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Do Insects Feel Pain?

By Dr. Shelley Adamo, Dalhousie University

Do insects feel pain?  Many of us probably ask ourselves this question.  We swat mosquitoes, step on ants, and spray poison on cockroaches, assuming, or perhaps hoping, that they can’t – but can they?  As someone who studies the physiology behind insect behaviour, I’ve wondered about it myself. Those thoughts motivated me to examine the question from the perspective of evolution, neurobiology and robotics.

Are these crickets angry? In pain from being whipped by antennae? How would we know?

To find out whether insects feel pain, we first need to agree on what pain is.  Pain is a personal subjective experience that includes negative emotions.  Pain is different from nociception, which is the ability to respond to damaging stimuli.  All organisms have nociception.  Even bacteria can move away from harmful environments such as high pH.  But not all animals feel pain.  The question, then, is do insects have subjective experiences such as emotions and the ability to feel pain?

We’ve probably all observed insects struggling in a spider’s web or writhing after being sprayed with insecticide; they look like they might be in pain. Insects can also learn to avoid electric shocks, suggesting that they don’t like being shocked.  However, just as I was appreciating how much some insect behaviour looked like our pain behaviour, I realized that Artificial Intelligence (e.g. robots and virtual characters) can also display similar behaviours (e.g. see (https://www.youtube.com/watch?v=YxyGwH7Ku5Y). Think about how virtual characters can realistically express pain in video games such as “The Last of Us” (e.g. https://www.youtube.com/watch?v=OQWD5W3fpPM). Researchers have developed circuits allowing robots and other AI to simulate emotional states (e.g. ‘joy’, ‘anger’, ‘fear’). These circuits alter how the robot/virtual character responds to its environment (i.e. the same stimulus produces a different response depending on the AI’s ‘emotion’).    However, this does not mean that robots or virtual characters are ‘feeling’ these emotions.  AI shows us that behaviour may not be the best guide to an insect’s internal experience.

Given that behaviour seemed an unreliable guide, I then looked for neurobiological evidence that insects feel pain.  Unfortunately, the insect brain is very different from the human brain.  However, once we understand how our brains perceive pain, we may be able to search for circuits that are functionally similar in insects.  Research in humans suggests that pain perception is created by complex neural networks that link up the necessary brain areas.  These types of networks require massive bidirectional connections across multiple brain regions.  Insect brains also have interconnections across different brain areas.  However, these interconnections are often quite modest.  For example, the mushroom bodies in the insect brain are critical for learning and memory. Although the mushroom bodies contain thousands of neurons, in fruit flies, for example, they have only 21 output neurons.  In humans, our memory area, the hippocampus, has hundreds of thousands of output neurons.  The lack of output neurons in insects limits the ability of the insect brain to sew together the traits that create pain in us (e.g.  sensory information, memory, and emotion).

Finally, I considered the question from an evolutionary perspective.  How likely it is that evolution would select for insects to feel pain?  In evolution, traits evolve if the benefits of a trait outweigh its costs.  Unfortunately, nervous systems are expensive for animals.  Insects have a small, economical, nervous system.  Additional neurons dedicated to an ‘emotional’ neural circuit would be relatively expensive in terms of energetics and resources.  If it is possible to produce the same behaviour without the cost, then evolution will select for the cheaper option. Robots show that there could be cheaper ways.

The subjective experience of pain is unlikely to be an all-or-none phenomenon.  Asking whether insects feel pain forces us to consider what we would accept as a subjective experience of pain.  What if it was devoid of emotional content?  What if cognition is not involved?  If insects have any type of subjective experience of pain, it is likely to be something that will be very different from our pain experience.  It is likely to lack key features such as ‘distress’, ‘sadness’, and other states that require the synthesis of emotion, memory and cognition. In other words, insects are unlikely to feel pain as we understand it.   So – should we still swat mosquitoes?    Probably, but a case can be made that all animals deserve our respect, regardless of their ability to feel pain.

