Salamanders have different ways of regenerating lost limbs

Regenerating complex tissues is an enviable ability. Salamanders have mastered this skill to perfection, but a recent study shows that two closely related species use different molecular strategies to regenerate their lost limbs.

The remarkable ability to regenerate body parts is fairly common amongst invertebrates. If you chop up a flat worm (planarian) in several bits, they will each grow into a tiny worm (scientists have even been able to grow flat worms from single cells!). When spiders (and some insects) amputate their own limbs because of an injury or as a defence against predators, a new limb identical to the original one will grow back. But for vertebrates like us, it’s a whole different story.

It’s well known that lizards and some other reptiles can regrow broken (or accidently squashed) tails. But the new tail isn’t a perfect replica (it doesn’t have bones or nerves), and lizards can’t regenerate limbs. In vertebrates, this kind of regeneration is unique to salamanders, and to some extent to fish and frog tadpoles (but not adults).

Lizards and geckos can regrow their tails, but the new tail 
isn't a perfect replica, and they can't regenerate limbs.

Salamanders are amphibians that live near lakes or in wetlands, but you may sometimes find them in your house or garden. They can regenerate any limb in all its complexity—with bones, nerves, muscle and skin—and no matter where the limb is amputated it will grow back exactly like the original one. And what’s even more amazing: salamanders can regenerate their limbs (and some organs) over and over again.

It’s not surprising then that salamanders are scientists’ favourite model system to study regeneration. But they come with a heavy baggage. Their genome is huge—about 10 times bigger than the human genome—and it has only recently been sequenced, and not completely. On top of this, genetic tools that insert or remove genes in salamanders are still scarce, especially when compared to other model organisms like fruit flies or mouse. Nonetheless, scientists have come a long way and we now have a good understanding of the basic steps of limb regeneration.

How to grow a new leg

Limb regeneration in salamanders (and frog tadpoles and fish) occurs in three main steps. Let’s say a salamander's leg is amputated. First, a thin layer of skin quickly covers the wound, and this is a crucial difference between salamanders and most other vertebrates, which develop thick scars. Second, this skin sends chemical signals to the cells underneath to instruct them to reverse their identity (bone, muscle, nerve…) to a stem cell-like undifferentiated state. Finally, these 'dedifferentiated' cells multiply and form the blastema—a pool of cells capable of turning into any cell type that will build a new, fully functional leg.

Diagram showing the steps in limb regeneration (credit: Whited and Tabin, Journal of Biology 2009)

The blastema is key for regeneration: if a blastema is grafted anywhere on the salamander’s body, on its back for example, it will grow a leg there. About a decade ago scientists discovered that blastema cells can also originate from ‘resident’ stem cells that hang around in tissues—satellite cells. Since then a question lingers: where do blastema cells come from? From dedifferentiated cells, satellite stem cells or both?

To answer this complex question, one would need to somehow track specific cells (like muscle satellite cells, for example) during blastema formation, which is a challenging thing to do in salamanders. But a collaborative research team from the Max Planck Institute in Dresden, Germany, and the Karolinska Institute in Sweden has now succeeded in doing just that, and what they found was quite unexpected.

"We show that in one of the salamander species, muscle tissue is regenerated from specialised muscle cells that dedifferentiate and forget what type of cell they've been, […] as opposed to the other species, in which the new muscles are created from existing [satellite] stem cells," said senior author of the new Cell Stem Cell study András Simon in a press release.

An ever so cute axolotl posing for the camera.

Simon and colleagues used genetic tricks to label muscle cells with a fluorescent marker in two closely related salamander species (newts and axolotls) and then tracked them under the microscope at different time points during limb regeneration. They showed that in newt, all blastema cells that form muscle tissue come from dedifferentiated muscle cells, while in axolotl they originate exclusively from satellite cells.

“It has always been assumed that in these animals muscle is derived from two sources during limb regeneration: satellite cells and dedifferentiation of myofibers [muscle cells]. The authors are making a radical departure from this idea,” says David Stocum, director of the Indiana University Centre for Regenerative Biology and Medicine and an expert in amphibian regenerative biology.

Limb regeneration in humans: fiction or reality?

The new findings imply that different species, even closely related ones, may have evolved slightly different ways to regenerate limbs “even though the process at an anatomical and histological level may look the same”, notes Stocum. But it remains to be understood why, and also whether this is the case for other vertebrate species. “It would be interesting to explore how similar are the mechanisms of muscle cell formation, overall blastema formation, and mechanisms of blastema development in different species” Stocum says “We might find some surprises there.”

