Evolution of amphibian genome size

When I was a kid, one of my favorite things to tell people was that the organism with the biggest genome was an amoeba. This is probably not true. Scientists used to think it was an amoeba; Polychaos dubium, however because that estimate has been somewhat contested, the current largest genome found may actually belong to Paris japonica, a Japanese flower.

Paris japonica: record sized genome

Which is probably still just as surprising to the average child. The largest animal genome almost certainly belongs to the marbled lungfish, with at least 130 billion bps (132.83 pg), followed by the salamander, Nexturus lewisi. So we have amoebas, some plants, and some fish and amphibians with giant genomes of over a hundred billion basepairs meanwhile humans have a baby genome in comparison of just 3 billion basepairs. Pathetic.

The marbled lungfish has a giant transposon-filled genome

Amphibian genomes are more varied than you might expect

Amphibians present an especially interesting case as not only are they tetrapods (where polyploidy is considerably rare— it pops up in fish more frequently), but amphibians also have the largest range of genome size of any group of vertebrates.

One would expect genome size to correlate with number of genes and organismal complexity, and to an extent that’s true. In prokaryotes, more genes means a bigger genome– and certainly eukaryotes have bigger genomes than prokaryotes, and are more “complex.” However, the relationship ends there. Eukaryotic genomes are full of complex regulatory regions, introns, and mobile elements or “junk DNA” (possibly a misnomer, as we really don’t understand the genome well enough to really determine what is “junk”). Genomes can undergo polyploidization, dramatic increases in tandem repeats and transposable elements, insertions/deletions, gene duplications etc., but there is absolutely a potential fitness cost to having a larger genome.

Amphibian genome size in relation to climate change, life cycle: not very related actually

A very cool paper that came out in nature Ecology & Evolution, delved into this conundrum of how closely related organisms have evolved such a huge diversity of genome size. They focused on amphibian genome evolution, with the expectation being that there’d be a strong relationship between genome size, life cycle complexity, and climate. What they ended up finding was that that’s not really the case. Despite having a huge range of genome size, amphibians actually seem to display macroevolutionary homogeneity generally within Anura (frogs), Caudata (salamanders), and Gymnophiona (caecilians), with only a few instances of dramatic shifts in genome size. 

The authors measured amphibian genome sizes (and I believe took from the, already vast record of, batrachian genome size data) and tested for rate heterogeneity, and whether amphibian genomes underwent a more Brownian motion pattern of evolution (random and gradual), or underwent dramatic changes (“saltation” as biologists say). They further wanted to construct an ancestral state of what the genome may have looked like for common ancestors of living taxa, and then they wanted to use their gigantic dataset to delve more deeply into the question of how life history, climate, and genome size relate to one another. If you’re into that kind of thing, I’d definitely read the paper, they made some very nice figures!

Salamander genomes are giant– but generally evolve gradually

Caudata have by far the largest genomes, with the smallest salamander genomes still exceeding Gymnophiona and Anurans. The authors were also able to perform an ancestral state reconstruction for genome size on a time scale (i.e. species 1 and species 2 diverged X amount of years ago and their common ancestor at that divergence point had a genome that was Y size). They estimated that the common ancestor of all salamanders had a genome of around 43 pg and then proceeded to evolve gradually as a function of time. This is a huge jump from the common ancestor of all amphibians which looks to be between 4.62 and 7.57 pg. The root of Anurans seems to be at about 200-225 million years ago, meaning they’ve been evolving gradually as a function of time for that long, and still have not even overlapped with the smallest salamander genomes. Gymnophiona (root at 50-275 mya) have also not reached the range of salamander genome size.

This dramatic genome size increase in salamanders is primarily the result of one a dramatic increase in Long Terminal Repeat transposable elements, as well as generally having more genes, longer introns, and a lower substitution and deletion rate. When measuring evolutionary Gymnophiona had the largest spread, with some species having rates around the lowest salamander rates and others having the highest across all amphibians by far. Salamanders had much lower rates than frogs.

Even though people tend to (wrongly) shove amphibians away in their minds as being “primitive” or “simple” tetrapods not worth our time, amphibians and their genomes are almost certainly worth our time (especially given they’ve been around since the Devonian)! They have the broadest range of genome size of any vertebrate group, they’re found all over the globe, and they have varying life cycles and climatic pressures.

Source:

Macroevolutionary shift in the size of amphibian genomes and the role of life history and climate. Nature Ecology & Evolution. H. Christoph Liedtke, David J. Gower, Mark Wilkinson & Ivan Gomez-Mestre. 24 September 2018.

 

 

 

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Microbe that could contaminate Mars

Recently there was an exciting discovery of evidence for a lake underneath the ice caps of Mars. Despite the already abundant evidence of water on Mars, this is evidence for an actual large stable body of liquid water. The water’s freezing temperature can be lowered with the huge amounts of salts present, as well as the ice caps exerting pressure. About nothing would be more exciting than finding life anywhere else in our solar system (or beyond!), but the possibility of life evolving and then being seeded on other planets is also pretty cool to imagine. But, if you’ve seen that Star Trek TNG episode about the terraformers, you’ll already know seeding a planet with life if the planet already contains its own life, violates the prime directive. So this would be out of the question (but it is still amazing that we could send a microbe-filled package to another star system and let evolution take over).

Some life on Earth is tolerant of such extreme conditions, that people once actually thought it couldn’t possibly have come from this planet. Even though most people jump to archaea when they think of extremophiles, the example that comes to my mind (that I believe everyone should familiarize themselves with) is a bacterium called Deinococcus radiodurans. 

