Wednesday, July 30, 2014

Does Life Come In XXXS?

Biology concepts – characteristics of life, archaea, bacteria, mycoplasma, synthetic biology, symbiosis, parasitism, nanobacteria, genome

As part of this blog, we have talked about some pretty small life. Wolffia globosa is the smallest flowering plant, only 0.6 mm long. We also talked about archaea, a different kingdom than bacteria, but still on the smallish side of life. The tardigrade is the toughest animal, but is also one of the smallest, at 100 µm (0.00394 inch).

The organism on the top is T. dieteri, and arthropod, just
as is any crab or spider. The size is deceiving. The pictures
on the bottom are to scale and are copepods, also
arthropods. The organism on the top is a parasite of the
organisms on the bottom. The small blue line? That would
be the scaled size of T. dieteri. So…. it’s SMALL.
The question for today is – is there a minimum size for life? Candidates might include bacteria or archaea; heck there’s an arthropod, Tantulacus dieteri, that's only 85 µm long! As long as we can keep finding smaller and smaller cells, we know that the minimum size for life is that small or smaller. So we keep looking – you’d be surprised how important it is to keep looking for smaller life.

Here’s one thing we should be able to agree on, viruses don’t get to play in our game. Viruses are very small, but they're not life! We’ve talked about this before - the seven characteristics of life (see this post). Viruses need a host in order to replicate, they don’t manage homeostasis, and they aren’t cells, so they aren’t life.

So how small has actual life become? Let’s assume that since tardigrades and T. dieteri are over 50 µm, huge when compared to some bacteria, our current minimum for life is probably a bacterium or archaea.

Let’s go straight to the genus of smallest bacteria we know about – the mycoplasma (from mykes = fungus, and plasma = formed). They were first described in 1898, but the observer didn’t have a clue what he was looking at; hence the fungal part of the name.

Mycoplasma don’t have the traditional cell wall of many bacteria, so they look different and this might be why they were mistaken for fungi. Whatever the scientists thought of them, they were confusing enough to be roundly ignored for 50 years. Rediscovered in the 1950’s-1960’s, this time they were thought to be L-forms of bacteria. L-forms are organisms that for some reason have lost their cell wall.

There are stable forms of L-bacteria; they can live divide and live on without their cell wall. There are also unstable L-forms as well; those that may revert to walled bacteria at any moment. Are mycoplasma simply bacteria that have lost a cell wall? Nope. They didn’t have a cell wall to lose. They have no cell wall genes, so if they had a cell wall, it was millions of years ago, before they became their own genus.

The difference between some free living cells. You can
probably see the E. coli in bright green, but you may have
to squint to see the mycoplasma above it. It’s pink. Really,
it’s there. Compare these sizes to those of the arthropods
above. 1 mm is equal to 1000 µm.
Mycoplasma is really, really, SMALLLLLLL.
Mycoplasma are generally described in the range of 0.2-0.8 µm in diameter. But this is a little misleading, because they are often not spherical. Even without a cell wall, they can take interesting three-dimensional forms and maintain them. Mycoplasma pneumoniae, which causes a form of …..….. anyone?…….. right, pneumonia, is pear shaped, so its 0.25 µm diameter is actually the measurement on its short side.

So mycoplasma are small, but they still have to play by the rules. They contain DNA and salts and proteins and ribosomes and other things that take up room. A single ribosome is about 50 nm in diameter (0.05 µm or 0.00000005 m), so there must be a certain volume required for the cell to function – a minimum size for life.

Which of the mycoplasma species is the smallest? Mycoplasma genitalium is considered to be the smallest mycoplasma known, and the smallest form of free-living organism - my gosh – you can fit about 400 M. pneumoniae inside one E. coli! As such, it is the current minimum size for life that we have. M. genitalium is 200 nm (0.2 µm) x 600 nm (0.6 µm), so they’re pretty dawg on small. Let’s put it this way, there are 25,400,000 nm in one inch – mucho small.

It is important to note that M. genitalium is free living, but does need some help. It uses cholesterol in its membrane but doesn’t make it itself. It picks it up from the cells that it lives near……wait for it….. your genital epithelium.

One of the human diseases that is becoming more
convincingly associated with M. genitalium is pelvic
inflammatory disease (PID). Resulting when many
different sexually transmitted diseases go untreated,
PID can cause permanent damage to the reproductive
organs of women. It is important to get treatment early.
The inflammation of PID may be associated with the
fallopian tubes or ovary, and will cause a chronic pain
in the lower abdomen, bleeding and pain on urination.
M. genitalium is a cause of non-gonococcal urethritis (inflammation of the urethra). A late 2013 review states that 1-3% of the general population is infected with M. genitalium, more than with gonorrhea. It is linked to pelvic inflammatory disease, and the review cites studies showing that people infected with this mycoplasma are more at risk for HIV and have more dual infections. It’s a sexually transmitted organism, just another reason for proper restraint. But even though it's helped out by your genital epithelium, it can live on its own and divide outside a host, so it's considered a free-living organism.

The idea of free-living is important because M. genitalium also has a very small genome (amount of DNA in one cell, including the list of all its genes). M. genitalium has about 580 kbp of DNA where kbp = kilobase pairs. Remember that DNA is doubled stranded (usually) so each base is paired with another. Knowing this, we count them as a unit. In all, M. genitalium has just 520 or so genes; it can make about that many proteins.