Adamo, S. (2019). Is it pain if it does not hurt? On the unlikelihood of insect pain. The Canadian Entomologist, 1-11. doi:10.4039/tce.2019.49 (Paper made available to read for FREE until Sept. 16, 2019 in cooperation with Cambridge University Press)

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A Nobel prize and the unknown benefits that come from saying yes

Aziz Sancar delivering his Nobel Lecture for his prize in Chemistry 2015. He said yes.

My early morning wakeup on Wednesday, October 7, 2015 began as usual with a, though admittedly not healthy, quick Twitter check. My internet-induced squint widened when I saw that Aziz Sancar was trending. Dr. Sancar had just been named co-winner of the Nobel prize in chemistry for his work on DNA repair mechanisms. Not at all surprised by the recognition of his career achievements, I was, however, flabbergasted because I actually know Aziz Sancar and in no small way, my career is what it is because of his generosity and kindness.

Twenty years ago, I was an MSc candidate studying the physiological ecology of amphibians at Trent University. At the time I was working with Michael Berrill on replicating and testing the findings of a 1994 PNAS paper by Andrew Blaustein and company. This was important work on declining amphibian populations in the Cascade Mountains. They found that these declining populations were characterised by low levels of a DNA repair enzyme called photolyase. This finding was intriguing because photolyase catalyses the repair of the principal form of damage to DNA from ultraviolet-b radiation. Because emerging ozone holes would result in natural populations experiencing an increased amount of UVB radiation, low levels of photolyase might be a “magic bullet” that explained which populations would be in decline in otherwise “pristine” areas.

Intriguing, but I was actually not ready to test it. With a potent combination of naïve enthusiasm, I figured I could simply contact the authors of the paper and ask them to teach me the methods that I needed to know to further their work. I tried email but could not find an address on the department website. So I phoned the Department of Biochemistry at the University of North Carolina at Chapel Hill. They explained that Dr. Sancar did not want or have an email address. I asked that the call be connected to his office. When he picked up the phone, I leapt immediately into my explanation that I was an MSc student from Trent University in Peterborough, Canada, and that I was hoping to visit his lab to learn methods of photolyase extraction that I would apply to my system. To my now weathered academic amazement, but, at the time, only to my joy, immediately and without hesitation, he said yes. If I could get myself to Chapel Hill, he would teach me what I needed to know.

Alex Smith with hair studying amphibian photolysase induction and concentration in the late 20th century.

Alex Smith with hair studying amphibian photolysase induction and concentration in the late 20th century.

So on my spring break of 1997, I rented a car (two cars actually – one died, another story) and drove from snowy Peterpatch to the flowering springtime of Chapel Hill, North Carolina to spend a week in Dr. Sancar’s lab. “Lab” didn’t quite cover it. Dr.’s Sancar (he and his wife, Dr. Gendolyn Sancar) had a floor of the building at UNC. Dr. Sancar met me on that Monday morning and arranged for a postdoc and a PhD student to help me all week and ensure that I could extract and purify the enzyme. He even arranged for another lab to give me some African clawed frog eggs to practice on! He met with me every day to see how I was progressing and answer any questions. I remember him encouraging me to take in a UNC NCAA women’s basketball game while in Chapel Hill (Tar Heels!), and I was very impressed that this academic superman was often watching soccer in his office when I arrived (the knockout phase of the UEFA Champions League, I think). A man of many interests! I left at the end of the week and proceeded to apply these methods successfully in my MSc. Three papers (Smith 2000, Smith et al 2000, and Smith et al 2002), eventually came from this project and one of the principal findings was that this enzymatic system could be induced in individuals from natural populations (previously not considered – and something that dramatically affects ones’ estimation of a populations’ photolyase level).

In my paper I was very critical of previous research – and not surprisingly, the manuscript received quite harsh and negative reviews. I had never written a response to reviewer comments before, and I did not craft them elegantly or with appreciation. Dr. Sancar was the editor at the journal handling the submission. He phoned me to suggest how I might better word my response. Connecting the phone call alone was no easy feat considering I was living in my car at the time, couch-surfing amongst friends on the west coast of North America – I’m still not sure how he managed to find me. But the advice was priceless and likely not something I would have come to on my own (let’s say it was something along the lines of…“I can hear that you’re angry by these comments, and they are not elegant – but you can’t say what you’ve said. What you mean is this……..so try expressing it like this….”). I was so appreciative, and now 20 years later I’m not sure I expressed my gratitude sufficiently.

