Showing posts with label thermosensing. Show all posts

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.

DEET

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

Wednesday, July 2, 2014

How Do Mosquitoes Find You?

Biology concepts – semiochemicals, hematophagy, proboscis, thermosensing, TRPA1

Sure, mosquitoes suck blood and pass along malaria

that kill more humans than any other infectious disease.

But would it be good to get rid of them. They provide

food for birds – one scientist suggests that elimination

of Arctic mosquitoes could reduce northern bird

populations by 50%. And mosquitoes pollinate flowers

too, like blueberries and cranberries. See, they’re

not all bad.

We can start our summer series of biology questions by continuing our discussion of taste and thermosensing. It seems that some people are bitten by mosquitoes if they peak out the front door, while other people can sit outside next to tall grass or ponds for hours with suffering a single bite. Unfortunately, I happen to be in the former category.

How do mosquitoes find some people and not others? Are some people just tastier than others?

First let’s get some common misconceptions and basic information out of the way. Do mosquitoes bite you (or any other animal)? No, they have no teeth, so they don’t bite in the traditional sense. What they do have is an elongated set of mouthparts called a proboscis. The sheath on the outside retracts as the longer parts inside pierce the skin like a hypodermic needle. Only this is a flexible hypodermic needle, small enough to go around individual cells and look for a small vein or venule.

On the left is a drawing of the mosquito proboscis parts. Most sit

in a groove of the labium, which retracts as the rest is injected

into the skin. The maxillae and mandibles are like our upper and

lower jaws. They are the sharp parts. The hypopharynx is what

delivers the anticoagulant saliva. On the right is the parts put

together. The fuzzy part is the labium and the sharp tips are

from the maxillae.

Take a look at these videos taken from a 2012 PLoS One study of a mosquito biting a mouse. The squarish objects are skin cells, and the red streaks are blood vessels. The second video in the sequence shows what happens when the proboscis finds a vessel and starts to suck out the blood. Makes you respect the mosquito a bit more – these are some determined females.

Of course it’s only the females that feed on blood. This suggests that feeding on blood is related to having babies. And it is – just not in a “gotta get the baby some food” sort of way. Most mosquito species require a blood meal in order to develop viable eggs. Females get energy from drinking nectar (full of carbohydrates), but they need protein to produce yolk for the eggs. They get the protein from feeding on blood. If the female doesn’t feed on blood, the eggs will be produced, but they won’t be able to hatch and become larva.

But here is one of our exceptions – some mosquitoes have gotten around the need for blood meals. All 92 species of mosquito in the genus Toxorhynchites (elephant mosquito) don’t need to feed on blood. Instead, their larvae feed on the larva of other mosquitoes, and the gather the proteins they need to lay viable eggs from their larval meals. They store the amino acids in their larval and pupal bodies, until they become adults and need them to lay eggs of their own.

Compare the sizes of the elephant mosquito (left) and

A. aegypti. I’m very glad that the females of the

Toxorhynchites genus don’t suck blood. They could drain

people dry! Even though their size is small, species like the

one on the right can consume 300 ml a day from every

caribou in a herd when they are swarming.

This suggests that the elephant mosquitoes could be used to combat disease spreading mosquitoes, like the Aedes aegypti mosquitoes that spread dengue fever, yellow fever and the current disease of interest, chikungunya fever. And the elephant mosquito has been used as a natural biocontrol agent. What's weird is that A. aegypti females actually help the situation.

A. aegypti, and many other mosquitoes that lay eggs in water, have larvae that eat bacteria. So they want to lay eggs where there are a lot of bacteria. Well, the eating of larvae by Toxorhynchites species leaves lots of little pieces of mosquito larva in the water, and this provides bacteria with a lot of food. A June 2014 study showed that A. aegypti females actually prefer to lay eggs in water that contain predators for their larva, because it increases the bacterial numbers so much. Thos that survive have lots of bacteria to feed on. It’s a calculated behavior – risk being eaten or risk starving.

