In chemical ecology, the devil--or the coral settlement cue--is in the details

This week we provide a quick overview of a suite of compounds found in Nature to highlight a central tenet in chemical ecology.

Small differences in the structures of molecules can result in vastly different bioactivities.  To quickly illustrate this concept, let's take a look at two familiar molecules: Splenda, many a dieter's favorite sweetener, and good ole sucrose, the calorie-full sugar in fruit and other foods. We can see that the only difference between these structures is the presence of three chloride atoms in place of hydroxyl (OH) groups in Splenda's structure. Those three halide atoms make the difference between your no-calorie drink and a sugar-laden treat.

Small differences in chemical structure make a big difference. For splenda and sucrose the difference is in three highlighted chloride atoms... and whether you lose weight or not! ;)

Small differences in chemical structure make a big difference. For splenda and sucrose the difference is in three highlighted chloride atoms... and whether you lose weight or not! ;)

Today's focus will be three different molecules that all have halide (e.g. chloride, bromide) atoms and planar (i.e. flat) rings of carbon atoms. Otherwise there isn't too much linking them. So enjoy each of these nuggets of chemical ecology like you would a fine cheese: Savor, enjoy and share with a friend.

The first compound comes from the paper that got me thinking about this concept to begin with. The paper was published in Nature Chemical Biology in 2017 and is largely the work of scientists in San Diego at the Scripps Institution of Oceangraphy. In this paper they report conclusive evidence that a very common metabolite found in marine sponges is actually produced by the bacterial symbionts that inhabit the sponges, not the sponge itself. This metabolite is a polybrominated diphenyl ether (PBDE). There are a range of highly similar compounds in this same class of PBDEs that are made by the bacteria and deposited in the sponge tissue. The structure of one of the common PBDEs found in sponges is right here:

Polybrominated diphenyl ethers are common marine metabolites and are made by bacteria that live in sponges. 

Polybrominated diphenyl ethers are common marine metabolites and are made by bacteria that live in sponges. 

Here are a few fun facts :

  • PDBEs can make up to 12% (!) of the dry weight of marine sponges within the family Dysideidae.
  • Despite its serious presence in sponge tissue, the biological activity of these compounds in the context of the sponge-bacterial symbiosis is not clear. Why do the bacteria make so much? 
  • PDBEs are abundant in marine ecosystems and are found across all trophic levels, from whales to bacteria. This is because PDBEs bioaccumulate: they are not easily broken down in living tissues and persist over the lifetime of many animals (including us!)

Interestingly, PDBEs look a lot like some of the nastiest human-made chemicals we know of! This brings us back to a class of compounds that we've discussed before on this blog: polychlorinated biphenyls (PCBs).

Shown here is the polychlorinated biphenyl PCB 156, which was Produced by many chemical companies until the 70s. In the 1970s, public awareness and expert knowledge about the harm caused by PCBs started to rise. The Toxic Substances Control Act banned the production of PCBs in the U.S. in 1978  thanks, Epa!

Shown here is the polychlorinated biphenyl PCB 156, which was Produced by many chemical companies until the 70s. In the 1970s, public awareness and expert knowledge about the harm caused by PCBs started to rise. The Toxic Substances Control Act banned the production of PCBs in the U.S. in 1978

thanks, Epa!

Previously at Chemical Intuition we've discussed PCBs in the context of how these and other persistent industrial chemicals have impacted the evolution of fish in highly polluted waters. That story focused on the brilliance of evolution to solve problems of survival in a hostile world. Let's focus on the chemistry of PCBs--why were they so popular with industry in the first place? What makes them bioaccumulate? Besides fish, what other organisms are affected by these compounds?

PCBs--why so popular?

Here are some physical characteristics of PCBs that help explain their popularity:

  • low electrical conductivity/insulating
  • non-flammable
  • chemical stability (i.e. these types of molecules don't easily break down when exposed to air, high temperatures)

These chemical and physical characteristics made PCBs good chemicals to help make:

  • electrical stuff, especially capacitors and transformers
  • paints and dyes
  • lighting fixtures: PCBs are part of the fluorescent light ballasts that make working inside in most office buildings such a natural-lighting joy :S
  • rubber and other bendy plastics

What makes them bioaccumulate?

The chemical stability of PCBs makes these chemicals difficult for our bodies to eliminate. And the chemical stability and lack of a biological route of degradation means that PCBs persist in the environment for very long periods of time. The half-life of elimination (i.e. the amount of time it takes for half of an initial amount to "go away" through direct elimination or degradation) of PCBs in the adult human body has been calculated to be around 10-15 years. That's a very long time if you compare it to how fast the human body can deal with alcohol (hours) or THC (a few days).

What organisms are affected by these compounds?

According to the EPA, PCBs have a wide-range of negative health effects on organisms, from fish to humans, at a range of doses, including chronic (small doses over a long period of time) and acute (high dose over a short period of time) doses. In terms of health effects on humans, the most widely studied question is whether PCBs are carcinogenic.  The EPA had a study that came out in 1996 to reassess earlier work studying this issue. On their website they state, "Studies in animals provide conclusive evidence that PCBs cause cancer. Studies in humans raise further concerns regarding the potential carcinogenicity of PCBs. Taken together, the data strongly suggest that PCBs are probable human carcinogens."

I'm so glad the US still has a regulatory agency that helps protect our environment. Well, at least at the time of this blog post, the EPA still exists. Here's the thing: PCBs are really useful. But they are also very bad for you and basically every other living organism on planet Earth. Sometimes capitalism can't have its way for the best of our bodies and our planet.

Speaking of what's best for our planet: If you care at all about Mother Earth I'll wager a guess that you already know about the massive amount of coral bleaching that's happening in the Great Barrier Reef right now. And that connection will take us to the third compound of today's aromatic medley: Tetrabromopyrrole, a coral settlement cue.

Tetrabromopyrrole, a coral settlement cue. It's got bromine atoms AND an aromatic/planar ring. Woo hoo!

Tetrabromopyrrole, a coral settlement cue. It's got bromine atoms AND an aromatic/planar ring. Woo hoo!


Corals are strange! Stony corals form the skeleton, literally, of the colorful corals that make some islands vacation destinations. When coral reefs are growing anew or recovering from damage, a key part of the growing process is the recruitment of coral larvae. These larvae can come from the coral that's already there (the brooders) or from the surrounding waters (broadcast spawners). In order to settle in a particular spot, the sponge larvae need a cue that it's a good location: the integration of physical and chemical cues is a complicated process that's not completely understood. After settling in the desired spot, the larvae also need to metamorphose from larvae into juvenile coral. "Complete settlement" involves both initial attachment and metamorphosis.

Researchers discovered in 2014 that a complete settlement cue for Carribean sponges (e.g. Porites astreoides) is tetrabromopyrrole. They found that this small molecule is produced by biofilm bacteria that naturally associate with coral communities, such as Pseudoalteromonas sp. PS5. Tetrabromopyrrole was a cue compound for both brooder and broadcast spawner coral larvae and has been found in other studies to induce metamorphosis in Pacific sponges. The authors speculate that given the lifelong association of proteobacteria like Pseudoalteromonas sp. PS5 with corals, and the ability of these bacteria to ward of coral pathogens through the production of other bioactive compounds, the tetrabromopyrrole cue may be a cue for the presence of a healthy and supportive bacterial biofilm community, which is essential to the survival of coral.

In conclusion: there are lots of bioactive compounds out there in the world and, like in great music, public policy, and the fine print on your iTunes agreement, the devil's in the details when it comes to chemical ecology.


In the Pink: thriving in a hostile environment

Lake Hillier in western Australia

Lake Hillier in western Australia

I recently found out that at there is such a thing as “millennial pink,” which refers to the color pink's rise in popularity coinciding with the dominance of the universally mocked millennial culture. Perhaps this explains why I was so thrilled and intrigued, and just tickled pink I guess, to learn that in Westgate Park, in Melbourne Australia, a lake turned hot pink!  And this is not an isolated phenomenon: pink lakes exist all over the world.  But why are they pink?  This turns out to be a rather complex and fascinating question.  

The key to the pink lakes has to do with the high salinity of these bodies of water and the microbial ecosystems that thrive in these conditions. Salt, in high concentrations, puts a lethal amount of osmotic stress on a cell – causing it to rapidly lose water and shrivel up and die.  This is why saline solutions are often used as an antimicrobial measure.  

So one would think that a body of water containing high concentrations of salt would be a terrible home for an organism. But as we have seen before, microbes are astonishingly adaptable, and numerous types of bacteria, algae and archaea, have evolved strategies to overcome the dangers of high-salt environments. These salt-loving (“halophilic”) organisms thrive in salty lakes because their salt-sensitive competitors have been killed off – leaving all the lakes’ bounty to the well adjusted.  Natural selection at its finest! 

The strategies employed by halophiles to overcome osmotic stress are numerous and diverse, but the general theme is this: osmotic stress is caused by a disparity in the concentration of a solute between the intra and extra-cellular sides of the cell membrane. Upon sensing high salt concentrations in their environment, halophiles either produce intracellular solutes (like amino acids or glycerol), or increase salt transport into the cell (using trans-membrane ion pumps). This allows them to match the concentration of solutes in their intracellular environment to that of their surroundings, decreasing osmotic pressure, and preventing water loss from the cell.  This generalized strategy is employed by halophiles (ranging from bacteria to plants) – but our explanation here really only skims the surface of a complex and deeply fascinating area of research.  In fact halophiles have even grabbed the attention of astrobiologists interested in adaptations that might allow an organism to survive in the extensive salt formations on Mars.

Many halophiles utilize photosynthesis to generate energy, and for halophiles this sunlight-induced metabolic activity is likely essential to generate the solutes or proteins required to maintain an osmotic balance in the cell. To maximize their sun exposure, some salt-lake dwelling halophiles float on the surface of the lake – and while this may increase cellular productivity, the unrelenting sunlight also increases their risk of UV-mediated DNA damage.  Yet these resourceful microbes have come up with a chemical strategy to overcome this environmental danger!  Some microbial photosynthesizers produce large quantities of anti-oxidants that scavenge the DNA-damaging free radicals that are generated upon UV exposure.  In other words, these microbes can produce their own SPF!



So what does this have to do with the pink color of the lake?  Well it just so happens that many of the halophiles residing in pink lakes produce a class of anti-oxidants called carotenoids, which are brilliant shades of orange and red. In fact, these molecules are often generated in such large quantities that the organism itself turns bright red, causing a pink-coloration of whatever broth they are growing in. One well-known example is that of Dunaliella salina - a halophilic unicellular green alga (so-called because it contains chloroplasts) that is commonly found in pink lakes. Researchers have shown that this microbe, when exposed to high salt concentrations, produces vast amounts of the carotenoid Beta-carotene. This well-studied anti-oxidant is a brilliant red-orange color and is a precursor to vitamin A, which is widely used in both the food and cosmetic industry (in the cosmetic industry, as retinol).  In the 1960s D. salina became a paradigm for how halophilic microorganisms could be mass-cultured for commercial production of Beta-carotene. 

Dunaliella salina

Dunaliella salina

But it turns out that while the commercial interests drove extensive study of D. salina, this organism is probably not the major contributor to the pink color of salty lakes. Sample collection from these lakes reveals diverse halophile populations that are often dominated by red archaea or bacteria, many of which are not well studied. These non-algal halophiles likely produce Beta-carotene or other carotenoids and could also be a source of previously uncharacterized red (or not red) molecules.  

Much of a flamingo's pink coloring comes from eating beta-carotene producing alga. 

Much of a flamingo's pink coloring comes from eating beta-carotene producing alga. 

The study of the microbial population dynamics of the pink salty lakes is pretty neat.  There are cool colors, fascinating environmental adaptations and potential for commercial production of useful natural products – but there’s still quite a lot to be determined.  Scientific efforts like the Extreme Microbiome Project aim to classify and characterize the fascinating organisms that reside in Lake Hillier, a well-known Australian pink lake, as well as numerous other extreme environments such as the Door to Hell gas crater and the toxic hot springs in Ethiopia. Through extensive sample collection and genome sequencing, this type of research aims to expand our understanding of the different types of organisms that thrive in diverse environments, and to examine the strategies these microbes use to overcome seemingly hostile environments. 