Deinococcus radiodurans

This polyextremophile can survive conditions that don’t even exist on Earth. It can survive extreme cold, acidity, dessication, a vacuum, and as the name suggests, ridiculous levels of radiation. It was discovered in the 50s when, after subjecting meat to high doses of gamma radiation, one bacteria species survived to spoil the meat.

It’s not that the radiation doesn’t hurt the bacteria. It does. It shatters the genome, but Deinococcus is able to stitch its genome back together–with high fidelity–extremely quickly. But given that no place on Earth is subject to as much radiation as Deinococcus can withstand, it’s suspected this bacteria evolved the radiation resistance trait as a result of adapting to dehydration which also causes DNA damage. Despite being able to survive intense environmental stress, Deinococcus does not form endospores, nor does it have some particularly magical or exciting genetic code.

While most bacteria only carry around one copy of their genome, Deinococcus carries between 4 and 10 copies consisting of two chromosomes, a megaplasmid, and a small plasmid, stacked on top of one another. Deinococcus also grows in tetrads which further helps protect it from damage. If you’re looking to isolate this bacterium, it helpfully produces the carotenoid pigment deinoxanthin, which makes it distinctively pink. It also is unique in that it is neither completely Gram-negative or Gram-positive, containing elements of both, with thick cell walls (characteristic of Gram-positive), and a second membrane (characteristic of Gram-negative). Having five layers as well as this pink pigment probably also helps protect them from stress.

One thing I could not find was whether Deinococcus radiodurans had any known phages. I mean, of course it HAS phages, but has anyone actually isolated any? I personally, would love to study a virus that’s able to infect this incredibly tough bacterium.

Help protect mice from gamma radiation – Deinococcus Mn(2+)-Decapeptide complex

When studying Deinococcus, researchers suspected the manganese (Mn)(II)-based antioxidant complex that could potentially be used to protect humans and animals from radiation from a future terrorist attack. It was reported to be highly radioprotective of proteins and was screened on cultured cells and enzymes and shown to protect them from ionizing radiation. The researchers from this PLOS one article exposed mice to high doses of radiation (enough to kill) and all the mice that took the MDP survived. The MDP protected white blood cells, helped stop damage to bone marrow from radiation, and also protected hematopoietic stem cells (which are cells that can turn into any type of blood cell). The MDP also helped in radiation recovery so could be used as a helpful post-exposure therapy as well as a prophylactic.

A fungus that can do the job Deinococcus couldn’t

As incredible as Deinococcus radiodurans is, it has not been as useful as scientists had hoped for cleaning up radioactive waste. Even though scientists have been able to genetically engineer Deinococcus to break down heavy metals and toxins, it has huge trouble actually growing at a very low pH and it can’t form biofilms under the extreme conditions.

What they eventually found was Rhodotorula taiwanensis, a red yeast that actually tolerates acidic conditions and forms biofilms in extreme conditions. It also tolerates–even happily lives around– insanely dangerous metals such as mercury chloride that would kill people no problem.

While the yeast are happy in heavy metals and low pH, they are pretty unhappy in high heat conditions. That being said, the yeast don’t need to be at the pit of the nuclear waste to get the cleanup job done, they can be nearby in a comfortable temperature and capture leaking waste.

The Koala genome: retroviruses, chlamydia, survival, and a eucalyptus diet

As reported in a recent 2018 Nature genetics article, some awesome scientists have just sequenced the complete koala genome and have been able to provide insight into how to hopefully conserve this vulnerable species. They have also provided the very important public service of reminding us all how cool koalas are.

Koalas seem to be unusually unlucky animals, constantly succumbing to lymphoma, leukemia, getting hit by cars, and chronic infections of chlamydia (if you didn’t know this already, then I’m very sorry to have ruined your day). Scientists suspected for a while that many koalas must have a suppressed immune system to allow for such a high rate of disease, and were reminded of HIV, hence the term KAIDS (Koala AIDS)

When scientists looked more closely at pieces of the koala genome and koala viral loads, they found something really terrifying: koalas are basically in the process of an evolutionary arms race between retroviruses that are endogenous and/or exogenous. So not only do they have transmittable retroviruses, they also have retroviruses in the process of integrating into the germline and being vertically transmittable. They found many similar koala retroviruses that are inherited in a Mendelian fashion (integrated into the germline) like your typical allele, so there is a spectrum in the population. BUT because they are both exogenous and endogenous, the koalas also transmit the retroviruses sexually.

I noticed while reading the new genome paper by Rebecca Johnson et al., that they mention Koala genomes are approximately 47.5% interspersed repeats and 44% of those are transposable elements. That is a lot, and as the paper mentions, they were able to study centromeres (the area in the center of the chromosome). Centromeres tend to have higher order satellite arrays, but when an animal has less, as in gibbons, transposable elements likely represent an important component of smaller centromeres instead. The reason I mention gibbons is because crazily enough, the gammaretrovirus, Gibbon ape leukemia virus (GaLV) is the closest relative of Koala retrovirus (KoRV). This is the result of a transspecies transmission, though scientists have not pinpointed the exact intermediate host (gibbons are placental mammals from Thailand, koalas are marsupials from Australia). Bats always come to mind as a possible candidate, but who knows.