So some mosquitoes will go a long way and risk death in order to get a good meal for their potential offspring. They’re looking for mammals usually, but even here there are exceptions. Some mosquito species, like Culiseta melanura, feed almost exclusively on bird blood – they say it tastes like chicken.

The picture represents the transmission cycle for

eastern equine encephalitis virus (EEEV). It can’t be

transmitted from mammals to other animals, so they are

called dead-end hosts. But it can produce disease in

them. Humans most often will present with a limiting or

subclinical disease, but horses have a hard time with it.

The major source is in birds, and the transmission from

bird to bird is by mosquitoes that rarely bite humans. The

way into dead-end hosts is by a mosquito that normally

bites mammals occasionally biting a bird, or the rare

occasion that a bird specific mosquito will bit a mammal.

But just because they feed mostly from birds doesn’t mean they aren’t important disease transmission. They are – for horses. Eastern equine encephalitis virus is passed from bird to bird by C. melanura, so the birds, especially cardinals, are a reservoir of virus. Then, when another species of mosquito that is less particular about its host species bite a bird then bites a horse or person, the disease can be spread. There are even cases where a C. melanura will occasionally feed on a human and spread the disease directly.

With this background, we still need to answer our question of the day – how do mosquitoes find a blood meal. Believe it or not, your socks help answer the question. For many years it was assumed that mosquitoes followed the heat of warm-blooded animals in order to find a meal, but this was an assumption that was not tested rigorously.

Then it was discovered that carbon dioxide (CO₂) is a strong cue for mosquitoes seeking sustenance. CO₂ means respiration, and respiration possibly means mammals. The mosquitoes have taste receptors in their antennae and mouths that will sense changes in CO₂ and they will follow the path of more carbon dioxide right to your nose and mouth (see this post).

Large people and pregnant women tend to exhale more CO₂, so they will be more attractive to mosquitoes. But there are large individuals who never get bothered by mosquitoes. Maybe there’s more to it.

Semiochemicals are part of the answer. Semio- comes from the Greek meaning signal, like in semaphore flags. So semiochemicals are molecules emitted by organisms for communication. Pheromones are the most famous of the semiochemicals – and we know that these are used in many animals, from helping to guide ants to follow the path of their predecessors, to influencing mate choices in many animals.

Semiochemicals might be attractants or repellants. In some cases, they can be both. Take human body odor – it contains dozens of semiochemicals, people find body odor repulsive, but mosquitoes enjoy it like the smell of fresh apple pie. Of course, body odor is only offensive nowadays; before the advent of deodorant, daily or three times daily baths and showers, perfume, aftershave, and of course Axe products – everybody smelled like that guy that lives under the bridge.

On the top of this image is a general idea of semiochemicals.

If they work on members of the same species (like mating

signals), then they are called pheromones. If they work on

other species, they are called alleochemicals. Each can be

either attractive or repellant. On the bottom is a homemade

mosquito trap. You might be able to see that it has been

baited with old shoes and grimy socks.

Bacteria feed on the sweat, sugars and proteins that mammals exude, and they give even more semiochemicals. This can make you more or less attractive to mosquitoes. In general, people with many types of bacteria on their skin are less attractive, while those with mostly a few attractive species will get bitten more often. Having a high number of bacteria is a turn off too, probably because that would expose the mosquitoes to more possible pathogens as well. Is it possible to be so disgustingly colonized that even mosquitoes won’t land on you?

Mosquitoes are attracted to several different semiochemicals, including octenol, CO₂, and nonanal. On mosquito antennae, especially the female antennae, there are receptors in the sensilla (see this post) for at least 27 different chemicals in human sweat.

Studies have shown that old socks are a good experimental attractant for mosquitoes. Instead of using an arm or other body part, scientists will compare the attractive ability of someone’s old sweat socks to individual chemicals or mixtures of chemicals. Of course, whose socks you use matters as well. Some people are classified as high attractors (HA) and some as low attractors (LA), so studies often include comparisons of chemicals or mixtures to both HA and LA socks.