So will it ever be possible to regenerate limbs in humans?

Frog tadpoles can regrow their limb buds but they lose this regenerative ability in adulthood, which means that the genes controlling regeneration must be shut down sometime during metamorphosis. So in theory it should be possible to trigger regeneration in frog adult limbs if we knew what’s blocking it (and we could then block that), and the same could be true for humans.

“I’m optimistic that it will eventually be possible, but how long it will take is anyone’s guess. […] Clearly, if species on this planet that have the capacity for appendage regeneration exist, understanding how they do it is a huge step forward in determining what is needed to make it happen in mammals, including humans,” Stocum says.

But keep in mind: you're not a salamander. If you were to cut your own leg off (don’t!), a thick layer of skin would close the wound and form a scar, and this would prevent regeneration (your leg would NOT grow back). Interestingly, when scientists grafted extra skin to a salamander wound after limb amputation, or when scaring was induced with genetic tricks, the limb didn’t grow back. Just as in humans.

Reference:

Sandoval-Guzmán T., Wang H., Khattak S., Schuez M., Roensch K., Nacu E., Tazaki A., Joven A., Tanaka E. & Simon A. & (2013). Fundamental Differences in Dedifferentiation and Stem Cell Recruitment during Skeletal Muscle Regeneration in Two Salamander Species, Cell Stem Cell, DOI: 10.1016/j.stem.2013.11.007

A shorter version of this article was originally published in Lab Times on the 10-12-2013. You can read it here.

Promiscuous female chickens choose who fathers their children... after sex

Sex is not much fun for female chickens. Even though they are likely to have many partners, female chickens have little choice over with whom they mate. On top of this, male chickens are anything but picky and will copulate with whoever comes their way, including their sisters. But female chickens can still have the last squawk—instead of choosing a partner, they select the sperm that fertilises their eggs.

Male and female red jungle fows (Gallus gallus)

It’s easy to understand why being promiscuous is advantageous for males: the more females they mate with, the more offspring they will produce. But female promiscuity (voluntary or forced) has long confused scientists. Mating is usually a dangerous affair for females; males are often so aggressive during sex that they seriously injure their partner. Besides, females (and ultimately their offspring) should in theory gain more from mating only with a champion male that carries the best genes—why bother with the others? In evolutionary terms, female promiscuity just doesn’t make sense. So why is it so widespread in nature?  

It appears that promiscuous females can pick who fathers their children after copulation. This so-called ‘cryptic female choice’ has been described in insects, reptiles, snails, spiders and birds. Which takes us back to chickens. After forced mating with several males, female red jungle fowl—the ancestor of the domestic chicken—can squeeze out unwanted sperm and keep only the sperm from their favourite mate in their reproductive track. Fowls use cryptic female choice to avoid inbreeding, for example, by selecting against sperm from their brothers. But it’s also possible that sperm is selected based on genetic compatibility of particular sets of genes.

Domestic chickens.

Researchers from the Universities of East Anglia and Oxford (UK) recently tested this hypothesis in fowls by looking at major histocompatibility complex (MHC) genes, which encode for key proteins involved in immunity. MHC genes come in a lot of ‘flavours’ that are linked to an effective immune response—individuals with a diverse mix of MHC genes are less likely to get sick and die from disease. 

Hanne LØvlie and colleagues asked whether fowls use cryptic female choice to make sure their offspring inherits a mixed MHC gene pool. They singly mated females with related or unrelated males after sequencing the MHC genes in all animals. They then calculated the fertilisation rate of each mating by scoring the number of holes made by sperm cells in egg yolk membranes.

The researchers found that more sperm reached the eggs when males were unrelated to the females, and this effect was even stronger when these males had a very different MHC gene mix from their partner. However, when the females were inseminated artificially, the fertilisation bias disappeared—eggs were fertilised at a similar rate by all sperm. These results suggest that female fowls somehow pick the male with the best set of MHC genes during mating, and then get rid of the sperm from other males by cryptic female choice. Evolutionary speaking, girl power wins.

References:

Lovlie H., Gillingham M.A.F., Worley K., Pizzari T. & Richardson D.S. (2013). Cryptic female choice favours sperm from major histocompatibility complex-dissimilar males, Proceedings of the Royal Society B: Biological Sciences, 280 (1769) 20131296-20131296. DOI: 10.1098/rspb.2013.1296

Images from Wikipedia Commons.

