Marked for Life

Conservationists working in Madagascar are doing the unthinkable—defacing the shells of endangered ploughshare tortoises—but it may be the animals’ last hope.

TOUGH ROW TO HOE: A male ploughshare tortoise released at Beaboaly, MadagascarLance Woolaver, Durrell Wildlife Conservation Trust

It was with a sense of great foreboding that Bronx Zoo herpetology curator John Behler complied with a mandate to return 33 young endangered ploughshare tortoises to the island of Madagascar in 1998. Behler had rescued the animals—less than half of those stolen from a conservation breeding colony in 1996—a few months earlier from a small zoo in the Netherlands, where the Dutch government had stashed them after an illegal exotic-pet dealer got cold feet. But Dutch commercial animal trader Olaf Pronk, who was well known for dealing in rare Malagasy reptiles, pushed the Madagascar government to sue to get the tortoises back.

“It became a messy legal battle,” recalls British conservationist Richard Lewis, director of Durrell Wildlife Conservation Trust’s Madagascar program, which originally bred the tortoises. “The Bronx Zoo was worried, rightly or wrongly, that the animals were going to just disappear again.”

A national Dutch court ruled that Madagascar still had a legal right to the tortoises, and required that the animals be returned to the island country. But before Behler and his colleagues complied, they made a bold move. Using a rotary electrical tool, they engraved “MEF” for Ministère de l’Environnement et des Forêts, Madagascar’s equivalent of the US Fish and Wildlife Service, on the carapace of each of the tortoises, which at the time were only the size of grapefruits. “Basically the Bronx Zoo said, ‘We’re in this legal tussle, we have to give the animals up, but we want to make sure that their resale value is trashed,’” Lewis explains.

Today, 14 years later, conservationists believe that at least 10 to 14 of those 33 tortoises are still alive. They are now just a couple years away from breeding age, and though the engravings are small relative to the size of the adult animal, they are still visible.

Of course it’s a shame that these beautiful animals have to be defaced, but it’s
done with their best interest
in mind.
—Rhett Butler, mongabay.com

“It was very controversial when he did it,” says Eric Goode of the Turtle Conservancy, which he cofounded with Behler, now deceased. On one hand, Behler’s actions defaced a beautiful and extremely threatened species. On the other, “he never wanted to have those animals enter into the trade.”

Goode, for one, is supportive of such practices, especially since the illegal pet trade continues to threaten the tortoises. “We’re at the brink with this species,” he notes. “Unless we take pretty radical measures it’s just going to go extinct in the wild.”

So Goode and his collaborators researched the exotic pet trade to determine the best way to deter smugglers. They even interviewed an infamous reptile dealer who served prison time for his involvement in the trade of Galapagos tortoises. “He basically said he would not buy [a marked] tortoise,” Goode recalls. “He [said] it was the kiss of death. If an animal is marked, especially with the initials of the government of origin, no one wants to take that and put that in their collection.”

Convinced it was a necessary step to save the ploughshare tortoise, Goode teamed up with Lewis in 2008 to start marking the shells of the animals at Durrell’s breeding colony in Madagascar, and more than 200 captive tortoises have been marked so far. The pair has also coordinated with locals, who started finding, marking, and releasing wild tortoises in January.

Like Behler, the researchers are using a rotary tool to do the engravings, which consist of MG (for Madagascar) and a number. Though it may sound invasive, the technique appears to be harmless to the animals. Tortoise shells are covered with layers of keratin, like fingernails, and the ploughshare tortoise has the thickest keratin of any tortoise in which it’s been measured, allowing the conservationists to drill quite deep into the carapace without hitting the bone below. “They’re not showing any real signs of pain whatsoever,” Goode says. “You do it, and the tortoise will just go about its day.” (Tortoises less than 20 centimeters long are marked using a primitive tattooing technique, similar to that employed by prisoners, Goode notes.)

The big question, of course, is whether it will work. “It remains to be seen,” Goode says. Some people might still buy the animals to breed them, for example, or try to find a way to conceal the mark. Others may kill the animals out of spite, as has occurred in rhinos whose horns have been cut off. “There’s no perfect solution,” he says. “It’s not going to end the trade, for sure, but we think it will at least do a lot.”

Not surprisingly, “[the project] was met with a lot of resistance,” says Goode. Even project participants have fleeting reservations. In the end, however, it’s gained the support of the conservation community. “I certainly feel that what Durrell is doing seems to make complete sense and is warranted,” says Mal Mitchell of Azafady, a conservation-oriented charity with a presence in southeast Madagascar.

“Efforts like this are a good short-term strategy,” agrees conservation journalist Rhett Butler, founder of the rainforest conservation site mongabay.com. “Of course it’s a shame that these beautiful animals have to be defaced, but it’s done with their best interest in mind.” Longer-term measures must also be taken to address the demand for such animals in the first place, Butler adds. “The obvious option would be establishing a captive breeding population for the pet trade, which would basically kill demand for wild animals.”

Microscopy Boot Camp

A researcher in Florida changes lives by showing struggling 20-somethings the ins and outs of life in the lab.

THE RECRUITS: Davidson lab members (from left to right) Riley Evans, Brittany Sell, John Allen, Kathy Malik, Michelle Baird, and Paula CranfillDavid Barfield

In the hours before they get hitched, some grooms eat, some pace, and some watch TV. Chris Murphy was in the lab.

While his bride, Brooke—a hairstylist he met while managing a pizza parlor next door to the boutique where she worked—prepared for the big day, Murphy prepared for his next experiment, plating and transfecting HeLa cells for subsequent photobleaching.

Cell culture is one of those tasks that is tough to entrust to others, Murphy explains, “as an infection can be costly.” But there were other upsides: “It was the only time I was alone the entire day,” he recalls. “It was kind of nice.”

Murphy’s wedding day was, in fact, his 300th consecutive day in the lab. Such dedication would be remarkable in a postdoc or grad student, but Murphy was neither. With an associate’s degree from a community college and two semesters of coursework at Florida State University (FSU), Murphy was a lab technician.

His boss was Michael Davidson, who runs a microscopy laboratory at FSU’s National High Magnetic Field Laboratory. The lab is a powerhouse, sporting more than 25 members and some 30 microscopes, seven of them top-of-the-line confocals. Focusing on live-cell imaging and the development and characterization of fluorescent proteins and molecular sensors, Davidson and his team have churned out more than 50 publications in the past decade, as well as microscopy-focused educational Web content for instrument manufacturers Nikon, Zeiss, and Olympus.

These days, Davidson staffs the place with people like Murphy—smart, hard-working twentysomethings who struggled in school and could use a break. “At my age, I don’t need more papers,” Davidson says. “I need to give something back, and that’s my way of doing it.”

Davidson exposes his recruits to what he calls “boot camp,” a no-nonsense environment in which they are expected to clock in daily at 8:30—not 8:45—work hard, and by the end of their tenure get a few publications. Most make it, a few don’t.

Davidson admits that he’s a micromanager, but claims to be no harder on others than he is on himself: he works 12 hours a day, 7 days a week. And Murphy, he says, was similarly driven. Tasks in Davidson’s lab run the gamut from washing dishes and feeding cells to cloning fluorescent proteins and doing microscopy. Yet he also encourages his employees to read and be independent. He trains them, but many techniques are self-taught.

The wage for that work is about $8.50 an hour, which Davidson pays out of the profits from his website contracts and photomicrograph royalties. For Murphy, a $7/hour starting salary was actually a pay cut, he says, but a worthwhile one. “It’s a bummer to make pizza for a living,” he explains. “I have an extremely active mind.” By the end of his tenure in Davidson’s lab, Murphy made $37,000 a year as a senior microscopist and had coauthored six publications. His experience helped get him his current job as a regional sales manager for microscopy camera company Photometrics, where he now makes “six figures.”

Davidson lab alumna Ericka Ramko had a similar experience. After graduating FSU with a bachelor’s degree in human sciences and “zero lab experience,” she worked at a local newspaper, but wanted get back into science. In her three and a half years in Davidson’s lab she became an expert in microscopy, tissue culture, and cloning, publishing papers in PLoS Biology and Nature with the likes of Roger Tsien and Clare Waterman. Ramko now works as a life science specialist at global lab supply company VWR, where she makes three times the salary she made in Florida.

Davidson’s lab, Ramko says, was intense, and new recruits face a steep learning curve. By all accounts Davidson is not the warm and fuzzy type. He’s direct and exacting, demanding excellence and hard work. “I describe these techs as an extension of myself,” he explains. Yet the ones who do best, he adds, are those who come to realize they really are working for themselves, not Davidson. “You have basically unlimited microscopy equipment, unlimited graphics capabilities, one of the best live-cell imaging labs on the planet, and the world’s largest collection of fluorescent proteins,” he says. “Try to take advantage of it.”