But there are other considerations as well. People with blood type O secrete different semiochemicals and are more attractive to many species of mosquitoes. Go ahead, try to change your blood type so you’re less attractive to mosquitoes.

Different species may aim for different body parts. Some seem to prefer feet and ankles, but this may be because they are closer to the ground. If convection currents created by the body heat rising suck the mosquitoes in from below, then it is really the fact that they are following their noses and not going after feet particularly. A small 1998 study showed that mosquitoes that went after feet and ankles preferentially did not do so when the volunteers lied on their backs and raised their feet high in to the air. But, what we have stumbled across in this discussion is body heat.

This is part of a complex figure from a 2011 scientific paper.

In addition to the pretty colors used, the message is that these

researchers identified TRPA1 ion channels on the proboscis

of a species of mosquito. They don’t just sense heat with

their antennae, but also their sucking parts. I wonder if the

interior parts also have TRPA1 to help them find a vessel

when the proboscis is inserted into the tissue.

But what was old is new again…. Scientists are again looking at heat as an attractant for mosquitoes. As compared to HA or LA socks, heat isn’t a strong attractor, but warm socks attract more mosquitoes than cold socks. On the other hand, a 2010 study says that heat and moisture is a greater attractor than heat alone, so it would seem that people working outside in the heat would be the perfect attractors for mosquitoes.

Since heat does seem to be an attractor, it would follow that female mosquitoes would have a receptor for heat. Voila, a new study shows that mosquitoes have sensilla on their antennae and palps that house TRPA1 ion channels. A 2011 study even showed that one malaria-carrying mosquito has TRPA1 receptors on its proboscis. We have talked before about how many mammals use this receptor to sense noxious cold as well as chemicals that cause irritation or pain.

On the left is a species of tick. You wouldn’t believe how big they

can get when feeding on blood. Look it up – I dare you. On the

right is a bedbug. The bedbug is not that closely related to the

tick, since the tick is an arachnid. Count the legs on each – spiders

(arachnids) have eight legs, insects have six. Both these animals

feed on blood, but no one has identified a heat sensor in them.

But in birds, reptiles and insects, TRPA1 is a heat sensor. The 2009 study showed that the TRPA1 were expressed on the female antennae only. But that isn’t to say that only female mosquitoes have TRPA1. A 2013 study indicates that A. gambiae mosquito larvae have functioning TRPA1 so that they can sense water temperature and stay in the most comfortable water.

So mosquitoes (most female mosquitoes) are finding suitable hosts for blood meals by using their senses of taste, smell, sight, and infrared detection. There are other vampire insects as well, ticks, bedbugs, etc. I wonder if they are using heat sensing too. These have yet to be reported on.

Next week, a related question – just how and why do mosquito repellants work?

Maekawa E, Aonuma H, Nelson B, Yoshimura A, Tokunaga F, Fukumoto S, & Kanuka H (2011). The role of proboscis of the malaria vector mosquito Anopheles stephensi in host-seeking behavior. Parasites & vectors, 4 PMID: 21272298

Albeny-Simões D, Murrell EG, Elliot SL, Andrade MR, Lima E, Juliano SA, & Vilela EF (2014). Attracted to the enemy: Aedes aegypti prefers oviposition sites with predator-killed conspecifics. Oecologia, 175 (2), 481-92 PMID: 24590205

Olanga EA, Okal MN, Mbadi PA, Kokwaro ED, & Mukabana WR (2010). Attraction of Anopheles gambiae to odour baits augmented with heat and moisture. Malaria journal, 9 PMID: 20051143

Liu C, & Zwiebel LJ (2013). Molecular characterization of larval peripheral thermosensory responses of the malaria vector mosquito Anopheles gambiae. PloS one, 8 (8) PMID: 23940815

Wednesday, June 11, 2014

What Cold Really Looks Like

Biology concepts – TRPA1, cold sensing, oxygen sensing, proteasome function, hypoxia, normoxia, hyperoxia, phototransduction, optogenetics, methyl anthranilate

Last week we learned that the TRPA1 ion channel causes you pain when you are too cold and helps you to avoid the cell damage that cold can produce. Even if that’s all it did, it would be pretty amazing, but there’s much more. It turns out that TRPA1 can do some amazing things - like keeping the right amount of oxygen in your cells.

