The Universal Sense (8 page)

But as with fish, you can’t just lump frogs together. Frogs are a highly diverse order: some totally aquatic, some mostly
terrestrial; some able to sit on the tip of your finger, others over 8 pounds and a foot long. One thing that does unite them is that in all known species, their social behavior and survival are dependent on their hearing. In fact, the presence of an obvious tympanic membrane (homologous with the human eardrum or tympanum) was one of the earlier ways that scientists used to differentiate frogs from toads, and its relative size compared to the eye is still how you tell the boy frogs from the girl frogs (males’ eardrums get much larger than their eyes, whereas female frogs’ ears are more petite). However, as with many things in the classification of animals by their external characteristics rather than their genetic relatedness, this turned out to be a problem, as some frogs have completely internal ears. This is one reason that
Xenopus laevis
, an aquatic frog, was initially called the African aquatic toad: being totally aquatic, they have evolved internal tympanic disks to allow them to hear each other calling in the muddy ponds that make up their natural habitat in Africa.

Xenopus laevis
has been the darling of a lot of different types of research since the 1930s. Its eggs are very permissive structures that will express proteins transplanted from other species to create functional structures—for example, DNA that codes for pieces of cells such as neuronal ion channels will undergo translation in an
X. laevis
egg or oocyte, creating an egg with neuronal ion channels in it, allowing researchers to carry out precise studies of neuronal kinetics or pharmacodynamics not otherwise possible. Its tadpoles develop in transparent external eggs, allowing a great deal of developmental research to be carried out on them with greater ease than in a shell-covered chicken or uterus-enclosed mammal. The tadpoles themselves are also transparent, allowing the injection of dyes and tracers to determine cell fate mapping, tracing the course of development
and migration of individually labeled cells even after they divide. And
X. laevis
was the first vertebrate species to be cloned. For quite a while it was the de rigueur amphibian for molecular and genetic studies, including hundreds of studies in how ears develop. However, with increasing knowledge comes the potential for the “oops” moment. While
Xenopus laevis
led the way for some of the most important work in developmental biology, it turns out that its genetics are very odd for a vertebrate—it is an
allotetraploid
, meaning it has four copies of each gene rather than two copies, as do humans and most other vertebrates, which are diploids. This means that
X. laevis
can never be selectively manipulated to delete or “knock out” genes or create specific mutations, and some of the genetic work done with the species is now in question. Much of this work that was done in this species is now being replicated in its closely related cousin
X. tropicalis
, a smaller, shorter-lived, but diploid species.

But
X. laevis
is an interesting animal for studying hearing. Although it is an amphibian, it is totally aquatic its whole life, from limbless swimming tadpole through four-legged carnivorous (and sometimes cannibalistic) adult. Like fish, it has a lateral line system, a series of external hair cells organized in interrupted lines called stitches across its head and sides to detect changes in water movement. Young
X. laevis
tadpoles use this system to determine the direction of the water current and orient themselves toward it to help maintain buoyancy and stability. This behavior, called
rheotaxis
, is used to help them maintain not only their position within a body of water but also their position relative to other tadpoles in their school and to detect sudden changes in water flow that might indicate the presence of a predator. Adults, which can get to be up to 10 inches long and weigh over a pound, typically lie near the bottom of a murky
pond and so have limited access to light. The adult’s lateral line is used to detect the motion of small insects or fish above them, which they then rush up and grab in their clawed fingers and shove into their wide spatulate mouths, their strange upward-looking eyes almost useless until they approach the water’s surface. And like most totally aquatic animals, they have no external ears.