Genome size could be another way of determining the minimum size of life - what's the minimum number of genes or number of base pairs of DNA for an organism to still meet all seven characteristics of life? As of summer 2014, no organism smaller in size than M. genitalium has been described, but there have been some other organisms discovered with smaller genomes.

Nanoarchaeum equitans was thought to have the smallest gene for a while, with only 491 kbp of DNA. It is an archaea that lives on the edge of hydrothermal vents at the bottom of the ocean. But it is an obligate symbiont with another archaea; it can’t survive without its partner, so can you say it has the minimal genome? It relies on another organism’s DNA.

On the left is the leafhopper in which N. deltocepahlinicola makes
his home. Well inside its cells that is. The leafhopper survives on
phloem and xylem; high in carbs but little protein. The bacterium
makes the amino acids the leafhopper can’t in exchange for energy
in the form of ATP. On the right is a colored photomicrograph of
the abdomen. The red is one type of endosymbiont bacteria,
the green is N. deltocephalinicola.
This is also true of Carsonella ruddii (159 kbp, 182 genes) and Nasuia deltocephalinicola. They are bacteria that must live inside insect cells, like those of grasshoppers. N. deltocephalinicola has the smallest known genome (112 kbp, 137 genes), but it doesn’t even make ATP, it steals it from the arthropod cells. This could hardly be considered free living, and so it can’t be considered the minimal genome for life. And even at that, their cell sizes are still bigger than M. genitalium.

So why is it important to find the minimal size and minimal genome for life? So we can use the information. J. Craig Venter (of the human genome project) wanted to develop a synthetic form of life (synthetic biology); a bacterium that could be developed to provide hydrogen for energy or eat waste to reduce pollution. Others say we need to know so that we can better recognize life on other planets, or life that may have come here from other planets (astrobiology).

Being J. Craig Venter, develop a synthetic form of life is exactly what he and his research institute did. It’s interesting that Venter was one of the scientists that first sequenced the entire M. genitalium genome in 1995. Some 15 years later, Venter’s JCVI-syn1.0 (2010) was the first synthetic life, housing 1000 kbp and 500 or so genes. The genome was based on that of another mycoplasma, M. mycoides. They modified the genome, and introduced it into a cell membrane that had been evacuated of all its constituents. The resulting cell was capable of growing, dividing, you know…. living.

If M. genitalium represents our current estimate for the minimum size of life, it’s only because we’re thinking of life as we know it. Perhaps we have already found life that is smaller, and the minimum size is actually much smaller than M. genitalium.

This is a photomicrograph of a meteorite from Mars. The
small spheres (like the ones the arrows point to, are
supposedly nanobacteria. Proof of life on Mars,
contamination from Earth nanobacteria, or just mineral
spheres that look a little like incredibly tiny bacteria?
The answer is C.
Something termed a nanobe and something else called a nanobacterium were described 20-30 years ago. Nanobes were first found in the rocks that came up during oil drilling in Australia, while nanobacteria were also found in surface rocks.  The size of both (about 1/20 size of M. genitalium) negates their use of ribosomes and DNA. They stain for DNA, but this may be artifact, the artificial result of other things picking up the stain.

But nanobes/nanobacteria have their proponents. Some scientists say that since no DNA has been exhibited, they are a completely different form of life, so size restriction (big enough to hold ribosomes) doesn’t apply. Nanobacteria are also claimed to be important in human disease, as these structures are found in many calcifications of diseased tissues.

On the other hand, nanobacteria are probably just mineral formations. A 2013 study showed that they form spontaneously from many different biological fluid samples, and their appearance in diseased tissues is more a sign of disease than a cause of it. We’ll just have to keep looking for something smaller.

Next week, another question tackled and dissected - think pink.

Manhart LE (2013). Mycoplasma genitalium: An emergent sexually transmitted disease? Infectious disease clinics of North America, 27 (4), 779-92 PMID: 24275270

Wu CY, Young L, Young D, Martel J, & Young JD (2013). Bions: a family of biomimetic mineralo-organic complexes derived from biological fluids. PloS one, 8 (9) PMID: 24086546

Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, & Venter JC (2010). Creation of a bacterial cell controlled by a chemically synthesized genome. Science (New York, N.Y.), 329 (5987), 52-6 PMID: 20488990

Wednesday, July 23, 2014

Let's Get Loud

Biology concepts – vocalizations, mechanical sounds, sonar, decibels, stridulation

Today it seems that truth is more complex than ever.
van Goethe was a German statesman and a very successful
writer. He wrote novels, scientific treatises, lyric poems, as
well as dramas. Born in 1749, one might say that his quote
was true for his day; it was a simpler time. But think how
simple our time will seem to those who live a hundred
years from now – unless we’ve found our way back
to the Stone Age.
I have worked for years in science, and I’m supposed to be a big boy and realize that things are complicated. But I still get frustrated when I can’t get a simple answer. It seems nothing's simple, every answer has a caveat – heck, I make a blog of the exceptions to answers!