Koala survival and conservation

During a wave of koala deaths about 100 or so years ago from disease, some were able to survive these exogenous retroviruses due to integrated ape leukemia virus in their genome. Because viral sequences were present at the same integration site in all koala cells, this clues us in that the virus had been endogenized and could now be inherited. New exogenous retroviruses do not have a preferential integration site in the genome for contrast. This endogenous process had to have been very recent, like as in, in the past century or so recent which is pretty amazing to think about given we generally think of endogenous retroviruses being ancient remnants of a once deadly disease. The endogenous viruses actually serve to protect the koalas from some exogenous retroviruses.

Gammaretrovirus genome

Koalas populations have undergone two especially dramatic decreases unrelated to disease. One, about 30-40,000 years ago when most Australian species suffered (so probably a habitat disaster or climate change rather than koala-specific disease). Then, again during European settlement when people decided to kill them– because humans seem to have a bizarre need to destroy anything cute and harmless.

The key to Koala survival is genetic diversity. We have destroyed much of their natural habitat which can result in population isolation which results in inbreeding and a decrease in gene flow and diversity. By looking at the genome and doing some coalescent analyses, the group was able to look at the koala population history and genetic diversity. The did find some koalas were able to maintain their genetic diversity and some level of habitat connectivity– but this is a delicate situation and it is absolutely vital to their species that they maintain gene flow. Southern koalas seemed to have significant inbreeding depression and were suffering from a lack of genetic diversity. Many small populations are in big trouble as they may suffer more from genetic abnormalities.

Retroelements, for background

In case you are unfamiliar with retroelements and other viruses that integrate into genomes: A provirus (or prophage as it’s referred to in bacterial viruses) such as HIV or Lambda phage, is a virus which integrates its genetic material into the host genome, so it can be passed down through cell generation. However, because HIV does not infect germline cells, and because it has an RNA intermediate that must be converted to DNA to integrate (hence the “retro”), it is referred to as an exclusively exogenous retrovirus and cannot be passed down to the next generation.

The retroviral sequences in our genomes are remnants of many, MANY retrovirus infections throughout our evolution that were passed through our germline (‘virus fossils’ if you will). These are termed endogenous retroviruses and have almost all lost their ability to be transmissible or exist outside the host at all after millions of years of genetic drift and host defense. While they once may have provided a selective advantage by providing some host immunity, they also would have been a tricky thing to deal with for the genome. Too many active transposable elements are detrimental to the genome stability, and in mammals we even have something called fetal oocyte attrition– where most oocytes in the fetal female are destroyed if they have too many transposable elements.

Retrotransposable elements that lack an extracellular phase in our genomes include LINES and SINES (Long/Short interspersed nuclear elements). Most other RT-retroelements do not have an extracellular phase and are not transmissible to other hosts so are not called retroviruses. They may have evolved from RT-retroelements to become extracellular via horizontal gene transfer from other viruses.

Back to the Koalas… Koala Retroviruses and Chlamydia

Another paper had previously shown that koalas infected with the retrovirus KoRV-B, rather than KoRV-A, were also more likely to have chlamydial disease. Chlamydia is quite common bacteria found in koala populations, but some koalas seem to just be carriers and do not progress to chlamydial disease. Other koalas have no chlamydia at all and of course do not chlamydial disease. Some unfortunate ones have chlamydial disease and the researchers hypothesized these koalas with chlamydial disease are more likely to have exogenous retroviruses suppressing their immune system.

Penny the Koala, getting treated for Chlamydia, from https://www.bbc.com/news/magazine-22207442

As I mentioned, koala retroviruses can be both endogenous and exogenous, so if the Koala only has endogenous retrovirus, it is immunocompetent and less likely to show disease or even be infected at all by Chlamydia. KoRV-B was a statistically significant predictor of chlamydial disease, as it seems to be only exogenous and cannot infect germ cells as it uses a different receptor.

This group’s data supported other researcher’s assumption that all northern Australian koalas carry endogenous KoRV-A strain, but they also found about 25% of koalas they tested carried the KoRV-B subgroup. Multiple studies have now shown that TOTAL KoRV gDNA load or KoRV viral RNA load was correlated with chlamydial disease, so it seems other KoRVs (which are present in all koalas) do not cause the immunosuppression that KoRV-B causes.

That being said, 46% of the koalas tested who actually had chlamydial disease were negative for KoRV-B and one koala did not have chlamydial disease despite being infected and being positive for KoRV-B.

New insights from the whole genome of koalas on diet, immune system, sex!

Koalas are now the fourth marsupial to have their genome sequenced (which makes me really want to sequence more marsupials in the hopes of getting my own nature paper—I love a good fishing expedition). The group was able to characterize novel lactation proteins that help protect the young while their still in the pouch. The proteins produced in koala milk have antimicrobial and antifungal effects, notably against Chlamydia, which would be important for the young who lack a developed immune system. They also found immune genes involved in the response to chlamydial genes (perhaps the koala retrovirus researchers should go back and look for links between these genes and their various retrovirus findings in relationship to chlamydial disease vs. carrier state vs. no chlamydia!).

But how koalas are able to handle their intense eucalyptus diet, may have to do with the expansion within a cytochrome P450 gene family. Bitter tastes help animals avoid potentially toxic plants. Koalas have more bitter taste receptor genes than most mammals, so they can detect toxic metabolites in eucalyptus, indicating they may be able to figure out which leaves to eat. While eucalyptus is toxic to most animals, the koalas tend to be selective in which leaves they eat and do manage to avoid as much of the toxic metabolite as possible and get the most nutrients as possible from the plant.