Casual Fridays: We Discuss Gardening Ants and Their Symbionts!

This week we bring you another “Casual Friday” post! While we aren’t actually posting this on a Friday, we are still bringing you a another casual discussion of a chemical ecology paper. This week’s paper is from the lab of Cameron Currie and is entitled “Black yeast symbionts compromise the efficiency of antibiotic defenses in fungus-growing ants.”

This paper is focused on a particularly fun ecological community: a three-part symbiosis between ants, fungi, and bacteria. Specifically, these are ants in the tribe Attini that cultivate fungus gardens. These farmed fungus-gardens are the ants’ primary source of food. Integral to health of the gardens are bacteria in the genus Pseudonocardia that live on the ants' appendages. The symbiotic bacteria are thought to make antibiotics that protect the gardens from invasion by parasitic fungi in the genus Escovopsis

Over the years Currie and co-workers discovered that in addition to the bacteria, the fungus AND the pathogen invaders, black yeasts are also very common colonizers of Attini ants. This paper was aimed at trying to figure out where in the ant-fungus garden-bacterial symbiont circle the yeasts fit in.

Specifically, they asked, what are the effects of a weak interaction from the yeasts on the ant/fungus/bacteria community? While chemical ecologists tend to focus on strong and long-lasting interactions between pairs of organisms, transient and non-specific interactions can have large ecological effects as well. Just ponder a moment the whole-body effects of a bacteria-induced case of food sickness: You are stuck in bed drinking fluids and your gut bacteria have to recover from having their neighbors flushed away!  


Dearest ladies,

I am really struggling with the experiment shown in Figure 1.

This is basically the piece de la resistance of this paper. It shows that the fungal cultivar fares worse when the black yeast is around. This result—that the black yeast are hurting the ecological success of the ants by making the fungal gardens more susceptible to Escovopsis attack—is the impetus for all of the other work in the paper. Yet, to my eyes, it seems like the effect is very small: the two lines’ final values seem so similar. Their end points only differ by 0.2 and the standard error is reported to be <0.01.  How many times was this experiment done? Given the complicated nature of the experimental set up, it seems amazing to me that the error is so small.


CAB:  Well, Alexandra, how should we feel about the data that supports the question at the heart of this paper? I think for the moment we need to just assume that this is a repeatable, significant difference in the ant garden health when black yeasts are present. If we can trust their experimental set-up, then we might conclude that though this interaction isn't intense, it is real and reproducible.

I have a few questions about the first experiment investigating a hypothesis as to how the black yeasts were negatively impacting garden health.

This experiment compared growth of the black yeasts on two different solid medias (solid media meaning agar-based, so sort of like a dry jello): agar containing ground-up bacteria collected from the ants and agar with no additives. They saw significantly more black yeast growth on the agar containing bacterial biomass. From these data, they conclude the black yeast can use bacterial-derived nutrients for growth.

To me, if you want to make a conclusion about the specificity of the black yeasts consuming the symbiotic bacteria you would need to compare growth on agar with biomass from different bacteria, and include isolates that don’t come from the ants but from the surrounding environment.



Hi ladies!

Interesting read - I always admire these types of experimental investigations. The natural world is incredibly complex!  Designing experiments to study these interactions is challenging and come with a caveat: the conclusions may not reflect what is going on in the environment as the interactions in Nature could be influenced by additional direct or indirect inputs, biotic or abiotic, that are not captured in the laboratory.  I suppose makes this an interesting paper to discuss! 

So onto your question Carolyn. I had the same thoughts when reading the experimental techniques. I guess I don't know too much about the feeding habits of fungi. Do they prefer very specific species/genera of bacteria? Or do they indiscriminately eat bacteria? If it is the former, then yes, the experiment you suggested seems very necessary.

So we are left asking if the Pseudonocardia bacteria are necessary for growth of the black yeasts on the ant or can the black yeasts obtain other food sources from the environment?

What happens to the fungal gardens when the Pseudonocardia bacteria are completely depleted from this network of organisms?


AMC: Regarding whether or not the black yeast like to feast upon Pseudonocardia: Are they really asking if the black yeast are eating the Pseudonocardia?  Because if they are, then why couldn't they do the experiment with live bacteria?  By adding dead Pseudonocardia bacteria to the media aren't you just enriching the media by adding more nutrients? Most laboratory media for growing bacteria contains ground-up yeast. As you two ladies have pointed out, it would be much more compelling if the black yeast opted for a Pseudonocardia bacteria meal over another bacterial option.


CAB:The questions keep coming while I consider another experiment. They wanted to figure out how the presence of the black yeasts impacted the general success of ant's bacterial symbionts. They grew the black yeasts together with Pseudonocardia on Petri dishes containing "nutrient medium" which allows for "good growth" of both the black yeast and bacteria. The two organisms were grown together on the plates, and the sizes of their colonies measured as an indicator of success. Their results lead to the conclusion black yeasts grow better with bacteria than alone, while bacteria grow worse with the black yeasts than they do alone.

How does the set-up of this experiment compare to Nature's set-up? I'm especially interested in the structure of the two communities on (around?) the ant. Are the bacterial colonizers well established on the ant before the black yeasts arrive? How do the inoculum sizes (i.e. the number of individuals cells of each organism present) differ? For instance, are there always 100-fold more bacterial cells than yeast cells (this would be my guess)? 

Second, we must take a lot for granted here in terms of the nutritional standards of the medium and their method for measuring growth. For instance, the yeast might obtain a limiting nutrient from the ants that isn't present in the nutrient medium; the ants might grow smaller colonies when the black yeast is present for a variety of reasons, such as the production of a diffusible factor by the yeast that inhibits large colony growth.

Alternatively, the ants might just take longer to get to the same colony size, and while their growth rate is slowed, perhaps that's never the issue in Nature as the ants are always well established by the time the yeasts show up.


KLS: I would imagine the ideal set up for this experiment would mimic the environment on the ant cuticle as best as possible. The authors mention that these cuticles are attached to ant glands that provide nutrients for the bacteria. What are these nutrients and how do they compare to the conditions used in this paper for growth?

I am fairly certain bacterial colonies are established early in an ants lifetime but the black yeasts may be established fairly early in the life of an ant as well. It seems we need answers to some central questions about the relative size and establishment of these microbial communities before we can think about a more “realistic” experimental set-up...


AMC: Since we are all about chemical ecology, maybe we can discuss the role of natural products in interpreting the results of this paper.  Of course there are the antifungal molecules produced by the Pseudonocardia - but what else? Are there other interactions that we think could be mediated by secondary metabolism?  Or do we think this interaction is more about competition for resources?  In other words, the black yeast is usurping nutrients used by the Pseudonocardia rather than actively trying to kill it.


CAB: OK, here’s me thinking more outside the box when it comes to the chemical ecology of this story. Maybe there are inhibitory compounds coming from the black fungus that stunts the growth of Pseudonocardia? Maybe the black yeasts physically block the ants from delivering their dosage of drugs to the fungal gardens to protect them from invaders. Not chemical, but it could be the culprit?


Life will find a way: a story from chemical ecology...and 2017?


2016 was a doozy. Merriam-Webster Dictionary nailed it when they named "surreal" the word of the year. That's because so many unexpected, upsetting and very serious events transpired in the past 12 months. Events that left us, at the very, very least, dazed and confused and wondering, when will this year end again?

So if you are are looking for some last-minute inspiration to carry you from the Year of the Crazy into 2017, Year of the ?, then you have come to the right place. Today I am here to provide an encouraging and optimistic note from the land of chemical ecology: As we've seen time and time again on our blog, life will find a way!

An article published in the New York Times on December 9th highlighted recent research that touches on common themes in chemical ecology. The research was published in the journal Science and is entitled "The genomic landscape of rapid repeated evolutionary adaptation to toxic pollution in wild fish." This work was a collaborative effort by scientists from the University of California Davis, the Woods Hole Oceanographic institute, and several other universities/institutes. The paper focuses on the question, how do organisms adapt to high levels of pollutants that humans have dumped into their ecosystem? How does a pollutant-sensitive organism become resistant to these toxic chemicals?

The researchers asked these questions in the context of Atlantic killifish populations, which, in certain geographic areas off the coast of the Northeast, have adapted to levels of halogenated aryl hydrocarbons and other persistent industrial chemicals that would normally be lethal to these fish. The questions posed by this paper are important to consider as many populations of animals throughout the world are in decline due to the activities of humans. The human-generated pollutants, often highly toxic to animals, have introduced new, strong selective pressures on organisms. Can we predict which organisms may be better equipped to adapt to these selective pressures by looking at the genetics of populations that have successfully evolved to live in polluted spaces?

Chemical pollutants are not created equal. Each chemical can impact health through different mechanisms, and that toxicity can be caused by widely different dosages. Chemicals even have very different stabilities-- some stick around environment for decades while others are degraded quickly. The same molecule can even impact organisms at different trophic levels (a trophic level indicates where in the food chain of a particular ecosystem an organism lives, from the large predators in an ecosystem, to the herbivores, plants and finally the bacteria at the bottom of the food chain) in different ways. Take, for instance, the case of neonicotinoids: these highly used pesticides effectively kill insects, of course, and can also impact the health of bird populations that rely on the bugs for their food.

The Atlantic salt-marsh estuaries where the Atlantic killifish (Fundulus heteroclitus) makes its home are polluted with a cocktail of persistent industrial chemicals including halogenated aryl hydrocarbons like the infamous polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). These pollutants bioaccumulate, meaning that fish and other animals that live in contaminated waters will accumulate the molecules in their tissues, much like mercury and tuna. PCBs affect fish growth at the embryo stage: susceptible fish do not develop normal tissues or organs and often do not survive to adulthood.

To ask the question, what genetic changes happened that allowed populations of Killifish to thrive in polluted waters, the researchers collected fish from eight sites, both contaminated and pristine (each contaminated site was paired with a nearby pristine site), where the killifish were either tolerant or susceptible to pollutants, respectively. One of the most contaminated sites is at New Bedford, Massachusetts, a so-called "Superfund site" where PCBs were discharged into the waters as industrial wastes for thirty years. The genomes of 50 fish from each of the sites were sequenced. First, the researchers found that the populations that were closest geographically had the most similar genetic backgrounds, and from this data they concluded that tolerant fish populations evolved independently. In other words, there was not one group of fish that evolved to be tolerant and then spread out to the other sites, but rather evolution happened at each of these sites independently.  

Next, the researchers analyzed the genomic data of the pairs of tolerant/susceptible groups of fish and looked for regions in the genomes with particularly high nucleotide diversity between the two groups; these outlier regions could give clues as to what gene changes differentiate tolerant from susceptible relatives. They found several genetic loci that were different across the four pairs of tolerant/susceptible groups. However there was a set of genes that was shared among the geographically separated tolerant populations and looked to be under strong selection. The genes are all involved in how the fish responds to aryl hydrocarbons, namely genes in the aryl hydrocarbon receptor pathway.

To follow up on these nucleotide changes shared by the tolerant fishes, the researchers measured the amount that these genes were expressed when the fish were raised for two generations in a clean environment and the embryos from the second generation were challenged with a PCB. They found that expression of genes in the aryl hydrocarbon receptor pathway was much lower in the fish derived from the tolerant populations than those from the susceptible populations. In essence, the fish that are tolerant to PCBs have become desensitized to these chemicals. From these results, the researchers conclude the aryl hydrocarbon receptor "signaling pathway is likely a key and repeated target of natural selection in tolerant populations."

From this work, we see that killifish from various polluted sites have undergone convergent evolution in order to adapt to ecosystem contamination. The killifish have converged on a solution to toxicity that involves dampening the cell's response to the presence of PCBs and related compounds. Essentially,  toxicity is mediated by decreasing the expression of genes that encode for the proteins that change cellular gene expression in response to PCBs.

