At Last: Mesothele Silk

September 28, 2012

Tags: Mesothelae, Liphistius, Mygalomorphae, Hypochilus, Latrodectus, silk, proteins, evolution, Starrett, Garb, Kuelbs, Azubuike, Hayashi

I’ve been waiting for years to learn something about mesothele silk genes and silk proteins. The 90 or so species of mesothele make up the oldest surviving spider lineage. They now live only in Southeast Asia. As we explain in Spider Silk, mesotheles are sometimes considered living fossils (a term biologists don’t like nowadays; it gives the false impression that the organism hasn’t evolved at all since some distant point in the past) because they display characteristics so similar to the characteristics found in a 290-million-year-old fossil mesothele and so dissimilar from more “modern” spiders. For one thing, unlike other spiders, their abdomens are segmented, an outward display of spiders’ descent from segmented ancestors. If we want a full picture of spider silk evolution, mesothele silk genes and proteins are an obvious place to focus.

James Starrett, Jessica Garb, Amanda Kuelbs, Ugochi Azubuike, and Cheryl Hayashi have, for the first time, looked at the sequence of mesothele silk proteins by synthesizing cDNAs from the silk glands of Liphistius malayanus, a mesothele. They also looked at the sequences of silk proteins of six species of mygalomorph spider—these are the spiders in the second major spider lineage, which includes the tarantulas.

Neither mesotheles nor mygalomorphs spin the kinds of webs most non-arachnologists think of when we think “web.” They are incapable, for example, of spinning the kind of silk araneomorph spiders (the third major lineage) use as dragline or rapelling rope. (This largely accounts for silk researchers’ relative neglect of mesotheles and mygalomorphs: there’s no obvious industrial or other money-making application of this research). Instead, these spiders use silk to create linings for their burrows, or above-ground tubes such as pursewebs, or sheets that spread away from the spider’s hiding place to create a prey detector.

The research team also looked at the sequence of silk proteins produced by Hypochilus thorelli, a lampshade spider, one of the earliest-evolved still-surviving araneomorph spiders. Unlike the mesotheles and mygalomorphs, Hypochilus produces different kinds of silk strands specialized for different tasks. The researchers state that the motive for their study is the belief that “characterizing silk transcripts in mesotheles, mygalomorphs, and a basal araneomorph lineage allows for a better understanding of the evolutionary transition from substrate-borne, general-use silk fibers to aerial webs with task specific fibers spun by orb-weavers.” In other words, ancient spiders may have produced multiple silk proteins, but they seem to have used them interchangeably. Later in spider evolution, spiders use specific silks for specific jobs. Can silk protein sequences tell us how spiders got from there to here?

Previous to this research, two different classes of silk protein had been identified: spidroins, or spider fibroins, which make up the bulk of spider silk fibers, and egg case proteins (ECPs). ECPs were identified only in 2005 (whereas spidroins have been studied for decades) and only in Latrodectus hesperus, the Western black widow. (Given how few spider families have been sampled, though, it’s quite possible other spiders also produce them.)

Back in the early 1990s, Joachim Haupt and Jacqueline Kovoor conducted histology and histochemical studies on mesothele silk glands that showed that these spiders produced more than one kind of fibrous protein. What makes Starrett’s team’s findings so interesting is not just that they confirm Haupt and Kovoor’s study, but also that they found such a large variety of recognizable silk proteins in the mesothele: a spidroin plus six ECP-like proteins.

This appears to confirm the hypothesis that spidroins evolved very early in spider evolutionary history. Arachnologists have long assumed that the “original” spider produced just one spidroin, and that the genes scripting later-evolved spidroins probably evolved from the gene scripting that original spidroin. This study found just one spidroin in a mesothele, which makes that assumption seem more likely.

At the same time, the presence of the ECP-like proteins in addition to the spidroin shows that silk proteins started to diversify quite early in spider evolutionary history. These mesothele ECP-like proteins, like the mesothele spidroin, contain little repetition compared to the araneomorph black widow ECPs. But the selection pressures on mesothele silks are quite different from the selection pressures on black widow silks. Mesotheles live underground in burrows, and their eggs are laid and covered in silk underground. Mesothele silk proteins may be selected to cope with dampness or dangers such as mold or interfacing with soil or underground predators and parasites, whereas black widow silks may be selected to cope with sunlight, aridity, attack by above-ground predators and parasites, and wind forces, among other challenges.