This week’s question is no exception – What living thing makes the loudest sound? Notice I said living thing, because I didn’t want to exclude anyone from the contest. Who knows, maybe some bacterium living in Wyoming makes a heck of a racket, it’s just that nobody is around to hear it. Or maybe a redwood falling down is the loudest. See, nothing’s ever simple.

This leads us to a second question – one which we have to answer first. What’s a sound? We are surrounded by air, and air has mass and density – it’s stuff. You can push stuff around. When you push the air, it moves away, which creates a wave, because the air you move then moves the air next to it, and so on.

A sound wave is generated when a force creates a vibration, and that vibration moves air, and that vibration is then propagated through the air. The air moves in the same direction the vibration was moving, and this makes it a longitudinal wave (see animation below).

A sound wave is a longitudinal wave, where the source moves
in the same direction as the wave. There is a compression of
the medium (air or water) and then a rarefaction with fewer
molecules as the compression moves on. The wavelength is
the distance between compressions and the frequency (how
high or low the sound is) is 1/wavelength.
So now we have a sound wave, but do we have a sound? It’s like the old question, if a tree falls and nobody’s there to hear it, does it make a sound? If you define a sound as a sound wave, then yes it does. Does that mean that every sound wave is a sound? There are sounds waves that dogs hear and we don’t because the frequency is too high – are they sound? There are waves that are too low or too soft for anything to hear, are they still sounds?

If you define sound as something your brain recognizes, then a sound doesn’t occur until the sound wave is transduced (changed in form) by a your ear to an electrical chemical impulse and that impulse is interpreted by the auditory cortex of your brain. See, nothing has a simple answer.

So, does a lonely falling tree make a sound? Scott McFarland of the University of Oregon installed microphones all over Crater Lake National Park 100 miles southeast of Eugene. In the remotest parts of the preservation land, he has heard everything from buzzing mosquito wings to, yes, falling trees. Unfortunately, he has also heard human intrusion.

Even from the most isolated areas, about 20% of every recording included airplane noise. He also heard cars, people, and detonations. It seems that no place is really naturally quiet anymore. But there were those falling trees -does that mean they do make a sound? Nope, it was a sound wave captured by recording equipment, transduced to digital information, stored, changed back to a sound wave by a speaker, and then to a neural impulse by our inner ear. It still isn’t interpreted as a sound until it reaches a brain, any brain. If a rabbit or bull moose is nearby, their ears would transduce the sound wave and they would “hear” something, even if they don’t think to themselves, “Hey, a tree just fell.” So again, it’s not so simple, maybe something with a functioning ear is in range.

The old tree falls in a forest question started out as a philosophical
question. It was meant as a thought experiment to students to
discuss the nature of what is real versus what is observable. However,
when it existed as a philosophical question, no mention of sound was
made, but was concerned with whether the tree existed at all if no one
was there to perceive it. It wasn’t until 1883 that the sound reference
was made, and then it was posed as more of a scientific question,
as we approach it in today’s post.
The human hearing sense is pretty sensitive. The pressure needed to generate a sound wave that a human could hear is about one billionth the value of atmospheric pressure. But this sound wave would be just barely audible (depending on the frequency), so it would be soft.

This brings us to a very short discussion of what it means to be loud or soft. We often measure loudness in decibels (db). A decibel is one tenth (deci) of a bel (named for Alexander Graham Bell), which is a unit of power or intensity.

Every 10 db increase represents a 10 fold change in intensity, so the scale is logarithmic. In acoustics (from Greek akoustos = hearing, and ic= pertaining to) this means that decibel is a measure of sound pressure, compared to a reference pressure (20 micropascals – we’ll get back to this).

Howler monkeys are the largest of the New World monkeys. But in
one way they are like old world primates. Howlers are the only new
world monkeys with color vision in both males and females. They all
see like we see, so they can discriminate different shades of green, red,
and blue. In most new world monkeys, the color receptor gene is on
the X chromosome, so females may get two types and be color vision
function, but all males get only one, and see only black and white.
A fairly recent gene duplication in howlers have given it color
vision again.
Examples of things that are loud (high sound pressure) would be a jet taking off 25 meters away (150 db), a clap of very nearby thunder (120 db), or a Harley Davidson 25 feet away (70 db). Note the distances; sound waves dissipate in power as they travel, the transference of energy along the wave goes in all directions and is not 100% efficient. This is why you can’t hear your brother playing with your Rock’em Sock’em Robots when you’re down the hall.

So who’s the loudest? The howler monkey (genus Alouatta, 15 species) has a great claim to being the loudest living thing. Used for communication over tremendous distances, the howl of this primate reaches 128 db from several feet away (hear it here). Howlers have an enlarged hyoid bone that is U-shaped. This creates an air sac on their throat that they can use to make their howl resonate.

The howl is really more of a growl for males but is higher pitched for females, and they can be heard more than 3.5 miles away. The question is why do they do it. A 2014 study concluded that the black howler monkey (Alouatta pigra) use their calls for several reasons, but most relate to defense.