Current lab member Paula Cranfill, an FSU biological sciences graduate, has been in the lab nearly two and a half years and published three papers. She typically works between 45 and 55 hours a week, but lately it’s been closer to 80 as she finishes up photobleaching studies with Vanderbilt University researcher David Piston. For Cranfill, that time is an investment: she wants to do graduate work with Piston, and is hoping her experience in Davidson’s lab will make her a better candidate.

That’s precisely the outcome Davidson hopes for: that his techs give him a few good years, and snag a good job or career advancement in return. For many in the lab, the experience is transformative. Nathan Claxton, a senior biosystems applications manager with Nikon Instruments, worked with Davidson for 3 years. Before that, he served drinks at a local bar.

“I think about what I might have been doing if I didn’t find that [job] ad in the paper,” he says. “Would I still be bartending at the Moose Lodge? I don’t know. I just didn’t have much direction

From Squeaks to Song

House mice sing melodies out of the range of human hearing, and the crooning is impacting research from evolutionary biology to neuroscience

Flickr, dullhunk

Dustin Penn eats, sleeps, and breathes sexual attraction. An evolutionary biologist at Vienna’s University of Veterinary Medicine, he studies the intricacies of courtship, focusing on those biochemical signals that help animals choose their mates. He’s studied them in songbirds, zebrafish, and humans—but his species of choice is the house mouse, Mus musculus.

Surrounded by cages in his lab, Penn has spent years hearing mice unmelodically squeak and chirp in the background as he worked to understand how they use smell to identify their mates. So when he first learned that male mice vocalize in the presence of females—singing, out of the range of human hearing, true songs that transcend the randomness of squeaks—he nearly flipped his lid.

“House mice are one of the most extensively studied organisms on the planet, and they sing like birds, and we don’t know anything about how it’s evolved or their behavior,” Penn says. Despite specializing in the hormonal aspects of behavior, he dove right in, figuring that “after chemical signals, acoustic signals are an interesting place to go.”

Up to that point, studies of ultrasonic mouse singing had been conducted exclusively in lab mice. Reasoning that the years of breeding for other traits might interfere with the songs’ melody and function, Penn went into the field to collect wild mice for his experiments. He recorded the males’ songs and played them back for females to see whether they’d respond. And they did: wild female mice were attracted to the tune of the murine Sinatras, preferentially hanging out in front of the speaker playing the vocal tracks (Anim Behav, 79:757-64, 2010).

IN TUNE: The experimental arena Penn used to test female mice’s attraction to male singing.Frauke Hoffmann

The females also lapped at their fur, preening themselves, while listening to adult male mice sing. But they stopped the behavior when their brothers’ songs were played, even though they had never heard their brothers sing in the flesh. “This suggested that the songs were used for distinguishing kin from non-kin,” says Penn. “What we want to know now is what the females are doing with this information. Can it help them identify better-quality mates?”

Charlie Walcott, a Cornell University ornithologist who studies birdsong, was surprised that he and the bird community had not yet heard of the singing mice, especially considering how similar the functions of each species’ song appeared. “The whole idea for a birdsong is to say, ‘Here I am, I’m a male songbird; if you’re a female I’d love to get acquainted, and if you’re a male I’ll pull your feathers out!’” he says. The mouse song “certainly has to do with attracting females, and the playback experiments make that quite clear. The question that I have is: What about male-male interaction?”

Penn hasn’t delved into whether the songs are used as signals of male-male aggression, but he has started picking apart the differences in song structure. Using the same software used to analyze birdsong, he compared the songs among brothers and between unrelated mice. The brothers’ songs were very similar—and previous work in lab mice suggested that the melodies are innate—but each also expressed distinct individuality (Physiol Behav, 105:766-71, 2012). These vocal “fingerprints” could be used to help mice tell one another apart, Penn says, and he plans to look into it.

Spectrographic analysis of the vocalizations also presented a fresh opportunity to scrutinize whether they really should be described as songs or just as patterned squeaks. “If they weren’t up there so high” in the ultrasonic frequency range, “I wouldn’t know that they weren’t birdsongs,” says Pam Rasmussen, an ornithologist at Michigan State University. “There’s so much we don’t know about groups we think we’re familiar with.”

Although mouse songs haven’t quite taken the ornithological community by storm, people who study the mouse brain are all ears. Neurobiologist Tim Holy, who published the 2005 paper that inspired Penn (PLoS Biol, 3:e386), hasn’t personally touched mouse-song research since. But his postdoc Terra Barnes at Washington University in St. Louis picked up where he left off. Using mice that are genetically modified to sing a disrupted song, she is trying to model stuttering, and she says other researchers are using the mice to better understand neurological diseases.

“A lot of really good research has been done in birdsong as it relates to the basal ganglia,” the region of the brain associated with speech disorders as well as diseases such as Parkinson’s and Huntington’s, Barnes says. However, bird brains are quite different from mammalian brains. “If we could join the two fields, taking what’s been learned in birdsong and applying it to mouse song, that would be really cool.”

It’s Raining Mice

GERONIMOUSE! Managers throw mice toxic to brown tree snakes from a helicopter.USDA APHIS Wildlife Services

A new brown tree snake control strategy takes to the skies as scientists scatter toxic rodents over Guam’s forest canopy.

Guam has brown tree snake problems. Alien, secretive, and extremely hungry, the reptiles snuck onto Guam as unintentional cargo on docking ships decades ago, possibly as early as the 1940s. And now snakes bite sleeping babies, cripple electrical grids by slithering into conductors, and wreak ecological havoc.

Although most island residents never encounter the nocturnal tree-dwelling snakes, they’ve have had a particularly woeful impact on Guam’s native bird, lizard, and flying-mammal species.
The Micronesian kingfisher now exists only in captivity, and the Guam flycatcher is extinct. Only one of Guam’s three native bat species has been sighted since 1968, and endemic lizards have been decimated.

Birds are critical for dispersing and digesting seeds, says Haldre Rogers, an ecologist at Rice University in Houston who studies the impact of bird loss on Guam. Rogers’s research has shown that seeds on Guam fall to the ground rather than being carried through the forest by birds, which means the forest may be shifting toward small homogeneous pockets of plant species rather than the robust, mixed plant communities seen on nearby, snake-free islands. Reductions in birds also allowed the island’s spider populations to explode.

To try and turn this tide of disruption, wildlife managers have deployed traps, detector dogs, barriers, and poison. But these efforts are labor-intensive and difficult to implement in deep forests, where most of the snakes can be found.

Now scientists with several US Department of Agriculture (USDA) agencies, including the Animal and Plant Health Inspection Service (APHIS), and its research arm, the National Wildlife Research Center (NWRC), together with the Guam Wildlife Services Program, are trying another angle: death from above. With grants from the Department of Defense, the researchers are testing a strategy that involves showering brown tree snake habitats with toxic mice.

FOREIGN INVADER: A brown tree snake, Boiga irregulariswikipedia, Soulgany101

An 80 mg-dose of acetaminophen, which is toxic to snakes, is stuffed inside the frozen carcass of a mouse neonate. The neonate is glued to a “flagger,” a cardboard square, attached to a “four-foot long streamer, like heavy-duty toilet paper” culminating in another unbaited flagger, says USDA Wildlife Services supervisory wildlife biologist Craig Clark. When the mouse is tossed from a helicopter, “it sort of rainbows,” he says, describing the arc of the poisonous mouse, and the streamer tangles in the forest canopy where the snakes reside. USDA wildlife biologists performed one drop in September 2010 as proof of principle and are planning a larger operation this year.

It’s the culmination of decades of research, says William Pitt, who coordinates brown tree snake research for the USDA. After USDA researchers showed, in a 1997 study, that dead mice were just as effective as live mice at baiting snakes, acetaminophen was identified as a possible brown tree snake poison. Then, in 2001, NWRC researchers demonstrated that seeding traps with acetaminophen-laced mice reduced the number of nontoxic mouse baits eaten by snakes by up to 80 percent.

Managers first attempted to scatter mice from the sky in 2002, but aerial bait dropping came with its own challenges. Paper parachutes were initially used to tangle the mouse baits in treetops, but in 2007 scientists tested hardier cardboard streamers. Bait persistence in the environment is a problem, says Pitt. The mice themselves only last about three days, prompting scientists to begin hunting for some kind of inanimate bait that will attract snakes for longer periods. The streamers are a different problem. Scientists hope to find more biodegradable materials, but they too must persist long enough to keep the baits in the treetops. Ground-dwelling species, such as coconut crabs, will scavenge mice that fall to the forest floor.

After tinkering with mechanical methods of deploying the mouse baits, it’s now done by hand. The mice are arrayed on trays, and one or two people sit in the back of a helicopter and drop the bait out. “You grab them and count: one thousand, two thousand…” to disperse the mice evenly, says Daniel Vice, the assistant state director for the agencies working to control brown tree snakes on Guam. The right cadence of counting and tossing was determined by driving a pickup truck at the appropriate speed through an empty field, explains Clark. An apparatus the team previously used to spray toxic mice into the forest caused the baited streamers to stick together, dropping two or three of them at a time, or tangling them in the wheels of the slow-flying helicopter. “It looked like toilet paper was stuck to the helicopter,” Vice recalls.

