They wipe them out as early as in the egg, switch their sex to female and kill them ‎in a variety of bizarre ways – Wolbachia bacteria, a common insect infection, do ‎not require males for their distribution – so they kill them and protect the females

The most common infectious bacteria in the world hate males and have a weakness for the true stronger sex - females. Named Wolbachia, they infect insects, arthropods like mites, spiders, scorpions and more - as well as parasitic worms. Seven out of ten insects carry the bacteria; a true epidemic. Once it infects an organism, the bacterium transforms it for its needs. It takes over the organism’s reproductive system, protects it from competing invaders and sometimes even grants its host special abilities.

The bacterium was discovered in 1924 by Marshall Hertig and Simeon Burt Wolbach in the reproductive cells of the common house mosquito (Culex Pipiens). Several years after its discovery, the bacterium was named Wolbachia Pipientis, after Wolbach and the mosquito. For decades after its discovery, Wolbachia was almost forgotten, partly because it does not seem to be able to infect humans. It was only when it became clear how common it is in nature, along with its strange properties, that intensive research began.

Wolbachia bacteria can only survive and reproduce inside their host’s cells. This type of relationship is called endosymbiosis, the most intimate relationship that can exist between two organisms, where one organism lives inside another. In fact, we humans owe our lives to such relationships. Most cells of our bodies contain organelles called mitochondria, which provide energy by breaking down sugars in the presence of oxygen. They also control cell death and processes related to aging of cells and of the organisms comprised of these cells.

Mitochondria are found in all multicellular organisms, animals, plants, and fungi, as well as in many unicellular organisms. They are missing in non-nucleated unicellular organisms such as, bacteria and archaea, since mitochondria were originally bacteria that penetrated approximately a billion and a half years ago into a large host cell, which ultimately became the ancestor of all nucleated cells. Today mitochondria cannot live outside the cell so this event is now thought of as the ancestor of all nucleated cells. These bacteria - which are distant relatives of Wolbachia - developed a partnership with their host cell. Over time they transformed from independent bacteria living inside cells into organelles. Although they retained some of their bacterial genetic material, they transferred some of it to the DNA in the nucleus of the host cell.

A very intimate relationship, which changes nature’s equilibrium. Wolbachia bacteria | Source: Science Photo Library

Who needs males?

Mitochondria are inherited solely from the mother. If you are male, your mitochondria are in trouble, since even if you have offspring you are a dead-end from their perspective; your mitochondria will die along with you without being passed on to the next generation. Wolbachia bacteria suffer from a similar problem. Although they occasionally might be able to infect new organisms “conventionally”, where a healthy animal is infected by another animal, their main way of transmission is from mother to offspring.

Wolbachia bacteria pass on from generation to generation via infected eggs. The sperm cells are just too small for them and there is no place for the bacteria to hide within them. So as it happens, only female carriers can ensure the continuation of the Wolbachia lineage. Males are the end of the road for it, and hence its “hatred of males”.

During evolution, the Wolbachia bacteria developed several ways to overcome this male problem and ensure their survival. One such way sees the bacterium behave as a reproductive parasite – it takes over the host’s reproductive system and changes it, so that the reproductive results will match its needs. In some species of Hymenoptera (ants, bees, wasps, etc.), mites and thrips (an order of tiny insects), in which males develop in ways other than from fertilized eggs, Wolbachia have completely eliminated the need for males. They enable their host to reproduce via parthenogenesis: the non-fertilized eggs the mother lays develop into females that are her clone, instead of into males. There is no need for fertilization and therefore males are useless.

This Wolbachia ability was discovered in 1990 when Richard Stouthamer from the University of California and his colleagues noticed that antibiotics “cured” the parthenogenesis of Trichogramma parasitic wasps. After antibiotic treatment, the males reappeared and even mated with the females. Three years after the discovery, Stouthamer and his colleagues identified the guilty culprits – the Wolbachia bacteria.

Nip them in the bud

In the arthropods that they infect, such as butterflies, moths, beetles, flies and pseudoscorpions, the bacteria kill males ‘the biblical way’, while they are still fetuses. Emily Dyson and Gregory Hurst from the University College London found in 2001, for example, that the nymph butterfly Hypolimnas bolina found on the Samoan Islands, Upolu and Savai’i consisted almost exclusively of females. Wolbachia bacteria massacred the males, so that for every 99 females there was only one male.