Back to the repeat elements in the genome: They were also able to fully characterize repeat-rich noncoding RNAs, including RSX. RSX in koalas mediates X inactivation in females which is what also occurs in placental mammals as a method of dosage compensation (so females do not get twice as many proteins from the X chromosome as males, one X is inactivated in cells). Sex determination and dosage compensation are really interesting to compare in birds, monotremes, marsupials and placental mammals, so I found that little tidbit pretty exciting. Beyond their sex determination, the researchers also found genes involved in induction of ovulation as koalas are induced ovulators.

Vaccine design and conservation

Annotating immune genes and studying diseased vs. healthy koalas has useful implications for designing vaccines against chlamydia. The research group found that differences in particular immune genes in koalas involved in a clinical trial for vaccines may have explained differences in their immune response to the vaccine. They were also able to look at the expression levels of different immune genes to see which were up-regulated or down-regulated in sick vs. healthy koalas. They were of course able to quantify and see where the retroviruses were integrating into the genomes, which will also help them hopefully produce vaccines and combat the resulting diseases from retroviruses.

Sources:

1. Adaptation and conservation insights from the koala genome. RebeccaN. Johnson et al. 2018. https://doi.org/10.1038/s41588-018-0153-5
2. Infection with koala retrovirus subgroup B (KoRV-B), but not KoRV-A, is associated with chlamydial disease in free-ranging koalas (Phascolarctos cinereus). https://doi.org/10.1038/s41598-017-00137-4

 

 

 

 

Banded Mongoose: mutualism, cooperative breeding, and vital adaptations

 Banded mongooses in Kruger National Park, South Africa (Peet van Schalkwyk ©)

“It is the hardest thing in the world to frighten a mongoose, because he is eaten up from nose to tail with curiosity. The motto of all the mongoose family is ‘Run and find out'” —Rudyard Kipling, Rikki-Tikki-Tavi

Cultural inheritance in banded mongooses

The banded mongoose is a bit of an anomaly in the mammal world. Instead of the offspring being raised by their parents (and behaving similarly to their parents), they instead inherit their behaviors from other adult mongooses. The adults are random, rather than closely related to the offspring, but take on a parental role in the sense that they sort of “adopt” a baby/juvenile (about one month old) and show it how to forage, hunt, stay safe from predators, and otherwise be a mongoose properly. These role models or “escorts” will carry around the pups and teach them closely for about two months. The plasticity of mongoose behavior is sufficient to allow for pups to behave more similarly to their role models than to their parents.

This kind of transmittance of behavior is known as cultural inheritance, and is actually quite common in the animal world– but the unique setup in mongooses provides an opportunity to easily decouple the direct genetic inheritance from parents and the cultural inheritance resulting from behavioral plasticity (social learning).

Cultural evolution is becoming one of the most popular topics in biology as more and more scientists are beginning to notice how new behaviors can sweep through a population in less than a generation. The classic non-human example of this would be humpback whale songs changing year to year.

While cultural evolution occurs more quickly than genetic evolution in one sense, it also allows for genetic diversity to persist. Higher behavioral plasticity results in higher variety of trait and higher variety of preference for those traits. It can actually slow down evolution towards physiological adaptation for an environment by slowing the adaptation of physiological change. That being said, it can allow for more rapid adaption and by relying on social learning, only one or a few members of a population need to “discover” a new behavior for it to spread through the population.

It’s possible the mongooses have evolved this escort system as a way to maintain diverse foraging methods and reduce competition in their groups. Maintaining plasticity in foraging behaviors would be useful for social animals that have a wide variety of food sources, as the mongoose does.

Timon and Pumbaa: based on a true story

So admittedly Timon is a Meerkat not a banded mongoose, but it’s the same family so it’s close. In a cool example of mutualism, Warthogs (which, by the way, are awesome animals and don’t get nearly enough love) can rid themselves of ticks and bugs by getting groomed by mongooses.

Warthogs–which should look kind of scary to a small mongoose, actually get along quite well with them. Wild pigs tend to have quite a few ectoparasites and bugs inhabiting their skin/fur which can provide an easy snack for the banded mongoose. Warthogs have learned to lay down when mongooses are nearby, so they can pick off the parasites. Besides allowing mongooses to groom them, they also welcome vervet monkeys to snack on their ticks.

Mutualism between mammals is somewhat rare, but wild pigs and mongooses (and vervets for that matter) are highly intelligent and it shouldn’t come as much of a shock that they are able to enjoy the company of other species.

Mutualistic foraging between dwarf mongooses and hornbills

In another species of mongoose, the dwarf mongoose of the Taru desert often cooperatively forages with large birds, particularly hornbills, who share the same prey. The hornbills will wait to start foraging around termite mounds if the mongooses are sleeping and the mongooses will wait for the hornbills to be nearby to begin their foraging. This is true mutualism, as, while many animals have instances of exploiting each other’s coincidental presence for their benefit, the hornbills and dwarf mongoose actually plan their foraging activities around the other.

The mongooses and the hornbills will warn each other of predators while they forage. The hornbills will even warn the mongooses when there are predators nearby that do NOT prey on the hornbills. The hornbills recognize predators specific to the mongoose as they will not warn against predators that do not prey on the mongoose. This is a pretty unique relationship as most cases of mutualism do not involve so much complex compensative behavior between species. The two species will communicate to one another with different vocalizations, and hornbills will sometimes wake up the sleepy mongooses when they’re impatiently waiting to start snapping up unfortunate insects.

Convergent evolution of neurotoxin resistance

Mongooses are mostly known for fighting cobras (partly because they’re exceptionally quick) but their resistance to the alpha-neurotoxin in cobra venom is what especially allows for this feat.