And so, fast forward 20 years when I wake to read that the world has recognised Aziz Sancar for his pioneering work in the broad field of DNA repair. It made me think about the often unappreciated or unintended effects that saying yes can have on those around you.

At the end of his Nobel Lecture in Sweden in December 2015, Dr. Sancar showed a slide acknowledging his lab and colleagues. In part, these people and their output are the metrics that the Nobel committee evaluated in awarding him the prize. It was an impressive, but I knew not an exhaustive, list, for Dr. Sancar’s direct effect on my career – and indirectly then on all the students I have worked with in the subsequent years – was invisible to the Nobel committee (and perhaps not even remembered by Dr. Sancar). But these effects are significant and they came from a busy scientist saying yes when confronted with a naïve but enthusiastic student. There were many reasons for him to not take my call, not encourage me to come to North Carolina, not host me while I was there nor mentor me through the review process later on. But he did. He did say yes and it had an immeasurable effect.

I now work with insects in the neotropics and Canada on questions of biodiversity. I don’t work with photolyase and I don’t work as a physiological ecologist. However, by saying yes to me 20 years ago, Dr. Sancar’s act of generosity enabled me to follow this path. In the over-scheduled and busy lifestyle that we lead, it is important to consider this ripple that saying yes can have. There are many intended and measurable outcomes of supervision and mentoring – however there are many, perhaps more, unintended and important effects that kindness can have. As Anne Galloway said on Twitter, “We’re all smart – distinguish yourself by being kind”. The Nobel committee judged Dr. Sancar’s academic output worthy of its highest award last year. They were likely unaware of the affect that he has had in other scientific disciplines through his generosity and kindness.

 

I don’t think I said it clearly enough before. Thank you Dr. Sancar.

 

Dr. Alex Smith
Department of Integrative Biology,
University of Guelph

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ESC Blog Classifieds – 2 Post-docs @ University of Alberta (Chemical Ecology & Ecophysiology)

Seeking Two Postdoctoral Fellows in Tree Responses to Insect Herbivores and Drought

Area of Research: Chemical Ecology & Ecophysiology

Location: Department of Renewable Resources, University of Alberta, Edmonton (Alberta, Canada)

Description of positions: The interdisciplinary project goal is to characterize the contributions that metabolomics and genomics-assisted tree breeding can play in comprehensive forest planning. Postdoctoral fellows (PDFs) sought for this project to assess the activities of tree defense and ecophysiological responses to insect herbivory and drought. The PDFs will characterize the secondary compounds, anatomy, and ecophysiology of two conifer species (lodgepole pine and white spruce) in response to insect herbivory and drought treatments in both greenhouse trials and associated progeny field trials in Alberta. The PDFs will be responsible for conducting and coordinating both lab and field investigations that include anatomical and chemical characterization of tree defenses, assessment of 13C, gas exchange, and chlorophyll fluorescence plant drought response, implementation of greenhouse and field experiments, data management, statistical analyses, writing reports and peer-reviewed journal manuscripts, and interact with industrial and government partners. The PDFs will also assist with supervision of full and part-time research assistants and undergraduate students. Even though each PDF will have his/her own research projects, it is expected that they work and collaborate together.

Salary: $50,000+ benefits per year, commensurate with experience.

Required qualifications: PhD in a relevant field is required. The ideal candidate should have background and experience in chemical ecology, ecophysiology, entomology, forest ecology, with strong analytical chemistry of plant secondary compounds (primarily terpenes and phenolics) using GC-MS and LC-MS, and writing skills. Suitable applicants with a primary background in one or more areas, plus interest in other research areas, are encouraged to apply.

Application instructions: All individuals interested in these positions must submit: (1) an updated CV; and (2) a cover letter explaining their qualities, including a list of 3 references along with their contact information (a maximum of 2 pages). Applications should be sent by email to Nadir Erbilgin (erbilgin@aulberta.ca) and Barb Thomas (bthomas@ualberta.ca) by the closing date. Please list “PDF application in Tree Responses to Insect Herbivores and Drought” in the subject heading.

