Lecture 27: The Insect Microbiome

“An inherited intracellular bacteria that live within the cells of the reproductive tracts of insects” • In alpha-proteobacteria class • Gram negative • Diverse - numerous species of Wolbachia • Though to be member of rhizobium • Cannot survive outside the host cell • Primarily; arthropods (~66% of species & insects comprise ~85% of all animal species) • Therefore may be most common endosymbiont in the world

• Primary mechanism for propagation is vertical transmission • (Can propagate horizontally) Vertical transmission: “passed parent to offspring with high efficiency” Horizontal transmission: “dispersal of bacteria between organisms living in same environment”

• Can manipulate cellular components so that it can move across spindles • Therefore when cell divides, it is split into both cells equally • can pass itself into germ cells, which ultimately lead to eggs and sperm

• Paternal transmission is very difficult (almost never) due to sperm being so small a. it can get into sperm heads, but not often at high enough dosages to infect offspring 2. It gets round this by various mechanisms tailored to different organisms of reproductive manipulation a. Feminization b. Parthenogenesis c. Male killing d. Cytoplasmic incompatibility

Despite the different techniques, they involve the end goal of producing more females than males. This means more propagation.

• A “toxin antidote system”? a. Bacteria inserts a toxin into sperm, despite not being able to fit itself, able to interrupt reproduction b. If bacteria is present in the oocyte when fertilised, there must be some form of antidote to counteract the toxin

• Identification is difficult: a. cannot be cultured outside insects hosts • Progress was made with whole genome sequencing and comparative genomics with “non-parasitic strains” a. allowed narrowing down of potential target genes • Analysis of proteins in sperm a. looking at proteins in infected & non-infected sperm LED TO IDENTIFICATION OF “cidA cidB Complex”

• A Wolbachia gene operon • Expression of cidA OR cidB on their own produces no effect • Males expressing cidA/cidB = sterile • Females expressing cidA/cidB = fertile • Female expression in cidA in ovaries can “rescue” sterile males Therefore a. production of cidA & cidB together = toxic sperm b. presence of cidA alone = antidote

• cidB causes “post-fertilisation embryonic defects” • When sperm fertilises oocyte and tries to form embryo, it fails at early stages of nuclear division PROCESS 1. Paternal nuclear envelope breakdown delayed 2. Male pronuclear DNA replication is incomplete 3. Nuclear division fails

• Once sufficiently diverged from each other, Wolbachia lineages can start to fail to rescue each other I.E., If sperm and oocyte are too evolutionary divergent, the females rescue operon may not match up to the toxic operon HOW CAN SOMETHING DIVERGE IF ITS DIVERGENCE IS A SELECTIVE PRESSURE

Conversion of hosts that would develop as males to female development

Induction of asexual daughter development

Death of male host during embryogenesis

Sterility of crosses between infected males and female that are either uninfected or infected with a different strain of symbiont

• Aedes aegypti - mosquito a. Primary vector of alpha viruses • Transmission from female mosquito to human when feeding on blood

• Somatic distribution - presence in other tissues in insects • It was found, that Wolbachia infection interferes with other pathogens • Oral infection with Wolbachia reduces the amount of DENV in mosquito saliva a. study show infection lasted less than two days compared to >8 in WT

• Incompatible Insect Technique (IIT) - reduces population sizes a. Unidirectional CI causes a population size decrease b. Make loads in lab and release lots of MALES - driving out uninfected lineages 2. Population Replacement - releases CI inducing and pathogen blocking symbionts a. Make loads in lab and release lots of MALES - driving out uninfected lineages b. Release both genders; infected population should still increase

• Found across Asia and Australasia • Infected with strain wBol1 - male killer • Only 1% of population is male • Male killing mechanism a. ZW = female b. ZZ = males c. Masculiniser gene (Masc) is downregulated in Wolbachia infected males leads to failure of dosage compensation and male death d. W chromosome produces small RNA called feminiser, which targets masculiniser to degrade WELL EXPLAINED IN VIDEO