A central question that follows from this work is, can we expect other populations to respond in the same manner as the killifish or are these common fish somehow special in their ability to adapt to a toxic environment? In the Science paper, the authors state that "selection on preexisting variants was important for rapid adaptation in killifish and that multiple molecule targets were available for selective targeting of a common pathway." The translation of this important concluding statement has two parts.

First, killifish may have been particularly successful at evolving to withstand pollutants because the killifish population was large and genetically diverse from the get-go. A larger population means there's a higher probability that an individual within that group has a mutation that lends it greater reproductive success in the face of a evolutionary pressure (in this case, toxic chemicals).

The second part of this concluding statement is also important and gets back to the "chemicals are not created equal" line of thought expressed above. For PCBs, the pathway in the cell that translates reception of the chemical signal into changes in gene expression involves many proteins. The moving parts of the PCB response in the cell means there are multiple targets in the pathway for evolutionary change. The authors suggest that because there are multiple proteins required for this pathway to function there are more chances for nucleotide changes to impact the cell's response to the chemicals. In contrast, if there was just one gene involved in responding to PCBs, there is a lower probability that a genetic variant in a population would be a variant in that one gene.

So, to wrap it up: the killifish have survived lethal levels of PCBs to swim another day. And while we should make efforts to clean up our messes when we make them, its encouraging, at least to me, to know that nature finds a way to make it work. Here's to making it work in 2017!



Ants and morphine

image (ANT) from jeff kubina (FLICKR)

image (ANT) from jeff kubina (FLICKR)

According to a study published this fall, ants can get addicted to morphine. The subject sounds absurd, but the study was interesting - or “novel” - for a few different reasons: first of all, behavioral studies related to addiction and reward-seeking behavior are almost always performed in mammalian system. Secondly, similar studies performed in invertebrate systems (such as the previously described bees and cocaine study), are always done in the presence of some sort of non-drug reward.  In other words, most studies tracks how an insect responds to a food-based reward (like sucrose) in the presence of drug, rather than how the insect responds to just the drug itself; this seemingly minor detail can potentially obscure the motivations behind drug-seeking behavior.  Finally, rather than simply perform behavioral studies on these ants, the researchers also acquired chemical data supporting their hypothesis that ants exposed to morphine are actually undergoing neurochemical changes analogous to changes undergone by humans addicted to morphine.   

In brief, Entler et al. performed a series of behavioral experiments to examine how ants previously exposed to morphine respond to rewards of morphine versus sucrose. Most of these experiments involved variations on the following:  ants that are systematically exposed to ("trained on") morphine are given access to dishes either containing morphine or sucrose, or a mixture. These morphine-trained ants consistently select the morphine dish - even in the absence of the more conventional ant reward, sucrose. Ants that were never exposed to morphine, however, showed no preference for the drug.  Additionally, researchers detected a 2-fold increase in dopamine levels solely in the brains of the morphine-trained ants – providing evidence that morphine exposure is likely affecting chemical processes in the ant brain. 

The take home message of this article is that, like vertebrates, invertebrates are inclined to become addicted to, and self-administer morphine. Furthermore, the chemical effects of this behavior are conserved across a broad and diverse range of animal species, suggesting that non-mammalian systems may prove valuable in untangling the neurochemical and behavioral underpinnings of drug use and addiction. 

From an ecological perspective this study is also rather thought-provoking.  As discussed in "bees on cocaine", plants are thought to produce psychoactive molecules as a deterrent to insect predators.  But do ants actually encounter morphine in their normal, non-lab lives? And what happens when an insect becomes addicted to this molecule?  Does it provide any benefit for the plant?  Does it significantly alter the lifestyle of the insect? While this particular paper does not aim to address these types of questions, I think they're worth considering.  The effects of addiction in a purely ecological context might actually hold some interesting insights into the evolution of this seemingly paradoxical behavior. 



The Cone Snail: beautiful but deadly

The cone snail shell is known for its beauty. The organism that resides inside is a highly adapted predator that uses a complex mixture of toxic peptides to trap and disable its prey. &nbsp;

The cone snail shell is known for its beauty. The organism that resides inside is a highly adapted predator that uses a complex mixture of toxic peptides to trap and disable its prey.  

The cone snail shell is a lovely and familiar item. Displaying a stunning diversity of patterns and colors, artists, jewelers and shell collectors have long admired these little ocean gems. In fact, in 1796, a cone snail shell (Conus cedonulli, shown below), measuring just 5 cm, sold at auction for over 5 times the price of the Vermeer painting Woman in Blue Reading a Letter. While innocuous as a display item, when occupied by the feisty cone snail, these shells become armor for one of the most venomous of the oceans critters. 

C.&nbsp;cedonulli  - the same type of shell that outshined a Vermeer at auction

C. cedonulli - the same type of shell that outshined a Vermeer at auction

When you look at a cone snail, it doesn’t really look that scary. Ranging in length from a few to ~10 centimeters, and displaying the aforementioned beauty, one might actually feel compelled to pick it up and peer closely at it. I would advise against this. These snails, which generally feed on small fish, worms, or other mollusks, are fierce hunters. Generally (though the exact order of events can vary from species to species), the snail attacks its prey by luring it in with an enticing-looking long proboscis. When the prey draws near, the snail shoots out a venom-filled “harpoon” thus rendering its target paralyzed, disoriented, and just generally helpless. Once it has immobilized its prey, the worm extends its large sucker-like mouth around the helpless victim and ingests it. Pretty grim stuff.  The venom injection of a cone snail is sufficiently potent to kill a human (though reports of fatalities from cone snail poisonings are rare); this toxic venom causes a range of neuromuscular effects such as paralysis, numbness, disorientation and difficulty breathing.

But what is in this venom? Why is it so potent? These are, of course, the first questions a natural products chemist might ask when learning about the extreme biological effects of this mysterious substance. Baldomero Olivera, a professor at the University of Utah, has dedicated his research career to these questions. Olivera first became aware of this marine organism while growing up in the Philippines where cone snails are ubiquitous. He has since published numerous research articles on a wide variety of neurotoxins (mostly called “conotoxins”) exuded in the cone snail venom. His research has revealed that the venom is primarily composed of peptides - strings of 10-30 amino acids - often containing multiple intramolecular connections (mainly disulfide bonds) that give the molecules their three dimensional shape.

Over the last 25 years the Olivera lab has identified a huge number of these cone snail neurotoxins. Perhaps not surprisingly, many of these molecules (mostly those identified from fish-eating species like Conus geographicus or C. magnus) have been shown to affect mammalian neurological and neuromuscular systems. The toxins are divided up into three major classes based on their identified vertebrate targets:  the α-conotoxins bind the acetylcholine receptor, µ-conotoxins block sodium ion channels and ω-conotoxins block calcium ion channels.

Two representations of the ω-conotoxin ziconotide (Prialt®). In the 3D form on the right the disulfide bonds that give the molecule its shape are marked in yellow.&nbsp;This analgesic is currently used in the clinic primarily for pain control in cancer patients. &nbsp;

Two representations of the ω-conotoxin ziconotide (Prialt®). In the 3D form on the right the disulfide bonds that give the molecule its shape are marked in yellow. This analgesic is currently used in the clinic primarily for pain control in cancer patients.  

What makes these molecules truly special, however, is that they can be tissue specific. Many molecules (including approved drugs) that target neurological transmission pathways cause severe side effects because they interact with closely related proteins in a large variety of tissues. Studies on the binding affinity of the conotoxins suggest that they may be able to discriminate between highly similar binding pockets of closely related protein subtypes expressed in different tissue types. For example, µ-conotoxins bind more strongly to voltage-gated sodium channels in skeletal muscle than the subtypes of these channels found in neurons, whereas previously identified sodium channel inhibitors, like tetrodotoxin (TTX), cannot discriminate between receptors in these two tissues.

Since selectivity is a desirable characteristic of human therapies, this property of conotoxins makes them an intriguing source of new drugs. In fact, ziconotide (or Prialt®) is a ω-conotoxin isolated from C. magnus, which specifically binds voltage gated calcium ion channels in neuronal tissues. This molecule is an FDA approved pain reliever and is often used in cases where morphine is ineffective or insufficient. Ziconotide, however, is not a first-line analgesic, as it has to be administered directly into the spinal fluid - a process that is invasive and presents additional risks to the patient. Currently researchers are hoping to develop variants of this molecule with properties that would allow for intravenous or even oral administration.  

To isolate novel conotoxins, researchers extract the venom and run it over a column that separates the components by physical property. Each peak is a different conotoxin, and as depicted above, each conotoxin may cause a different phenotype when injected in mice. &nbsp;Note: the trace shown above is just a representation, not data from a real experiment.

To isolate novel conotoxins, researchers extract the venom and run it over a column that separates the components by physical property. Each peak is a different conotoxin, and as depicted above, each conotoxin may cause a different phenotype when injected in mice.  Note: the trace shown above is just a representation, not data from a real experiment.

The toxin delivery strategy of cone snails is also fascinating because of its molecular complexity. The venom is a mixture of a variety of conotoxin subtypes (termed a “nirvana cabal”), meaning these molecules are delivered in concert and can hit multiple targets simultaneously. In other words, cone snails were using the drug cocktail approach long before combination therapies were put into use by humans. Further understanding the full biological effect of the venom could provide insight into the interconnectedness of the distinct molecular pathways targeted by each conotoxin.  

Analysis of the many venom contents of a diverse species of cone snails has uncovered a large variety of novel peptides that may one day provide novel therapeutics for pain relief or neuromuscular disorders. Yet cone snail venom has also been shown to contain the familiar. In a recent study by the Safavi-Hemami lab at University of Utah researchers found that some fish-eating cone snails inject their prey with insulin (as a component of the venom mixture). Much like in humans, this rush of insulin causes a plummet in blood sugar making their target fish lethargic and susceptible to cone snail predation. It’s remarkable to consider how varying the ecological context in which a molecule is produced could so completely alter its biological role.

Just within the genera Conus there are already >500 distinct known species.  But there are also other families of marine gastropods closely related to cone snails that could provide hundreds of thousands more unique venom sources. These small, resourceful marine creatures are a gold mine for short peptides with potent neurological effects. And whether they become drugs, or biological probes, or just molecules that can cause weird phenotypes in mice, they have the potential to broaden how we view the beauty of the cone snail.


Molecule of the Moment: Tetrodotoxin (TTX)

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Name: Tetrodotoxin (TTX)

Source: Tetrodotoxin is a highly potent neurotoxin found in a surprisingly large, phylogenetically-diverse group of organisms and has a wide eco-geographical distribution. Organisms known to contain TTX include: 

1.     marine animals such as tetraodon fish (see: puffer fish, blowfish, porcupine fish, globe fish), blue-ringed octopusesmoon snails, starfish, and crabs

2.     terrestrial amphibians such as newts and frogs

3.     microscopic organisms such as algae, plankton, and bacteria

The widespread occurrence of this highly complex molecule in such phylogenetically distinct organisms has puzzled many. Which of these organisms are biosynthesizing and producing TTX? Are the higher organisms – such as fish and amphibians- sequestering TTX from their diet? Do any of these animals harbor symbiotic microbes that supply host organisms with the compound? Or - is it possible that the higher organisms have evolved to make TTX themselves?

The answer to the true source(s) of TTX appears to be quite complex. Many different types of bacteria, including both symbiotic and environmental microbes, have been found to produce TTX, though no biosynthetic genes have been identified (yet!). In order to investigate whether certain organisms sequester the TTX from their food or obtain it from other sources (symbionts, etc), scientists have conducted isolation experiments with higher organisms raised on nontoxic diets. Interestingly, certain animals, such as puffer fish, decrease toxicity during isolation (suggesting the toxicity primarily derives from a TTX-containing diet), while others, such as newts, have been found to increase their toxicity (though no TTX-producing symbionts for amphibians have been identified to date). Much of this data is inconclusive, however, and the mystery of the origin and ubiquity of this highly unusual toxin is still very much an active research area.

(a) Tetrodotoxin (TTX)&nbsp;has a (b) adamantane core with a (c) guanidinium Motif.&nbsp;

(a) Tetrodotoxin (TTX) has a (b) adamantane core with a (c) guanidinium Motif. 