This article was originally published in Lab Times on the 19-11-2013 (print).

Cocoons protect ants from disease – and from nest eviction

For ant larvae and pupae, getting sick is a death sentence—when adult ants spot an infirm individual in their spotlessly clean nest, they simply chuck it out and leave it to die. But some pupae have worked out a way to avoid nest eviction. Scientists have discovered that in some ant species the pupae spin bug-proof cocoons that help them dodge disease.

Credit: Alexander Wild (www.alexanderwild.com)

Ants are tormented by all sorts of nasty bugs, from bacteria to fungi and parasites. Because larvae and pupae lead a sedentary lifestyle inside jam-packed nests, they’re particularly vulnerable to disease. To make matters worse, unlike the adults, larvae and pupae have thin cuticles (outer skin) that can be easily pierced through by some deadly fungi.

So adult ants have come up with a complex sanitary behaviour to protect the brood from disease. Besides keeping the nest immaculate, adult ants obsessively groom eggs, larvae and pupae to remove any trace of rubbish or microbe. In some species, they even spread disinfectant (an anti-fungal poison produced by special glands) on themselves and on the brood.

When infection can’t be avoided, adult ants take a more radical approach: they get rid of the sick, no questions asked. This extreme “hygienic behaviour,” as it’s technically called, is an effective way of containing disease outbreaks in crowded insect colonies. It was first described for honeybee colonies in the 1960s, and only recently observed in one ant species by Sylvia Cremer’s research team at the Institute of Science and Technology, in Austria.

Now, Cremer’s team reports in a new study published in BMC Evolutionary Biology that for some ant pupae, being sick is not always grounds for eviction.

Scientists have long wondered why in some ant species the pupae spin silk cocoons around their bodies, whereas in others the pupae are “naked”. In a few odd cases, ants can even swing both ways: in the same species, some pupae build cocoons but others seem to live happily without one.

Other insects, such as fleas, moths or wasps, use cocoons mostly as camouflage or for protection from predators, though some studies suggest cocoons may also work like an air conditioning system, to control the temperature and humidity around the pupae. But ant pupae are well secluded from predators and atmospheric changes inside their nests. So why do some of them bother weaving cocoons?

Credit: Alexander Wild (www.alexanderwild.com)

Cremer and colleagues suspected that ant cocoons act as shields against fungal invasion.

“The fungal infectious stages require contact with the insect cuticle […]. We therefore suspected that the cocoon silk protein would not be a good target for fungal penetration, and would represent a mechanical barrier that stops the fungus from reaching the pupal cuticle, therefore preventing or delaying fungal infection,” says Cremer.

To test this, her research team exposed larvae and pupae from five ant species (with naked, cocooned or indecisive pupae) to a highly infectious fungus—the kind that can penetrate thin cuticles— and then watched how the adults managed the outbreak.

In all species, the adults seemed to detect the fungus within a couple of days, and then quickly removed the contaminated brood from the nest. This finding shows that this type of hygienic behaviour “is actually a widespread behaviour in ants”, Cremer says.

But there was an unexpected result. Even though the contaminated brood was taken out of the nests, the cocooned pupae were often left behind.

To work out why, the researchers looked at how far the disease spread in the colonies. They found that the brood removal strategy was so efficient that in all species, only about 4% of larvae and pupae left inside the nest got sick. In contrast, most of the brood that was tossed out of the nest died from the fungal infection—except cocooned pupae.

“Ant cocoons can form a protective barrier against fungal infection,” Cremer explains. And what is even more remarkable, the adult ants seem to be aware of this, as “fungus exposure only leads to a fast and effective removal of the susceptible naked brood from the brood chamber, but not the non-susceptible cocooned pupae.”

It’s a win-win situation for the ants: the pupae don’t get sacrificed, the adults don’t waste energy carrying them around, and the colony stays safe from an epidemic.

It remains unclear though “by which mechanism the cocoon protects the pupae from infection”, Cremer notes, and this is what her team plans to investigate next.

Reference:

Tragust S., Ugelvig L.V., Chapuisat M., Heinze J. & Cremer S. (2013). Pupal cocoons affect sanitary brood care and limit fungal infections in ant colonies, BMC Evolutionary Biology, 13 (1) 225. DOI: 10.1186/1471-2148-13-225

A shorter version of this article was published in ScienceNow on 18-10-2013. You can read it here.