Closing date: November 30, 2016.

Supervisors: Nadir Erbilgin (https://sites.ualberta.ca/~erbilgin/) and Barb Thomas (http://www.rr.ualberta.ca/StaffProfiles/AcademicStaff/Thomas.aspx)

Expected start date: January 2017 (with some flexibility)

Terms: 1-4 years (1st year initial appointment, with additional years subject to satisfactory performance).

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ESC Blog Classifieds – Postdoc opportunity with Dr. Brent Sinclair

Postdoctoral Fellow – Functional genomics of insect overwintering

Applications are invited for a funded postdoctoral position in insect functional genomics as part of a collaborative project between labs at Western University and the Canadian Forest Service, both in Ontario, Canada.

The project will involve coordinating work between two laboratories to identify and validate candidate molecular markers associated with diapause and cold tolerance in the Asian Longhorned Beetle, Anoplophora glabripennis using a combination of RNA-Seq, high-throughput metabolomics, and RNAi. The ideal candidate will be creative, and enthusiastic, with an ability to work both independently and as part of a team.  We will prefer someone with a background in insect physiology or molecular biology, and with a strong publication record in RNAi (in insects), bioinformatics, transcriptomics and/or metabolomics analyses in non-model systems.  Because of the geographic separation of the CFS and Western labs, excellent oral and written communication in English is a must, as is a valid driver’s license.

The successful applicant will be primarily based in London, Ontario, Canada in the Department of Biology, Western University.  The Sinclair lab at Western is a diverse, vibrant, and globally-collaborative group of low temperature biologists with broad interests in insect ecology, physiology, and molecular biology.  Please visit http://publish.uwo.ca/~bsincla7/ to learn more about the group; informal communication with Dr. Brent Sinclair prior to application is welcomed and encouraged; he will be at the ICE in Orlando, and will be happy to discuss the opportunity in person at the meeting.  The project is in collaboration with Drs. Amanda Roe and Daniel Doucet at the Great Lakes Forestry Centre, Sault Ste. Marie (http://www.nrcan.gc.ca/forests/research-centres/glfc/13459), and will make particular use of the insect rearing and quarantine facility.

The initial appointment will be for one year with opportunity for a two-year extension.

To apply, please send a cover letter, detailing your fit to the position, a CV, and the names and contact details of three referees to Dr. Brent Sinclair bsincla7@uwo.ca by Noon (EST) on Monday 3 October.

We are committed to diversity, and encourage application from all qualified candidates.

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When you’re a spined soldier bug laying eggs, they can be “Any Colour You Like”

By Paul Abram
PhD Student, Université de Montréal

When Pink Floyd recorded their epic, psychedelic instrumental “Any Colour You Like” for the classic album Dark Side of the Moon, were they inspired by a predatory stink bug?

Three spined soldier bugs happily eating a mealworm.  Their voracious appetite makes them a widely-used biological control agent of insect pests (Photo credit: Andrea Brauner).

Three spined soldier bugs happily eating a mealworm. Their voracious appetite makes them a widely-used biological control agent of many different insect pests (Photo credit: Andrea Brauner).

Well … probably not.

The spined soldier bug (Podisus maculiventris), can’t actually lay any colour of egg it likes – but the real range of possibilities is pretty impressive.

The range of possible egg colours that can be laid by a single female spined soldier bug (Photos: Paul Abram/Eric Guerra)

The range of possible egg colours that can be laid by the spined soldier bug (Photo credit: Paul Abram/Eric Guerra)

Almost three years ago, when I started working with stink bugs and their parasitoid wasps, I noticed this astounding variation in the colour of the eggs of the spined soldier bug. I was surprised to find that nobody had looked into the cause of this variation or its potential functions. In fact, the function of insect egg colouration seems to have been a bit neglected in general. While I was initially hesitant to start on the dangerous path towards a PhD “side-project” (code for “I would like to take much longer to finish my degree, please”), I eventually caved.

In 2013, I was visiting a colleague’s lab where newspapers are used as a laying substrate for these bugs, and I noticed that there seemed to be a loose correspondence between the colour of the egg masses and the darkness of the paper, especially in high-contrast places like crossword puzzles. I wondered – could stink bugs actually adjust the coloration of their eggs to match the darkness of the laying surface? If so, this would be the first case of an animal able to selectively control the colouration of its eggs.