Chemical structure: TTX is a cage-like compound with a positively charged cyclic guanidinium moiety fused to a diox-adamantane skeleton (read: four connected 6-membered carbon rings with hydroxy groups attached). Several analogues have been found in Nature, though the toxicity of these derivatives is often lower. Interestingly, TTX and its derivatives are the only known natural products that incorporate the 10-atom cage architecture of adamantane, though a few synthetic drugs are known to contain this motif.

Discovery: TTX was initially identified in pufferfish, whose potent toxicity was documented as far as back as the first or second century BC (the therapeutic use of tetradon fish eggs were even described in the earliest known Chinese pharmacopeia, The Book of Herbs!). Anecdotes of pufferfish poisoning – intentional and otherwise- were recorded over the following centuries, including this fabulous description in the journals of Captain James Cook (who luckily survived the incident) in 1774:

“We were seized with the most extraordinary weakness in all our limbs attended with numbness of sensation like to that caused by exposing one’s hands and feet to a fire after having been pinched by much frost. I had almost lost the sense of feeling nor could I distinguish between light and heavy objects, a quart pot full of water and a feather was the same in my hand”

These intriguing properties of the toxic puffer fish inevitably caught the attention of scientists and in 1889, the first comprehensive study on the pharmacology of the pufferfish poison--then termed "tetrodotoxin"--was completed in Japan. Isolation of the active compound, however, was not achieved until 1950 and the molecular structure was not elucidated for another 14 years. In 1964, chemists from three independent research groups (K. Tsuda, T. Goto, and R.B. Woodward) all reported identical structural characterizations of TTX at a natural products symposium in Kyoto. 

While many organic chemistry groups around the world were racing to identify the pufferfish toxin, scientists in California were preoccupied with studying of a toxic, paralyzing substance found in Taricha newts. With the help of organic chemists, they were able to isolate sufficient quantities of the toxic substance for characterization (using a mere 1000 kilograms of newt egg clusters). Surprisingly, they found that not only the pharmacological properties of this substance were oddly similar to tetrodotoxin, but the chemical properties were as well. This finding was reported in Science in 1964 (the same year as the puffer fish reports!); both the newts and the puffer fish contained the exact same unique, highly toxic defense chemical.

Since the initial discovery in newts and tetradon fish, TTX has been identified in many several additional organisms (see: Source).

Biology: TTX’s high toxicity is due to its ability to selectively bind voltage gated sodium channels of muscle and nerve tissues. Binding of the toxin inhibits the flow of sodium ions through the channels, halting virtually any function dependent on electrical excitability of nerve and muscle tissues (think: locomotion, cognition, etc.).


For organisms producing/containing TTX:

·       Defense. TTX is generally thought to serve as a deterrent to predation, though this has rarely been tested experimentally. The antipredation role of this toxin has been characterized, however, in Taricha newts, whose “arms race” with its often TTX-resistant predator, the garter snake, has been extensively studied by evolutionary ecologists (highly resistant garter snakes are only found near highly toxic newts, suggesting the evolutionary pressure to evolve resistance is stronger in these populations).

·       Other ecological roles. TTX has also been found to serve as a pheromone for certain pufferfish, blue-ringed octopus, and arrowworms and has been shown to be used for prey capture by flatworms. Given the sheer number of organisms containing TTX, other functions for this compound are bound to be elucidated in the future.

For humans:

·       Neurobiology. TTX has been an incredibly valuable chemical tool for the study of neurophysiology. Classic experiments carried out in the mid and latter part of 20th century with TTX and other natural toxins helped elucidate the role of ion channels in membrane physiology. These early experiments greatly facilitated the identification and characterization of ion channels, as well as the overall electrical behavior of cell membranes. In the following decades, TTX and its interaction with sodium channels have become one of the best documented toxin/receptor pairs in science (a search for "tetrodotoxin" in PubMed yields >18,000 results!).

·       Medicine. Scientists have explored the potential of the potent analgesic properties of TTX for treating migraine, withdrawal symptoms in heroin addicts, and pain in cancer patients.

Dangers: TTX is one of the most powerful neurotoxins known (over 1000x more toxic to humans than cyanide!) and has no antidote. Responses are dose dependent and symptoms include tingling of the tongue and lips, headache, vomiting, muscle weakness, and ataxia. The onset and severity of the symptoms are dose dependent but TTX ingestion can result in DEATH due to respiratory and/or heart failure.

As tetradotoxin has the potential to pose a severe threat to both human and animal health, it is listed a "select agent" by the US Dept of Health! 

Interesting fact: Despite the very serious dangers listed above, pufferfish is still consumed, primarily in Japan, where it is considered a very tasty delicacy known as fugu. Part of the appeal of fugu is said to be the sensation of oral numbness due to just the right amount of TTX blocking the sensory nerves. To make sure patrons enjoy meal without grave consequences, fugu must be properly prepared and served carefully so there is rigorous training and licensing of chefs who are permitted to do so. Despite the precautions taken by the Japanese government (and ours!), incidents of poisoning and death by miscalculated and ill-prepared fugu dishes still occur (in particular by thrill-seeking “foodies” who specifically ask for organs known to contain high levels of TTX, such as the liver, and are granted it illegally). Eat at your own risk!!!



Summer Booklist

We are well into summer which we all know is a great time to chip away at the old book list.  Here are some of my book reading goals for this summer:  

Lab Girl - By Hope Jahren

This book sounds like a wonderful mix of lab-life anecdotes and personal reflection, with a sprinkling of interesting facts about plant science.

I Contain Multitudes:  The Microbes Within Us and a Grander View of Life - Ed Yong

This will not be available to buy until August  so it's a good one to work into the latter half of the summer.  With our ever-expanding awareness of the ubiquity of microbes and their undeniable impact on the human existence, this will likely be a pretty eye-opening exploration of the human microbiota and beyond.  You can find more info about this book and Ed Yong's science writing on his website.

In the Company of Microbes - Elio Schaechter

Elio Schaechter is a (retired) microbiologist who runs the wonderful blog Small Things Considered. This book is a selection of entries from his past 10 years of blogging as well as his personal reflections and musings. Sounds delightful.

The Gene: An Intimate History - Siddhartha Mukherjee

This book not only details the history of genetics, it explores the future of this field as our regard for the significance of epigenetics expands and our ability to manipulate the human genome fall within reach. Sure to spark some lively debates!  Also Mukherjee is the Pulitzer Prize-winning author for the book The Emperor of All Maladies.  

On the Move - Oliver Sacks

This is a somewhat sentimental choice but it will undoubtedly be a thought provoking and emotional read.  Oliver Sacks, who wrote one of my favorite books, The Man Who Mistook His Wife for Hat (consider this a recommendation), died last summer.  His last book, On the Move, is a memoir.  See a more eloquently written review here.

When Breath Becomes Air - Paul Kalanithi

I actually cried just reading the NYT review of this book so proceed with caution.  When Breath Becomes Air is also a memoir of sorts, written by a doctor, diagnosed with terminal cancer, during his last year of life.  Absolutely astounding reviews from both “experts” and friends.  You can check out an excerpt here.

Quiet:  The Power of Introverts in a World that Can’t Stop Talking - Susan Cain

A book about the importance of introverts in a society that undervalues them.  Interesting stuff!  Mostly I want to read this because I am an introvert and am looking for some positive reinforcement. 

My Brilliant Friend - Elena Ferrante

The number of people who have recommended this book (and series: The Neapolitan Novels) to me is astounding.  So, this summer, it’s finally happening.  This series of books explores the complexity of female friendships in the background of 20th century Naples.  These books are basically an international phenomenon, though the author herself is apparently quite a mysterious figure.

The Girls - Emma Cline

A “coming of age” story about a teenage girl drawn into the intoxicating world of a charismatic cult-leader.  Loosely based on Charles Manson’s following during the summer before the Tate-LaBianca murders, this sounds like a pretty thrilling read.  

Swann’s Way - Marcel Proust

This is on my booklist every summer. 

Any recommendations?  Want to criticize my taste in books? Make a note in the comments section! 


Relevant or not: Return to the Plastisphere!

Depending on how closely you follow Chemical Intuition, you may or may not remember our article from way back in June of 2015 entitled "Welcome to the Plastisphere." In that article, we talked about the Texas-sized mass of microplastics in the Pacific Ocean and the huge amounts of plastic accumulating in the world's waters. We discussed the possible ecological impacts caused by the presence of so much plastic in the ocean. One of the conclusions from this piece was that microplastics are a magnet for pollutants present in the ocean, and if organisms ingest these microplastics, toxicity can occur through the high dose of pollutants adsorbed to these tiny pieces of trash. However, one conclusion from this piece was that direct evidence for ecosystem impacts were lacking. 

In today's "Relevant or not" post, we'd like to quickly highlight a few recent articles that lend serious credence to the theory that, yes, microplastics significantly and negatively impact ecosystem functioning. 

First, a paper in the Proceedings of the National Academy of Sciences from Sussarellu and co-workers demonstrated that when oysters ingest microplastics during gametogenesis, the feeding and reproductive capacities of the oyster are negatively impacted. This leads to decreases in the oysters' individual fitness. This is problematic as oysters are incredibly important for the role they play in filtering water; oysters form reefs in the ocean, and they function to increase both the water quality and biodiversity.

A commentary on the above article by Galloway and Lewis noted a calculation performed using data from another research paper that "the average dietary portion of six oysters would contain around 50 plastic particles." Oysters are highly effective filter feeders and remove around 70% of the microplastics in the surrounding seawater. The result? Your fancy seafood dinner is full of plastic debris.

The plastics we pump into our environment travel full circle, ultimately returning to us, the consumers. We can't escape our wasteful ways.

A recent article in the journal Science also discovered negative impacts of microplastic ingestion on organismal fitness, this time in the European perch. In this paper, they show that exposure to relevant concentrations of microplastic polystyrene particles resulted in lower growth rates and altered feeding behaviors in the European perch larvae. Most worrisome, perhaps, was that ingestion of plastic impacted the ability of the fish to respond to natural chemical cues; the larvae exposed to microplastics did not respond to olfactory threat cues, meaning that many more died at the hands of their predators.

These papers are part of a growing body of scientific literature pointing to the very real and negative impacts microplastics in the ocean have on organismal fitness. The impacts on organism fitness impact ecosystem functioning. Combined with other human-driven environmental perturbations (including ocean acidification as a result of larger concentrations of dissolved carbon dioxide and rising water temperatures as a result of global climate change), we should be very worried about the collapse of marine ecosystems.

What are we to do? As individual consumers, we can do as much as we can to reduce our plastic consumption. But much more is needed. Galloway and Lewis closed their commentary with strong words we should listen to: "Given the impossibility of removing all microplastics contamination from the oceans, the impetus is on all of us--governments, scientists, and individuals--to reduce our utterly ridiculous levels of plastic consumption and waste before we induce permanent alterations to our fragile marine ecosystem."



Casual Fridays at Chemical Intuition: Moth wedding gifts?

With (human) wedding season fast approaching, we felt that it made sense to shed some light on the wedding traditions of other species. This week, we're reading another paper from the dynamic chemical ecology duo, Meinwald and Eisner, entitled "Pheromonal advertisement of a nuptial gift by a male moth (Utetheisa ornatrix)." The paper was originally published in the Proceedings of the National Acadamy of Sciences in 1991 and is freely available here.


Carolyn: So, I will admit that I chose this paper because of its intriguing title. Also, I knew that a classic Meinwald and Eisner paper promised to be both strange and highly informative.

This paper deals with the study of the nuptial gift of a male moth to the female moth during mating. First, let's just call the nuptial gift what it really is: it's a spermatophore. A spermatophore is a package of sperm plus nutrients and, in this case, defense compounds, transferred to the female upon mating. It had been found previously that male moths have circulating pyrolizidine alkaloids that they transfer in the spermatophore, and these alkaloids are potent defense compounds capable of warding off predators. The pyrolizidine alkaloids are derived from a plant (specifically the seeds of Crotalaria) that the moths eat.