Prolyl hydroxylase domain enzymes (PHD1, 2, and 3) are the primary mammalian sensors for oxygen in the blood and tissues when oxygen levels are normal (normoxia) or low (hypoxia). Oxygen binding to the PHD acts as an "on" switch for the binding of another molecule to PHD, a molecule called 2-oxoglutarate (2-OG). 2-OG can only bind to PHD if O₂ is already bound.

On the left is a cartoon that shows how ubiquitin and PHD proteins

help mediate the destruction of unneeded damaged proteins. On the

right is the proteasome, the structure which recognizes the proteins

targeted for destruction and carries out the cutting that

releases amino acids.

The function of 2-OG bound to a PHD is to trigger the destruction of another type of protein, called hypoxia inducible factors (HIFs). They do this by modifying some of the amino acids of the HIF so that cell thinks the HIF is old or defective. Old and defective proteins are recycled for their parts by a big complex of proteins called a proteasome.

When there is sufficient oxygen in the blood and tissues, the PHD is bound by O₂ and 2-OG, so the HIFs are degraded. But when there is hypoxia – little O₂ and 2-OG are bound to the PHD – and there is no destruction of HIFs.

If they aren’t targeted for the proteasome, HIFs are free to do their job. They turn on genes that work to increase oxygen in the blood and tissues, including making more red blood cells, using more iron for heme, stimulating the production of new blood vessels, and triggering cells to use glycolysis instead of the citric acid cycle because glycolysis doesn’t need O₂ (here’s a review).

When oxygen levels in blood and tissue return to normal, more O₂ will be free to bind to the PHDs and the HIFS will then be degraded. Their effects on genes will be turned off as their concentration decreases.

Oxygen sensing in the cells is important at all stages of life. At one

time it was believed that very premature infants, with less than

mature lungs, would benefit from a high oxygen environment.

What it actually did was make them blind due to oxygen mediated

damaged to the retina.

This is all well and good for normoxic or hypoxic situations, but how would it help in times of too much oxygen (hyperoxia)? And believe me, too much oxygen is a very bad thing. Short bursts of high oxygen can be disorientating, with central nervous symptoms that affect breathing and may cause myopia.

Prolonged exposure to high levels of O₂ (or short exposures to very high levels) can cause cell destruction, collapse of lung alveoli, retinal detachment, and seizures. Scuba divers, firemen, and anyone else using oxygen tanks must be aware of the dangers of either running out of oxygen or breathing in too much.

This is where TRPA1 ion channels enter the picture. A 2008 study showed that TRPA1 can be activated by O2. In hyperoxic situations, there is too much oxygen to be bound up by the available PHDs, so some is left to interact with TRPA1 channels in the membranes of the vagus and sensory neurons, as well as in tissue cells. The O₂ can open the TRPA1 channel directly and lead to firing of the neuron.

The more O₂ there is, there more activation of TRPA1, and this is good. In hyperoxic situations, the body constricts many blood vessels to limit the excess oxygen getting into the tissues. Hyperoxia also ramps up the cells’ protective mechanisms against reactive oxygen species damage.

What we don’t know is which responses to hyperoxia are controlled by the TRPA1 channel activity. A 2011 study goes onto show that the PHDs lose their inhibitory function on TRPA1 in both hyperoxia and hypoxia. This is a similar conundrum to the one we saw last week - where TRPA1 senses cold, but responds to many of the “hot” agonists of TRPV1. Here, the same sensor is stimulating different responses to too much or too little oxygen.

Some situations with low oxygen tension can create big problems.