But the odd, flattened four-legged-fish appearance of these creatures hides the fact that they represent a major step in the evolution of hearing. While they share the fish’s saccule (which may play a role in their hearing, particularly when they are tadpoles), they also have additional inner ear organs, called the amphibian and basilar papillae, small hair-cell-rich structures dedicated to hearing underwater. The amphibian papilla consists of a membrane stretching across the inner ear with hair cells that respond to lower-frequency sounds, from about 50 to 1,000 Hz, arranged in a loosely
tonotopic
, or frequency-specific, order. The basilar papilla is a smaller cup-shaped organ full of hair cells that respond to higher-frequency sounds, typically up to about 4,000 Hz. And while the saccule is still there,
X. laevis
, unlike fish, has a middle ear, consisting of internal cartilaginous tympanic disks, homologues of our eardrums, which are different enough in density from the surrounding tissue and water to allow pressure changes from sound to vibrate them and is connected to the inner ear by a piece of cartilage called a
stapedium
.

This sounds like
X. laevis
would be a peculiar species to use if we’re trying to understand anything about humans. But strange and ancient as it is,
X. laevis
is an amazing model for acoustic social behavior, because it tells the story of sound and sex.

Xenopus laevis
frogs live for love songs. Like all frogs, they depend on
phonotropism
—homing in on the calls of the opposite
sex—to be able to find each other in the murky ponds they call home in the wilds of southern Africa (or in the somewhat less murky water of the lab aquarium). Unlike in most other species of frogs, the females do as much calling as the males, and it’s the females who are in control.
Xenopus laevis
doesn’t have complex singing apparati—it makes calls by using its laryngeal muscles to snap two cartilaginous disks together to create castanet-like clicks. It doesn’t offer much in the way of tonal repertoire, but this type of signal doesn’t require passing air over the vocal system and messing up the sound with bubbles, and clicks spread well through water, without distortion. Besides, as with all love songs, it’s in the timing. Males produce a relatively wide range of calls, from slow amplectant calls—ticking away every few seconds—when they are in a loving embrace with a female to occasional chirps (especially when they are picked up) and growling click trains. But their most important call in mating is their
advertisement call
, a half-second-long sequence of clicks, slow at first and then followed by a rapid burst, repeated at rates of up to a hundred times a minute.

An advertisement call is exactly what it sounds like—it’s a signal to try to attract females in the area and to warn off other males, and it is heard most often in response to a female’s call. Female members of
X. laevis
have only two different songs—rapping and ticking, also made up of differently timed clicks—but they control the males’ behavior. Ticking calls are quite slow, only about four clicks per second, and females sing this when they are not sexually receptive. A male hearing a ticked-off ticking female will often move away from her, for reasons that should be obvious. Rapping is a call females sing when they are sexually receptive; it is a series of clicks about three times faster than ticking, eleven to twelve clicks per second, that acts
like an acoustic aphrodisiac for any male in the area. Even playing a recording of a female
X. laevis
rapping song will make any male in the area approach the source and try to mate with whatever is making the sound. This often requires the lab tech to pry it off an underwater speaker and do extensive cleanup afterward.

While in human singing, the ability of the singer is based on a great many physiological, cognitive, and behavioral factors (especially practice, or else Auto-Tune), in frogs the males’ and females’ songs are based more on physiological hardwiring. One of my favorite lab experiments of all time was called the
vox in vitro
or “song in a dish” by Darcy Kelley and Martha Tobias. Tobias and Kelley removed the larynx from male and female frogs along with part of the laryngeal nerve. When they stimulated the nerve at the appropriate rates, they found that they could actually make the disembodied larynx create sexually specific songs without the rest of the frog, but that even by changing the stimulation rate, they could not make an adult female larynx call quite as fast as a male’s larynx. This is due to sexual differentiation in the type of muscle fibers in the larynx. Males’ laryngeal fibers are
fast-twitch
or Type II muscle fibers. This type of fiber is metabolically suited for high-speed but relatively short-duration activity. Female laryngeal fibers are primarily
slow-twitch
or Type I muscle fibers and have greater endurance but contract more slowly. The difference between the two is based on developmental exposure to sexual hormones. Exposure to greater concentrations of androgens (of which the best-known is testosterone) during development changes the type of muscle fiber that will be expressed. However, sexual differentiation of the larynx is not the driving force behind differences between male and female frog calling—it is more of a co-effect. What drives
the differences between male and female calls are the sexually differentiated vocalization regions of the brain.