To signal to females that he's carrying a load of alkaloid, the male moth converts a pool of the pyrolizidine alkaloids to a volatile pheromone called hydroxydanaidal. Hydroxydanaidal is emitted from a pair of brushes, also called "hair-pencils" or "coremata.” The male uses the hair-pencils to waft the pheromone toward the female in order to seduce her. According to this paper, "males endowed with hydroxydanaidal have a higher mating success than those devoid of the pheromone," so the strategy works.

The question at the center of this paper was whether the amount of pheromone on the hair-brushes (and advertised to potential mates) was correlated with the amount of alkaloid in the moth (i.e. the systemic alkaloid) or the amount transferred to the female via the spermatophore.

So my question for the ladies of Chemical Intuition is the following: given that the hydroxydanaidal is derived directly from systemic alkaloids, is there any reason to hypothesize that the pheromone content would not be correlated with the amount of alkaloid systemically or in the spermatophore, ahem! nuptial gift?

The male uses the hair-pencils to waft the pheromone toward the female in order to seduce her.
— Carolyn

Alexandra: So given the assumption that these systemic alkaloids are precursors of the pheromone, then, at least in my extremely humble opinion, it seems highly likely that the quantities of the two would be correlated positively. In other words, their results did not make me fall out of my chair or anything.

Something that might be vaguely related though, and could be interesting to consider, is the potential evolutionary advantage of utilizing all precursor alkaloids for pheromone production. This is related to behaviors that evolutionary biologists call "cheating.”* Basically, if the male moths produced a ton of hydroxydanaidal, leaving no protective alkaloids for the marital gift, the females would basically be tricked into mating with them and would receive none of the advantages implied by their impressive pheromone endowment or whatever. Horrid! Do we think this could happen? And if we think deeply about how this would affect relative quantities, could this be related to the original question posed by the authors?


*This paper defines cheating as “engaging in behavior that exploits the cooperative behavior of conspecifics by imposing fitness costs on them, while providing fitness benefits to the cheater.”


Kristen: Do the precursor alkaloids serve any other purpose for the male beyond protection from predation? Is there huge energetic cost to converting these alkaloids to the pheromone? And how is the pathway to pheromone from alkaloid regulated? For instance, could an organism, as Alexandra suggested, increase pheromone production perhaps upon receiving some signal (perhaps a chemical signal that a single lady moth is flying about), leaving no protective alkaloids and effectively "cheat" just in time?


Carolyn: Wow! So many questions and so few answers :) 

I think that many of our questions are centered trying to understand the possible reasons why the pheromone titre is positively and linearly correlated with the titre of systemic alkaloid. In other words, we're generally wondering what are the energetic costs and regulatory systems that lead to this result and why isn't cheating (i.e. over representation of systemic alkaloid content) prevalent? 

To get at the energetic costs, I think the best route to explore this topic is to investigate how the alkaloid, which is ingested from the favored food source of the moths, is converted to the hydroxydanaidal. Just looking at this structure (and using my chemical intuition!!) it looks as though there needs to be two hydrolysis reactions (to remove the linker bound by two ester groups) and two oxidation reactions (one to convert the alcohol to an aldehyde and another to oxidize the pyrolizidine ring to the pyrrole oxidation state). I think that this could conceivably occur nonenzymatically, but my bet would be that there are three, perhaps four enzymes that are involved in this biosynthetic pathway. Given that this reaction involves the oxidation of the ingested alkaloid, it could be possible that the production of the pheromone contributes to redox balance in the insect, i.e. the regeneration of reduced cofactors like FADH2 and NADH.

Regulatory systems: If we knew the genes involved in the biosynthetic pathway, we could look at the elements that regulate transcription of these genes. Are they constitutively expressed? Or only expressed when mating is imminent? In the paper we read, they compared the amount of hydroxydanaidal in both once-mated and virgin males. Based on these data, it looks as though the levels of pheromone are similar in these two groups. While I'm not an expert on moth physiology, this would imply to me, at least, that even without a female around to mate with, males produce significant quantities of pheromone that they display on their hair-pencils. 

Evolutionary reasons to not be cheaters: I think the number one reason I can think of in this regard is that if the males convert all of their pyrozilidine alkaloids to pheromone, then they have no defensive compounds left to defend themselves. I think that the amount of pheromone produced is probably the result of an evolutionary trade-off between self-protection and successful advertisement to potential mates. Another reason to not be a cheater in this scenario is that the alkaloids transferred in the spermatophore go on to protect the eggs of the nascent moth baby; thus, the nuptial gift is, in a way, an insurance policy of the male to ensure that his progeny survives and goes on to propagate the genes of Papa Moth.


Alexandra: Great points Carolyn. You sent me on a whirlwind of thoughts that I hope are relevant.  

Given the many advances in science technology since these experiments were performed, it's interesting to think about what experiments we would do now that were not feasible in 1991. I think Carolyn alluded to a few of them:

(1) DNA sequencing to determine the biosynthesis of the pheromone

(2) mRNA sequencing to determine when these pheromones are being produced.

(3) The scientific community’s new(ish) appreciation for the prevalence and importance of bacterial symbionts could lead us to investigate whether these molecules are actually being produced by a bacterial resident on the moth.

Finally, lets get to the role of the pyrolizidine alkaloids, about which I know very little. Apparently they are a pretty ubiquitous class of molecule made by a number of different plants. They are quite toxic (especially to hepatocytes, i.e. liver cells) because they can cross-link DNA with itself or with other proteins in the cell nucleus, causing intracellular mayhem. I think the assumption here is that these are defensive compounds to prevent herbivores from feeding on the plant; in fact grazing animals (like sheep or cows) can become fatally ill from feeding on pyrolizidine alkaloid-producing plants. Interestingly, the moth does not suffer from the cytotoxic effects of these compounds. Why? Are the alkaloids specifically toxic to hepatocytes or other cells found in, say, mammals, or do the moths have a self-defense mechanism?And what types of predators are moths warding off? 

Given the many advances in science technology since these experiments were performed, it’s interesting to think about what experiments we would do now that were not feasible in 1991.
— Alexandra

Kristen: Often organisms that sequester toxic chemicals from their diet for their own defense have methods for enzymatic detoxification and/or specialized organs for storage. Enzymatic detoxification, however, appears to not be at play in this system since the monocrotaline can be extracted directly from the males. And there’s no mention any specialized storage locations where the systemic compounds are localized…. Mystery unsolved.

Re: moth predators? it appears that these alkaloids are very important for warding off spiders and birds.


Carolyn: Well, here's to another Casual Friday, done come and gone. I think humans getting hitched this summer could learn a thing or two from the mating behaviors of moths. Mostly, that they should be honest about what they are each bringing to the relationship!


Further reading: Tom Eisner has some great narratives describing his observations in his entomology studies and how they led to his fruitful explorations into chemical ecology (and his decades long collaboration with Jerry Meinwald), including this nice little paper where he describes his initial observations and studies with these moths. For more stories, check out his book!

Casual Fridays at Chemical Intuition: The Science of Leg Swabbing

This is the second installation of a new feature here at Chem Intuition, called "Casual Fridays," in which we discuss seminal papers in chemical ecology. Our conversations are driven by our own chemical intuition and curiosities of the moment. We encourage you to check out the paper we're reading for yourself and join the conversation! 

This week we're reading "Defense of Phalangid: Liquid Repellent Administered by Leg Dabbing" which was published in Science in 1971. This is a paper from the labs of the dynamic chemical ecology duo of Cornell University, Professors Eisner and Meinwald. The paper deals with the identification and biological study of the repellant used by Vonones sayi toward their ant aggressors. Read it here!

Kristen: Another great classical entomology and chemical ecology paper chock full of amazing descriptions of the experimental procedures used to obtain and characterize the unknown glandular product, including descriptions of the "milking" of the Vonones (a daddy longlegs-like insect). I loved the simplicity of their initial experiments to assign the source and rough identity of the defensive fluid, such as the experiment where they fed dyed water to the insects and were able to conclude that the clear droplets formed upon agitation are in fact regurgitated fluid. Likewise, I loved the inclusion of the deductive reasoning that since the additive is brown, has a characteristic odor and tans human skin it is likely quinonoid. Genius. 

So it appears these spider-like insects have evolved a rather unique method for distributing their defensive fluid:

1) regurgitate droplets of water
2) mix in a brown, repulsive quinonoid glandular substance
3) dip forearms into the mixture
and finally, 4) use forearms to rub- or "dab"- it against the offender.

The authors hypothesize that due to the general instability of quinones in water, the dilution of the repulsive substance immediately before use may be an evolutionary trick to retain the potency of quinones (as opposed to storing the quinones as an already dilute solution). But why dilute the quinones in the first place? Why not just "dab" the quinone solution itself? Any thoughts?

In panel  A &nbsp; Vonones sayi  has freshly extruded the quinone Goo. in panel  B &nbsp;it has dipped its leg into the mixture and is ready to swab at some ants! &nbsp; This &nbsp;Image was taken from Eisner, T.; Rossini, C,;Gonzalez, A.; Eisner, M. (2004) CHemical Defense of an opilonid (acanthopachylus aculeatus). Journal of experimental biology,&nbsp; 207 ,&nbsp;( 8 ), 1313-1321. Doi: 10.1242/JEB.00849

In panel A Vonones sayi has freshly extruded the quinone Goo. in panel B it has dipped its leg into the mixture and is ready to swab at some ants! This  Image was taken from Eisner, T.; Rossini, C,;Gonzalez, A.; Eisner, M. (2004) CHemical Defense of an opilonid (acanthopachylus aculeatus). Journal of experimental biology, 207, (8), 1313-1321. Doi: 10.1242/JEB.00849

 Carolyn: I considered the same questions while reading this. The authors state that the clear liquid serves only as a substance that dilutes the quinones, and has no activity on its own. Since the spider needs to move the substance from its point of excretion to its prey, I figured that the watery stuff aids in quinone delivery. In other words, I think that without dilution by water, the brown stuff, i.e. the mixture of the quinones, is either too viscous or too small a volume to be effectively transferred from the mouth area to the legs and finally, onto the victim. 

I did however, enjoy the note that described the fact that as purified substances the quinones are crystalline at room temperature, but when they are mixed together the melting point of the mixture is depressed such that the brown stuff is liquid at room temperature. Go Nature, finding some good solutions to problems of states of matter. 

Go Nature, finding some good solutions to problems of states of matter.

My favorite part of this paper was the description of the experiments in which the Vonones were placed in an enclosure with a bunch of ants. The authors state, "During the several hours that Vonones were confined in the crowded arenas, only seven assaults took place, although casual encounters with ants occurred continuously and the ants were demonstrably aggressive (mealworms offered concurrently were promptly killed)." 

First, why are ants so aggressive? Second, and maybe an easier question to consider, what is the mechanism by which the quinones are toxic to the ants (and other insects for that matter)?


Alexandra: A fun paper for sure.  I have to admit, I got vaguely irritated at times with the excessive use of insect anatomy jargon, which I found unnecessary, and ultimately vaguely distracting from the main points of the paper.  Once I got over that though, I thoroughly enjoyed the read.  I especially liked imagining those feisty Vonones slappin' that regurgitated fluid on other insects.

Ditto Carolyn's assertion that the dilution is most likely to increase diffusion or to increase the volume so delivery is easier.  

The question about toxicity and mechanism is interesting.  Do the authors say in this paper that the quinones are toxic to the ants?  Or just repellant?  Toxic quinones are produced by many plants to deter insects and apparently these are usually secreted through the roots or leaves and are toxic upon ingestion.  In the case of the Vonones the quinones are likely not being ingested, though I guess it's possible they are taken up through the exoskeleton.  So I wonder if these quinones are similar to the insecticide quinones produced by plants?  Additionally, is it possible that these quinones are basically mimicking the plant-produced quinones, in effect acting as a repellant without having to incur any sort of toxic effect?

Side note:  quinones are known to have a large range of biological mechanisms:  DNA interacalation, generation of free radicals, metal sequestration….but I haven't read up on which of mechanisms are the most common in insecticides.  