Acetylcholine is a very important neurotransmitter, so there are acetylcholine receptors all over your muscle cells that need to be free to bind (or not bind) acetylcholine, allowing your muscles to expand/contract. This neurotoxin, called alpha-bungarotoxin (alpha-BTX), works by binding to these acetylcholine receptors and blocking them up resulting in paralysis and eventually death. However the mongoose, the snakes themselves, and several other animals have independently evolved to alter the shape of their acetylcholine receptors so the neurotoxin, alpha-BTX, doesn’t bind.

Tweaks to the nicotinic acetylcholine receptor to prevent snake neurotoxin binding have been shown to have evolved at least four separate times in mammals (the honey badger, pigs, mongooses, and hedgehogs), but in the mongoose, the tweak involves a glycosylation site on the receptor matching the site present in snakes.

Syncing up birthdays

Despite being highly social and altruistic, meerkats are a more vicious member of the mongoose family and are especially well known to participate in infanticide. As meerkats live in a matriarchy with intense dominance hierarchies, many babies do not stand a chance from more dominant pregnant females. Banded mongooses however, have managed to evolve to sync up their birth so they’re all born on the same day. This prevents infanticide as all females are on essentially identical schedules (hormonally and in how they spend their time), so pups are never left alone with other females.

Photo credit: Feargus Cooney

While most mammals have adapted to differentiate their own offspring very well, the banded mongoose benefits from having all pups be treated equally by the adult mongooses. Syncing up birth to the day or few days is a useful strategy, also seen in flamingos, where both male and female flamingos will even produce and feed crop milk to young who are not their own. However unlike flamingos, syncing up birthdays seems to be more about preventing infanticide and having a more lax dominance hierarchy.

This is a pretty unique strategy as usually species tend to fall on a continuum of high to minimal parental care. In this case, the banded mongoose receives a lot of “parental care” but not necessarily much from their parents. In almost all of nature, the level of paternal parental care is based on certainty of paternity, yet the mongoose has no idea who its close kin are. The behavior where a species mentors and takes care of young they know are not their own is, of course also seen in humans. Mongooses are exceptional in their array of cooperative behaviors– displaying reciprocity, altruism, cooperative breeding, and mutualism. But knowing humans, most will probably continue to believe we are special and entirely disconnected from these evolutionary adaptations.

Sources:

1. Dwarf mongoose and hornbill mutualism in the Taru desert, Kenya. O. Anne-E. Rasa – Behavioral Ecology and Sociobiology – 1983
2. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor. D. Barchan-S. Kachalsky-D. Neumann-Z. Vogel-M. Ovadia-E. Kochva-S. Fuchs – Proceedings of the National Academy of Sciences – 1992
3. Decoupling of Genetic and Cultural Inheritance in a Wild Mammal. Catherine Sheppard-Harry Marshall-Richard Inger-Faye Thompson-Emma Vitikainen-Sam Barker-Hazel Nichols-David Wells-Robbie Mcdonald-Michael Cant – Current Biology – 2018
4. Reproductive competition and the evolution of extreme birth synchrony in a cooperative mammal. S. Hodge-M. Bell-M. Cant – Biology Letters – 2010

New Rhabdoviruses in bat ectoparasites: as told by a rabid supporter of bat research

vesicular stomatitis virus, taken by the CDC

Few things disturb me more than rabies. The thought of a small, bullet-shaped virus encoding only five genes, causing an incurable, creeping infection along the nervous system, into the brain, then spreading everywhere, all while causing the host to become violent and delirious– is a bit overwhelming to dwell on. Even for someone who reads about infectious disease everyday. But rabies, for all the attention it gets, belongs to a group that seems almost neglected otherwise.

Rabies, a member of the Lyssavirus genus, is unquestionably the most famous Rhabdovirus (which is fair– it’s the most deadly virus known to man), so it might be surprising to hear that most members of the rhabdovirus family don’t even seem to infect mammals.

Despite bats being well known reservoirs for some of the world’s most deadly viruses (Henipaviruses, Marburg, and Ebola to throw some names out there), bat ectoparasites have been pretty neglected by viral ecologists. This is especially surprising as arboviruses (viruses transmitted by an arthropod vector e.g., Dengue), have only been spreading further geographically as our climate heats up. Even more surprising, these bat parasite viruses are often close relatives of rabies, yet so far seem relatively harmless.

Most rhabdoviruses that actually infect bats (that we know of) have been lyssaviruses, but that could partly be because detection methods for rhabdoviruses in bats (and their ectoparasites) were often restricted to lyssaviruses. Now that we’re starting to look a bit harder, we’re realizing there’s probably a lot more viral diversity than we’d planned for.

Kanyawara virus: how much of a concern are rhabdoviruses, really?

The Kanyawara virus, a nycteribiid bat fly virus found in western Uganda, is raising some questions about rhabdovirus diversity and potential pathogenicity. A phylogenetic analysis placed the virus within a genus containing bat rhabdoviruses; Ledantevirus. If the most recent common ancestor of these viruses was bat-associated, this means is that it is likely this virus infects bats as well and is not an arthropod-specific virus, but rather and arthropod-vectored virus. Viruses that infect bats can result in bat-borne zoonoses, examples include basically all the worst things we usually think of when thinking about viruses.