Seeing is believing? An optical illusion that challenges decades-old assumptions

Our brains are wired to make things up. To make sense of the physical world around us, the brain takes bits of information received from the senses and, like an artist painting a landscape, creates a unique mental picture shaped by its experiences. Without this ability to process sensory information (called perception) we wouldn’t be able to see in three dimensions, understand someone speaking in a noisy room, or even watch a film at the cinema. But there is a caveat: the brain can sometimes make mistakes, and optical illusions are one example.

Optical illusions are not only entertaining but they can also help scientists learn more about how our brains work. Researchers from the University Paris Descartes, in France, have now discovered an optical illusion that challenges decades-old assumptions about how the brain perceives movement.

You see it, but it isn’t there

Long before Walt Disney brought cartoons to the wider public in the late 1920s, motion pictures and animations were being made by quickly running a series of images in some sort of projector device. This rapid display of still images creates an illusion of movement, or as it’s technically called, ‘apparent motion’.

 

"A couple walzing" by Eadweard Muybridge (1830-1904) projected with a phenakistoscope.

Several decades of research on how people perceive apparent motion have established a few solid principles on the way our brains process movement. However, an optical illusion discovered serendipitously as a bug in a computer program recently challenged a couple of these principles.

“The minimal-motion principle has extensive support not only in psychology but also in neurophysiology, and underlies nearly every computer motion-detection and motion-perception algorithm. What we have found is a blatant violation of this principle,” says Mark Wexler, an experimental psychologist at the University Paris Descartes who first reported the so-called ‘high-phi’ illusion a couple of years ago.

In this strange illusion, when a moving scene on a screen is interrupted by a random image, the observer sees an illusory fast backwards ‘jump’, as though the image has a hiccup (you can see the illusion here). Somehow, although our eyes detect the still random image, our brain turns that visual cue into fast motion (high-phi jump). But how does the high-phi illusion challenge the minimal-motion principle?

This principle states that when we look at many different motions simultaneously (which happens pretty much all the time), our conflicted brains will ‘choose’ to see the slowest one. We can spot this effect in the barber pole illusion- the pole’s movement is horizontal and quite fast, but what we see is a vertical slow movement. In the high-phi illusion, the brain always perceives the fast jump, even though it has the choice of perceiving slow motion, or even no motion at all.

The best way to understand what’s going on is to watch the illusion in slow motion (by pressing the button on the right), which reveals what we should really be seeing.

 

High-phi illusion. The fast jumps are an illusion, in slow motion you see
the real image (Credit: Mark Wexler et al. PNAS 2013)

Breaking the limit

To better understand the high-phi illusion, Wexler and colleagues asked volunteers to watch blob patterns rotating on a computer screen and then measure the size of the high-phi jumps they saw by using a visual probe. The results of these experiments were published in March in the journal Proceedings of the National Academy of Sciences.

The team found that high-phi jumps could be triggered using different tricks, for example, by reverting the contrast of the blob pattern, or by rotating it in large steps, rather than continuously. And this is where the high-phi illusion breaks yet another paradigm.

The theory predicted that the observers wouldn’t be able to detect any movement when the patterns were shifted in large steps, above a certain cut off distance- the upper displacement limit or d_max. However, this is not what Wexler’s team found. “Below d_max, the steps should be seen as what they are, more or less. Above d_max, on the other hand, you're supposed to not perceive motion, just noise. This is not what happens, though: you perceive the high-phi jump,” Wexler explains. Strangely enough, the high-phi jumps have a maximum size that is “very closely correlated with the d_max limit: people who have higher d_max limits, also see a larger high-phi jump.”

John Perrone, an experimental psychologist from The University of Waikato, in New Zealand, who was not involved in the study says “The study describes a really interesting motion phenomenon that helps constrain theories and models of motion processing in humans. […] The authors have spent a lot of time also carefully testing the various parameters that influence the [high-phi] effect. These results will be useful to theoreticians and modellers and will help provide clues as to what mechanisms underlie the effect.”

So what are the neural mechanisms behind this optical illusion?

“Good question: if we only knew! We can only describe what’s happening: the brain seems to have a default of very fast motion. I wish that a physiologist would find a neural correlate to this effect,” Wexler says.

Reference:

Wexler M., Glennerster A., Cavanagh P., Ito H. & Seno T. (2013). Default perception of high-speed motion, Proceedings of the National Academy of Sciences, 110 (17) 7080-7085. DOI: 10.1073/pnas.1213997110

This article was published in Lab Times on 4-10-2013. You can read it here. 