The lesser water boatman (Micronecta scholtzi) is only 2 mm
long, but packs a big auditory punch. It’s a freshwater (aquatic
rather than marine) insect. Many marine animals have loud
calls, but here is an example of one that lives in slow moving
streams and ponds. You can hear it when standing on the bank.
This means the sound is so loud it can traverse the water/air
boundary, which usually stops most sounds.
They howl most often in defense of feeding areas. The volume of the howl makes the monkey seem bigger than he/she is. They also use it to defend infants or mates from males that are not part of the group. It calls attention and help comes a runnin’.

If we look at smaller land animals, some of them are loud too. Cicadas can produce stridulations (see this post) that reach 100 db from a foot away. But the king of the small animals would be the lesser water boatman.

This small freshwater insect (Micronecta scholtzi) can put out a stridulation of 105 db, even though it’s entire body is only 2 mm (0.078 in) long! A 2011 paper in PLoS One broke down the song of the male into three different parts, each with its own peak intensity. The loudest part could be heard from a riverside, even though the insect is underwater. By comparing peak db to size, the male outcalls every other organism we will discuss. 

But it’s only the male that calls so loud. Why? Because he’s looking for a mate, and it’s his penis rubbing against his abdomen that makes the stridulation. This sort of eliminates the females from participating in the contest.

But back to the bigger animals. Bats are extremely loud, but their calls are of such high frequency that we can't hear them. Parrots can call to each other in the range of 100 db, but we can go bigger. In fact, the biggest animal may have the biggest voice as well.

The blue whale is the heaviest animal to yet live on Earth.
Being this large, it still has two natural enemies – man, who
hunted it to near extinction, and the orca. Orcas coordinate
their attacks on the blue behemoths, often trying to separate
babies from their mothers. Nearly 25% of sighted blue whales
have scars from either orcas or monumentally stupid octopus.
The blue whale (Balaenoptera musculus) is the largest living animal and the heaviest animal ever to live on Earth (yet). The whale’s song can reach 188 db! Some of the frequencies are too low for us to hear, but the higher pitched (higher energy) sounds can be detected 800 km (497 miles) way (hear it here).

Interestingly, a 2009 study shows that blue whale songs are getting lower in frequency. Since the whaling ban of 1966, male blue whale songs have been using lower and lower tones. The authors suggest several reasons for this. Males may not need to call out so loudly to find females because the ban has resulted in higher numbers of whales. But it is also possible that more man made noise in the ocean is forcing them to use lower frequencies.

The sperm whale is right there too. It doesn’t really vocalize, but it makes clicks to echolocate each other and prey. The clicks are made by forcing air through two lips (folds of tissue) in front of their blowhole. These clicks come in several varieties, but the “usual” click can reach 230 db. This makes them the kings of sound, if you don’t limit your choices to sounds made with the mouth.

Sounds in water are louder than in air, because the density is higher and the transmission is more efficient. So decibels in water are relative to 1 millipascal instead of the 20 micropascals in air. If you want to compare directly, you need to subtract 61.5 db from the water sounds. This still makes the sperm whale clicks 170 db and the blue whale song about 127 db, just about the howler monkey level. So the sperm whale wins our contest.

The different pistol or mantis shrimp are not very large, but
they have second largest sound to size ratio in the natural
world. Only one claw is the pistol. The right image is an
enlargement of the claw. S= socket, pl = plunger, D= dactyl,
p = propus. The muscles of the dactyl close it so fast, that the
water displaced in the socket by the plunger shoots out at
100 km/hr. This produces the cavitation bubble.
But for interesting and functional loud noise, I like to go with the pistol shrimp (family Alpheidae, hundreds of species). A special engineering twist allows them to cock one pincher like a gun. When they release it, the sound wave travels so forcefully and fast that the water around it turns to vapor and a cavitation bubble is produced (see video). The temperature in the immediate vicinity reaches 4000˚C and prey within 1.8 meters is stunned or killed! The sound, from several inches away, is 218 db (before subtracting the 61.5 db to match air measurements) when the bubble collapses.  

A 2006 study describes that the hoods of the carapace (shell) that partially cover the eyes of the pistol shrimp are to protect themselves from their own explosive snap. The study suggests the hoods evolved first, and that allowed for the development of stronger and stronger snaps. If this continues, the pistol shrimp may start taking humans out!

Next week, another question about the extremes of life.

Van Belle S, Estrada A, & Garber PA (2014). The function of loud calls in black howler monkeys (Alouatta pigra): Food, mate, or infant defense? American journal of primatology PMID: 24865565

Sueur J, Mackie D, & Windmill JF (2011). So small, so loud: extremely high sound pressure level from a pygmy aquatic insect (Corixidae, Micronectinae). PloS one, 6 (6) PMID: 21698252

McDonald, M., Hildebrand, J., & Mesnick, S. (2009). Worldwide decline in tonal frequencies of blue whale songs Endangered Species Research, 9, 13-21 DOI: 10.3354/esr00217

Anker A, Ahyong ST, Noël PY, & Palmer AR (2006). Morphological phylogeny of alpheid shrimps: parallel preadaptation and the origin of a key morphological innovation, the snapping claw. Evolution; international journal of organic evolution, 60 (12), 2507-28 PMID: 17263113

Wednesday, July 16, 2014

East To West And Back Again

Biological concepts – carbohydrates, heliotropism, monoecious, dioecious

I’m trying to think of a situation where quantity is better than
quality. Perhaps some could argue that since quality is subjective,
one person’s quality would be another person’s attempt for
quantity. In friends and experiences, I go with quality. You can
travel to every place on Earth, but if you don’t come back
changed, there was no quality. You can have many
acquaintances, but you really need only one true friend.
When it comes to the number of economically important plants, the Americas have not got many to show off. But what the two continents lack in number they make up for in quality. We have talked before about the biology of corn from North America and how it has been important for the development of molecular medicine.