Nycteribiid: Why does this thing even exist though

The researchers tested nine bats for the Kanyawara virus but no virus was found in any of them. The researchers mentioned they suspect this is due to transient viremia rather than that the virus never infects bats, but they don’t really know.

darling member of the same genus as the host of the parasite carrying Kanyawara virus

Rhabdoviruses infect a huge range of hosts, but it seems arthropods play the biggest role in rhabdovirus ecology. Plant rhabdoviruses are usually transmitted by arthropods, fish rhabdoviruses are often transmitted by aquatic arthropods, and many viruses have been found in both vertebrates and arthropods or are vectored by arthropods. That being said, many rhabdoviruses are insect-only viruses which would make them significantly less concerning. However bats are way overrepresented amongst non-arthropod hosts in terms of how many rhabdoviruses infect them as a preferred host.

Host-parasite coevolution

Previous phylogenetic analyses done by a group at Cambridge show rhabdoviruses as closely coevolving with their hosts. When a host phylogeny closely matches a virus phylogeny this is strong evidence for coevolution. The more detailed phylogeny in this study, shows a less mirrored picture. Basically there’s a positive correlation between genetic distance of the hosts and genetic distance of the viruses, but the viruses seem to switch easily between closely related species.

The group found only two major host switch types in the phylogeny (a host switch being more broad in this case, when a virus from one host cluster such as plants, arthropods, or vertebrates switches over). Three phylogenetic transitions were from an arthropod-vectored vertebrate virus to a vertebrate-specific virus. Two were arthropod-vectored to arthropod-specific. One arthropod-vectored, to arthropod-specific group, the sigma viruses, actually lost their vertebrate hosts and are now vertically transmitted.

This type of analysis is useful because it allows us to predict with some confidence whether a virus isolated from a host is a likely pathogenic threat based on where it might fit in a phylogeny. These types of large scale phylogenetic analyses are becoming even more useful now that we are discovering so many viruses but can’t necessarily keep up with understanding their biology.

If most rhabdoviruses are harmless, but rabies (a rhabdovirus) can infect basically anything, and is the most deadly pathogen known to man, you can imagine how confusing it is when a new rhabdovirus emerges. We really don’t know what’s out there, even when it comes to human rhabdoviruses. Recent studies have isolated new rhabdoviruses in apparently healthy people in West Africa, some of which were extremely similar genetically to Bas-Congo virus (a virus associated with a hemorrhagic fever outbreak in 2009)– yet we don’t know how they were transmitted, how common this is, and why even closely related rhabdoviruses of vertebrates can switch between asymptomatic and deadly.

Bats are exceptionally diverse, so their parasites are quite diverse with them. These parasites’ parasites (the viruses) are then unsurprisingly quite diverse as well. Bats are then an ideal reservoir for some truly horrific viruses to circulate and happily evolve in, as bats have a unique immune system allowing them to harbor zoonotic viruses and remain asymptomatic. As interactions between bats and humans increase there’s now further selective pressure and opportunity for viruses to expand their host range to hosts with less fine-tuned, dampened immune systems–like, us for example.

Sources:

1. Kanyawara Virus: A Novel Rhabdovirus Infecting Newly Discovered Nycteribiid Bat Flies Infesting Previously Unknown Pteropodid Bats in Uganda. Tony Goldberg-Andrew Bennett-Robert Kityo-Jens Kuhn-Colin Chapman – Scientific Reports – 2017

2. The evolution, diversity, and host associations of rhabdoviruses. Ben Longdon-Gemma Murray-William Palmer-Jonathan Day-Darren Parker-John Welch-Darren Obbard-Francis Jiggins – Virus Evolution – 2015

Bioluminescence- the immense diversity of organisms that glow

Bioluminescence is a beautiful evolutionary phenomenon which has aided organisms in defending against predators, attracting mates, attracting prey, communicating, and even coping with metabolic stress. A ton of groups contain bioluminescent members (fungi, echinoderms, cnidarians, the list goes on and on) including some real evolutionary stand-outs.

In most cases (but not all!), bioluminescence results from enzyme-catalyzed oxidation of luciferins—light-emitting compounds—by luciferases. There can be many different luciferase compounds used even in closely related species.

New luciferin found in glowworms

A newly identified luciferin was discovered in caves in New Zealand (because of course it would be in a cave in New Zealand) in glowworms. This luciferin uses Xanthurenic acid and tyrosine as the two precursors to the glow. This particular glowworm is Arachnocampa luminosa, a species of fungus gnat that, in its larval stage, produces sticky threads by building a long muscousy tube and moving along the tube sort of vomiting up little sticky threads to trap insect prey. How disgustingly beautiful nature can be!

Waitomo caves 

Glowworms are not really worms, but rather, larvae of several families of beetle and fungus gnat–however the bioluminescence is not homologous among the groups (so it’s arisen independently many times over). While it’s not always just the larvae that glows, the larvae emits the brightest blue-green glow. The glow helps the glowworms attract insects, attract mates, and protects them from predation (it also inspired James Cameron to make the blockbuster hit, Pocahontas with Glowworms Avatar).

Deep sea Cephalopods like to flash each other

An especially cool evolutionary example is of a deep-sea octopus, whose “suckers,” which still retain a sucker appearance and sucker-like traits, have had many of their muscle cells replaced with light producing cells. Researchers suspect this may have occurred as the result of once being a shallow-water bottom dwelling octopus, and moving to a deep open-ocean environment where suckers were less necessary.

Deep sea octopus

Now it appears, the octopi use these glowing suckers for communicating to one another via visual signaling. They may also be using them for attracting a favorite prey item of theirs—copepods (small crustaceans). This is an unusual prey item for an octopus, but the copepods are attracted to the bioluminescence.