Baby zebras can suckle for psychological needs, not just for feeding

All mammals are born with a sucking reflex - an instinct on which their lives depend - but human babies are unique in that they also need to suck for comfort. Or so it was thought. A new study now shows evidence suggesting that baby zebras can suckle for psychological needs, rather than just for feeding.

Grévy's zebra foal

The use of soothers is a sensitive topic amongst parents. Soothers (also known as pacifiers or dummies) comfort babies and help them sleeping, but many parents go through great lengths (and many sleepless nights) to avoid using a soother. These concerned parents may fear their baby will develop crooked teeth, or have problems breastfeeding, or they may simply find soothers unnatural. Regardless of their individual choices, all parents will agree that their baby has a need to suck- whether it’s a soother, a thumb or an old rag. 

There are many studies showing that so-called 'non-nutritive sucking' can comfort babies, help them to settle, reduce the risk of sudden infant death syndrome (SIDS) and even increase tolerance to pain. The use of soothers is therefore often recommended in intensive care units for premature babies and sick newborns who sadly may need painful medical procedures. But is ‘comfort sucking’ widespread amongst mammals or a specific evolutionary adaptation of our species?

Scientists previously assumed that the duration and frequency of suckling reflected the energetic needs of the young – the longer an infant spent suckling, the more milk it drank. But studies in mammals directly measuring infant weight gain and time spent suckling have shown no correlation between the two. So why do babies and mums across so many species invest so much time and energy nursing? One of the reasons is bonding, but there is more to it.

Grévy's zebra mare with her foals

In a new study published in the September issue of the Journal of Zoology, a research team from the Institute of Animal Science and Czech University of Life Sciences, in Prague, Czech Republic, compared suckling behaviour in three zebra species - mountain, plains and Grévy's zebras. These species are closely related but have very different social organisations, so the researchers could ask whether time spent suckling might reflect the social needs of the young. 

Mountain and plains zebras live in stable groups, or ‘harems’, of several females, their babies (or foals) and only one male, while Grévy’s zebras prefer to graze on their own and form loose social bonds. Zebras are far from being docile creatures - to defend their position in the harem social hierarchy, mountain and plains zebra females (called mares) take their gloves off and become very aggressive. Mountain zebra mares are especially hostile, and can sometimes even harass unrelated foals. Grévy’s zebras are the least aggressive of the three species, perhaps because of their more solitary nature.

The researchers observed the suckling behaviour of 30 foals of mountain, plains and Grévy’s zebras at the Dvůr Králové Zoo throughout several years. After watching the zebra herds for an impressive total of about 1,500 hours, the results were clear: mountain zebra foals suckled for longer and more frequently, followed by plains and Grévy’s zebras. As mountain zebra herds have the highest aggression rates and Grévy’s zebra the lowest, the team concluded that baby zebras spend more time suckling in species where there is higher social tension.

Plains zebra foal suckling (Credit: by Chadica/Flickr)

Because the study was performed on zebras held in captivity - and so all zebras were exposed to the same living conditions - these differences in sucking behaviour can’t be explained by water or food availability, or by a specific adaptation of each species to its unique environment. But the authors of the study are nevertheless cautious about over-interpreting their results:

“I don’t think that the foal initiated suckling to seek comfort and stress reduction. What I think is that the suckling bout duration reflects the psychological needs of the foal rather than the milk transfer. Thus, prolonged suckling can reflect social tension”, says Jan Pluháček, an ethologist at the Institute of Animal Science and leading author in the study.

Previous studies in primates and rodents had shown a link between maternal care and social organisation, but Pluháček and colleagues provide new evidence suggesting that, at least in zebras, suckling may not only be a means for the young to feed or to bond with mum- it could also reflect their psychological needs.

“We suppose that when any tension in the herd occurs then the young try to stay for longer with the mother, and longer suckling can strengthen the bond between young and mother in all mammalian species” says Pluháček “[…] in zebras 98% of suckling is initiated by the foal. So it’s the foal who seeks soothing via suckling.” 

Reference:
Pluháček J. et al. (2013). Time spent suckling is affected by different social organization in three zebra species, Journal of Zoology, DOI: 10.1111/jzo.12077

Rare embryo discovery gives hints on how dinosaurs reproduced

Sometime in the Late Jurassic era, a dinosaur nest was hit by a fatal tragedy and its eggs never hatched. Whatever killed the baby dinos - perhaps a hungry predator or a flood - was a stroke of luck for the team of paleontologists who, about 150 million years later, stumbled on the crushed eggs and embryo remains in the Lourinhã geological formation, in Portugal.  