The next drop will tell scientists more about the challenges of scaling up the operation. They’ll drop mice over fenced and nonfenced areas of 100 acres each. Baited (but unpoisoned) ground traps will be set, and biologists will track success at eliminating snakes by measuring how many traps are emptied. This attempt will cover several hundred acres, with a field biologist sitting in the back of the helicopter, counting and tossing.

An island-wide mouse drop using a mechanized deployment system is in the works, but for now the cumbersome manual drops are the best strategy, says Pitt. The principle has been proved, and current work is focused on smoothing out the kinks: “It’s definitely working,” he says.

Mighty Moth Man

An evolutionary biologist’s posthumous publication restores the peppered moth to its iconic status as a textbook example of evolution.

SALT AND PEPPER: Camouflage is the key to peppered moth predator avoidance.Michael Majerus

It must have seemed terribly ironic to late University of Cambridge evolutionary biologist Michael Majerus, after dedicating nearly half a century to the study of peppered moths (Biston betularia), that in the late 1990s his name became central to an increasingly contentious campaign to strip the peppered moth of its status as the prime example of Darwinian evolution in action.

It’s a well-known story: The moth’s ancestral typica phenotype is white with dark speckles. In the decades following the Industrial Revolution, a new, soot-colored form, known as carbonaria, flourished and displaced the typica moths in the heavily polluted woodlands of Europe.

Although scientists hypothesized as early as 1896 that the increase in carbonaria frequency could be explained simply by the fact that soot-covered tree barks camouflaged the dark-colored moths against predation by birds, it wasn’t until the 1950s that an Oxford University lepidopterist named Bernard Kettlewell performed the key experiments that provided persuasive evidence that bird predation was indeed the selective agent at work.

Kettlewell placed live carbonaria and typica moths on tree trunks in polluted and unpolluted woodlands in the U.K. and counted how many of each type survived predation. Moths with a coloring that blended better with the tree trunks survived in greater numbers.

But beginning in the 1980s, peppered-moth experts, including Majerus, began noting flaws in Kettlewell’s experimental designs. The most serious of these was that limited research into peppered moth behavior seemed to suggest that tree trunks were not the insect’s preferred resting place. That alone threatened to put a serious dent in the validity of Kettlewell’s setup—and in the bird predation theory itself.

In his 1998 book, Melanism: Evolution in Action, Majerus discussed these shortcomings in the context of a critical dissection of all the peppered-moth case evidence that had accumulated.

MOTHS ON TREES: Majerus confirmed that wild moths (typica above and carbonaria below) did indeed rest on tree trunks.Michael Majerus

For Jerry Coyne, a University of Chicago evolutionary biologist who had been teaching the case to university students for years, learning that “Kettlewell’s experiments weren’t really that carefully done” came as a shock, he says. In a review of Majerus’s book, published in a November 1998 issue of Nature, Coyne concluded that “for the time being we must discard Biston as a well-understood example of natural selection in action.”

Coyne’s review dismayed Majerus, who, despite his criticisms, did believe there was strong evidence to back the case for the peppered moth as evolution’s poster child.

In no time, the popular media as well as the creationist movement pounced on the review, calling the classic peppered moth story “fraudulent,” a “blunder,” and an “embarrassment” to science. Even The Scientist contributed to the hysteria, publishing a 1999 opinion piece by then-cell biology postdoc and current intelligent design advocate Jonathan Wells on rethinking the peppered moth story.

“Through innuendo and through public smear campaigns, this case study was put into disrepute undeservedly,” says Ilik Saccheri, an evolutionary biologist at the University of Liverpool.

Not one to shy away from controversy, Majerus was nonetheless distressed by the fallout from Coyne’s book review. “[Majerus] was a cocky bastard,” says Laurence Cook, a retired geneticist from the University of Manchester. “He liked to have arguments of this kind around. But I think he did feel, because what he had written had been misinterpreted, that it was up to him to try and put it right.”

And that’s exactly what, in 2001, Majerus set out to do.

Over the course of 7 years, Majerus systematically recorded the fates of 4,864 peppered moths released into a 2.5-acre plot of unpolluted rural land in Cambridgeshire, U.K.

This time, instead of artificially placing the moths on tree trunks during the daytime, as Kettlewell did, Majerus released the moths within the hour before sunset into netting sleeves set up around the lateral branches of trees—which, through years of observation, he had identified as the moth’s preferred resting site. (He somewhat vindicated Kettlewell by observing that wild moths could in fact be found resting on trunks at least a third of the time.)

Majerus would then leave the moths to flutter about inside the enclosure throughout the night, removing the netting only after sunrise, when the moths were already settled in their resting positions for the day. After 4 hours or so, he counted the number of moths that were still at their resting sites. Those that were missing were presumed eaten. He also saw some of them being eaten by birds.

The results of his ambitious predation experiment showed that, in the unpolluted parkland of Cambridge, carbonaria moths had a daily survival rate 9 percent lower than that of their light-colored kin. This was significant enough to explain rapid directional changes in color frequencies.

In 2007 Majerus presented his results at a conference in Uppsala, Sweden. But before he could publish them, he passed away from a sudden and aggressive case of mesothelioma in 2009.

After realizing that Majerus’s results would not be published, Cook, Saccheri, and evolutionary biologists James Mallet from University College London and Bruce Grant from the College of William and Mary published the detailed account of Majerus’s work in the journal Biology Letters in February (Biol Lett, doi:10.1098/rsbl.2011.1136, 2012). “We really felt that we had a duty to try and make sure that all his efforts were not wasted,” Cook says.

Upon seeing Majerus’s results in a peer-reviewed journal, Coyne resumed teaching the peppered moth story after a 14-year hiatus. “He did everything he needed to do to take care of the problems in Kettlewell’s experiment,” he says.

Correction (May 4, 2012): This story has been updated from its original version to correctly state that Kettlewell used only live moths in his experiments, not live and dead moths as originally stated. The Scientist regrets the error.

The Sound of Color

A completely colorblind musician and painter perceives the world in a new way with help from technology

ORANGE TONES: Neil Harbisson wearing his “eyeborg,” a device that converts light waves into vibrations that lend a touch of color to his worldDan Wilton/RedBulletin

As a kid growing up in Barcelona, Spain, Neil Harbisson could tell you that the sky was blue, the grass was green, and a lemon was yellow. But he couldn’t tell you exactly what all those descriptions really meant. Born with a rare inherited condition similar to the one that plagued the Pacific islanders neurologist Oliver Sacks wrote about in The Island of the Colorblind, Harbisson sees only in shades of gray, and had simply memorized the colors he thought he was supposed to know. But it wasn’t until he was 11 years old that he learned that he didn’t perceive the world in the same way as most people.

“I noticed that other students at school could identify colors easier than me,” he recalls. “Then I knew there was a problem with color.”

A decade later, as a music composition student at the Dartington College of Arts in England, Harbisson discovered there was hope that he might see things differently. In 2003, he attended a lecture about using technology to change the way we see the world. After the talk, Harbisson approached the speaker, young cybernetics innovator Adam Montandon, then at the University of Plymouth, to describe his condition and ask if there might be a way to help him perceive color.

On his train ride home, Montandon thought about the possibility of using a system that would assign different musical instruments to colors—drums for red and violins for blue, for example. But realizing that this would introduce his own interpretations of color, Montandon then considered a device that would simply say the names of colors aloud, but this didn’t sit right with him either. “I wanted to give him something a bit more magical,” Montandon recalls. Finally, he thought about the physical similarities of light and sound. “Light is a wavelength that moves very fast,” he says. “[If] you slow it down enough, it stops becoming visible. It starts becoming audible.”

In just 2 weeks’ time, Montandon and Harbisson created a device that translated the light waves that correspond to different colors into sounds with different pitches. The prototype, constructed from an inexpensive computer webcam, a laptop carried in a backpack, and a pair of old headphones, was a bit “crude,” Montandon admits. “It was fairly primitive, but it was good enough,” he says.

EYES AND EARS: The original version of the eyeborg used a computer webcam to detect light, a laptop to compute the conversion to sound, and headphones to transmit that sound to Harbisson’s ears.an Wilton/RedBulletin

Harbisson tested the device in a school hallway in front of a big red notice board as a friend pointed to different objects, identifying their colors. He quickly learned the color/pitch associations. “Then he just ran off down the corridor,” Montandon says. “I couldn’t stop him. He went to listen to absolutely everything.” And when Montandon returned to the school 2 weeks later, he learned that Harbisson “hadn’t even switched off the computer.”