I especially liked imagining those feisty Vonones slappin’ that regurgitated fluid on other insects.


Kristen: The mode of action of the quinones is definitely an interesting question, especially since the activity assay in this paper is more or less a qualification of "repelled ants or didn't repel ants” and the paper doesn’t provide much of any information about the actual mechanistic effects of the quinone mixture on the ants. Quinones are commonly used by other insects, such as millipedes, beetles and cockroaches, as defense compounds. Although I couldn't find too many studies on the mechanism of action of these compounds on their predators, I did come across a hypothesis that quinones, which are highly irritating and sometimes toxic to vertebrates, could act on predatory insects as irritants through “common chemical sense," which I like to think of as "chemical street smarts." This aligns with your hypothesis Alexandra, where the negative/toxic effects of quinones –whether plant or insect derived– could deter predators regardless of whether the specific quinone actually has toxicity.

The ubiquity of quinones as defensive chemicals in both plants and insects is interesting to contemplate. I also wonder if quinones are relatively easy to access from other essential metabolites and thus their use could have evolved independently in these different types of organisms?


Carolyn: Without doing a deep literature search to help answer the question of how exactly do Vonones biosynthesize or obtain their quinones used to repel insects my chemical intuition makes me want to say that the quinones could be biosynthesized either from shikimate pathway intermediates (the shikimate pathway is involved in the biosynthesis of aromatic amino aicds) or through some sort of polyketide synthase (PKS) pathway that uses acetate as a building block. What's interesting is that the shikimate pathway is found only in bacteria and plants, and insects are not generally known to utilize large, multimodular PKS type enzymes to biosynthesize secondary metabolites. So, from my perspective, it seems likely that there are bacterial symbionts of Vonones that may either completely biosynthesize the quinones or could provide late stage intermediates that the bug then, perhaps, oxidizes or hydrolyzes. 

To address the question about the mechanism of action: what baffles me is that it seems as though the ants are repelled by the quinone excretion even when they are not in contact with it. So my question is, are the ants somehow getting volatile signals from this excretion (which seems unlikely, given the fact that the quinones are solids are room temperature) or, perhaps, is the ant that is put in contact with the gross brown goo sending out a secondary "alarm" signal to warn the other ants away? 

We are perhaps unveiling all sorts of fun chemical ecology interactions beneath the surface of this paper.... 

Bottom line:  Vonones are feisty!  Who knew that insect secretions could lead us through such a winding discussion. While this "classic" paper pokes at the complex interactions between Vonones and their enemies, there is still much to be learned about how these chemical secretions work their repellant magic.  



Molecule of the Moment: Cyclopamine

creative commons (sheep: By  Kreuzschnabel ; corn lily:  DcRjSr )

creative commons (sheep: By Kreuzschnabel; corn lily: DcRjSr)



Name: cyclopamine

Source: corn lily (Veratrum californicum)

Chemical structure: steroidal alkaloid (in other words, steroid-based molecule containing a nitrogen)

upsetting image of a cyclops lamb ( via USDA-Agricultural research services )

upsetting image of a cyclops lamb (via USDA-Agricultural research services)

Discovery:  The search for cyclopamine began in 1957 after the birth of a one-eyed sheep on a farm in Idaho.  Unfortunately this was not an isolated incident, and once several more cyclops lambs emerged, scientists set out to uncover the causative agent behind this striking birth defect.  It took 11 years (involving multiple summers of literally living with the sheep) for researchers, led by Lynn James, to pinpoint corn lilies as the culprit.  By closely observing sheep behavior, James realized that during times of drought the herd would move to higher ground and graze on the flowers and roots of the innocuous-seeming corn lily.  It took several more years for chemists to determine that the cyclopia-inducing agent was a small molecule, which they, appropriately, dubbed cyclopamine.  

Biology: Cyclopamine blocks a protein involved in the hedgehog signaling pathway (named after Sonic the hedgehog - for real).  This pathway is involved in numerous processes related to development and cancer.  Before researchers uncovered the relationship between cyclopamine and hedgehog, mutations in proteins along this pathway were implicated in developmental malformations. 

Odysseus and his guys kill the cyclops polyphemus, eleusis museum (via  napoleon vier  from  nl )

Odysseus and his guys kill the cyclops polyphemus, eleusis museum (via napoleon vier from nl)

Uses: It turns out that in multiple types of cancer, components of the hedgehog pathway are over-expressed - or hyperactive.  Since cyclopamine is known to block this pathway, several pharmaceutical companies have worked towards developing this molecule (or derivatives) as an anti-cancer therapeutic.  As of yet nothing notable has emerged.  

Dangers:  This seems obvious, but do not ingest this molecule if pregnant.  It causes severe birth defects.  

Fact that can be used to impress friends/foes at a cocktail party:  

Cyclopamine is a molecule that recalls one of the best scenes from The Odyssey.  It has an awesome name, a cool history, and potential as an anti-cancer agent.  


Molecule of the Moment: Acylhomoserine lactone

Source: Many species of Gram-negative bacteria use acylated homoserine lactones (AHLs) to regulate and control a number of different phenomena, including the production of light and the expression of virulence factors. 

Chemical Structure: The basic structure of AHLs features a lactone (here, a five membered ring in which an ester is contained within the ring) connected through an amide bond (or N-acyl linkage) to a flexible carbon chain, usually featuring a ketone group. Twelve different analogues of this basic structure have been found in Nature. These differ in the length of the acyl chain and all share the lactone ring.

this is an acylhomoserine lactone.

this is an acylhomoserine lactone.

Historical background:  According to a 2001 review by Greenberg et al., when AHLs were first discovered in the 1960s, "biologists weren't ready for the idea that bacteria were talking to each other. Nevertheless, that is exactly what they do."

Since these seminal discoveries,the biosynthesis and use of AHLs to mediate signaling has been discovered in various species of bacteria that inhabit diverse niches. AHL signaling is associated with a process known as quorum sensing. Quorum sensing is the process through which organisms respond to signals in a cell-density dependent manner. Quorum sensing is mediated through a variety of signal molecules, including AHLs with various acyl chain lengths, as well as two other chemically distinct molecules called furanosylborate and  cyclic thiolactone. Quorum sensing is a common signaling strategy when coordinated gene expression is needed by a community of bacteria. For instance, quorum sensing using AHLs is used by bacteria, such as Vibrio cholera, to switch on virulence genes under certain conditions that favor a pathogenic lifestyle.

In the case of AHLs, bacteria biosynthesize a species-specific AHL at a basal level. As the local population of bacteria increases, AHL accumulates in the surrounding medium. At a certain cell density (and corresponding concentration of AHL), there is enough AHL in the cell cytoplasm that a complex forms between AHL and a protein called LuxR. When the AHL-LuxR complex is formed, this complex acts as a promoter of gene expression at certain genetic loci. In all bacteria that use AHLs to do quorum sensing, one of the loci that gets upregulated by LuxR-AHL complex is the gene that is responsible for the biosynthesis of AHL.  In this way, as the local concentration of bacteria increases, AHL production increases in an exponential manner. Other genetic loci that are regulated by LuxR include those involved in turning on light production in the symbionts of the bobtail squid (see below) and virulence factors (such as the production of proteins that allow bacteria to invade human cells) in the case of pathogenic bacteria.

Uses (just a few of many):  At Chemical Intuition, our favorite use of AHL is by the bacteria that live in the light organ of the bobtail squid. These symbionts include the species Vibrio fischeri and Vibrio harveyi. In these bacteria, AHL biosynthesis and the protein LuxR (mentioned above) are encoded in a set of genes clustered together on the bacterial chromosome. These genes are called the lux genes for the enzyme called luciferase, which is also encoded in this cluster of genes. The luciferase protein performs a reaction that results in the production of light, making these bacteria bioluminescent organisms!  By coordinating gene expression through AHL-mediated quorum sensing, the bacteria can quickly turn on light production when cell density is high.

Dangers: In contrast to our last molecule of the moment, atropine, one could likely consume a reasonable quantity of purified AHLs and survive to tell the tale.

Fact that can be used to impress friends/foes at a cocktail party:  You can impress your friends by telling them the strange details of the Bobtail squid's camouflage strategy. The squid does a maneuver known as "counter illumination," a strategy in which the squid tries to match the intensity of light coming from the moon to reduce its silhouette in the water.  The squid produces light so that it doesn't cast a shadow at night under moonlight, which helps the squid evade predators. The bacterial symbionts are key to the squid's camouflage strategy. The squid's symbiotic bacteria live in the squid's so called "light organ." This bacterial-filled light organ is tricked out with accessories that give the squid fine-tuned control of its light production. These accessories include special tissues that reflect or focus light and an ink sac that allows the squid to dial in the intensity of the light its light organ emits.  




Casual Fridays at Chemical Intuition

We're starting a new format here at Chem Intuition, in which we discuss seminal papers in chemical ecology. Our conversations are driven by our own chemical intuition and curiosities of the moment. We encourage you to check out the paper we're reading for yourself and join the conversation! 

This week we're reading "Purification of the Fire Ant Trail Substance" which was published in Nature in 1965. This is a paper from Harvard Professor Chris Walsh during his undergraduate days at Harvard University where he worked in the lab of renowned ecologist and ant researcher E. O. Wilson. The paper deals with the isolation of the active substance that ants use to mark their trail to food. Check it out here.

Alexandra: I really enjoyed reading this article. The whole description of how the ants are isolated and extracted is fantastic. The explicit use of a tea strainer to separate the ants from the debris: Love it. What really delights me about these classic papers are the in-depth descriptions of the methods. When I read recent papers I feel like I’m frantically looking for how the experiments were done and it's always hidden in some outlandishly long supplementary file that takes 20 minutes to download. Then you read it to find that they’ve actually just directed you to another paper. Repeat process. This is why PhDs take so long. 

This is why PhDs take so long.

Kristen: Agreed- it’s a great paper. I loved that ~200,000 ants were used for the purification. That's a terrifyingly large number! It always amazes me in these classic chemical ecology and entomology papers how much biomass is required to isolate a minute amount of active compound. You can't help but admire the heroic efforts put forth by these authors to obtain the material of interest.

Carolyn: Hello! Jumping on the ant bandwagon, or perhaps I should say trail…

The first thing I loved about this paper was the wonderfully detailed description of how the authors gathered hundreds of thousands of ants and proceeded to obtain about 250 micrograms of "apparently homogeneous trail substance." Having come of (science) age in an era in which papers have six figures in the main text and twenty-plus figures relegated to the supplementary information, I felt that reading this brief report was a blast of fresh air. 

Alexandra: So this paper ends with the isolation of an "active trail substance" but do we know if this substance has since been identified? Is the assumption that this substance is actually a mix of molecules? Or do they think that the activity is attributable to just one single molecule? 

Kristen: An (admittedly) quick search did not turn up any papers further characterizing the "active trail substance" of this particular species (S. saevissima), but it appears some further work has been pursued with a related species, S. invicta. A series of farnesene compounds were isolated from the trail hormone of S. invicta, none of which were very active alone. However, when a subset of these compounds was combined at physiologically relevant concentrations, recruitment activity was observed. Perhaps such chemical synergy is also at work in the "active trail substance" of this species? 

On another note, I love the hypothesis put forth at the end of the paper that the instability of this "active trail substance" may be evolutionarily advantageous for the ant: after the trail has served its purpose, it will disappear. As a chemist, I associate the instability of a chemical of interest with lab frustration and challenges, but it’s interesting to contemplate how this instability may be useful and selected for in Nature.

As a chemist, I associate the instability of a chemical of interest with lab frustration and challenges, but it’s interesting to contemplate how this instability may be useful and selected for in Nature.

Alexandra: The stability hypothesis is definitely intriguing. It also makes me wonder if other species of ant – or even other insects – can recognize this same substance. I imagine that upon locating some amazing food source they probably want to keep it as exclusive as possible. 

Kristen: And exclusive or not, one could imagine they probably don't want old trails leading to depleted food sources lying around-- no need for workers to waste energy pursuing an obsolete trail.