Potatoes, cocoa beans, peanuts, and vanilla are also from the New World and deserve posts of their own. We’ll hear about vanilla later this summer. But one plant from the Americas has been important for food, oil, and decoration – the sunflower.

If we are going to talk about sunflowers, one question immediately comes to mind. Do sunflowers really turn to follow the sun?  The answer is more complicated than it would first seem, and the answer is just part of the amazing biology of this plant.

First things first – the sunflower (genus Helianthus, about 50 species), as named in Carolus Linnaeus in 1752, does not refer to their tendency to follow the sun. Instead, he called them sunflowers because, ”Who could see this plant….without admiring the handsome flower modeled after the sun’s shape.”

Analysis of nearly fossilized human waste from the caves of Arizona (4000 BCE) show that sunflowers were an important part of the Native American diet. Sunflowers were tough, so they could grow in the Great Plains and other environments that got little rain and lots of sun. They could also grow in temperate environments. Basically, all of North America was there home.

The buffalo would trample huge swathes of land in their migrations, and the torn up ground was perfect for germination of the sunflower seeds. Slowly, this rapacious weed became a cultivated crop. Hybrids were grown, crossing prairie species with forest species and such. In modern science, the sunflower has been used extensively to study genetics of hybrids, much of this work being done at Indiana University in Bloomington, IN – my alma mater, thank you very much.

Number two - the sunflower isn’t a flower, it’s an inflorescence. This is a scientific word for a group of flowers bunched together on the same stem. We talked long ago about the Philodendron selloum inflorescence that controls it’s own temperature and gets hot to attract pollinating beetles.

Sunflowers actually have two types of flowers, the rays and the
discs. The ray florets have a longer petal, they are yellow because
bees see yellow best. The rays are fertile, and have very small
stamens and pistils that provide pollen and ovules. The disc florets,
when male, may have a sterility gene, and this makes sunflowers
very good for studying hybridizations. They also have a naturally
occurring restorer gene, so that they can again make
functional pollen.
In the sunflower, there are two types of flowers, the ray florets around the edge that every one thinks are the only flower petals, and the disc florets, which everyone assumes are the seeds. The ray florets are sterile and therefore for show only; they attract the pollinators.

The ray florets are usually bright yellow, but the disc florets are different colors in different species. They can be yellow, maroon, or even red. The red varieties all stem from a single mutation, but that isn’t the weird part. The disc florets start out male, and produce stamens and pollen, but then turn female as they mature, with the stigma pushing its way up through the middle.

This makes the flowers “perfect” and the sunflower monecious, meaning that have both male and female structures on one plant, but it also makes them smart, as the different timing reduces the chances of self-pollination (pollen and stigma aren’t around at the same time).  For more discussion of monoecious (meaning “one house – male and female flowers on same plant, maybe even as the same flower as with the sunflower) and dioecious plants (male and female flowers on separate plants), see this post.

But even in this, the sunflower can be an exception. The florets mature from the outside discs to the inside discs over time. So while the inner ones may still be male, the outer ones may have become female. In times when pollinators are more rare, if a disc floret remains unpollinated, its stigma may bend down enough to touch the pollen of the still male florets more towards the center of the inflorescence! This is rare, but does occur in species that are annuals.

An achene is a type of fruit that has a hard shell and the seed is
inside. Strawberries are accessory fruits, where the accessory
organs from many achenes join together. The achenes are the
little pieces on the outside. The papery husk (exocarp) of the
sunflower achene is  made from the ovary wall and protects
the seed until it is ready to germinate, like being stuck in dry,
hard, cold ground, or in the belly of a bird.
And third, the disc florets each produce a single fruit (achene), which we call (incorrectly by the way) a sunflower seed. Inside the achene shell is the sunflower seed that we eat. A single sunflower inflorescence can have as many as two thousand disc florets, so that’s a lot of fruit. In species that have more than one inflorescence, each inflorescence will have many fewer than two thousand. Flowers are energetically very costly to produce. Incidentally, almost all the wild varieties have more than one inflorescence, the domesticated versions are bred to have one.

Now for the answer to today’s question – do sunflowers follow the sun? Well, yes and no. Young sunflower plants, including the very small, juvenile flowers, have the capacity to grow very quickly. This means lots of cell growth, and the need for lots of sunlight (to produce ATP and carbohydrates by photosynthesis).

The ability to follow the source of sunlight, called heliotropism (helio = sun, and tropic = loving) requires lots of cell growth. The flower stalks don’t turn so much as they grow in a different direction. As long as the cell growth is rapid enough and the stalk is small enough to respond to changes in cell size, the plant can appear to turn.