“Most of the time what happens is that you find eggs without embryos, to find them together is really a matter of chance,” says Ricardo Araújo, lead author in the recent Scientific Reports study describing the Lourinhã fossils.

 

Lourinhã geological formation (Credit: wikipedia commons)

Findings of fossilised eggs with embryos are extremely rare - no more than a handful have ever been found. But without embryos it is virtually impossible to link an egg to a specific dinosaur species. So although many dinosaur eggs have been discovered all around the world, sadly we still know little about how dinosaurs reproduced and looked after their babies.

Araújo and colleagues at the Museum of Lourinhã didn’t immediately realise the importance of their fossil discovery; it wasn’t until they prepared the specimens in the lab that they saw tiny teeth and bones amongst the broken eggshells. “That’s when the good news really happened,” Araújo says.

The dinosaur baby teeth had nothing cute about them - they were long, pointy and sharp. Together with other bone features, the shape of the fossilised teeth gave away their identity: they belonged to Torvosaurus, the top meat-eating predator of the Late Jurassic era.

Sketch of the anterior part of the embryonic maxilla, showing the sharp teeth.
(Credit: Museu da Lourinhã)

Torvosaurus is an older, or 'primitive', dinosaur of the theropod family, which includes large carnivorous predators like the famous Tyrannosaurus and also modern birds. Even though Torvosaurus lived (and was extinct) well before Tyrannosaurus was around, it looked a lot like its cousin- it had huge jaws and walked on two legs, but it had longer and stronger arms. Torvosaurus was also nearly as big as Tyrannosaurus, measuring up to 11 meters and weighing about two tons.

Partial fossils of adult Torvosaurus have been found in North America and in the Lourinhã formation, but no embryos had ever been discovered. Up until now that is. The new specimens are the oldest theropod embryos found to date.

"Before we had examples of eggs and embryos of very advanced theropod dinosaurs, but we didn’t know anything at all of what was happening at the base of the family tree," says Araújo “[…] this finding is one of the oldest in the world, and it’s certainly the oldest for theropod dinosaurs,” he adds.

So what do these new fossils tell us about how primitive theropods lived?

By using a bunch of high tech methods, like high-power electron microscopy, the Lourinhã researchers looked in extreme detail at the microstructure of the eggshell pieces. They found that Torvosaurus’ eggs had a single structural layer, in contrast to advanced theropods that have more complex eggshells with two or even three layers (including modern birds, which are technically living dinosaurs).

It was known that primitive dinosaurs from other families had single-layered eggs, but theropods were the missing piece in the puzzle. “Now we have the evidence that eggshells of primitive dinosaurs only have one structural layer,” Araújo says. It appears that eggshell complexity increased throughout dinosaur egg evolution. But this isn’t all.

Torvosaurus is a basal or 'primitive' member of the theropod dinosaur family (Credit: Vladimir Bondar
& GEAL - CIID - Museu da Lourinhã)

The Torvosaurus’ eggshells have another interesting (and strange) feature- they have huge pores, or holes. Bird, reptile and dinosaur eggshells have pores so gases can be exchanged between the inside and the outside of the egg, so the embryos can breath. As a rule of thumb, eggs with larger pores are laid in a moist substrate, while eggs with smaller pores are incubated in nests exposed to air.

The Torvosaurus eggshells have large pores that “interconnect in a network towards the top of the eggshells,” explains Araújo “this is really different from what was found to date”. The eggshell pore size suggests that Torvosaurus buried their eggs, just like most modern reptiles. 

Another indication that Torvosaurus’ eggs were buried is the fact that the fossilised eggshells and embryos are exceptionally well preserved. "The eggshells are nearly exactly the same as they were 150 million years ago," says Araújo. Being underground would have protected the fossils from bacteria and atmospheric erosion.

“Dinosaur embryos are very rare and also challenging to identify,” says David Varricchio, a paleontologist from Montana State University “This study provides an important addition to our understanding on dinosaur reproduction.”

The Lourinhã formation is very rich in Late Jurassic fossils; it has many dinosaur nests and footprints, and countless invertebrate fossils. Araújo notes “There have been more discoveries and […] they will give further insights into dinosaurs and other types of vertebrates from 150 million years ago in Portugal. Finding other eggshells and embryo associations from other groups of dinosaurs would be really helpful to complete the picture.”