Back in Montreal a few months later, I started working on this question with an undergraduate summer student, Marie-Lyne Desprès-Einspenner. We did the simple experiment of putting individual females in Petri dishes painted white, black, or black on one side and white on the other.

Petri dishes housing spined soldier bug females, along with a mate, prey, and some green bean.  Everything a stink bug needs! (Photos: Paul Abram)

Painted dishes housing spined soldier bug females [right], along with a mate, prey, and some green bean [opened dish shown on the left]. (Photos: Paul Abram)

To our surprise and excitement, we got some nice results. First of all, it was clear that individual stink bugs could lay eggs across the whole spectrum of egg colours, and that the egg colour variation wasn’t just a result of advancing egg development. Additionally, stink bugs tended to lay darker eggs in the black petri dishes than the white ones; and, in the bi-coloured dishes, overall darker eggs on the black side than the white side. These effects were subtle, though, compared to the most important and unexpected factor: where the eggs were laid. Eggs tended to be lighter when laid on the underside of the lid (which was lit up from above) than when laid on the side or the bottom of dishes.

So, individual stink bugs can lay eggs of a range of colours, depending on where they are laying. Our next question was: how does this capability express itself on natural laying surfaces? We did some experiments using soybean plants, and figured out what seems to be the key to this whole thing: the stink bugs have a very strong tendency to lay darker-coloured egg masses on the tops of leaves (which have a relatively low surface brightness, like our black dishes), and lighter-coloured masses on leaf undersides (which have a high surface brightness due to light passing through from above, similar to the lids of our white dishes).

Light eggs laid on a leaf underside (upper panel), and dark eggs laid on a leaf top (lower panel). Photo credit: Leslie Abram.

A light egg mass laid on a leaf underside (upper panel), and a dark egg mass laid on a leaf top (lower panel). Photo credit: Leslie Abram.

Because leaves are excellent filters of ultraviolet (UV) radiation from the sun (protecting most insect eggs, which are usually laid on leaf undersides), and dark pigmentation often acts as a ‘sunscreen’ in nature, we wondered if dark colouration would protect developing stink bug eggs from a lethal sunburn when they are laid on the tops of leaves. Eric Guerra-Grenier (another undergraduate researcher in the lab) and I tested this in the lab by exposing differently coloured eggs to different doses of sun-mimicking UV radiation.

The results were crystal clear – darker eggs are better-protected from UV radiation than light eggs, with a strong dose-dependency with respect to UV radiation intensity and egg colouration.

This was an exciting find, but begged the question: what is the pigment that makes eggs dark, anyway? The clear answer was that it must be melanin, which is responsible for most dark animal pigmentation, including in us humans, and is also really good at protecting against UV radiation damage.

Eric and I did the obvious thing, sending hundreds of (freezer-killed) stink bug eggs to two melanin biochemists in Japan. Our collaborators ran a suite of tests to confirm that the egg pigment was melanin. But…it turned out that the egg pigment wasn’t melanin! Right now, we simply don’t know what this “mystery pigment” is (maybe something totally new to science?).

As is common in research, we are left with more questions than answers. What is the physiological mechanism that allows stink bugs to selectively apply pigment to eggs? In evolutionary terms, why lay eggs on UV-exposed leaf tops in the first place? And why still lay some light eggs on leaf undersides? Could the pigment also have a role in camouflage, thermoregulation, or water retention? Do other, closely related (or why not distantly-related) insect species also have this capacity? We’re currently working on some of these questions, and I hope that we get to try to answer all of them eventually.

If you’d like, you can find a lot more details about our findings, including the answer to “does UV radiation affect the control of egg colour?”, in a newly published paper (remember to listen to the accompanying song while reading) – and stay tuned for more results in the coming months.

In the meantime, fellow entomologists and naturalists, look closely at insect eggs – is there anything interesting about how they’re coloured/patterned?