Carolyn: I don't think that the authors assume that the substance is a mix of molecules or one single molecule. Rather, they leave this as an open question by stating "While it is clear that the active material is contained within the peak observed by gas chromatography, the sample may still be contaminated with a large amount of an inactive compound with similar chromatographic properties." The authors had a limited amount of information from which to draw conclusions and they don't over interpret or stretch the data to support theories to questions like these. 

The stability question is, to me, the most fascinating part of this paper. It seems as though the ants have evolved to make a disappearing ink trail substance: something that sticks around while they need it and degrades quickly when it's no longer benefiting the ants.

It made me think that this chemical instability is just one solution to a more general problem in chemical ecology. Specifically, chemicals are made as signals to indicate something to members of the same, or in some cases, different, species. Generally, the organisms need the signal to dissipate as soon as the indicated event is over. What are other solutions to this problem in Nature that you all know of? Signal instability and quick degradation is one solution. What are others? 

Alexandra: Great point about the hesitancy of the authors to over-state their findings.  Unfortunately, it seems as though, these days, the norm is to do the opposite. Researchers often over-hype the implications of their research in order to maximize the impact of the publication or to help obtain funding.  To some extent this over-hyping is understandable. On the other hand, it can be very misleading to readers who may not have the expertise to judge the data for themselves.  

Moving on. So you’re asking, what are other solutions to this problem in Nature? That's a tough question. Instability definitely seems like the best way to erase a chemical cue, but I suppose there could be more "deliberate" strategies - like enzymatically converting the molecule to an unrecognized derivative once it is no longer needed.  This seems way more energetically unfavorable.

Kristen: In the same vein as signal instability, how about chemical volatility? Organisms can rely on diffusion for signal transport and only respond at a critically "higher" concentration i.e. when the signal is still freshly produced or when the organism receiving the cue is close to the signal’s source.  

Methyl jasmonate is a volatile signaling molecule used by many species of plants. Its production is induced by herbaceous damage and can signal to neighboring plants to expend energy in order to raise their defenses. One can imagine these sorts are mechanisms are particularly abundant in/important for immobile organisms (such as plants) that can't disperse (or move away from) the chemical signal they produce.

Carolyn: Acylhomoserine lactones come to mind when thinking about the need for a certain concentration threshold for signaling to happen. AHL’s are used by a variety of bacteria in quorum sensing processes, most notably by the light-making symbionts of the bobtail squid

So, to recap, right now we have listed the following as ways that organisms control turning chemical signaling on and off:

·  chemical instability, in which the molecule is unstable and breaks down (either enzymatically or spontaneously) into an inactive compound after some period

·   volatility, in which the chemical, presumably a gas, is lost through the process of evaporation or diffusion

· concentration effects, in which signaling is triggered on or off when the concentration of the signal surpasses or does not reach threshold concentration

Alexandra: Another good example is cyclic AMP, which is a small molecule produced by amoeba upon starvation. This molecule signals to neighboring amoeba to chemotax (move) and differentiate (commencing their “social lifestyle” - a way for the organism to disperse and locate a new food source). However, rather than the amoebal response increasing linearly with the concentration of cAMP, the amoeba become desensitized to the signal once it reaches a certain concentration. So the response curve of amoeba to cAMP is like an upside-down U, like this “n”. This type of receptor desensitization is another adaptation that can allow organisms to respond differently to a signal based on concentration rather than any kind of change to or loss of the signal itself. 

Carolyn: So did they purify the amoeba pheromone by getting the amoeba to sniff the HPLC fractions? 

Alexandra: They evaluated their response by having an amoeba focus group fill out surveys after being exposed to different concentrations of cAMP.

Carolyn: "How did it make you feel?"

Understanding how ants use chemicals to communicate with their nest mates remains a very active area of research today.  If you want to know more about these critters, E.O. Wilson and Burt Holldobler, pioneers in this field, have written some fascinating and comprehensive books about the social lives of ants and the evolutionary implications of their behaviors (as well as numerous other books on equally engaging topics).

The Ants -  Burt Holldobler & Edward O. Wilson

The Superorganism: The Beauty, Elegance, and Strangeness of Insect Societies - Burt Holldobler & Edward O. Wilson

Journey to the Ants: A Story of Scientific Exploration - Burt Holldobler & Edward O. Wilson

Atropine - molecule of the moment

Atropine structure (left) and atropa belladonna (via Flickr, Don macauley)

Atropine structure (left) and atropa belladonna (via Flickr, Don macauley)

Name:  Atropine

Source:  Members of the nightshade family, most famously Atropa belladonna (or deadly nightshade).  It is produced in high quantities in both the foliage and the delicious-looking berries. 

Chemical structure:  Alkaloid. Atropine is derived from the amino acid phenylalanine. Its core structure is common to many psychoactive molecules isolated from plants.

via WELLCome images

via WELLCome images

Historical background:  Like many natural products, atropine has probably been in use for longer than we give it credit.  Atropine-containing plants, like the nightshades, were commonly used over the last five centuries for a variety of medicinal purposes such as headaches, muscle spasms, night sweats etc. (the list is basically endless). Of course, these plants contain a wide range of molecules called alkaloids that are chemically similar to atropine but can have significantly different biological activities. It wasn’t until the 1830s that german chemists isolated and characterized atropine in its pure form. From that point forward researchers started to explore the diverse biological effects of pure atropine.  

Uses (just a few of many):

Long-lasting pupil dilation (not for your standard eye exam)

Prevention of excessive sweating (hyperhidrosis).  Also decreases salivary secretions leading to drymouth - though this is more of an irritating side effect than a medicinal property.  

Heartbeat stimulation.  Atropine is an anticholinergic molecule; in other words, it prevents acetylcholine from performing its normal tasks in the body, one of which involves speeding up your heart rate (fight or flight!)

Antidote?!?!  Atropine serves as an antidote for potent nerve gases like sarin. This is because many nerve gases prevent acetylcholine breakdown leading to an overload of acetylcholine in the system.  Since atropine blocks acetylcholine it alleviates the toxic effects of these noxious chemicals.  

Dangers:  POISON!  Nightshade berries are quite poisonous (due predominately to atropine content), causing extreme dryness of the mouth, dizziness, hallucinations and eventually death.  As a poison, belladonna has a pretty glamorous history. It was rumored to be used by Emperoror Augustus and Agrippa  (wife of Claudius) to poison their enemies, and it also pops up in many an Agatha Christie-esque murder mystery.  In all seriousness though, these berries are to be avoided - their ingestion seems pretty traumatizing

Fact that can be used to impress friends/foes at a cocktail party:  

Atropa belladonna was named after Atropos - of the three fates (from Greek mythology).  Known as the “inevitable,” her role was to the “cut the thread” of a person’s life, and determine their manner of death.  

During the renaissance era women often used belladonna cosmetically. Since atropine causes the pupils to dilate, a drop of nightshade extract in the eyes gives that desirable wide-eyed look. This use for nightshade is why it is called “belladonna,” which means “beautiful woman” in Italian.  

Atropine is both a poison and an antidote.  Does it get cooler than that?  No. 


Is there (or will there be) life on Mars?

In 1971, David Bowie sang a question that has become only more relevant in the 45 years since the release of Hunky Dory: is there life on Mars? Several recent scientific discoveries have elevated the pondering of this question from the lowly lands of fantastical thinking to the high plains of Serious Scientific Endeavors. In this installment of the ongoing “Life, uh, finds a way” series we will attempt to place these discoveries in the context of chemical ecology and, in the process, remind everyone that yes, space is cool.

For many years, it's been known that there is plenty of ice water on Mars. The poles of Mars are covered with ice caps that are a mixture of frozen water and carbon dioxide (aka dry ice, the stuff you used in high school to lend low budget theater productions a creepy mood). However, life on Earth requires water in the liquid phase: even microbes that live in glaciers are only metabolically active when and where there is liquid water in their environment. Specifically, the bacteria that inhabit Antarctic glaciers actually live within small (microns wide) veins of liquid water that form in the ice where the local salt concentration is very high. This high salt concentration decreases the freezing point of the water to a degree such that, even at temperatures much below freezing, liquid water can still flow. The microbes that are capable of living in these glacier veins with very high salt concentrations are known as halophiles, meaning “salt-loving.” Bacteria that have adapted to environments such as the veins of glaciers can handle the osmotic stress that is placed on the cell membrane in high-salt concentrations- conditions that would likely kill a bacterium that lives in a fresh-water lake, for example.

So microbiologists and origin-of-life scientists alike were extremely excited when NASA announced in September of 2015 that they'd discovered clear evidence for the presence of liquid water on Mars. All of Mars' known liquid water is localized to features named “recurring slope lineae” (RSL): dark, 100-meter long streaks found in multiple locations across the planet that appear in the warmer months (when the temperature exceeds -10°F) and disappear in colder months. The RSL are composed of liquid brines, aqueous solutions of salts including magnesium perchlorate and sodium perchlorate. Much like the liquid water veins that can form in Arctic glaciers, liquid water can only form on Mars due to the presence of high concentrations of salts. But you may be asking, why perchlorate? Perchlorate is a highly abundant compound on Mars (it makes up about 0.6% of the weight of Martian soils), and is hygroscopic, meaning it readily binds to water to form hydrates.

NASA took this image, but don't get too excited, the colors are fake. The dark streaks seen in this image are the RSL, which are roughly the size of a football field.&nbsp;  image credit:   NASA/JPL-Caltech/Univ. of Arizona

NASA took this image, but don't get too excited, the colors are fake. The dark streaks seen in this image are the RSL, which are roughly the size of a football field.  image credit: NASA/JPL-Caltech/Univ. of Arizona

The abundant perchlorate on Mars is what makes liquid water possible on the planet. But could anything live in this perchlorate brine?

To help answer this question, let's examine the state of perchlorate on our planet. Perchlorate is not a naturally abundant compound on Earth, which is good for us because it is fairly toxic to humans. Perchlorate blocks iodide uptake by the thyroid, which can lead to a host of thyroid-related metabolic issues. Despite its toxicity, there is a big demand for man-made perchlorate salts. Ammonium perchlorate is used as an oxidizer in modern day rocket fuels, which make such explosive things like spacecraft and expulsion seats possible. While some perchlorate ends up in fireworks and pesticides, about 90% of manufactured perchlorate is destined for aerospace and military applications. Unfortunately, every time the military updates or retires rockets, massive amounts of perchlorate are disposed, increasing the chances of its release into the environment. Perchlorate is highly water soluble and is thus easily dispersed in environmental waterways (rivers, streams, groundwater etc). It is also a very stable chemical, so once in the environment it tends to stick around. To help protect people from perchlorate toxicity, the Environmental Protection Agency (EPA) has set the limit of the allowable concentration of perchlorate in tap water to 6 parts per billion, which is approximately six orders of magnitude less concentrated than the perchlorate levels in Martian soil.

The interesting part of the perchlorate story is how we've decided to clean it up. The most efficient way to detoxify perchlorate-contaminated water is through a process called bioremediation, defined by the EPA as “a treatment process that uses naturally occurring microorganisms (yeast, fungi, or bacteria) to break down, or degrade, hazardous substances into less toxic or nontoxic substances.” It has been known for about fifty years that naturally occurring bacteria can degrade perchlorate, and ill-defined microbial communities that include these perchlorate-reducing bacteria have been used to treat contaminated water successfully. It's only been in the past fifteen years or so that the biology of perchlorate-reducing bacteria has been studied in any detail. The results of these studies have revealed that some strains of bacteria can actually grow through the reduction of perchlorate to generate ATP (the primary energy source of cells). The reduction of perchlorate results in the production of two harmless end products, oxygen and chloride. Interestingly, perchlorate-reducing strains only perform this chemistry under anoxic (oxygen free) conditions, as these bacteria preferentially utilize oxygen over perchlorate for energy production.