Harbisson listened to the colors of the houses in the street. He went to local grocers’ shops to listen to the sounds of fruits and vegetables. “It’s like listening to electronic music,” says Harbisson, who now wears a refined version of the device, which he calls an “eyeborg.”

“And it’s constant,” he adds. “I wasn’t expecting that I would be listening to colors all the time, but there’s color absolutely everywhere.”

The device has since been through many iterations, with the help of Montandon; a software developer from Kranj, Slovenia, named Peter Kese; and most recently, Matias Lizana, a computer engineering student at the Universitat Politècnica de Catalunya in Barcelona. Today, the system comprises a camera that sticks out above Harbisson’s head like an antenna, and a small computer chip that converts light to sound. Harbisson has since also forgone the need for headphones, mounting the chip to the back of his head, where it transmits the sound vibrations directly into his skull bones. “I receive color through the bone, and I’m listening to you through the ears,” Harbisson says. This helps him “differentiate what is a visual sound and what is an audio sound.”

Harbisson can also hear many more colors than the handful portrayed by the original device. He can even hear colors of wavelengths not visible to the human eye, such as those in the infrared. This year, he plans to add ultraviolet. The project is the ultimate demonstration of the promise of cyborgs, says Montandon, now a digital technologies professor at Erhvervsakademiet Lillebælt in Denmark. “It’s very easy with the technology we have now to explore different parts of the spectrum that we don’t normally experience,” Montandon adds. “You’re not just enabling someone with a disability, but you’re enabling someone to be more than a regular human.”

Of course, the device is not the most practical medical solution to color blindness, its creators recognize. First, wearing a camera around all day is inconvenient. Furthermore, the device is really designed for those with true color blindness, or achromatopsia, which affects only about 1 in 30,000 people—not for those who simply are unable to distinguish red from green, a far more common condition. And people with achromatopsia “also have a whole host of other vision problems,” says Medical College of Wisconsin neuroscientist Joseph Carroll, including involuntary eye movements called nystagmus, very poor acuity, and photophobia. “The ability to perceive color is the least of their worries,” he says. A more promising treatment for these patients is gene therapy to restore cone function, and thus increase acuity and reduce photophobia, he adds. Human trials for these treatments are expected to start this year.

That said, Carroll adds, “this is really cool and adds a dimensionality to your visual experience.” Indeed, Harbisson’s eyeborg is catching the attention of some musicians and artists. The pianist Jools Holland, for example, used a version of the device to accompany live concerts held in 2009–2010 across the U.K., Montandon says. “He likes to improvise, [so] we created a reverse system that would turn his music into colors and lights as he played.”

And Harbisson himself, who now works full-time as a freelance “colorologist,” pairing colors with music, uses the device for artistic inspiration, often painting what he hears. Listening to his world has definitely changed how he perceives it, he says. “People said that cities were gray—they’re not. They are actually extremely colorful. I’m discovering color in a different way.”

Finding Phasmids

Researchers rediscover a giant insect, thought to have gone extinct a century ago, and plan to reintroduce it to its native island off the coast of Australia.

A BIG STICK: Nick Carlile, seen here with the Lord Howe Island phasmid, discovered the thought-to-be extinct insect in 2001.Patrick Honan, Zoos Victoria

Australian government ecologist Nicholas Carlile was often asked to review requests from outdoors climbers to visit the normally restricted island of Ball’s Pyramid off the nation’s east coast. An extremely narrow, 562-meter (1,844-foot) high spire of rock, it is the tallest sea stack in the world and a perfect challenge for ambitious climbers.

Some requests were made under the guise of looking for a phasmid, a stick insect the size of a human hand that went extinct from nearby Lord Howe Island in the early 20th century after a supply ship ran aground and released a swarm of hungry—and prolific—rats. There had been rumored sightings of the insects’ skeletal remains and freshly dead specimens on Ball’s Pyramid, and climbers often claimed they were part of a scientific expedition in search of the lost species.

But Carlile, who specializes in island and seabird ecology for the state of New South Wales (NSW) Office of Environment and Heritage, had his suspicions. “You’d look at the list of people who were in the crew, and there wasn’t a scientist,” he recalls. “We knew this was just a ruse for a climbing troop.”

PHASMID HOUSE: Entomologist Patrick Honan in the phasmid enclosure he established at the Melbourne ZooNicholas Carlile, OEH NSW

Convinced that the phasmids were extinct—the bare rock of Ball’s Pyramid simply didn’t have the creature’s preferred humid habitat—Carlile and his colleague David Priddel decided they needed to mount their own survey of the island to prove it once and for all. “Then we could tell these people they had no basis for their climbing,” Carlile says.

So he gathered up a small team, planned for 4 years, then set out for Ball’s Pyramid in February 2001. The team carefully leapt from the pitching boat onto the rocky island, then traversed its jagged perimeter while slowly ascending to GanNet Green, the largest patch of vegetation on the island, about 150 meters, or nearly 500 feet, above sea level. Finding no evidence of the Lord Howe phasmid, Carlile and company began to make their way back down to base camp. During the descent, Carlile spotted some old insect excrement near a bush. Large insect excrement. But, hot and out of water, the team continued downward.

After several cups of a tea and a rejuvenating swim, Carlile decided he would return to the bush where he’d spotted the feces that night, when the nocturnal phasmids might be moving about. Just as it got dark, Carlile set out with Lord Howe ranger Dean Hiscox. When they reached the shrubs, they saw an adult female phasmid—the first sighting of the insect in nearly 80 years. “It was such an amazing experience,” Carlile recalls. “I’d not seen such a large invertebrate. [It was] like going back to the Jurassic, when insects ruled the world.”

When he returned home, Carlile contacted entomologist Patrick Honan at the Melbourne Zoo about starting a breeding program. After 2 years—“both to overcome the logistics of moving invertebrates as carry-on luggage that people weren’t allowed to open up, as well as dealing with the bureaucracy of wanting to collect what is potentially the rarest invertebrate in the world,” Carlile says—government approval was granted and Carlile returned to Ball’s Pyramid to collect four individuals; two males and two females. One pair went to Honan, while the other went to NSW entomologist Stephen Fellenberg, who had bred other species of phasmids.

ISLAND FORTRESS: Ball’s Pyramid, a rocky volcanic remnant jutting out of the Pacific Ocean, where the stick insect was rediscoveredNicholas Carlile, OEH NSW

But starting a breeding program was easier said than done, Honan writes in an e-mail. “We knew almost nothing of their biology, let alone their husbandry.” Determined to make it work, he stayed with the phasmids every night for the first month. “With only one [pair], and little chance of collecting another, we have to make sure [to get it right the] first time,” says Honan, now a manager of live exhibits at the Melbourne Museum. Today, the captive population is thriving with more than 500 individuals, and Honan and his successor at the Zoo, Rohan Cleave, have sent phasmids to other institutions to start their own colonies. In total, there are more than 1,000 adults and 20,000 eggs now in captivity all over the world, Carlile says.

Studying the phasmids has revealed “an enormous amount about a highly unusual species, whose behavior, morphology, ecology, and reproductive strategy is unlike just about anything else,” says Honan. Eventually, Carlile and his colleagues hope to use the captive-bred phasmids to repopulate Lord Howe. “The phasmid was the major consumer of vegetation on the island, and therefore a key component in the ecosystem in terms of turning over nutrients,” Carlile explains.

The Lord Howe phasmid is “a really important case study in terms of rearing and reintroduction,” says Aaron Dossey, an entomology postdoc at the United States Department of Agriculture’s Agricultural Research Service and the founder and owner of All Things Bugs, a company that promotes the use of insects as a human food source. But before the phasmids can be reintroduced, the rodents that drove them off the island in the first place must be eradicated. The project, which is slated to start later this year, will take some $10 million and 3 years to complete.

“This is an enormous task and has been in the planning stages for about 7 years,” Honan says. “It would be one of the biggest and certainly the most complicated eradication project ever.”

Once the rats are gone, Carlile will begin reintroducing the phasmid, as well as a biological control, such as an owl species, to avoid overpopulation by the phasmid. “It’s part of the rebuilding of the ecology of the island,” Carlile says. “You’ve got to deal with a number of components to get the pieces to fall back into place.”

From Bones to Brains

With the help of a mother, one researcher uncovered a common link between autism and a devastating bone disease.

Yu Yamaguchi’s Ext1 knockout mice avoid social interaction, an autism-relevant behavioral phenotype.Fumitoshi Irie and Yu Yamaguchi

In 2003, glycobiologist Yu Yamaguchi received a phone call that would change the course of his scientific career.

At the time, the Sanford-Burnham Medical Research Institute scientist was studying heparan sulfate (HS), a polysaccharide active in all animal cell types. He scoured the brain for HS and found it concentrated around synapses after they had fully developed.