Heliotropism is seen in many plants; they need the sun for their
very lives, so it isn’t surprising that their biology would evolve to
maximize sun exposure. The reason the cartoon uses grass – that’s
the plant in which heliotropism was first studied. What scientist
discovered this marvel of nature? Charles Darwin.
The sunlight causes destruction of a plant hormone group called auxins, so they build up in the cells of the shady side. Auxins like indole acetic acid (IAA) promote cell growth and division, so there is much more growth (longer cells and more cells) on the shady side. The uneven growth pattern makes one side longer than the other and forces the stalk to turn (see picture).

So, immature flowers will face east in the morning and west in the afternoon. But that is only part of the answer. By morning, they’re facing east again. How does that happen? A current review (2014) suggests that there may be a diurnal rhythm of several plant hormones, or a natural easterly face that is altered by light signaling. The actual mechanism for the daily turning waits to be identified.

But even this is only half the story. As the stalk gets larger and the heavy inflorescence matures, there can’t be enough cell division or hormone action for the plant to move this massive flower. The mature flowers face east all the time. But why east? Maybe they just can’t bring themselves to move one morning, and since they start out facing east, they stay that way when they give up.

Maybe, but I would imagine there’s a more biologically reason than surrender. The 2014 review cites a study that hypothesizes that facing east protects pollen from the mature florets from sun damage. Final answer, sunflowers follow the sun until it’s time to make little sunflowers, then they settle down and face the rising sun.

So young sunflowers turn with the sun, but how about another question – Why? It’s an inflorescence, not the most efficient photosynthesizer (more about this soon), so why would that structure turn to keep facing the sun? It seems like it would keep the flower in one place and turn the leaves to the sun. Hmmmm.

Now that we’ve answered the question of the day and raised another, let’s talk about the sunflower and world history. But for some unfortunate biology, you might eat sunflower roots like French fries.

The Jerusalem artichoke tuber (top) looks a little like ginger root,
but it is sweeter and not so fibrous. See the text for why you almost
grew up eating McDonald’s sunchoke fries instead of potato fries.
One species of sunflower, Helioanthus tuberosus, has an edible tuber root that is often called a Jerusalem artichoke. Since the sunflower is from North America, you know that the Jerusalem part of the name is wrong. And it’s not an artichoke either.             How it got its name

Around 1600, the Jerusalem artichoke became a popular foodstuff. Easily grown and propagated, the sunflower tuber was a great source of carbohydrates and protein. It was easy to prepare, lasted a long time in storage, and didn’t taste like dirt or wood. Cultivation of the Jerusalem artichoke took off, and it became the primary food for many poor people and a delicacy for the rich.

The South American potato filled the same role, so who would win out as the food of the day? The Jerusalem artichoke (also called a sunchoke) had one big drawback, and it lost the battle. The potato won out, and 250 years later the great potato famine changed the immigration/emigration and ethnic patterns of the world.

What was this thing that cost H. tuberosus the war? It gives you gas. Among the many carbohydrate molecules produced by the Jerusalem artichoke is inulin. This polymer of six carbon sugars is one of those sugars that humans can’t digest, like cellulose. But our gut bacteria can.

Inulin is a branched chain of six carbon sugars. They come in several
varieties and together are called fructans. The “n” means there can be
any number of these units in the chain. They are a good source of
natural fructose, and chicory (right) is the most commercial source of f
ructans. Chicory has been used as a coffee substitute, a salad green
(endive and radicchio are types of chicory) and even in brewing beer.
In breaking down inulin, bacteria produce fructose monomers. They use these monomers as an energy source, and in doing so, produce carbon dioxide. In Central Europe, where the potato vs. Jerusalem artichoke battle was taking place, about 30-40% of the population have a genetic predilection for poor fructose absorption. This means more fructose stays in the gut….more bacteria food. This means much more carbon dioxide and …. flatulence. 

In U.S. finer restaurants and gastropubs, the sunchoke is making a comeback, mostly because Americans can usually absorb fructose just fine. And the fructose helps diabetics too. Many diabetics use the high fructose:glucose ration to even out their glycemic indices.

What’s more, a 2014 study found that mice fed a high fructose diet over time do develop type II diabetes and/or fatty liver. Preceding the disease development, many specific genes change their expression patterns. If their diet was supplemented with extract from Jerusalem artichoke, many of the genes showed normal expression, and the diseases did not develop. Not bad for a sun chasing flower.

Next week, another question to investigate - what/who makes the loudest noise in life?

Vandenbrink JP, Brown EA, Harmer SL, & Blackman BK (2014). Turning heads: The biology of solar tracking in sunflower. Plant science : an international journal of experimental plant biology, 224C, 20-26 PMID: 24908502

Chang WC, Jia H, Aw W, Saito K, Hasegawa S, & Kato H (2014). Beneficial effects of soluble dietary Jerusalem artichoke (Helianthus tuberosus) in the prevention of the onset of type 2 diabetes and non-alcoholic fatty liver disease in high-fructose diet-fed rats. The British journal of nutrition, 1-9 PMID: 24968200


Wednesday, July 9, 2014

What’s So Repelling About Repellents?