While various strains of perchlorate-reducing bacteria have been isolated from anoxic water-treatment plants contaminated with perchlorate salts, others have been found in pristine environments. This raises the important question as to why these unrelated bacteria found in diverse environments have evolved the ability to reduce perchlorate. You may recall from our previous installment of the “Life, uh, finds a way” series that Deinococcus radiodurans (affectionately named rad) was able to withstand crazy high levels of ionizing radiation. After much wild speculation as to why rad had evolved to withstand radiation that is not found in any natural habitat on Earth, scientists discovered that rad's resistance to radiation was a side effect of it's adaptation to survive dessication, an ability that fits in with the needs of its known terrestrial lifestyle. Given the paucity of perchlorate on Earth, and the fact that perchlorate-reducing bacteria are not closely related in terms of genetics or habitat, it's likely that perchlorate-reduction has arisen from adaptation to some other requirement. For instance, based on the enzymes implicated in the reduction of perchlorate, biochemists studying the issue have posited that perchlorate reduction has arisen from the evolution of these strains to reduce nitrate, a common oxidant on Earth.

To review: First, there is liquid water on Mars that isn't pure water, but rather a brine that contains high levels of perchlorate salts, which are toxic to humans. Second, there are bacteria on Earth that can reduce perchlorate and even use perchlorate to grow. Given these pieces of information, I think it's not so crazy to think that, if the only hurdle to life on Mars was the requirement to utilize perchlorate for energy production, there could be or could have been life in the RSL. However, since we are pondering these issues while pacing back and forth in the leather armchair and pipe smoke-filled land of Serious Scientific Endeavors, there are a few more issues we should consider when we ask the question, is there life on Mars?

Let's consider one such hurdle to life on the red planet. Mars lacks a global magnetic field, meaning that solar wind (high energy, charged particles released by our Sun) has been bombarding the planet for about 4 billion years, making a protective atmosphere like that of Earth impossible. As a result, if you were to spend a year on Mars, you'd receive about 500-times more ionizing radiation than if you'd stayed on your couch reading this article (hopefully it doesn't take you a year to read this, but hey not judging). The constant onslaught of ionizing radiation on the surface of Mars would kill off even the amazing rad, because the bonds found in organic molecules are simply not sturdy enough to withstand these energy levels over long periods of time.

Finally, let's turn to a big issue that needs to be discussed whenever we start theorizing about the possibility of life outside of our beautiful green world. While we mostly understand the basics of what is required for life on Earth, that understanding only applies to the version of life that appears on our planet, which happens to be the only version of life we know. If we are searching for life on other planets and we think that it should take the form of life we have on Earth, than we can clearly see if the checklist for life (which includes, among other things, liquid water, lack of ionizing radiation that breaks apart organic molecules, and a supply of elements with which to build cellular structures) is satisfied on that planet. However, at least in my mind, I really don't believe for one second our version of life, which comes down to DNA inside of a membrane, is the only version out there. And when we start thinking that life on another planet may take another form altogether, it becomes pretty much impossible to even make that “need for life” checklist, let alone check it.

So, at least for now, I think that we can pretty safely conclude that, no, there is most likely no life as we know it on Mars right now. But there might be life (read: humans) on Mars in the future, and it is useful to think about what elements of the Martian environment need to be changed to make the planet habitable for us weaklings. For instance, could we send engineered bacteria to Mars to clean up the soil and make it habitable for us? Perhaps we could use perchlorate-reducing bacteria to detoxify the soil and generate oxygen for our breathing needs in the process. Ultimately, our study of the strange ways of microbes on Earth could impact future space exploration and may enable us to colonize another planet. If that planet is Mars, there will definitely be ample fireworks supplies. So, Happy Mars Day! Cue explosion.


Bees on caffeine!

Previously we explored the effects of bees on cocaine so it seemed appropriate to highlight a recent study by Couvillon et al. delving into the complex relationship between bees and caffeine.   Much like nicotine or cocaine, plants produce caffeine as an insect deterrent.  While its bitterness and general toxicity make caffeine-containing leaves unpalatable to most insects, the presence of caffeine in the seeds and nectar of these plants is more puzzling.  Plants rely heavily on pollinators to help them generate offspring so it is in their best interest to keep their pollinators happy and coming back for more.  So what exactly does caffeine-containing nectar do for bees?  Similar to our cocaine example, caffeine seemed to increase bees’ general enthusiasm for their nectar source.  The waggle dancing increased and the bees convinced their hive mates to return to this delectable resource.  Taken at face value this seems like a real win-win situation – the plant provides a resource for the bees, the bees help the plant reproduce – but there’s something vaguely nefarious at work here.  While the bees are happy to feed solely on this caffeine-containing nectar, they neglect to notice that it may not actually be the best quality (based on sugar content).  In other words, rather than shop around, they limit their focus to the caffeine-containing nectar, ultimately decreasing the honey production of the hive.   While brief, this study raises some interesting points:  For one, it may inspire some to reassess their own complex relationship with caffeine – or more specifically their perceived productivity after ingesting this alluring yet deceitful substance.  More generally though this study reveals the convoluted nature of symbiotic interactions.  We generally view the partnership between bees and plants as mutualistic, and while this perception is probably true overall, like (many) relationships, one partner is benefitting at the slight cost of the other.  


Nobel committee recognizes the impact of natural product-based drugs

Image: flickr/ Thomas fisher library

Image: flickr/Thomas fisher library

This year’s Nobel prize in Physiology or Medicine was awarded for the discovery of 2 natural products that have set a paradigm for the treatment of infectious diseases.  The Nobel committee recognized Satoshi Omura and William C. Campbell for their part in the discovery of the natural product Avermectin – a drug that is essentially responsible for the abolishment of roundworm infections (such as river blindness or lymphatic filariasis).  Dr. Omura is an expert in laboratory cultivation of a class of bacteria called Streptomyces, which are profilic producers of bioactive molecules (such as the cephalosporins, vancomycin, rapamycin – and many more).  By testing molecules extracted from Dr. Omura’s diverse library of bacteria, Dr. Campbell, a parasitologist, was identified an extract that could successfully kill parasitic worms in mice.  The active component of this bacterial soup was named Avermictin and this class of molecules (avermectins) continues to be a critical treatment for parasitic diseases. 

avermectins are a class of anti-helminthic drugs &nbsp;

avermectins are a class of anti-helminthic drugs  

Tu Youyou is the third recipient of the prize for her work in the discovery of the anti-malarial drug artemisinin.   Dr. Tu discovered artemisinin while investigating the bioactive components of traditional chinese medicine (interestingly this endeavor was part of a secret funding effort started by Mao Zedong - read more here or here).   She isolated this molecule from wormwood (Artemisia annua) – a plant always suspected to have anti-malarial activity.   Since Dr. Tu's discovery of the anti-malarial properties of this molecule, derivatives of artemisinin have gone on to become crucial in controlling the malaria epidemic.  

the anti-malarial drug artemisinin was isolated from wormwood ( Artemisia annua )

the anti-malarial drug artemisinin was isolated from wormwood (Artemisia annua)

Over the last few decades many researchers’ enthusiasm for drugs derived from natural sources has waned.  Yet with the rapid influx of sequenced bacterial, plant and fungal genomes, the extensive diversity of naturally produced small molecules is apparent.   This year’s Nobel Prize in Medicine underscores the major impact natural products have made on world health and will perhaps advance the ongoing resurgence in natural products research.  


The bacterium at the end of the Earth

If there’s one thing we can all agree on, it’s that SPAM is definitely not alive. So it must have been surprising when someone yelled, “there’s something alive in my SPAM!” 

To be fair (and accurate), it wasn’t exactly SPAM, but rather a tin of corned beef that had been irradiated with x-rays to prevent microbial spoilage. In 1953, researchers accidentally discovered living in this tin of meat the amazing bacterium Deinococcus radiodurans, the star of today's installment in the ongoing "Life, uh, finds a way" series. 

D. radiodurans– which we’ll call rad for short–has an insanely high resistance to ionizing radiation. It can grow under chronic gamma (γ) radiation of 50 grays (Gy)/hour and can survive doses greater than 10 kGy, which explains why it was able to thrive in that irradiated can of beef. But you may be wondering, should I be impressed? 

Before we address this question, let’s cover some basics about radiation. The unit gray (Gy) describes a dose of radiation: the amount of absorbed energy per unit mass of tissue (1 gray = 1 Joule/kilogram). The type of radiation we are talking about is γ radiation: electromagnetic radiation of very high energy, with wavelengths on the order of 10 picometers, which is a little smaller than the diameter a hydrogen atom. Now let’s put 10 kGy into context. A PET scan delivers a whole-body dose of about 0.01 Gy. Two-hundred times that amount (2.0-3.5 Gy) causes severe nausea and hemorrhaging, and –without medical treatment- has about a 25% chance of killing you. This dose is still 20-times less than that which rad can survive... per hour

Why are γ rays so life destroying to most organisms? γ rays easily penetrate biological materials (like your skin) to produce intracellular reactive oxygen species (ROS). ROS are highly reactive, oxygen-based radicals that destroy proteins and DNA through oxidative reactions. While cells have repair mechanisms to deal with the slow burn of oxidative damage that arises from life on our oxygen-rich planet, the onslaught of chemical degradation that happens with a dose of γ rays is often too much for a cell to handle, resulting in cell death.

Over the years, scientists have pieced together the molecular mechanisms by which rad can survive 10 kGy of radiation. While some researchers probably believed they would stumble upon a fundamentally new strategy for handling DNA damage, the mechanism that rad uses to recover from the chemical destruction of γ rays is actually not so different from the known strategies. It’s just much, much more efficient. While most organisms can repair about ten double-strand DNA breaks per cell, rad can repair hundreds. Rad does this by keeping ROS levels low through the use of multiple anti-oxidant small molecules. Lower ROS means less protein degradation through oxidative reactions. More intact proteins means more enzymes to repair messed up DNA. Those enzymes repair DNA through the same mechanism as in other bacterial cells, via synthesis-dependent strand annealing.

Since the discovery of rad’s ridiculous propensity to survive radiation, researchers have sought to understand the ecological role of this trait. What evolutionary pressure lead to rad’s extreme resistance? Since there's no natural environment that has the kinds of levels of radiation rad can survive (and could thus apply some pressure to evolve this trait), some turned to outer space for ideas. One paper, entitled “Was earth ever infected by martian biota? Clues from radioresistant bacteria” concluded that, while the evolution of highly radioresistant bacteria couldn’t take place on Earth due to low levels of background radiation,

the “process could take place in a subsurface layers of polar regions of Mars. This is the only place of the Solar System, where this process could take place. A few number of discovered high radioresistance terrestrial bacteria can be transferred from Mars on martian meteorites.”

While we love any hypothesis that invokes space-traveling microbes, there is substantial, countervailing evidence that rad are related to terrestrial bacteria and did not originate in the polar regions of Mars.

The most convincing explanation for rad’s radiation resistance came from a paper published in 1996 in the Journal of Bacteriology. Their work grew from the fact that radiation-resistant bacteria can be isolated from natural microbial populations by selecting for resistance to desiccation. When bacteria become extremely dry, double-strand breaks and other DNA mutations are created, which is similar to what happens when cells are exposed to γ rays. To explicitly connect desiccation and radiation resistance, the authors evaluated 41 radiation-sensitive rad mutants, and found that these mutants were significantly less viable than the wild-type strains after desiccation.

But why would rad evolve resistance to extreme dryness? Rad is found in diverse environments all over the world and it gets to all of these exotic locations using the wind. Carried on dust particles, rad reaches great heights up in the stratosphere. Here, it mingles with other dust-borne bacteria and is exposed to short-wave UV light 100-1000 times more intense than at the surface. When it rains or snows, rad becomes rehydrated and falls back to Earth. Since hanging out on dust particles miles above the Earth's surface is a major part of rad's ecology, it follows that it has evolved resistance to desiccation. 

From a chance discovery in some questionable meat, scientists were given the gift of a biological oddity, which has provided fodder for basic research for decades. After reading so much about rad, the thing that strikes me the most is my newfound–though strange– sense of optimism. When humans have destroyed the environment and each other using nuclear weapons, I'm confident that rad will survive. I imagine that, after floating carefree with some dust above the clouds, it will serenely fall back to Earth on radioactive snow to live another day.