At first glance, a bacterium killing its male hosts seems to be a waste of time, because the Wolbachia dies with it. However, when the infected females hatch – after their brother died as an egg – they then do not have to compete with them anymore for food. Also, in many cases the dead brothers become the first meal of the young females, and a dead brother also reduces the risk of incest. All these slightly improve the chance of survival for the female, so it can reproduce and transmit the bacteria to the next generation.

According to the theory of evolution it is widely expected that such mass killings will lead to a strong selection towards the appearance of a mutant male that is resistant to such killing. Indeed, already in 2007, Hurst reported on the islands of Upolu and Savai’i that the ratio between males and females was again balanced, at 50:50. In less than ten generations the butterfly population showed a resistant gene; an example of very rapid evolution. Hurst says the gene might have developed within the population of butterflies on the Islands or might have come from the same species population in Southeast Asia, who were resistant to the male slaughter by Wolbachia.

At some point there were 99 females for every male. Female Hypolimnas bolina | Source: Wikipedia

Preference for female carriers

Despite the return of males, Wolbachia have not lost the war. The bacteria have other tricks and are still thriving in the population of butterflies despite resistance. When they allows males to live, or when males are able to develop resistance and survive, the bacteria move to a different tactic called “cytoplasmic incompatibility”. The bacteria may not be able to penetrate sperm, but they can affect their production. They change the sperm cells so that they cannot fertilize eggs that are not infected with the bacteria of the same strain. Therefore, when a healthy female – without Wolbachia – mates with an infected male, her eggs will not be fertilized. In contrast, when a female carrier mates with another male – carrier or not – it will lay fertile eggs and the offspring will carry the bacteria.

This situation provides a huge advantage to female carriers over healthy females. Female carriers can mate and produce offspring with all males, whereas healthy females can do so only with healthy males. As a result, the infected females become increasingly common as generations pass, and soon much of the population will be comprised of male and female carriers. This manipulation is the most common one performed by the bacteria and can be found in almost all organisms that the Wolbachia infect.

Sex reassignment surgery

The fourth way in which the bacteria deal with males is by turning them into females. This phenomenon is seen in some species of butterflies, bugs and isopod crustaceans such as the pill-bug (Armadillidium vulgare).

In pill-bugs, like us, the sex is determined according to the sex chromosomes. In humans, the Y chromosome determines the sex of the fetus. Except in extraordinary cases, females will have two identical sex chromosomes (XX) and males will have two different chromosomes (XY). In pill-bugs the situation is reversed, females have two different sex chromosomes (ZW) and males have a pair of identical chromosomes (ZZ).

However, in many populations of pill-bugs females are the majority. In these populations the Wolbachia bacteria damage special glands that the males carry, and induce hormonal changes that cause fetuses with genetic male ZZ chromosomes to develop into fertile females. In all these populations all the infected fertilized eggs develop into females, regardless of their genetic composition. In coming generations, the female sex chromosome that is not being used in the infected population, will disappear: the females are infected genetic males without female chromosomes, so they do not pass it to their offspring. The sex is determined only by the infection status of the Wolbachia – females are infected pill-bugs and males are healthy pill-bugs.

However, a population of pill-bugs were found that were not infected with Wolbachia and still have a female majority. According to their chromosomes the females are males, but they do not carry the bacteria.

According to a study presented in September 2016 at the International Conference of Entomology, the cause of this strange situation is still Wolbachia. It turns out that the bacterium does not release its grip easily. The Wolbachia make female pill-bugs by donating DNA directly to the pill-bug genes. The genetic fragment from the bacteria that is responsible for turning male pill-bugs into females, is incorporated into the sex chromosomes of the males. In fact within this pill-bug population there are two sex chromosomes – the old Z chromosome and another Z chromosome that contains the bacterial DNA that makes it a female. This is an evolution of a new chromosome, one might call it W.

Sex reassignment surgery: bacteria turn males into females via a hormonal change. Pill-bug | Source: Wikipedia

The virus within the bacteria

The transfer of DNA fragments of Wolbachia into the genome of hosts occurs occasionally and is probably mediated by a bacteriophage (phage, for short) - a virus that attacks the bacteria. Jonathan Swift wrote at the time, “Big fleas have little fleas, upon their backs to bite 'em, and little fleas have lesser fleas, and so, ad infinitum.” This sentence is true for all organisms: phages called WO, after the bacteria, can be found in almost all Wolbachia that live inside cells of arthropods.