Yamaguchi was surprised to hear a woman on the other end of the phone identifying herself as a mother. However, Sarah Ziegler isn’t just any mother. When she learned that her son had a rare bone disease called multiple hereditary exostoses (MHE), in which unchecked cell growth forms hundreds of painful though benign tumors in a child’s bones, she founded the MHE Research Foundation to fund scientists working on treatments. She called Yamaguchi because she thought his research could help her son and other children with the disorder.

Two-thirds of MHE cases are caused by a mutation in the gene Ext1, which normally encodes a protein that assists in the formation of HS. Ziegler asked Yamaguchi flat-out if he knew that his polysaccharide was linked to the devastating disease. “At that time I was aware of it, but I did not pay much attention to it,” he recalls. After all, what would a bone-growth disorder have to do with the synapses formed in the brain?

But Ziegler was not calling to talk about osteology. She suspected that her son not only suffered from MHE but was on the autism spectrum. She explained the symptoms to Yamaguchi: avoidance of social interaction, repetitive behavior, and communication problems. “Teachers and physicians think that the shyness or peculiar personalities [of kids with MHE] may be the consequence of this traumatic experience,” he says. “So they didn’t pay any attention to these other symptoms.”

SUGAR-FREE MICE: Normal mice display social behavior, nesting together in a corner of their cage.Fumitoshi Irie and Yu Yamaguchi

Yamaguchi developed a mouse model to study the effects of mutant Ext1in the brain. But he and his lab mates had no experience studying behavior, so they had to train themselves “entirely from scratch,” he says.

The mice seemed normal to Yamaguchi. But as they sexually matured, he noticed that they weren’t building nests like normal mice. And more complex behavioral tests detected all three core symptoms of autism. The male mice didn’t sing to females, suggesting communication issues, and were uninterested in exploring, instead repeating the same motions over and over again.

These mice don’t have autism per se, says behavioral neuroscientist Caroline Blanchard of the University of Hawaii, who also studies autism. “We call it an autism-relevant behavioral phenotype,” she explains. “They made a very good case for an autism-relevant behavioral phenotype.”

Yamaguchi found that, during social interactions, his model mice had significantly lower activation in their amygdalae, the brain region associated with emotion and memory. Zooming in even further, Yamaguchi identified a problem with the placement of glutamate receptors in the brains of these mice. Glutamate, an amino acid and the major excitatory neurotransmitter, has recently been linked to several autism-spectrum disorders, including the relatively common Fragile X syndrome.

“Just looking at electrophysiology and glutamate, you can’t say that it’s autism; it could be anything,” says pharmacologist Shashank Dravid of Creighton University Medical School. “But you could say that these mice probably have some behavioral deficits associated with social or emotional behaviors.”

That changes in such a seemingly insignificant sugar as HS could cause such a variety of problems is not entirely surprising, says Jerry Turnbull, a glycobiologist at the University of Liverpool. “Heparan sulfate is very multifactorial in its functions, so one single deficit or change in the overall structure could impact a number of different pathways.”

But Blanchard suspects that this isn’t just a coincidental finding. In her autism studies she uses the widespread BTBR mouse model, which is inbred and asocial. Recently, she found that these mice also have a problem with their HS, hinting at a common mechanism. “I don’t know if heparan sulfate is going to turn out to be the mother lode or not,” she says. “But at least for right now, it has a way of focusing our attention on the possibility of some common mechanisms that—nevertheless—can have a range of important effects. It’s a very good place for autism research to go.”

A Can of Worms

Scientists at the American Museum of Natural History use DNA barcoding to show that even sardines infected with nematodes can still be kosher.

aNDRZEJ kRAUZE

How can you tell if there are microscopic worms in your sardines? “It’s not simple,” Rabbi Chaim Goldberg says about distinguishing nematodes from other, innocuous white stringy bits in the fish. So Goldberg’s nematode-expert friend, armed with a toothpick and good light, gave him lessons in his synagogue’s kitchen over several Sunday mornings in March 2011.

Goldberg was hunting nematodes because he works for the New York City-based Orthodox Union (OU), one of the largest certifiers of kosher food in the world. Only some kinds of parasites are allowed in kosher fish, and the type of worms that sometimes show up in canned sardines might have been the kind that renders them unkosher. So Goldberg, OU’s “fish expert,” was brushing up on his worm-finding skills before he and several of his colleagues brought their problem to parasite experts at the American Museum of Natural History (AMNH) in New York City.

A complex set of rules, which are periodically re-interpreted in the face of new information but ultimately rooted in a pre-microscope, medieval understanding of the world, governs whether a food is kosher. Worms, for instance, are not kosher, says Rabbi Chaim Loike, a colleague of Goldberg’s at OU who collaborated on the nematode project. But fish infested with nematodes belonging to genus Anisakis is kosher according to the Talmud’s rules for parasites.

KOSHER CAVIAR: The capelin roe from which AMNH researchers extracted sequenceable DNAAMNH/M. Siddall

There are also three categories that can help classify whether a parasite renders a kosher host unkosher, says Loike: visible, barely visible, and invisible. Invisible organisms cannot make a kosher food unkosher. Otherwise, bacteria, which are not kosher in isolation, would render virtually all food inedible under Jewish law. So a fish that swallowed a parasite larva, invisible to anyone without a high-powered microscope, is still kosher. If the larva goes on to grow in the muscles of the fish, it’s considered “from” the fish, and the piscine host remains kosher, according to ancient Jewish texts. But other parasites, like those found in the nostrils, behind the ears (for example, in a sardine’s brain), or in the alimentary canal, clearly developed outside the fish and will render it unkosher, explains Goldberg.

Goldberg’s friend, who insisted upon remaining anonymous for the purposes of this story, conducted research showing that the worms turning up in the flesh of the sardines might have migrated there after growing to visible size in the gut, meaning they might not belong to the genus Anisakis, the larvae of which are known to develop in their host’s muscles and gut walls after the fish eats zooplankton harboring microscopic Anisakis larvae.

So Goldberg, Loike, and another OU colleague bought various brands of canned kosher-certified sardines at supermarkets and brought them to biologist Mark Siddall at AMNH. Siddall planned to use DNA barcoding—where a single mitochondrial gene is sequenced to determine species—to identify the nematode species and ascertain whether the sardines could be certified kosher.

The rabbis had arrived at AMNH after a thorough literature search and conversations with relevant researchers, who had all pointed them in Siddall’s direction, says Sebastien Kvist, a graduate student at AMNH’s Richard Gilder Graduate School who works with Siddall. “They were really well informed,” he says.

The scientists were startled at the number of tiny worms they could see in the fish samples brought by the rabbis. But there was a stumbling block—because canned sardines are brined and cooked, the nematode DNA was degraded, making it difficult to accurately sequence. Luckily, the rabbis had also brought along several pounds of fresh, uncooked capelin roe, so the researchers used the parasites in the roe to extract high-quality DNA.

But when Kvist and his colleagues started sequencing, they discovered that the database of the gene they were barcoding, cytochrome c oxidase subunit I (COI), was spotty for parasites, and the comparisons gave inconclusive results. So they also sequenced cytochrome c oxidase subunit II (COII), using universal and Anisakis-specific primers. Comparison with the more complete COII database showed that most nematodes found in the roe were indeed Anisakis larvae. This insight combined with the fact that the nematodes found in the fish were larval and therefore likely developed Anisakis-like outside of the gut, alleviated the rabbis’ worries that the sardines were rendered unkosher by the parasites.

This wasn’t the first time that OU turned to science to test food quality, says Goldberg. When high levels of tartaric acid in kosher pomegranate juice suggested the product had been adulterated with cheap grape juice, scientists were able to determine that it was actually made from pomegranates with higher levels of tartaric acid than most. But the sardines presented a unique problem. Unlike some contaminants that render food unkosher but are easily washed away, like bugs from fruit, the worms can’t be completely removed from the sardines. If the worms in the fish had not been kosher, explains Loike, kosher certification would not merely have been removed from certain brands: “we couldn’t certify any fish.”

Space Rocks

DANGER PAY: Not only do they have to brave the perils of leaving Earth, astronauts can also be prone to developing kidney stones (above) and other ailments during extended stays in space.Istockphoto.com, Pgiam

Orbiting ultrasound machines are being used to diagnose and treat astronauts’ kidney stones.

In November 1982, Soviet cosmonaut Anatoly Berezovoy, floating 475 kilometers above the surface of the Earth aboard the Salyut 7 space station, was seized by a sharp and sudden pain in his left side. For a couple of agonizing days, Berezovoy, who had spent more than 180 consecutive days in space, bent double in intense pain as crew member and onboard engineer Valentin Lebedev watched in distress.

Fearing that the cosmonaut was suffering from appendicitis, Soviet space program officials began preparing for a costly emergency evacuation. But just as they were getting ready to launch the rescue, Berezovoy passed a small kidney stone, and his symptoms dissipated.

The kidney-stone episode aboard Salyut 7 still serves as a stark warning of the unique medical challenges that human space travelers face.