The big question is: Why did these silk proteins start to diversify in the first place? It’s possible that mesotheles are not quite so indiscriminate in their use of different silk proteins as we think. In black widows, the ECPs knit with a specialized spidroin called tubuliform silk to form egg casing. Neither mesotheles nor mygalomorphs produce tubuliform silk. The research team believes that, because both ECPs and the ECP-like proteins are rich in the amino acid cysteine, and because cysteine plays a role in the knitting together of black widow ECPs and tubiliform spidroin, the mesothele ECP-like proteins may knit with that spider’s spidroin. Does the mesothele tailor its protein mix to lay down, for example, an egg blanket versus a trap door?

That isn’t an easy question to answer. Araneomorphs’ specialized silks are produced in specialized glands, and they can be observed as they emerge from distinct spinnerets. Mesothele glands don’t seem much different from each other. And closely observing a mesothele laying down silk requires unusual dedication: they undertake housekeeping chores in the dark, stop when exposed to light, and also, unlike mygalomorphs and araneomorphs, their spinnerets are inconveniently (for observers, not for the spiders) placed under their abdomens instead of exposed out at the end. So whether he or she chooses dissection and RNA extraction or focused observation or both, some researcher is going to have to contract an obsession with this question before we get an answer.

It’s also possible that the early diversification of these proteins is related to the different amounts of energy required for their synthesis. Spiders not entirely dependent on any one protein in their heavy dependence on silk may have been able to weather fluctuations in prey capture more easily.

What is clear is that this early diversification of silk proteins shows once again that silk genes are evolvable; that is, they have a propensity for evolution—they can duplicate and change slightly but still be useful. Eventually, under the right circumstances, accumulated changes may result in proteins suited to specific tasks.

The research team’s analysis of the mesothele spidroin, the newly found mygalomorph spidroins, the newly found Hypochilus spidroin, and previously known spidroins supports this scenario. They believe that their analysis shows that the common ancestor of the opisthotheles (the combined mygalomorphs and araneomorphs) had a minimum of five related spidroin genes, meaning that this gene duplication occurred before the mygalomorphs and araneomorphs went their separate ways. Mygalomorphs, like mesotheles, have pretty uniform silk glands. So this would mean that spidroins diversified before glands evolved to be specialized.

ECPs and ECP-like silk proteins are probably also much more diverse—it strains belief that they are produced only by mesotheles and black widows, which are pretty distant relatives. It’s much more likely that, due to the way the cDNA synthesis process works, it’s possible the team sampled their mygalomorph and Hypochilus specimens at a time when the genes scripting ECP-likes or ECPs weren’t being expressed. We’ll only know for sure when genome sequences—rather than just cDNA sequences—are available for various spiders. Then we’ll also be able to see what, if any, evolutionary connections there are between ECPs and spidroins.

Besides these basic findings, this study found a number of other interesting details that should be of interest to silk specialists. And like all good research, it points the way to a number of further research questions, which the authors point out: We know that the number of repeats has an influence on silk fiber formation and mechanical properties, but what influence does the length of repeats have? Do the silks of different mygalomorphs have different mechanical properties, and how are those properties related to the molecular architecture of their silk proteins? And finally, once the mechanical properties and functional properties of mesothele and different mygalomorph silks are understood, how have those properties been influenced by the natural selection pressures exerted by life underground?

Once you get tangled up in spider silk research, there’s really no end to its fascination.

Foot Silk: True or False?

March 10, 2012

Tags: silk, tarantulas, Foelix, Rast, Peattie, Mesothelae

Last summer, a team of researchers reported they had indeed found that tarantulas secrete silk from their feet. This appeared to settle a controversy that had started in 2006 when a different team reported finding foot silk. The original finding was bound to be controversial: no one had ever before observed the secretion of silk from the feet of any kind of spider. In 2009, a team investigating the first claim couldn't find evidence of foot silk. Last summer's report, which included a micrograph that seemed to show a blob of silk protein forming at the tip of a purported foot silk spigot, appeared to validate the 2006 report. (For a discussion of these three reports and of the questions concerning silk evolution they raise, see this earlier Spider Silk blog post.)