Reference:

Araújo R., Castanhinha R., Martins R.M.S., Mateus O., Hendrickx C., Beckmann F., Schell N. & Alves L.C. (2013). Filling the gaps of dinosaur eggshell phylogeny: Late Jurassic Theropod clutch with embryos from Portugal, Scientific Reports, 3 DOI: 10.1038/srep01924

This article was publish in Lab Times on the 13-08-2013. You can read it here.

The strange virus from the sewage

Viruses can infect all types of organisms. Unable to multiply on their own, viruses parasitise animals, plants, bacteria and even other viruses, in order to propagate. Bacteria-killing viruses, called bacteriophages or simply phages, are the most abundant and diverse organisms on the planet. It is estimated that there are over 100 million different phages, but only about 0.0002% of phage genomes have been sequenced. 

Sewage-polluted waters, like some lakes and ponds, are a sample haven for virologists- they are filled with organic material on which bacteria thrive; and where there are bacteria, there are bacteriophages. It was in one of these bacteria broths, about 200km northwest of Vilnius, in Lithuania, that a research team led by Rolandas Meskys and Laura Kaliniene found Rak2, a phage unlike any other.

 

Water contaminated with sewage is a sample-haven for virologists. 

(Credit: Flickr/eutrophication&hypoxia)

Merciless lifestyle
Bacteriophages latch on to bacteria and then transfer their genetic material into them. In a matter of minutes, the bacterial cellular machines replicate and translate the phage genes into viral proteins, which assemble into hundreds of new viral particles. Merciless to their host, the new phages burst the bacterium to free themselves and move on to infect other victims.

Despite the phages' tiny size - about 100 times smaller than bacteria - with the help of high-power electron microscopes (EM), scientists can see them in quite some detail. The most abundant types of phage are by far the Caudovirales, or tailed phages, which have a maraca shape, with a head (containing the genetic information) and, as the name suggests, a tail. The phage tail is a versatile lethal weapon. First, it recognises the right bacteria host by protein matching, like a barcode reading machine. Second, it works as an anchor, firmly attaching the phage to the bacterial surface. And finally, the phage tail acts as a syringe, by piercing the bacterial cell wall and pushing viral DNA through it.

It was the shape of the Rak2 phage that first intrigued Meskys and his colleagues at the University of Vilnius. "The morphology of this phage is amazing," he says. Detailed EM images revealed that Rak2 is a tailed virus from the Myoviridae family, which typically have a contractile tail with six fibres at the end. But Rak2’s tail is very special. “The EM shows that the tail fibres contain spikes, this is only known in a few phages,” explains Meskys. Rak2’s tail structure, with its spiky fibres, resembles the tails of some myoviruses, but other features, like the absence of prongs and the intricate pattern of the spikes, set Rak2 aside from any other known phages.

 

Typical myovirus bacteriophage (Credit: wikipedia)

A giant phage

The other unusual thing about Rak2 is its genome- it’s huge. With about 534 predicted genes, Rak2 is the fourth largest myovirus sequenced to date, and the largest phage known to infect Klebsiella sp. bacteria, Rak2’s only host. But size isn’t everything; Rak2’s genome is truly unique. About half of its genes don’t have any similarity to other viral genes, and a significant proportion of its predicted proteins have an unknown function. The 117 genes that do encode for well-described proteins, such as tail or DNA repair proteins, show similarities to genes of different phage families, but also to some bacterial genes. Meskys says “Philogenetic analysis shows that this phage is quite mosaic, some parts [of the genome] are more similar to the Myoviridae group and other parts to the Podoviridae group, maybe there was some horizontal transfer of genes.”

Horizontal gene transfer occurs when genes ‘jump’ from one species to another. For instance, different bacteria strains can exchange antibiotic-resistance genes between them in a process called conjugation- the closest thing bacteria have to sex. Viruses can exchange genes between them and also with their host. Instead of killing their host cell, some viruses, including phages, insert their DNA into the host’s genome so it replicates with its DNA. When the viral DNA leaves the host’s genome, it can carry along some chunks of it, or, more often, it can leave some of its own DNA behind. Because several viruses can invade the same host, genes from one virus might end up in another virus’ genome. It is estimated that a whopping 8% of the human genome is made of viral DNA, and it seems that most species, from bacteria, to mammals and plants, carry viral genes in their genome. Bacteriophages are experts in this kind of inter-species gene mixing, and Rak2 appears to have an extreme mishmash genome.