Sexual differentiation of the brain has been a contentious issue in science for centuries. Early nineteenth-century anatomists cited the smaller size of the average human female brain to claim that women were inherently less intelligent; later, scientists graduated to debates on whether gender orientation is genetic or behavioral. But across all these years, the connection between sexual behavior and the brain has remained about as complicated a topic as you get in science.
⁸
And so
Xenopus laevis
, with its relatively limited but sexually differentiated vocal repertoire, is a wonderful animal for studying the basics of an extremely complicated subject. For example, if a male
X. laevis
is castrated, it stops calling altogether after about a month, but if provided with testosterone, it will begin giving out sexually appropriate calls again. If adult females are given testosterone, they will begin giving faster and faster trill calls, not able to match the highest-speed calls of normal adult males due to limits on their laryngeal muscles, but definitely masculinized. This is a different and arguably simpler system than that observed in birds and mammals, which usually only change their calling behavior in specific critical periods.

To try to get some kind of objective handle on the basic properties of the sexually differentiated brain, Ayako Yamaguchi and colleagues figured out how to remove the entire brain of male and female
Xenopus laevis
and keep them alive and active for quite a while. With the removal of all the superfluous
external gunk that animals spend so much time worrying about, an isolated brain in a dish, kept alive by an oxygenated bath of artificial cerebrospinal fluid, has time to focus on the really important things, like sex. Yamaguchi and her colleagues found that if she applied serotonin, a neurotransmitter involved in many complex behavioral tasks, the brain would begin to produce what is called a “fictive song”—sending neuronal signals from a song generator in the brain down the laryngeal nerve at rates appropriate to the sex of the frog whose brain was currently on vacation from its body. Subtle sexual differences between distribution and function of serotonin receptors, specifically those called 5-HT2sc receptors, change the rate at which signals are sent down the nerve, yielding different songs from male and female brains.

But so far we’ve still been talking about underwater life, which, as your experience with listening to the radio while you’re in the bathtub shows, is not the same as life that hears and communicates through the air. Here we move from the aquatic
Xenopus laevis
to ranid frogs such as the American bullfrog (
Rana catesbeiana
), a species that listens and makes noise both in
and
out of the water. Bullfrogs are the largest North American frog—I’ve worked with older females who have weighed over 2 pounds and had no trouble fighting me off when I tried to pick them up.
⁹

Like
Xenopus laevis
, the American bullfrog’s social behavior is centered on calling and being heard. But unlike
X. laevis
, these are animals that spend a lot of time out of the water (or at least with their heads out of water). A typical night in a bullfrog
chorus, where hundreds of males may be sitting around the edges of a pond making their “jug-o-rum” advertising calls, can reach deafening levels of 100 dB or more, a seemingly chaotic wall of sound. But it makes sense to the frogs. These calls both advertise the males’ territorial claims, warning off other males, and try to lure females to their clammy green embrace.

If you go out to a bullfrog pond on a summer night armed with a flashlight, you can get an instant cue to the differences hearing in air entails as well as a quick way to tell the difference between male and females—look at their ears.
¹⁰
Just behind their eyes, there are plate-like structures called tympani, the bullfrog equivalent of your eardrum. The female’s eardrum gets to about the same size as her eye, half an inch or so. But in a male bullfrog, the eardrum keeps growing throughout the frog’s life. A full-sized adult male’s eardrums can be an inch and a half across, sometimes giving it the appearance of wearing rather flat studio headphones. This is not a sexual signal like a peacock’s tail. The size of the tympanum gives you an idea of what frequency the ear will be most sensitive to—the larger the area, the lower the most sensitive frequencies.