Toyama Bay, Japan where firefly squid gather to spawn. Credit: Brian J. Skerry/Getty Images

For a more flashy light show, I’d recommend the firefly squid. Their deep blue lights (produced by photophores) are used for communicating with mates and perhaps rival squid. The light can also be used to break up the body pattern to confuse predators and attract small fish to prey on (because deep sea fish just cannot seem to learn which glowing lights mean danger). The really cool thing about firefly squid though, is not so much the light they produce, but the evolution of their eye that seemed to come with it. They are thought to be one of the only cephalopods to have color vision (by the way, cephalopod eyes: a fascinating topic). They have three visual pigments while other cephalopods only have one, and this may be so they can better distinguish ambient light from bioluminescent light, and perhaps because their light color is pretty unique from other bioluminescence emitted in the deep sea.

Symbiotic bacteria- lux operon helps flashlight fish and bobtail squid

In perhaps the most of obvious function of bioluminescence: The flashlight fish (Anomalous katoptron in this case, though there are several species), produces light using symbiotic bacteria. The fish’s light organs are located under it’s eyes so it can turn the light on and off by blinking. These organs are packed with bioluminescent bacteria to produce a greenish-blue light. Researchers found that the fish blink less (meaning their organs are open) in the presence of their planktonic prey indicating they use their bioluminescence for finding prey.

 

Quorum sensing and a beautiful tale of symbiosis

One of my favorite bioluminescent evolutionary excerpts is that of Vibrio fischeri and Euprymna scollops (the Hawaiian bobtail squid). V. fischeri is a symbiotic bacterium that produces bioluminescence through the lux operon (which involves another luciferase oxidizing a compound to produce blue-green light). The Vibrio interact with the squid (using type IV pili) which starts the maturation of light organs in the squid. These bacteria help the squid conceal its shadow while its foraging for food under the moonlight. This protects the squid from predators while providing the bacteria with a stable home. 

simplified diagram of lux operon at low and high cell density

What makes this bacterium especially notable, is that it was one of the first bacteria to be discovered to use quorum sensing. Quorum sensing is a gene expression regulation tool (often called “bacteria communication” and totally going to be on your exam tomorrow) where the Vibrio’s gene expression responds to changes in bacteria cell density. A signal molecule- N-acylhomoserine lactone (AHL), is synthesized by LuxI (a protein produced by the lux operon I mentioned earlier) and leaves the bacteria cells. LuxR forms a complex with AHL and binds the lux box causing the activation of luminescence genes. The bacteria colonize the squid’s light organ at a very high density producing lots of this AHL molecule.

Millipedes: Glow first used for coping with climate, co-opted for warning signal

 

If you’re ever in California, be on the lookout for the Motyxia millipedes. They’re pretty easy to spot as they emit a teal glow from their entire body. They also produce poison cyanide which many other millipedes do as well. Instead of concentrating their glow to one light organ and instead of emitting light from a luciferase reaction, they glow all over their exoskeleton using a photoprotein whose homology is unknown.

M. sequoiae (left), M. bistipita (right)

But what’s REALLY cool about the Mytoxia is that for a while it was thought that bioluminescence evolved in the millipedes as a way to warn predators. However, when researchers discovered that another species (previously Xystocheir bistipita, now reclassified as Mytoxia bistipita) glows, but much more faintly, they looked more into it.

They found that Mytoxia may have actually evolved to cope with hot, dry climates (this species is found in the Sierra Nevada Mountains). The glow of M. bistipita is much less intense and they also have fewer predators than other species. Millepedes have difficulty metabolizing oxygen in hot, dry climates which creates toxic byproducts (like peroxide). Their bioluminescent photoprotein actually helps to neutralize these toxic byproducts. The researchers concluded that the millipedes colonized higher elevations more recently than the bioluminescence evolved, and that with that colonization came more predation. Only then did they co-opt the trait for warning predators of their poison cyanide production. The brighter the millipede, the more cyanide it contained!

Sources:

1. Paul E. Marek, Wendy Moore. Discovery of a glowing millipede in California and the gradual evolution of bioluminescence in Diplopoda. Proceedings of the National Academy of Sciences, 2015.
2. Jens Hellinger, Peter Jägers, Marcel Donner, Franziska Sutt, Melanie D. Mark, Budiono Senen, Ralph Tollrian, Stefan Herlitze. The Flashlight Fish Anomalops katoptron Uses Bioluminescent Light to Detect Prey in the Dark. PLOS ONE, 2017.
3. Quorum Sensing in the Squid-Vibrio Symbiosis. Subhash C. Verma and  Tim Miyashiro. Int J Mol Sci. 2013 Aug.

Botulinum toxin found in new, common bacteria

Clostridium botulinum, Photo: Eye of Science

Evolutionarily, horizontal gene transfer between bacteria is generally not so groundbreaking. In this case however, it’s a little more newsworthy. A lot of bacteria carry plasmids, which are mobile little DNA structures that are not part of the bacterial chromosome and can be transferred to other bacteria. Often plasmids contain genes that may provide some selective advantage (otherwise why bother keeping them?), such as a toxin or an antibiotic resistance gene. Enterococcus happens to be a frequent plasmid-carrier, which makes it an especially good candidate for frequent horizontal gene transfer. But first, Clostridium. 

Clostridum is an especially fun group of bacteria including some really famous germs:

• C. tetani (Tetanus),
• C. difficile (the bacteria in your gut that gives you stomach issues after you take antibiotics as it swarms in on all the spaces other bacteria used to be, before they were killed by antibiotics),
• C. perfrinigens (perhaps the nastiest of all, gas gangrene),
• the less famous C. sordelli (which is a rare–but plenty horrific, pregnancy and abortion-associated infection almost always resulting in death by toxic shock syndrome)
• And of course, C. botulinum, the primary culprit of botulism.