A spined soldier bug female having a drink and contemplating the future of insect egg colour research (Photo credit: Leslie Abram)

A spined soldier bug female having a drink and contemplating the future of insect egg colour research (Photo credit: Leslie Abram)


Postscript:

I would like to suggest additional Pink Floyd song/entomology paper pairings (feel free to suggest your own!):

“Breathe” //  “Active Regulation of Insect Respiration”

“Run Like Hell” //  “Mechanics of a rapid running insect: two-, four- and six-legged locomotion”

“Mother” // “Parental care trade-offs and the role of filial cannibalism in the maritime earwig, Anisolabis maritima

“Echoes” // “The adaptive significance of host location by vibrational sounding in parasitoid wasps”

“Time” // “Short interval time measurement by a parasitoid wasp”

“Us and Them” // “Boundary disputes in the territorial ant Azteca trigona: effects of asymmetries in colony size”

“Comfortably Numb” // ”Effects of carbon dioxide anaesthesia on Drosophila melanogaster

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Physiology Fridays: Trace-metal phantoms

Living in metal-contaminated lakewater is just another day’s work for phantom midge larvae. 

In the lakes surrounding Sudbury, Ontario and Rouyn-Noranda, Quebec, over 75 years of smelter operations have left their mark by contaminating soil and water with the trace metals cadmium, nickel, copper, and zinc.

This contamination led Maikel Rosabal, Landis Hare, and Peter Campbell, all from the Institut national de la Recherche scientifique in Québec, to study how aquatic animals tolerate these contaminants.  To do so, they needed a study organism that was abundant, easy to collect, and could accumulate and tolerate trace metals.  The best option turned out to be larvae of the phantom midge Chaoborus.

“The lakes in the area, and their watersheds, have been contaminated by the deposition of atmospheric aerosols and particles.  Metal concentrations in lake water tend to be higher in the lakes that are downwind from and close to the smelters, than in lakes that are upwind and distant from the smelter stacks,” says Dr. Peter Campbell. “The presence of Chaoborus in lakes with high metal concentrations implies that they are highly metal tolerant.”

The researchers chose a total of 12 lakes around Sudbury and Rouyn-Noranda with differing concentrations of trace metals, and collected water samples, using diffusion samplers that excluded particles, and midge larvae using a plankton net. After homogenizing the larvae, the researchers used a series of centrifugation, heating, and sodium hydroxide digestion steps to separate the subcellular components of the larvae.  They then measured the amount of metal in each fraction as well as the concentrations of dissolved metals in samples of lake water.  This allowed them to relate the concentrations of each trace metal in lake water to the concentrations in larvae.

They found that the majority of each metal accumulated in the cytosolic heat-stable protein fraction that they isolated from the larvae—a fraction that contains large amounts of metal-binding proteins. And while other fractions also contained small amounts of metals, it was in the heat-stable protein fraction that metal concentrations responded most obviously to the increasing metal concentrations in lake water. This suggests that the Chaoborus larvae were able to bind and detoxify increasingly large amounts of these potentially toxic metals.

“This suggests an important role for these metallothionein-like proteins in the detoxification of metals,” says Dr. Campbell.  “Presumably this contributes to the presence of this insect in highly metal-contaminated lakes.”

While laboratory studies usually focus on the effects of exposure to a single trace metal (usually dissolved in the water), animals in this study were exposed in the field to many trace metals both in the water and in their planktonic food. The researchers suggest that Chaoborus larvae would be effective “sentinels” for estimating trace-metal exposure to lake plankton, which is a key component of ecological risk assessments.

“Rough estimates of trace metal exposure are often obtained by measuring total metal concentrations in the water or the sediment.  Such values usually overestimate metal exposure because much of the metal present is not available for uptake by organisms because they are bound to substances such as organic matter or iron oxides,” explain the researchers. “For these reasons, measurements of trace metals in organisms are increasingly used to estimate exposure in risk assessments.”