On the left is pneumonia; the filling of the alveoli with edema fluid

limits the amount of oxygen that can get to the bloodstream. In the

middle is Payne Stewart; at altitude, his plane lost cabin pressure,

everyone on board basically went to sleep, and the plane flew in a

straight line until it ran out of fuel and crashed. On the right is a diver

who specializes in going as deep as possible on one breath. Many

participants drown.

The same channel that senses really cold temperatures in your skin and tells you that this bad by stimulating pain also helps you keep a normal amount of oxygen in your tissues – a system that has little or nothing to do with pain. Weird enough for you? Well it gets weirder.

It seems that your TRPA1 cold sensing channels are important for tanning in the summer! A 2013 study shows that a process called phototransduction uses G-protein coupled receptors to stimulate TRPA1 in melanocyte and keratinocyte membranes and results in an influx of calcium. This destabilizes the membranes and facilitates the transfer of melanosomes from melanocytes into the surrounding keratinocytes, as we have talked about before.

Phototransduction in general is where light energy is changed (transduced) into another form of energy. The best example is in the retina of the eye, where rods and cones turn visual light into neural signals that are then processed in the brain as images – that’s how you see. In the skin, the energy of the UV light is turned into chemical energy (flow of ions in and out of channels) to stimulate cellular activity.

The retina is the most obvious example of phototransduction. Light

energy is converted to chemical energy and information. Light

strikes the retina, and excites the rods and cones. On the level of

the membrane, the light (hv) set in motion a series of reactions that

results in the opening or closing of ion channels, including

the TRPA1 channel.

A second 2013 study from the same group says that retinol (hear the word retina in there? As in the retina of your eye?) is the photoactive chemical that starts the G-protein couple receptor cascade that then results in TRPA1 activation and melanin synthesis in melanocytes.

A new photosensitive protein called optovin has been identified in zebrafish. It mediates TRPA1 activation via a sensitive part of the TRPA1. Optovin allows for optical control of TRPA1-expressing neurons, meaning - optovin absorbs light, generates singlet oxygen radicals and these interact with the the oxygen-sensitive cysteine residues on TRPA1 and activates the receptor. Sound familiar? This is similar to the binding of oxygen that helps the body recognize and respond to hyperoxia.

In an effort to take advantage of this great system, the research field of optogenetics has been born. Let’s say you want to study what happens when a set of neurons fires. Introduce optovin and TRPA1 (or a similar system, like opsin or retinal) into the appropriate neural pathway and then you can fire them at will just by shining a light on them. Imagine, shine a laser pointer on a mouse and instantly he starts to jump, or drool, or tell a joke.

Optogenetics was the method of the year in 2010! On the left

is how it works, using phototransduction molecules to stimulate

responses in target cells. On the left is how it works in a live rat.

The light is positioned so that it can illuminate the altered

neurons so that processes can be turned on and off with light.

One last exception for the day – one that will lead to some amazing stories for next week. In mammals and many other animals, TRPA1 senses noxious cold (in addition to the amazing things we just talked about), but in some species it acts completely the opposite.

In mosquitoes, TRPA1 senses heat instead of cold. A 2013 study shows that larval A. gambiae (the mosquitoes that carry malaria) rely on TRPA1. If you decrease the amount of TRPA1, the mosquito larvae don’t exhibit thermal locomotive behaviors that would normally keep them in the preferred temperature of water. This might be important for killing mosquito larvae in water; if you can use antagonists to TRPA1 to move them away from their optimum temperature, they’ll die as babies and never become bloodsuckers.

In fruit flies, TRPA1 is also important for circadian (daily) locomotor activity patterns (2013 study). Instead of having a light/dark drive their activity, the temperature fluctuations between day and night can also serve to entrain circadian cycles. There are activity cells in fly brain for morning, daytime, and evening activity levels. TRPA1 isn’t expressed in morning neurons, so it’s activation by heat is what increases activity in day and evening.

TRPA1 gene function (painless in fruit flies) controls many

circadian behaviors. Depending on the amount of firing, it

tells the fly what time of day it is, and this time controls

which behaviors will be favored.