Biology concepts – thermosensing, repellent, odor receptors, gustatory receptors, semiochemcials

Science explains our world, and then technology and engineering
build a model of that for our use. The better we know how our
universe works, the better we can make use of it. In the 1985
film Real Genius, this difference is stated when the scientist
students ask what a 6 megawatt laser might be for, one student
says, “Let the engineers figure out a use for it.” In this case, they
used it to fill a house with popcorn.
Science exists to describe our universe in terms of rules and mechanisms; what is and how it comes to be. Knowing that something exists is only half the equation. Science seeks to explain how something exists in terms of the rules of the universe. Observation is good, but it only shows us the question – mechanisms of action and interactions show us the answers.

As an example – we know that certain naturally occurring oils and well as some man made chemicals keep mosquitoes from feeding on us. This is the observation. But the question is – how do mosquito repellents work? The answer is more interesting and more complicated than you would initially think. Repellents rarely repel.

Investigating how chemicals keep us from getting bitten will teach us about how the living systems work, will give us a better understanding of our universe, and then give us better insect repellents. Don’t think that’s important? Consider the hundreds of millions of people who are infected every year (several million die) with mosquito-borne diseases (malaria, encephalitis, dengue fever, yellow fever, filiariasis). So yes, we need more repellents.

Mosquito borne diseases can be unpleasant at best. Top left is
filariasis, a worm is transmitted via mosquito and it clogs up
your lymphatic vessels, so that body parts swell from excess
fluid. Top right – malaria can result in so much red blood cell
lysis that your spleen (the guy who cleans them up) can
rupture. Bottom left – Dengue fever is often called breakbone
fever, the pain is not something an image can express. But the
hemorrhagic form of the disease can produce some bleeding
in weird places. Oh, and it can kill you too. Bottom right –
yellow fever is caused by a virus transmitted by mosquito. Your
liver breaks down and causes your whole body to turn yellow
and you bleed into your skin.
We should start with the repellents for which we have good ideas of their mechanism of action. But there aren’t any. We have some hypotheses and working ideas of the modes of action of mosquito repellents, but nothing is definitive yet. Let’s look at two of them and see if we can find some common pathways.

Citronella oil
Citronella is a combination of many different natural oils produced in lemongrass plants (Cymbopogon nardus and Cymbopogon winteratu). As a natural oil and a flavoring in Asian cooking, one would think that citronella oil would be considered just about the safest insect repellent this side of a slap with an open palm.

But no, Canada says that one small component of citronella oil called methyleugenol, can increase the likelihood of tumor formation in rats. Of course this was when methyleugenol was distilled from the oil, given by itself in large doses, and introduced directly into the stomach. But Canada is still in the process of banning citronella oil as an insect repellent. Of course, you can still eat thai food in Canada, which is often flavored with lemongrass.

The EU, on the other hand, said that the repelling function of citronella oil hadn’t been proven and it was deemed illegal to use in the EU in 2006. Oh, you could eat it, and use it soap or perfumes, you just couldn’t use it to keep mosquitoes away. They reconsidered in 2014 and some restricted uses of citronella oil as a repellent are now allowed.

Citronella oil comes from the lemongrass plant (Cymbopogon
nardus or Cymbopogon winteratu). There are two major species
for acquiring the oil, and the oil from each is a little different in
the percentage of each chemical. Lemon grass is also used in
cooking, the woody stalks are used with extra long cook times.
The torches that burn citronella oil work pretty well, but you
have to stay in the volatilized cloud of oil for them to be efficient.
Despite these issues, the U.S. Environmental Protection Agency (EPA) says citronella is safe and effective as an insect repellent. One weird side issue – you can take all the lemongrass you want from the US to Canada, where its oil is under attack, but you can’t bring any lemongrass from Canada to the US, where it is considered safe. Hmmmm.

Citronella oil probably works in a couple of ways. It's strong and sweet smelling, so it covers up and dilutes the odors that mosquitoes use to find you. If they’re detecting all the citronella in the air, then they aren’t smelling you. But research also shows that citronella oil activates TRPA1 ion channels. In us, they detect cold and noxious chemicals and are interpreted as pain. It is very possible that the detected signals in mosquitoes just come through as something unpleasant and to be avoided.

In this way, citronella would be an actual repellent. It repels on contact as well, as the taste is thought to activate bitter taste receptors and contact greatly reduces feeding time.

But citronella only seems to work when you are in the cloud produced by burning the candles or torches, or within the area of the spray. And if you’re using an oil or cream with citronella, it should really be reapplied every 30-45 minutes - not the most user-friendly method for discouraging pests.

World War II in the Pacific was an insect nightmare for the US Army. In response to the plethora of insect-borne disease that ran through the allied forces, defense scientists starting looking for better insect repellents. In 1946, their efforts produced N,N-Diethyl-meta-toluamide, or DEET.

Just how they came up with DEET is a mystery to me, it must have been a massive exercise in trial and error. Why? Because we know less about how DEET works than we do about citronella oil. And that’s with the benefit of 40 years of research. They didn’t have a clue how it worked or even what systems it was targeting when developed in the 40’s.

Guess which hand has been treated with DEET. The
mosquitoes come very close to the hand that was treated,
but don’t land on it. This argues that DEET is less repelling,
than it is disguising. On the right, the structure of DEET is
similar to several human semiochemicals, it fits into the lock
and key system of several odor receptors and activates or
inhibits them.
Originally it was believed that DEET disrupted the mosquito’s ability to detect semiochemicals (octenol) produced by mammals, especially humans, so mosquitoes couldn’t find a mammalian host to feed on. Then they played around with the idea that it blocked detection of CO2.