Rosabal, M., Hare, L. & Campbell, P.G.C. (2012). Subcellular metal partitioning in larvae of the insect Chaoborus collected along an environmental metal exposure gradient (Cd, Cu, Ni and Zn), Aquatic Toxicology, 120-121 78. DOI: 10.1016/j.aquatox.2012.05.001

Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/22647479

Chaoborus larvae

Photo: Maikel Rosabal

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Physiology Fridays: From boozy breath to colony control: ethyl oleate production in honeybees

Honey bee flying with pollen - Photo by Alex Wild

Honey bee flying with pollen – Photo by Alex Wild, used with permission

Honeybee colonies are famous for their orderly divisions of labour.  As worker bees grow up, they transition from housekeepers (cleaning the colony) to nurse bees (feeding young bees), to finally switching to foragers who go out and collect nectar and pollen for the rest of the colony.   To maintain a healthy colony, bees need to decide how many foragers and how many nurse bees are needed, and control of these numbers is accomplished by pheromone levels within the colony.

In honeybee colonies, there are pheromones like the alarm pheromone that cause immediate behavioural responses (called releaser pheromones) and others that trigger physiological changes like hormones do (called primer pheromone).  From previous work, it seemed that ethyl oleate functions as a primer pheromone, produced by foragers, that delays the maturation of nurse bees into foragers.

“Ethyl oleate does not elicit any noticeable behavourial responses in recipient workers,” says Dr. Erika Plettner, who supervised a recent study on the synthesis of ethyl oleate at Simon Fraser University in British Columbia.  “Yet it has a profound physiological effect”.

To understand how this chemical is produced in the individual bee and then distributed in the colony, Carlos Castillo and colleagues from Simon Fraser University in British Columbia and the Laboratoire Biologie et protection de L’Abeillie in France looked at several ways to identify the source and synthesis of ethyl oleate.  This chemical can be produced by a reaction between oleic acid (a common fatty acid in insects) and ethanol.  While you might not think of honeybees as heavy drinkers, it turns out that yeasts in flower nectar ferment the sugars present into ethanol, and so the forager bees have much higher exposure to ethanol than nurse bees.

To figure out if ethanol and oleic acid can be made into ethyl oleate by honeybees, the researchers incubated different honeybee body parts from forager and nurse bees with these precursors.  They found highest production of ethyl oleate in the head tissues, and that both nurses and foragers could produce ethyl oleate when given ethanol.  In addition, in whole bees, they found that the ethyl oleate migrated from the gut to the exoskeleton of the bees where it would exude into the colony.

Taken together, these results suggest that making ethyl oleate, while it is useful for colony control, might also be a way to deal with the occupational hazard of consuming toxic ethanol.  “Foragers have much higher occupational exposure to ethanol than nurses do,” says Dr. Plettner.  “This is why they make ethyl oleate in nature”.

Ethyl oleate molecule

Ethyl oleate

To track down where exactly the ethyl oleate was synthesized, they coupled oleic acid to a chemical that would produce fluorescence when the oleic acid was combined with ethanol to produce ethyl oleate.  Under the microscope, areas that fluoresced showed where ethyl oleate was being made.  They found that ethyl oleate was made in the esophagus, honey crop and stomach.

The authors were also able to identify the genes responsible for the synthesis of ethyl oleate in the honeybee and the resulting enzymes that catalyze the reaction between oleic acid and ethanol.  These enzymes are then secreted into the gut fluid, where they produce ethyl oleate, which is then transported to the cuticle.

The biosynthesis of ethyl oleate then can be thought of a way of providing updates to the colony about the availability of flower nectar in nature.  “EO might be some kind of ‘resource meter’ that tells the nurses in the colony how many nectar and pollen resources are coming in,” says Dr. Plettner.  “If lots of food is coming in, then it makes sense to inhibit nurse to forager transition, as the nurses would be more needed in the brood chamber than as foragers.  Conversely, if few resources and/or foragers are coming in, then it makes sense to speed up development of nurses so that they can forage and fill the need.”