Although kidney stones are commonplace on Earth—1 in 10 people get one over the course of a lifetime—they pose “a very real threat in space flight,” says Jonathan Clark, a neurologist at the National Space Biomedical Research Institute (NSBRI). Astronauts have a higher risk of developing the renal condition because the gravitational forces that stimulate the deposition and building of bones are not active in microgravity environments. As a result, bones demineralize, releasing calcium, phosphate, and other salts into the bloodstream. These salts ultimately concentrate in the kidneys, where they can crystallize into “stones.”

This risk is compounded by the fact that astronauts in the microgravity of space experience a decrease in plasma volume and often do not drink the approximately 2 liters of water necessary to stay fully hydrated, explains Jeffrey Jones, a urologist at Baylor College of Medicine and a former NASA flight surgeon. This decreases the likelihood that stones will get excreted in the urine, thus increasing the risk that they will continue growing to sizes that require surgical removal.

There have been at least 14 kidney-stone cases documented in US astronauts. Luckily, they all occurred shortly after they returned from space. But as NASA gears up for longer, exploratory manned missions beyond the Moon, the kidney-stone problem will only become more threatening.

For the past decade, the agency has been testing commercial ultrasound machines aboard the International Space Station (ISS) for monitoring a wide range of physiological processes in astronauts. “Ultrasound is so lightweight, so easy to use, and it doesn’t introduce any kind of radiation,” says Dorit Donoviel, deputy chief scientist at NSBRI. “It’s the modality of choice for doing diagnostics and all kinds of things in space.”

With funding from NSBRI, engineers Michael Bailey and Larry Crum of the Applied Physics Laboratory at the University of Washington have been adapting the diagnostic imaging tool for detecting kidney stones. Over the past 3 years, they have developed a prototype ultrasound machine the size of a desktop computer that can be used not only to spot small kidney stones, but also to apply short bursts of sound waves that push the stones out of the kidney.

“Most urologists usually send patients to an imaging clinic where an X-ray technician does the imaging of their kidneys,” Bailey says. “We wanted to make a faster and easier way to detect these stones.”

Using custom-built, user-friendly software dubbed “Rolling Stones,” the researchers can image kidney stones using the standard ultrasound black-and-white “B” mode, which creates an image of the anatomy, or the Doppler mode, which because of acoustic signals unique to kidney stones, shows them “twinkling.” Combining and tweaking these, the team developed a new imaging algorithm, called “stone mode,” which can display a stone in color against the black-and-white background of its anatomical location.

“With our technology and imaging modality, we can now see stones much easier and [see stones that are] somewhat smaller than [those in] an ordinary ultrasound grayscale image,” says Crum.

Importantly for NASA and future space flight missions, the equipment Bailey and Crum developed has an open-architecture—meaning they can adapt the hardware and software at will. “In space, it’s not just about delivering health care, we’re also trying to do research to understand some fundamental biology,” Donoviel says. “It will be nice to have an ultrasound capability that can be variable if we have additional needs in the future.”

Ultimately, the goal is to have a single ultrasound machine aboard future NASA spacecraft that can image anything from internal organs to eyes, teeth, bones, and muscles by switching probes or different software modalities.

Rolling Stones is expected to enter human clinical trials this year, pending FDA approval. Once it’s proved safe for use in humans, it will take another 1 to 3 years to adapt it for space travel.

Microbiology Goes High-Tech

ROBOT CENTRAL: A grad student examines a yeast colony array from the 192-plate carousel of one of eight BioMatrix robots housed together in Charles Boone’s lab at the University of Toronto, where his lab studies gene networks in yeast.Karen Founk

Charlie Boone’s lab at the University of Toronto doesn’t think small. His 21-member group is trying to map gene networks in yeast at the genomic scale. To do that, they introduce two different, relatively benign mutations into the same haploid yeast strain, then look for lethal combinations.

“That’s a genetic interaction,” explains Michael Costanzo, a senior research associate in the lab, “a genetic relationship that tells us that the two genes probably work together and somehow compensate for each other.”

The process is simple enough. S. cerevisiae of one mating type and bearing one mutation are mated to cells of the opposing mating type harboring a different mutation, and then driven through meiosis to create double-mutant progeny, whose fitness is assessed by growth on solid agar.

But there’s a catch: to fully map the organism’s genetic circuitry, Boone’s team must be comprehensive, testing every possible pairwise combination—some 18 million, in duplicate. At that scale, manually manipulating colonies with toothpicks and pipettors won’t work. The team needed a different kind of tool.

Enter automation.

Inspired by University of Washington geneticist Stan Fields, who first automated yeast two-hybrid analyses of protein-protein interactions, Boone worked with engineers to design custom-made robotics equipment to handle two key aspects of his workflow: replica plating and re-arraying.

“Robotics became important the minute genomes were sequenced,” Boone says, “because we now saw all the genes and we could start thinking of manipulating them all at once in various assays.”

Today, Boone’s lab is tricked out with a battery of microbiological robots (which you can check out on his groovy Sgt. Pepper’s Lonely Hearts Club Band-inspired website), including machines from Singer Instruments (RoToR, ~$80,000 USD) and S&P Robotics (BioMatrix BM3-SC ~$110,000 to $180,000, depending on options). Most labs can get away with a far smaller investment, if they need any at all. If you’ve been on the fence about whether your microbiology lab would benefit from automation, read on; we’ll walk you through some of the key considerations.

Is automation right for you?

Unfortunately, there’s no easy answer to this question. It depends on the application, the scale of the experiments, expected number of colonies and conditions (such as pairwise combinations) being tested, frequency of those experiments, and anticipated future needs—not to mention finances.

CUSTOM MODEL: Bart Deplancke of the Swiss Federal Institute of Technology worked with Tecan to build this custom robotics system.Bart Deplancke, University in Switzerland in Lausanne

Bart Deplancke, an assistant professor at the Swiss Federal Institute of Technology who uses yeast one-hybrid assays to detect protein-DNA interactions, automated his lab from the get-go. He used some 450,000 Swiss francs from his startup package and an equipment grant from the Swiss National Science Foundation to buy a custom-built Tecan robotics system to run the lab’s transformation assays. With his anticipated workload, that investment made sense.

“I envisioned screening literally hundreds if not thousands of different [DNA] elements,” he says. “If you’re only interested in, say, a couple of genes or a couple of DNA elements, then obviously this is clearly not worth it.”

Still, plenty of microbiology labs use automation for smaller-scale work. And not just for speed; automation ensures consistent quality and accuracy. Gray Crouse, professor of biology at Emory University in Atlanta, studies DNA repair mechanisms in yeast. The work involves lots of colony counting, a tedious effort that is not so much a learning experience as a time sink. Crouse felt that time could be put to better use, so he ponied up about $15,000 for an automated colony counter—a Synbiosis ProtoCOL 2. Now, counting that used to take hours is done in minutes, he says.

But Crouse’s purchasing decision wasn’t only about time savings, he says; he also factored in who was doing the counting. Undergrads, he explains, are relatively inexpensive, and they are paid to perform such menial tasks. Wasting hours of grad student or postdoc time is different, he says. “That becomes more expensive and it becomes more of an issue.”

What are my options?

Just about any routine and repetitive microbiology task can be automated. Besides the standard plate-filling robots and other liquid handlers, there are also automated colony counters, colony pickers, replica platers, and more.

At the Center for Cancer Systems Biology (CCSB) at the Dana-Farber Cancer Institute, researchers run proteome-wide yeast two-hybrid screens to identify protein-protein interactions in humans, yeast, worms, and plants. This is a group that does yeast two-hybrid work on a grand scale; David Hill, Associate Director of the CCSB, estimates the lab has performed hundreds of thousands of cloning reactions in the past few years just to feed its pipeline.

The CCSB has a pair of Qiagen Bio-Robot 8000 instruments dedicated to automating mini-prep work, plus traditional liquid-handling robots (Tecan Genesis and Tecan Freedom EVO, for which they paid approximately $250,000 each) that can duplicate plates, set up matings, cherry-pick and re-array clones, and so on—tasks that drive the lab’s yeast two-hybrid experiments. (The BioRobot 8000 is no longer available, but a comparable Qiagen BioRobot Universal System costs about $110,000, the company says.)

In cherry-picking and re-arraying, selected clones from a set of source plates are picked and transferred to fresh plates, to collect all positive clones from a primary screen, for instance. The colonies in this case are not randomly distributed, as they would be on a streaked agar plate or a pour plate; instead, they are arrayed as on a microtiter plate, from which they can easily be picked by pin tools.

TEDIUM, BE GONE: In Gray Crouse’s lab at Emory, which studies DNA repair mechanisms, Gina Rodriguez counts yeast colonies using a Synbiosis ProtoCOL 2 automated counter.Gary Crouse, Emory University

Replica plating is just what it sounds like, making a duplicate of the colonies on one plate by transferring to another, possibly at a higher density (e.g., copying four 384-well plates to one 1536-well plate), to test growth on different media, for instance.