More recent studies have been more rigorous, but haven’t helped solve the puzzle. A 2008 study suggested that DEET was actually repellent; the mosquitoes didn’t like the smell and would avoid it. But other studies have shown different mechanisms of action.

A study in the journal Nature in 2011 found that mosquito odor receptors could be confused by DEET. The receptors for octenol were less responsive in the presence of DEET, but other receptors more more responsive.  The conclusion of the study was that odorants from humans could be detected, but their pattern was confused, so the mosquito didn’t recognize the target as a target. It’s as if we disappear from the mosquitoes radar when we wear DEET.

A 2010 study showed similar results. DEET activated certain odor receptors but not others when given alone, but the opposite effects were seen when DEET was given in the presence of things from human sweat that would normally attract a mosquito. Once again, the signals were confused. This is really more of a chemical disguise for us, not a repellent. Next time your kids go outside, you should insist that they apply their mosquito confusant.

However, a 2013 study in the Journal of Vector Ecology found that heat and moisture were critical elements for recognition of targets by female mosquitoes, and that DEET messed not with odor, but with detection of heat and/or moisture. Different from the other studies, but still more of a masking than a repellent.

Something a little disturbing. Mosquitoes can learn to ignore
DEET. Most mosquitoes will be confused by DEET and never
find you. But if they do and then are repelled by the taste, they
learn from that and the second taste is not repellent. Hopefully
they just don’t find you a second time.
There was an interesting study from 2013 that showed that if you mutate or knock out Orco, one of the co-receptors (a protein that works with many different odor receptors so that they can function properly), then two things happened. One, DEET didn’t have any effect on the mosquitoes, and two, mosquitoes that normally preferred humans greatly would then settle for any mammal.

Weird - Orco is needed for both DEET to work and for mosquitoes to find humans more attractive. I haven’t figured that one out yet. The researchers showed that DEET only maintained an effect on the Orco mutant mosquitoes when they landed on a DEET covered surface, and then they didn’t like it at all.

This suggested that DEET might have more than one mechanism, confusion in the air and repellent taste on contact. Older studies supported this idea, as a couple of studies in 2005 and 2006 showed that contact with DEET would reduce feeding behavior in mosquitoes and one in 2010 showed that fruit fly bitter taste receptors are activated by DEET.

So, we have studies that say DEET is a confusant rather than a repellent, others that say it is a true obnoxious smell that they can’t stand, and yet others that say DEET is confusing to the smell and repellent to the taste. But there are more. Other studies suggest that DEET actually inhibits the smelling of anything, while others say that it inhibits an important protein called cytochrome p450.

Used commercially since the 1950’s, DEET has been the gold standard for efficiency for many years. Although it has to be used at fairly high concentrations, it can keep mosquitoes away for 4-6 hours at concentrations where citronella oil might work for less than an hour. At 100% concentration, DEET is active for more than 12 hours. What’s more, if you combine DEET with 5% vanillin, it works two hours longer!

A lime with cloves stuck in it as a mosquito repellent – really?
Well, lime is kind of like citronella oil, and clove has eugenol,
which acts on TRPV1 ion channels. But how many would you
have to have, or do you wear them like earrings? Penny royal
contains menthol and mosquitoes stay away from it. But it
also has toxins that will kill you.
As good as DEET is, people still question whether it’s safe. The EPA in a 2014 review said that DEET is safe for human use and poses no identifiable risks for human health, even in children. But this doesn’t keep people from suspecting chemical usage of carrying negative effects.

On the other hand, DEET dissolves plastic, foam rubber, spandex, gore-tex, and nylon. I can see where this might make people leery about slathering it on their skin for hours at a time. And a few people are allergic to DEET, so the best current repellent isn’t without some negatives.

One last point – a newer repellent called picaridin is almost as effective as DEET and doesn’t eat your back packing equipment and clothes. The interesting point is that picaridin is a synthetic version of piperine, the spicy chemical in black peppercorns. Add to this that menthol is also a fairly decent mosquito repellent, and we have some good arguments that TRP receptors might be involved in repelling activity – as with citronella oil. Piperine is a TRPV1 agonist, and menthol activates TRPM8 and TRPV1. All our talk about spicy food and heat/cold receptors has an impact even in the spread of malaria and other deadly diseases!

Next week, another question to answer - do sunflowers really turn with the sun?

DeGennaro M, McBride CS, Seeholzer L, Nakagawa T, Dennis EJ, Goldman C, Jasinskiene N, James AA, & Vosshall LB (2013). orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET. Nature, 498 (7455), 487-91 PMID: 23719379

Stanczyk NM, Brookfield JF, Field LM, & Logan JG (2013). Aedes aegypti mosquitoes exhibit decreased repellency by DEET following previous exposure. PloS one, 8 (2) PMID: 23437043

Klun JA, Kramer M, & Debboun M (2013). Four simple stimuli that induce host-seeking and blood-feeding behaviors in two mosquito species, with a clue to DEET's mode of action. Journal of vector ecology : journal of the Society for Vector Ecology, 38 (1), 143-53 PMID: 23701619