EM images of Rak2 phages (Credit: PLoS ONE/Rolandas Meskys)

An alternative to antibiotics?

Meskys plans to continue working on this phage. He would like to understand the function of its unique proteins predicted by sequence analysis. “We found a gene that predicts a huge protein with no functional homology in any other phage. What is this protein doing?” he asks. There are also potential applications for Rak2. Historically, phages have been used in medicine to treat bacterial infections, such as dysentery and cholera, but with the discovery of antibiotics this approach was mostly abandoned. Now, with the dangerous rise in antibiotic-resistance bacteria strains, phage therapy is coming back in vogue.

There are many advantages for using phage therapy. Unlike antibiotics, phages target specific bacteria strains, so the ‘friendly’ bacteria in our guts are left unharmed. If bacteria become resistant to a phage, it can quickly change to overcome the new resistance, while new antibiotics take over ten years to be developed. Besides, new phages targeting multi-resistant bacteria can easily be identified in sewage samples. 

Another key difference between using antibiotics and phage therapy is that, in contrast to antibiotics, which normally just prevent bacteria from multiplying, phages actually destroy bacteria. And they do it with finesse- at low dosage (phage dose is increased ‘naturally’ by replication in the bacteria) and with negligible toxicity for the human patient. 

A number of pharmaceutical companies are also developing phages for other applications, such as veterinary, agriculture, food control and drug delivery, just to name a few. “If we could identify which type of tail spikes are involved in the recognition of a specific bacteria strain, […] maybe we will be able to change the spike proteins so that the phage attacks other bacteria that are more important for medicine or food,” Meskys says.

A productive department

Meskys currently runs a research department at the Institute of Biochemistry of the University of Vilnius, the country’s capital. With six research groups working on several aspects of bacteriophage diversity and biocatalysis, the department operates as a huge lab. “If we have a particular problem to solve, we can involve different members of the department to solve it.” Meskys has a strong creative input in the department’s research and plays an essential part in getting intra-departmental collaborations going. “I am involved in all research groups […] I need someone to implement my crazy ideas,” he jokes. 

A biochemistry graduate, Meskys began his research career as a PhD student in Valdas Laurinavicius’s lab at the Institute of Biochemistry, where he later established himself as an independent researcher and finally was promoted to head of department in 2002. Despite having spent his entire career in Lithuania, Meskys started multiple international collaborations and has, in several instances, been invited to teach or visit labs in other countries. There are fruitful relations established in the department with local and foreign biotech companies. “We are cooperating in the screening for new enzymes for chemical synthesis, diagnostics, food processing etc. Our expertise is in development of new screening technologies,” he says.

Laura Kaliniene, lead author of the Rak2 study, holding a phage plate.

Research in Lithuania

The main source of research funding in Lithuania is the Lithuanian Research Council (LRC). Like many research institutions in Europe, the LRC gives priority to applied research. Most research grants are allocated to projects with potential industrial applications, or to groups with high number of publications and patent submissions. “There is pressure to show that you are achieving something,” says Meskys, but there are also smaller grants for projects “where you can do what you want,” but the competition is high.

Despite Lithuania’s fast growing economy, rising unemployment and low salaries continue pushing highly skilled Lithuanians abroad. "We are losing the bright and intelligent people, emigration is a huge problem for Lithuania.” Meskys adds that there is a ‘narrow market’ in research in Lithuania, so students prefer to do their PhDs in countries like the UK, Denmark or the USA. But there is some world-leading research in Lithuania, especially in the fields of biochemistry and laser technology (a certain type of laser produced in Lithuania accounts for 80% of the world market), and the number of biotech start-ups is on the rise; for example, Fermentas, which was bought by Thermo-Fisher in 2010, was originally a Lithuanian company. “Some research fields are well established, Vilnius University is more than 400 years old, […] in some specialities there are long traditions.”

Reference:

Šimoliūnas E., Kaliniene L., Truncaitė L., Zajančkauskaitė A., Staniulis J., Kaupinis A., Ger M., Valius M., Meškys R. & van Raaij M.J. & (2013). Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae, PLoS ONE, 8 (4) e60717. DOI: 10.1371/journal.pone.0060717.s003

This is a modified version of my article published in Lab Times on 5-07-13. You can read it here.