Another 2013 study shows that TRPA1 is used as thermoregulatory control of circadian rhythm in drosophila. Loss of TRPA1 altered behaviors and changed the expression of an important circadian rhythm protein called Per in the pacemaker cells.

In this one regard, birds are a lot like mosquitoes and flies. Chickens for example, have TRPA1 channels that induce pain due to high heat, just like their TRPV1 channels (which, you remember, don’t react to capsaicin).

A 2014 study showed that chicken TRPA1 is a heat and noxious chemical sensor – it acts opposite to the TRPA1 in humans, even though we are both homeotherms (maintain a body temperature within a small range). Chicken TRPA1 is almost always co-expressed with TRPV1, so they double up on heat but might have less cold sensing – why? - cold can still be damaging to cells so they need to know to avoid it.

The weird TRPA1 of birds lends itself to a bizarre use for us. Methyl anthranilate (MA) is a non-lethal bird repellent, and the same 2014 study shows that it works by activating bird TRPA1 pain sensors. MA doesn’t activate TRPA1 in other species, like humans; three amino acids critical for its MA activity are different in bird and mammal TRPA1.

Since MA doesn’t work on human TRPA1, it can be sprayed on crops to keep birds away – it’s a chemical scarecrow - if it only had a brain! It can also be sprayed on surfaces to keep birds from congregating. MA works as a repellent by stimulating the trigeminal nerves via TRPA1 in the bird’s beak, eyes and throat.

Grapples (pronounced grape – ple) are apples soaked in

methyl anthranilate (MA) so they taste like grapes. MA is

sensed as hot in birds by TRPA1 but does not activate

the human TRPA1. We taste it as grape flavor and smell.

For me – if you want to taste grapes, eat grapes!

Very similar to MA is dimethyl anthranilate (diMA). It also activates bird TRPA1 and can be used as a repellent, but we use it for flavoring grape Kool Aid. Too bad Jim Jones wasn’t leading a flock of chickens – they’d all still be alive.

Both MA and diMA are naturally occurring in concord grapes, strawberries, other fruits, and are especially important for flavor of apples. Maybe that’s why they make grapples – grape-flavored apples. Actually, grapples are apples soaked in MA, not a genetic hybrid. DiMA is also released from musk glands of foxes, and is produced in rotting flesh – does spoiling meat taste like apples or grapes to you?

Next week – odd changes in TRPA1 and TRPV1 turn animals into better hunters.

Bellono, N., & Oancea, E. (2013). UV light phototransduction depolarizes human melanocytes Channels, 7 (4) DOI: 10.4161/chan.25322

Bellono NW, Kammel LG, Zimmerman AL, & Oancea E (2013). UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes. Proceedings of the National Academy of Sciences of the United States of America, 110 (6), 2383-8 PMID: 23345429

Saito S, Banzawa N, Fukuta N, Saito CT, Takahashi K, Imagawa T, Ohta T, & Tominaga M (2014). Heat and noxious chemical sensor, chicken TRPA1, as a target of bird repellents and identification of its structural determinants by multispecies functional comparison. Molecular biology and evolution, 31 (3), 708-22 PMID: 24398321

For more information or classroom activities, see:

Oxygen sensing –

http://www.nature.com/scitable/content/oxygen-sensing-by-hif-hydroxylases-57249

http://www.cell.com/developmental-cell/abstract/S1534-5807%2813%2900353-5\

http://www.cell.com/cell-metabolism/abstract/S1550-4131%2808%2900318-5

http://www.uchicago.edu/research/center/center_for_systems_biology_of_oxygen_sensing/

http://proteopedia.org/wiki/index.php/Molecular_Playground/Prolyl_Hydroxylase_Domain_%28PHD%29_Enzyme

http://www.sdbonline.org/sites/fly/genebrief/hph_fatiga.htm

http://www.dailymotion.com/video/xqzjjn_oxygen-animation_news

Proteasome –

http://www.hhmi.org/biointeractive/proteasome

http://www.hhmi.org/biointeractive/proteasome-and-protein-regulation