What is Botulinum toxin?

C. botulium is known for releasing botulinum toxin (BoNT), a toxin which interferes with the release of the neurotransmitter—acetylcholine, from axon terminals at the neuromuscular junction resulting in flaccid paralysis (you can’t breathe).

The toxin (a protein) works by binding to receptors at the neuromuscular junction. It is then taken into the cell by receptor-mediated endocytosis. A conformational change occurs inside the early endosome (due to a pH change). A piece of the protein (the light chain) is translocated to the cytosol when another piece of the protein (the heavy chain) forms a sort of channel. This light chain can then cleave these important complex-forming “SNARE” proteins, which are vital to normal vesicular transport and neurotransmitter (which in this case is acetylcholine) release. So without these SNARE proteins, the little acetylcholine vesicles don’t get to where they need to be. In normal neurotransmitter release, SNARE proteins would work to tether the vesicles to the membrane so the cell and vesicle membranes could fuse to allow the release of neurotransmitters.

Visualized by me on Pymol- PDB file: 3BTA, BoNT, Serotype A. You too, can play around with it in pymol/swissPDB/Chimera/some other visualizer!

Botulinum toxin is the deadliest toxin known, requiring just two billionths of a gram to kill one person. It’s so dangerous that when type H (one serotype of the toxin) was discovered and failed to be controlled by typical botulinum antibodies, the type H toxin became the only example of a genetic sequence that was hidden from public databases due to security concerns. I say “may be” because how would we really know?

Not just in C. botulinum anymore

While we used to take comfort in knowing we could probably avoid Clostridium botulium by just avoiding puffed up canned food, rotten meat, and organic honey, that may no longer be the case. For the first time, the botulinum toxin has been found in a completely different (and very distantly related) bacteria: Enterococcus.

This new variant of the toxin called BoNT/En, was found in South Carolina, in an E. faecium strain isolated from cows. And it wasn’t just the toxin that was found. Several associated proteins that prevent the toxin from being degraded were also found. That being said, the BoNT/En variant didn’t give the cow it was found in, botulism, and it was not actually very harmful initially in mice. Researchers had to manipulate the toxin to better target mice before it was able to actually kill them (I know. Trying to give mice botulism is upsetting, and the reason why I stick to cells and microbes for my research). They are testing the toxin on human neurons to see how toxic it is to humans.

Enterococcus

What makes this particular jump so terrifying is that Enterococcus is a highly abundant group of bacteria all over our bodies. It is ubiquitous in animals and actually a significant human pathogen. E. faecium is a very hardy bacterium easily evolving antibiotic-resistance and actually pretty difficult to get rid of in hospital surfaces. The thought of a dangerous neurotoxin being easily transferred to a ubiquitous and antibiotic-resistant bacteria is somewhat frightening. That being said, plasmids containing incredibly dangerous neurotoxins would likely need to provide a selective advantage to the host to stick around (and it’s possible in E. faecium’s case, it could even provide a slight disadvantage). BoNT can be carried in a bacteriophage, in a plasmid, or on a chromosome, but carrying a sizable gene cluster you don’t need in evolution has usually resulted in a “you don’t use it, you lose it” outcome. As plasmids by themselves are slightly disadvantageous to carry, what keeps them around is any selective advantage the genes they contain provide.

Where did these toxins come from?

Researchers found that the homolog of BoNT in Clostridium, seemed to be a flagellin gene. These flagellins contain collegenase which breaks down peptide bonds in collagen, so they are also a proteolytic toxin family like the Clostridia neurotoxins. They believe that the neurotoxin and adjacent genes, evolved from an ancestral collagenase-like gene cluster.  This gene cluster was likely duplicated and evolved separately to become the BoNT we know today.

My case against Botox: amazing, but maybe take it easy?

Botox can be useful for treating cerebral palsy, urinary incontinence, wrinkles, sweating, spasms, headaches, and tinnitus. While no one’s reportedly died from cosmetic Botox use (presumably because people are probably extra careful with such a terrifying substance), there’s a risk of the toxin spreading beyond the injection site and causing respiratory paralysis. While ordinarily I’m totally accepting of people’s desire to do strange or expensive things in the name of beauty, my (probably illogical) fear is that all it takes is one new guy thinking a smudged decimal point is a comma and you’re dead of flaccid paralysis.

Consider that a muscle with a cut nerve will quickly result in atrophy which will definitely make you look worse, not better. Not using your face muscles isn’t going to prevent you from wrinkling because the issue is not the muscles, but the soft tissue decline that comes with age. Neuromodulators like Botox are very expensive, usually require a high enough dose to actually be effective and wear off in a few months, meaning you would have to get several injections a year to stay smooth and shiny, and the repeated injections are when people tend to see more negative side effects and require a higher dose.

Paralyzing muscles over and over again is not great for you long-term so maybe try sunscreen, a nice skincare routine, and drink plenty of water instead? Mistakes happen, people misplace decimal points, I’d rather not be near it personally. Just a friendly PSA to remind you to definitely do your research if you want to mess around with botulinum toxin!

Sources:

1. Sicai Zhang et al. Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium. Cell Host and Microbe, 2018
2. P.K. Nigam, Anjana Nigam. Botulinum Toxin. Indian Journal of Dermatology. 2010 Jan-Mar; 55(1): 8–14.
3. Doxey AC, Lynch MD, Müller KM, Meiering EM, McConkey BJ. Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster. 2008.