Well, there is now more evidence that tarantulas actually DON'T produce foot silk. Rainer Foelix is a leading spider anatomist and author of the must-have Biology of Spiders. When he examined the micrographs included in last summer's report, the alleged foot silk spigots looked like chemoreceptor hairs he had studied intensively in the 1970s. Kathryn Knight summarizes what Foelix did next. He, Bastian Rast, and Anne M. Peattie report their full findings in the April 1, 2012, issue of the Journal of Experimental Biology.

Foelix, Rast, and Peattie explicity address the questions concerning silk evolution that these earlier studies raised for us. They note that--even if the silk allegedly secreted through the foot "spigots" really is silk--all the tarantulas tested tend to stay close to the ground. It's not clear, then, what survival advantage foot silk would give them, particularly when the tiny amounts produced would add little cling compared to the spiders' already clingy adhesive setae, or hairs.

Most interesting to us, the team compared the feet of the tarantulas in their study to the feet of Liphistius desultor, a mesothele. As readers of Spider Silk know, mesotheles make up the oldest extant branch of the spider family tree. They live in burrows, rarely venturing more than a few inches beyond the burrow's trap door. Foelix, Rast, and Peattie state that Liphistius has no adhesive hairs on its feet. But it does have the same hairs that earlier researchers identified as silk spigots but that Foelix et al. are pretty convinced are chemoreceptors. The tiny amounts of "silk" produced from the hairs in question wouldn't allow the mesotheles to climb, even if they wanted to.

Next step in deciding whether the hairs in question on tarantula feet are silk spigots or chemoreceptors: testing them for sensory innervation with transmission electron micrography. Down the road, we wonder whether anyone will find any evolutionary connection at all--given the evolutionary relationship between limbs and spinnerets--between the proteinaceous fluid that apparently oozes from these hairs (which are also found on the spinnerets and all extremities) and the protein silk that is secreted through spinneret spigots.

As usual, the best research leads to more questions.

Venomous Mesotheles

December 6, 2010

Tags: Mesothelae, Foelix, venom

If we're fortunate enough eventually to write a second edition of Spider Silk, we'll have to make a change: contrary to what we wrote, we now know that mesothele spiders do have venom glands.

Mesotheles are the oldest branch of the spider evolutionary tree. Arachnologists consider them "living fossils": these spiders remain outwardly virtually identical to a 290-million-year-old mesothele fossil found in France, so their characteristics and behaviors can provide insight about spiders' early evolutionary history. They share a common ancestor with the much more numerous mygalomorphs (tarantulas and their closest relatives) and araneomorphs (the "true spiders" most of us are more likely to come into contact with), which in turn share a more recent common ancestor that they don't share with the mesotheles. Mesotheles are difficult to study. They live only in Asia, they're hard to find, and they're long-lived, which means any researcher wanting to study their life cycle has to be awfully patient.

With the exception of the araneomorph family Uloboridae, (which clearly lost them during evolution) all other spiders have venom glands. So whether or not mesotheles also have them is an important spider evolution question, especially as researchers work to figure out how spiders first evolved along a different path from their closest arachnid relatives. Depending on the answer, researchers would look for the origins of the venom glands in one of two different time spans. If the mesotheles don't have venom glands, it probably means that this characteristic evolved after the original mesotheles but before the mygalomorph and araneomorph lineages diverged, between about 300 million years ago and 250 million years ago. But if all three major branches of the spider evolutionary tree (mesotheles, mygalomorphs, and araneomorphs) have venom glands, it means that the common ancestor of all the spiders probably also had venom glands. This common ancestor lived in its burrow at least 300 million years ago, and perhaps even more than 350 million years ago. (A third possibility, that the common ancestor of all spiders had venom glands but the mesotheles lost theirs during evolution, has usually been considered much less likely than these other two possibilities.)

The best single source of information on mesotheles is Joachim Haupt's 2003 monograph. In his studies, Haupt couldn't find either venom glands or the duct openings in the fangs that would indicate the presence of glands.

But now Rainer Foelix (author of the standard text Biology of Spiders) and Bruno Erb report in the latest issue of the Journal of Arachnology that they have indeed found such openings in nine different mesothele species by using stronger magnification. It wasn't easy. The openings measure only 5 to 10 micrometers in diameter (about the same size as a single red blood cell), and they lie not at the tip of the fang, where they would be easier to locate, but along its length. Having found the opening, Foelix and Erb were pretty sure they would find the gland, but this also proved challenging. Their description of how they had to tease out individual muscle fibers with watchmaker's forceps in order to expose the gland makes performing root canal look easy.






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