Commercial re-arrayers/cherry-pickers and replica platers are widely available, including the Genetix QPix2, SciRobotics’ Pickolo (available on Tecan Freedom EVO robots), and the S&P Robotics BM3-BC. Some systems can handle both tasks. The BM3-BC, for instance, can be outfitted with a cherry-picking tool with 96 “separately addressable” pins.

John Reece-Hoyes, a research associate professor in the laboratory of Marian Walhout at the University of Massachusetts Medical School, uses a Singer Instruments RoToR HDA replica-plating robot in his work studying protein-DNA interactions. (It features, among other things, an optional corkscrew or beer bottle opener “free of charge,” according to Anna Singer, the company’s director.)

According to Reece-Hoyes, robotics enable the lab to work with 1,536-colony plates, a format that is simply too dense for use with manual pipettors or re-arrayers. Now, screens that used to require 40 plates can be completed with just three, he says. In one case, the lab tested 50 bait strains against some 1,000 transcription factors, a task that took a PhD student in the lab two years to complete manually, but which could be repeated in just two weeks using robotics.

Also available for microbiology labs are colony counters and automated plate pourers. For those working with standard petri plates (most robotic systems use microtiter plate-formatted dishes), the MP-1000 automated dish-filling system (for 100-mm plates, list $19,735) and MM9 bench-top media sterilizer (soon to be discontinued, but currently available for $22,615) from New Brunswick Scientific (now a division of Eppendorf) autoclave and dispense some 7L of culture medium at a time. Crouse, whose lab uses both instruments, estimates his team pours probably 300 to 600 plates per week. The system, for which he paid “a bit under $36,000,” enables him to maintain consistent volume from plate to plate, and to minimize contamination.

What are the variables

If you do decide to automate, there are several variables you should consider before making a purchase.

First, what exactly does your protocol require? Will you need to transfer colonies only from one solid medium to another (as in a mating protocol), or do you plan to migrate back and forth between liquid and solid cultures or solid cultures and PCR or sequencing reactions? Pin-based systems—picture a block with 96 or 384 nails poking out—work differently from traditional liquid handlers, so make sure whatever configuration you choose will meet your needs.

What about other processing steps, such as incubation, shaking, or centrifugation? Automating such tasks requires a more sophisticated robotics system. Deplancke’s custom Tecan installation, for instance, can transform about 750 DNA sequences simultaneously into yeast on two 384-well plates. The system features two on-deck incubators set at 30˚C and 42˚C , a centrifuge, and an arm to transfer plates from one station to the other.

Other key considerations are speed and throughput. Some robotic systems can transfer an entire plate’s worth of cells at a time using a 96- or 384-well pin array; others use 1 or, say, 8 pins (one column of a 96-well plate). Some employ disposable tips (which cost money, increase waste, and must be kept on hand), whereas others must be cleaned after every step in which the pins or tips are dipped in a solution (a necessity that adds time to each transfer operation). Also, not all systems offer “walk-away” automation. If plate stackers aren’t standard equipment, a technician will need to be on hand to exchange plates as the run proceeds.

Make sure colony counters and pickers can accurately identify your colonies of interest. Can they differentiate colonies based on size, color, sheen, or shape, if necessary? And, how quickly do they work? Tecan’s colony picker can pick at a rate of 800 colonies/hour and can integrate that step into a workflow of automated cloning experiments. But some pickers can move nearly an order of magnitude faster.

Perhaps the biggest decision is whether to invest in a more flexible, programmable system, such as a Beckman Coulter Biomek or Tecan Freedom EVO, or to stick with what Food Network personality Alton Brown calls a “unitasker”: a dedicated system with a specific function, such as a replica plater.

If you anticipate performing the same task over and over again, a unitasker might make sense, says Hill. “But your money may be better spent looking . . . at an open-architecture system that has many more capabilities and that will give you the flexibility to do more than just one thing, albeit with a steeper learning curve.” Indeed, Deplancke spent nearly a year and a half getting his custom platform just right, he says. “Everything just took more time than I anticipated.”

What about inexpensive alternatives?

Most labs have no need to automate microbiology tasks. But that can easily change. A lab dealing with just a handful of genes may suddenly find itself wanting to test a few hundred. Fortunately, there are alternatives to robotics.

The simplest option is a multichannel pipettor. It’s tedious, to be sure, but for a one-off experiment, such an approach may be sufficient. Thermo Scientific’s Matrix Equalizer Pipettes, for instance, are available in 9- and 12-channel models with adjustable tip-to-tip spacing, and the Matrix Electronic Multichannel Pipette also comes in a 16-channel configuration. Either model can accommodate up to 384-well plate formats. (See “Little Squirts,” The Scientist, February 2012.)

For block transfers of 96 or 384 colonies at once, manual plate replicators, such as V&P Scientific’s Library Copier tool, provide templates for copying one plate to another. Crouse uses a Boekel Microplate Replicator ($466 from Fisher Scientific) to transfer colonies into solution for PCR analysis. “We are not doing it at a scale where we could justify any fancy robotic equipment,” he says.

Each lab must find its own balance, all agree, and it’s not always obvious where the tipping point between manual and automated lies. Generally speaking, if a process is only performed occasionally or is only mildly tedious, it’s probably not worth it to automate. Says Reece-Hoyes, “If you are only doing a few screens or assays at a time, you could perfectly well use velvet [manual plate replicators] or 96-spot replicators that are vastly cheaper than the system that we use.”

Rapid Bird Flu Test

AITbiotech's H5N1 Bird Flu Diagnostic KitAITbiotech

New PCR assay can detect more than 40 strains H5N1 in a single go.

Researchers in Southeast Asia have developed a diagnostic kit that can detect all known H5N1 influenza strains within a few hours with almost 100 percent accuracy, according to a press release by the Agency for Science, Technology and Research (A*STAR), in Singapore, where the kit was designed. The real-time reverse transcription polymerase chain reaction (RT-PCR) assay, which is being distributed by AITbiotech, is reported to detect the more than 40 distinct strains of H5N1 that have been identified by the World Health Organization (WHO).

The new assay was designed to be compatible with AITbiotech’s existing “4-plex” Influenza Kit, and when used together will detect a range of influenza types including the H1N1 strain that caused the 2009 pandemic, H3N2, H5N1, Flu A, and Flu B.

“With this latest H5N1 assay, we can easily combine it with our previous 4-plex Influenza kit to differentiate which strain of Influenza is present with one test, giving a definite diagnosis and faster turnaround for our patients and our colleagues in infection control and public health,” Timothy Barkham a clinician at the Tan Tock Seng Hospital in Singapore who helped design the new assay, said in the press release.

The kit is currently being tested in several Southeast Asian hospitals.

25,000 Sign OA Petition

A petition to require researchers funded by US federal science agencies to share their results now has enough signatures to prompt a response from the White House.

By Bob Grant

Wikimedia Commons, PLoS

Open access advocates are declaring a small victory after collecting 25,000 signatures on a petition that urges the federal government to require scientists receiving federal research funds to make their findings available on publicly accessible websites—a policy already adopted by the National Institutes of Health. The petition made its way to the White House a week and a half ago, at which point The Guardian reported that President Obama’s science advisor, John Holdren, was meeting with open access advocates to discuss the matter.

“Requiring the published results of taxpayer-funded research to be posted on the Internet … would speed the research process and increase the return on our investment in scientific research,” reads the petition, which started circulating last month (May 13). With their stated goal of collecting 25,000 signatures now reached, the petition’s backers hope that the White House will respond to the request that other federal agencies adopt open access policies similar to that of the NIH, which requires the researchers it funds to submit manuscripts to the open-access PubMed Central archive within 12 months of publication in a scientific journal.

Illegal Genes

Wikimedia Commons

California mulls a state law that would criminalize the unauthorized sharing, storing, or analyzing of genetic data.

By Bob Grant | April 30, 2012

Late last week, a committee within the California State Senate passed legislation that would protect the genetic information of the state’s residents, according to GenomeWeb. The Senate Judiciary Committee passed the California Genetic Information Privacy Act, which would levy civil and/or criminal penalties against anyone who collected, stored, or analyzed genetic material or information from Californians without their consent.

“As genetic testing becomes more accessible there is an increased risk of this information being used without consent,” state Senator Alex Padilla, who introduced the bill, said in a statement. “We have laws to protect the privacy of our financial information, our medical records, and even the books we check out from the local library. We need genetic privacy protections because nothing is more personal than our DNA.”

The bill, which will now move on to California’s Senate Appropriations Committee for consideration, comes on the heels of a genetic anti-discrimination bill that the state passed last year. That state law, which makes it illegal to discriminate against a California citizen based on their genetic makeup, mirrors the federal Genetic Information Nondiscrimination Act, passed in 2008. Last year, Massachusetts also passed a “Genetic Bill of Rights.”