Castillo, C., Chen, H., Graves, C., Maisonnasse, A., Le Conte, Y. & Plettner, E. (2012). Biosynthesis of ethyl oleate, a primer pheromone, in the honey bee (Apis mellifera L.), Insect Biochemistry and Molecular Biology, 42 (6) 416. DOI: 10.1016/j.ibmb.2012.02.002

Corresponding author: Erika Plettner (plettner@sfu.ca)

Further reading:

Castillo, C., Maisonnasse, A., Conte, Y.L. & Plettner, E. (2012). Seasonal variation in the titers and biosynthesis of the primer pheromone ethyl oleate in honey bees, Journal of Insect Physiology, 58 (8) 1121. DOI: 10.1016/j.jinsphys.2012.05.010

Stick Insect Baculum extradentatum
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Physiology Friday – Nitric oxide causes a sticky situation

Physiology Friday is a monthly column by UWO PhD candidate Katie Marshall and will feature new Canadian research on insect physiology.

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Nitric oxide (NO) is usually overshadowed in fame by its more famous cousin laughing gas, but it’s difficult to think of many simple molecules that have such a variety of important biological functions.  While NO only lasts a few seconds in the free gaseous state in the blood, it has been implicated in processes that involve everything from immune function to neurotransmission.  One important role for NO is in the cardiac system, where it functions as a vasodilator and in vertebrates it slows heart rate, while in insects it has the opposite effect.

Stick Insect Baculum extradentatum

Baculum extradentatum photo by Sara da Silva

Most of the research about the physiological functions of NO has focused on vertebrates, but recent work published in the journal of Cellular Signalling by graduate student Sara da Silva and her postdoctoral fellow mentor Rosa da Silva in the lab of Angela Lange (University of Toronto Mississauga), has shown that, unlike other insects, the Vietnamese stick insect Baculum extradentatum can respond to NO like a vertebrate.

“Our initial research interests in cardiac physiology were influenced by earlier work indicating that stick insect hearts are innervated and can be modulated by endogenous chemicals [like NO],” says study director and University of Toronto Biology professor Angela Lange.  “It is for this reason that we chose this understudied organism, which contains a simplified cardiovascular system that can be considered a model for work on other cardiac systems.”

The researchers first attempted to find the natural source of NO in the stick insect by removing hemolymph (blood) samples and staining for the presence of an enzyme that produces NO.  Then they examined the effects of NO on heart rate by dissecting the dorsal vessel out and maintaining it in a Petri dish with physiological saline.  They could measure heart rate through the placement of electrodes on either side of the dissected heart, and monitor the effects of various chemicals on the cardiac activity of the stick insect.   They also could examine whether heart rate was mediated by the central nervous system by leaving the nervous system attached or not.

insect heart rate

The effects of nitric oxide on the heart rate of B. extradentatum. Figure 3 from da Silva et al. 2012

They found that the hemocytes (blood cells) of the stick insect were producing an enzyme that was similar to the enzyme other animals use to produce NO.  In addition, the more of a chemical called MAHMA-NONOate (which produces NO) they added, the slower the stick insect hearts beat.  This surprising effect was completely opposite to what had been found in other insects and was more like the response of the vertebrate heart.

“Insects have evolved different strategies depending upon life history, and have co-opted different messenger systems for this success,” says study author da Silva. “We need to understand the full ecology of all species to finally appreciate the factors involved.”

Using the same setup, they also tested other components of a system of compounds that they thought might be involved in the pathway that produces NO that leads to decreased heart rate in B. extradentatum.  They believe that NO is produced in the hemocytes, travels to the wall of the heart, and then leads to the production of a messenger molecule that decreases heart rate.

Schematic diagram of the proposed regulation of cardiac activity in B. extradentatum by the gaseous signaling molecule, nitric oxide (NO)

Schematic diagram of the proposed regulation of cardiac activity in B. extradentatum by the gaseous signaling molecule, nitric oxide (NO). Figure 7 from da Silva et al. 2012.

“This study further emphasizes the evolutionary links between the physiological processes of vertebrate and invertebrate systems,” says da Silva. “Our findings suggest that signaling molecules (such as NO) common to both types of organisms can have similar effects on cardiac activity.  These novel findings demonstrate that the study of vertebrate systems can be complemented with studies in model invertebrate organisms.”

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da Silva, R., da Silva, S.R. & Lange, A.B. (2012). The regulation of cardiac activity by nitric oxide (NO) in the Vietnamese stick insect, Baculum extradentatum, Cellular Signalling, 24 (6) 1350. DOI: 10.1016/j.cellsig.2012.01.010