genotype to dominate the population by infecting the undesired genotype (e.g. Xi et al. (2005)), or by shortening lifespans to prevent sexual maturity (e.g. Moreira et al. (2009)). Wolbachia can also lead to population bottlenecks with very few individuals becoming able to reproduce (Nice et al., 2009), which is another way to control a pest population.
In non-pest studies, Wolbachia leads to increased susceptibility to parasitoids in Drosophila (Fytrou et al., 2006). It also leads to a less effective immune system in the pillbug Armadillidum vulgare, as seen by a lower density of haemocytes and higher density of bacteria (Bracquart-Vanier et al., 2008). These would be other avenues for pest control if a similar effect is seen in pest groups.
Positive Effects of Wolbachia
Interestingly, the effects of Wolbachia aren’t all negative. In the Cimicidae (bed bugs), Wolbachia is a mutualist; getting rid of it with antibiotics reduces the amount of food the host gets (Hosokawa et al., 2010). In mosquitoes, Wolbachia was found to boost their immune system and cause resistance to dengue virus (Bian et al., 2010). Pinto et al. (2012) describe how this happens at the genetic level. This is another potential use of Wolbachia as a biocontrol agent for disease vectors. In Drosophila, Wolbachia has been shown to confer resistance to several RNA viruses (Teixeira et al., 2008). In a Drosophila lab culture, Weeks et al. (2007) showed that the Wolbachia went from being a parasite to being a mutualist within two decades.
At the extreme end, nematode-infecting Wolbachia are needed for nematode development and fertility (Foster et al., 2005), so Wolbachia antibiotics could be used to control their populations (Taylor & Hoerauf, 1999). This is useful knowledge, given that some of the affected nematodes are vectors for very serious diseases like elephantiasis and onchocerciasis. It’s a similar story with the wasp Asobara tabida, wherein no ovocyte can even be produced when Wolbachia isn’t present (Dedeine et al., 2001) because its absence promotes excessive apoptosis in the ovarioles (Pannebakker et al., 2007).
Other effects of Wolbachia on sexual physiology have been documented, for example an increase in sperm competition in Tribolium beetles (Wade & Chang, 1995), or changes to the spermathecal duct in female Allonemobius crickets (Marshall, 2007).
An interesting point can be made about the process of molecular evolution in Wolbachia. As I said in the introductory paragraph, Wolbachia is a nematode mutualist and arthropod parasite (generally speaking). One of the Wolbachia genes involved in interaction with the host is wsp, which codes for cell membrane proteins. It was found to be undergoing divergent selection when it is in a parasitic relationship, but not when it’s in a mutualistic relationship (Jiggins et al., 2002). This is in line with what we expect: wsp being involved in host recognition means it theoretically should experience heightened evolutionary rates, and this is confirmed by the empirical data.
Many, if not all, negative and positive effects of Wolbachia have evolved by natural selection in order to maximise the transmission of the strain, either by allowing the bacterium to survive in the host (depressed immune system), or reducing competition by blocking the transmission of other pathogens (as Teixeira et al. (2008) suggest for the viral resistance effect). By extension, this means that parthenogenic arthropods aren’t expected to be Wolbachia hosts, since the manipulations are useless there. In terms of evolutionary theory, they can be treated as nothing more than selfish genetic elements.
When I first heard of Wolbachia, my intuition was that it played a sizeable role in speciation, since it promotes reproductive isolation, or by selecting for subdivided populations (Hatcher et al., 2000). Some analyses showed it not to be true (Rousset & Raymond, 1991), but more and more recent studies are supportive of the idea (Bordenstein, 2003), so it’s accepted as a cause of speciation. It definitely has been demonstrated (Thompson, 1987), and in some cases has also induced rapid speciation (Bordenstein et al., 2001).
On a general evolutionary synthesis level, Wolbachia is pretty interesting as a very recognisable case of inheritable symbiosis, one of the few proper examples that lend credence to the view that symbioses are a driving force behind evolution.
Milder effects of large-scale Wolbachia infection and sex ratio-skewing include altering dispersal ability – many insects have dispersing females and non-dispersing males, or vice versa. On a more influential level, there is also evidence that they can play a role in sexual selection (Jiggins et al., 2000), since sexual conflict gets reduced when levels of polyandry fall (Arnqvist & Rowe, 2005).
Wolbachia can sometimes present a methodological stumbling block for molecular phylogenies based on mitochondrial DNA, since mtDNA will also hitchike maternally, favouring the maternal mtDNA haplotype, eventually leading to the entire dataset being worthless; see Ballard & Rand (2005) for more information. However, Arthofer et al. (2010) tested this idea using infected bark beetles and found no significant effect from Wolbachia, so it is still unsure just how significant this slight inaccuracy is.
Where it is definitely a problem is in barcoding initiatives using mtDNA. For successful barcoding of a species, a stable molecular marker needs to be used that is guaranteed not to vary across individuals, populations, or ecomorphs. However, there are some studies that show that Wolbachia causes divergences in mtDNA sequences even among individuals of the same species, e.g. in the butterfly genus Hypolimnas (Galtier et al., 2009). The reason for this is that mitochondrial genes are transferred only maternally, so only the maternal set plays a role in evolution. Given the ubiquity of Wolbachia, this is definitely a large problem that should be studied carefully before proceeding with mtDNA barcoding.
Wolbachia alone can’t be cultivated, but it is possible to keep a Wolbachia line using host cell lines (Noda et al., 2002), so experimental evolution studies are possible with them.
For the entomologists among you, make sure to check any colonies for Wolbachia infections, as they could invalidate your results, especially if you’re doing population biology. They can be gotten rid of using any antibiotic. I hear that tetracycline is recommended; if that’s not possible, high heat is enough, since Wolbachia is sensitive to temperature. If you’re sequencing your insects as well, using DNA from the legs is probably the safest way to avoid getting contaminating Wolbachia DNA amplified (this is standard procedure anyway).
Other symbionts that alter the reproduction of their arthropod hosts include Buchnera and Cardinium – but I’ll leave them for other posts.
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R. Stouthamer, J. A. J. Breeuwer, & G. D. D. Hurst (1999). WOLBACHIA PIPIENTIS: Microbial Manipulator of Arthropod Reproduction Annual Review of Microbiology DOI: 10.1146/annurev.micro.53.1.71