Oil Spill Changes Microbe Communities

Belleair Boulevard on Dauphin Island, Alabama, in September 2010Holly Bik

The beaches around the Gulf of Mexico harbor different nematodes, protists and fungi now than they did before the Deepwater Horizon disaster in April 2010.

More than 2 years following the largest oil spill the Gulf of Mexico has ever seen, scientists are still discovering consequences on the surrounding ecosystems. Most recently, researchers at the University of New Hampshire’s Hubbard Center for Genome Studies (HCGS) and colleagues found that microbial communities in the marine sediments around the Gulf changed significantly as a result of the disaster, according to the study published this week (June 6) in PLoS ONE.

The researchers sampled five shore sites, near Dauphin Island, Alabama, and Grand Isle, Louisiana, at two time points—just after the spill began (before oil had reached the beaches) and again in September 2010. “In that short time period, we saw a drastic change in the microbial community,” lead author Holly Bik, then a postdoc at HCGS, said in a press release. “We were shocked.”

The team found that the microbial eukaryote communities, once dominated by nematodes, as most beaches are, shifted to being comprised predominately of fungi—specifically, fungi with an appetite for oil. That is, the fungi are believed to utilize the hydrocarbons found in oil, thriving in such hostile, polluted environments.

The authors suspect that these changes in the microbial makeup of the beaches along the Gulf will impact other marine and short animals as well. Microbes “underpin the entire ecosystem,” said Bik, now at the Genome Center at the University of California, Davis. “If you knock out the base of the food pyramid, you’re not going to have food higher up in the food chain.”

Fetal Genome in Mom’s Blood

FLICKR CREATIVE COMMONS, LUNAR CAUSTIC

Scientists have sequenced the genome of an 18.5 week-old fetus using blood and saliva samples taken from the parents.

By Hayley Dunning | June 7, 2012

Researchers used DNA circulating in a pregnant mother’s blood to predict which genetic variants were inherited by the fetus, with a reported 98 percent accuracy when compared to cord blood collected after birth, according to a study published in Science Translational Medicine (June 6). The University of Washington, Seattle, team also identified 39 of the 44 de novo mutations that occurred, but also identified false positives. Their new technique has the potential to replace invasive methods of investigating the fetal genome that sample the placental tissue or amniotic fluid, which can trigger a miscarriage.

“This work opens up the possibility that we will be able to scan the whole genome of the fetus for more than 3,000 single-gene disorders through a single, non-invasive test,” co-author Jay Shendure told BBC News.

About 13 percent of the cell-free DNA in a pregnant mother’s blood plasma is from the fetus. Shendure and colleagues deep-sequenced this DNA and first compared it to the mother’s genome sequenced from her blood cells. They then sequenced the father’s genome using saliva samples to see which traits the fetus had inherited from him. After the baby was born, they sequenced DNA from the cord blood to determine the accuracy of their method.

The researchers also used a shallower DNA sequencing technique on another fetus at 8.2 weeks into the pregnancy, and predicted its inherited variants with 95% accuracy.

Because such tests may prompt parents to abort fetuses with severe developmental abnormalities, they come with a suite of ethical questions. “Catching everything in the basket” may have its drawbacks, Françoise Baylis, a bioethicist at Dalhousie University in Halifax, Canada told Nature. “As technology moves on, so does our willingness to think of difference as a defect.”

Parkinson’s Vax Enters Clinical Trials

Researchers in Vienna are starting a Phase I trial on the first ever vaccine with a potential to treat the neurodegenerative disease.

By Bob Grant | June 7, 2012

Alpha-synuclein accretions (brown spots)
accumulate in the brains of Parkinson's patientsWikimedia Commons, Jensflorian

A new vaccine that has the potential to treat Parkinson’s disease is entering a Phase I clinical trial in Vienna. The first-of-its-kind vaccine, PD01A, aims to ramp up an immune response to the alpha-Synuclein (alpha-syn) protein, which is involved in the onset and progression of Parkinson’s. Austrian biotech AFFiRiS is testing the vaccine with support from the Michael J. Fox Foundation.

“Worldwide, for the first time immunotherapy is applied for the treatment of Parkinson’s,” AFFiRiS CEO Walter Schmidt said in a statement. “PD01A is the first medication worldwide aiming for clinical efficacy by modulating the metabolic pathway of alpha-syn.”

Hat tip to Medical News Today.

The DSM-5 Alcohol Debate

stock.xchng, jsanders

The new version of the diagnostic manual will do away with alcohol dependence and abuse categories in favor of a single “alcohol use disorder” diagnosis.

The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5)—slated to be published in 2013—has been causing a stir in the psychiatric community over the proposed revisions to the previous edition. Now, that controversy continues with a new debate on how to diagnose alcohol and other substance use disorders.

Under the current DSM-IV guidelines, different criteria are used to determine whether a person has an alcohol dependence or an alcohol abuse problem. Under the new guidelines, however, the separate categories have been melded into a single “alcohol use disorder” diagnosis, which requires a person to meet two criteria out of a list of 11.

“Our goal was to try to make the criteria easier for the usual clinician to use, and so we’re no longer asking them to remember one criteria set for abuse and a separate set for dependence,” Marc Schuckit, who served as a member of the Substance Use Disorder Work Group for the DSM-5, said in a press release.

A major concern of the revision has been that it may lead to more people being diagnosed as having an alcohol use disorder—with a recent study published in Addiction predicting a 60 percent increase in the rate of alcohol-use disorders under the new diagnostic guidelines.

“The further watering down of definitional standards will make psychiatric diagnosis so ubiquitous as to be almost meaningless—and divert scarce resources away from those who do need them,” Allen Frances, professor emeritus at the department of psychiatry of Duke University School of Medicine and chair of the DSM-IV Task Force, wrote in a Psychology Today blog post.

But Schuckit asserts that an evaluation of more than 100,000 cases using both the DSM-IV and the DSM-5, did not result in a significant difference in the number of diagnoses.

CT Scans Increase Risk of Cancer

wikimedia commons, NithinRao

Exposure to doses of radiation used in some diagnostics in childhood can triple the risk of brain cancer and leukemia as an adult.

Children under the age of 15 who received two to three CT scans of the head have three times the risk of developing brain cancer as adults compared to those who have never received the scan, according to new research published yesterday (June 6) in The Lancet. In addition, children are also three times as likely to develop leukemia as adults if they receive 5 to 10 scans.

The study drew on data from nearly 180,000 patients who had CT performed between 1985 and 2002. A total of 74 of the patients were diagnosed with leukemia, and 135 with brain cancer. Although absolute numbers are small, the increase in risk over the general population justifies exploring alternative imaging options such as ultrasound or MRI, the researchers argue. “Of utmost importance is that where CT is used, it is only used where fully justified from a clinical perspective,” first author Mark Pearce of Newcastle University, said in a press release.

While the findings are in line with exposure estimates that researchers developed based on atomic bomb survivors in Japan, some experts debated whether the risks were real, given the low doses used for the scans. In an accompanying commentary, Andrew Einstein of Columbia University Medical Center, wrote that this study, and others like it that are expected to be completed in the next few years, should put an end to the debate and reaffirm that “justification of any CT scan is important because good evidence suggests that 20–50 percent of such examinations could be replaced with another type of imaging or not done at all.”

Camel Pharmacies?

WIKIMEDIA COMMONS, TOBY HUDSON

Researchers create transgenic cells that may help camels produce milk full of therapeutic proteins.

Camels’ highly adaptable nature and resistance to disease has always made them essential to desert-dwelling cultures, and with a little help from genetic engineering they may one day provide us with cheaper drugs. A team of researchers at Dubai’s Camel Reproduction Centre have created transgenic camel embryos to which they introduced non-human genes “similar to those of humans,” according to United Arab Emirates newspaper, The National. They haven’t yet been able to introduce human genes into the embryos, but the head of the Centre’s reproductive biology lab, Nisar Wani, told The National that he and his team have taken an important first step. If human genes that code for proteins such as insulin could be added, the camels could produce milk laden with pharmaceuticals to fight diabetes, obesity and emphysema.

“Patients with genetic disorders need these proteins, which are very costly today because companies are producing them by bacterial cultures in their labs,” Dr Wani said. “But if we’re successful at producing them in the milk, say in 15 to 30 litres, we can get a huge quantity of protein and that will drastically decrease their cost worldwide.”

Wani’s group is currently working on increasing the ratio of implanted embryos that survive to delivery, and introducing new genes from other species to improve milk production. Increased lactation could bring the cost of milk-borne drugs down, but Wani cautions that mass-production is still at least five years off.

The Centre’s success with camels, including sequencing its genome and producing the first cloned camel in 2009, prompted Wani to predict that this new innovation could one day make camels ideal candidates for growing human organs for transplant.

“Soon we will have organs that will be like universal tools for anybody who has a kidney failure or heart problems,” he said. “He can get the organ from the animal.”