These phages can multiply inside the bacteria in two cycles. One of them is called the lytic cycle, where the phages recruit the bacterial systems to generate more copies, finally breaking out as they dissolve the bacteria and kill it. The second cycle, called the lysogenic cycle, is when the DNA of the phage integrates into the genome of the host bacterium and multiplies as the bacterium multiplies and divides. Sometimes a dormant lysogenic phage is awakened and switches to the lytic cycle.

When the phage breaks out of Wolbachia bacteria into their host cell, it might find itself in trouble. If there is no Wolbachia bacteria “free” in the area it must get out of the host cell, and infiltrate another cell, find in it Wolbachia bacteria and penetrate it. This is not simple. Viruses are almost always limited to one domain of organisms. For example, viruses that infect bacteria are not able to penetrate archaea (a type of unicellular organism) or nucleated cells (such as humans and other multicellular organisms) and infect them. However, it seems that WO found a way to overcome the eukaryotic cells on the way to infect its bacterial host.

To find out how it does this, Seth and Sara Bordenstein of Vanderbilt University in Tennessee mapped the genome of the phage. To their surprise, they found that half the genetic material of the phage originated in animals. One of the DNA sequences revealed it is part of a gene responsible for production of the components of the venom of widow spiders that can pierce cells of animals.

The Bordenstein couple identified other genes derived from animals related to causing cell death, identifying pathogens and controlling the immune system. They speculate that the stolen genes and their new combinations they generated in the phage, allow them to leave and enter animal cells and evade the immune system and other defense mechanisms of the organisms that hosts the bacteria.

The ability of the phage to adopt genes from the host of the Wolbachia may be beneficial to the bacteria themselves. After all, when the virus is latent, its DNA is integrated in the genome of the bacterium, and could therefore be considered as a passing of segments of DNA from the hosts to the Wolbachia. In an interview with Ed Yong, the Bornstein couple discussed evidence that some of the genes involved in the ability of bacteria to control the reproductive system of hosts lies in the genome of the phage and not the bacteria. However, these findings have not yet been published in the scientific literature.

Fertile cooperation

When Wolbachia are not playing in the reproduction system of their host, they help them in many different ways that enhance their chances of survival: moth larvae from the Phyllonorycter Blancardella genus are leafminers and live on leaves of apple trees. They prevent the section of leaf they live on from turning yellow and dying. The Wolbachia bacteria living in the hungry larvae secrete substances onto the leaves creating such “green islands” – green patches of living tissues that allow the larvae to continue to eat and reach maturity; bed bugs get B vitamins from the Wolbachia bacteria, which they generally lack in their blood meals.

The genes for the production of B vitamins does not exist in most Wolbachia. You can only find them in those that live within bed bugs or close relatives of the bat bug. These genes were transferred to them from the genome of other bacteria. Their presence has helped these insects and therefore the bacteria that infect them, and so the relationship between Wolbachia and bed bugs became mutually exploitative – a partnership.

Even people can take advantage of the bacteria. Although Wolbachia is not able to infect vertebrates such as humans, it is certainly able to influence organisms that cause disease in them. For example, parasitic worms that host the bacterium are unable to live without it. Treatment against worms of this sort, such as those that cause river blindness or lymphatic filariasis, is difficult and can harm the patients themselves, because worms and humans are quite similar. However, we can use antibiotics that destroy the bacteria living in the worms, and thus stop the worms without hurting the person.

The most important feature to us of the Wolbachia bacteria is to protect their host against other pathogens. In mosquitoes, for example, it activates genes that enhance the activity of their immune system to make it difficult for viruses and other parasites to infect the mosquito. In addition, the bacterium directly competes, and successfully, with pathogens for nutrients and so can inhibit pathogens that eluded the mosquito's immune system. Of course, mosquitoes that are not infected with pathogens will not transfer them to the person off whom they feed.

All is needed to exploit this bacteria in order to help us is to bring it to the affected areas, for example with zika or dengue, and infect a group of mosquitoes with Wolbachia. Its ability to control the reproductive system of its host will ensure that it will soon spread and infect the majority of the population, if not the entire population, and thus will halt the spread of the disease-causing agents.

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