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Physiology Fridays: A feeding frenzy–Insulin signalling in larval brains

Insulin is perhaps best known as the crucial molecule whose lack leads to diabetes.  It’s a hormone that regulates carbohydrate and fat metabolism, and signals cells to increase uptake of glucose from the blood.  What most people don’t know is that insects use insulin too.

“Insulin signalling is a very conserved pathway which has been investigated extensively in humans as well as more recently in Drosophila melanogaster,” says Dr. Ana Campos, a researcher in the Biology Department at McMaster University.

And it turns out that in both insects and humans, insulin plays a much broader role in the brain than previously thought.  In a recent paper Dr. Campos and her technician Xiao Li Zhao published in the Journal of Experimental Biology, they showed that insulin signalling in the mushroom body (a critical region of the insect brain) regulates feeding behaviour in fruitfly (Drosophila melanogaster) larvae.

“Insulin has been implicated in a wide variety of biological processes. Its importance goes beyond its well-known role in the regulation of carbohydrate and fat metabolism, says Dr. Campos.  “In addition, it has been implicated in synaptic plasticity and cognitive function in humans and relevant animal models.  Recent findings indicate that abnormal insulin levels contribute to the development of neurodegenerative diseases.”

Image: Mushroom body in D. melanogaster (from Jennett et al. 2006, BMC Bioinformatics, doi:10.1186/1471-2105-7-544)

Investigating the role of insulin signalling in the mushroom body came about by a chance observation in their lab: they found a mutation in the Ran-binding protein M gene (RanBPM) that disrupted feeding behaviour in D. melanogaster larvae also inhibited insulin signalling.  Since this gene is also highly expressed in the mushroom body, it made sense to the researchers to investigate how the mushroom body influenced feeding behaviour and whether insulin signalling mediated it.

The researchers created a series of D. melanogaster strains with different parts of the known insulin-signalling pathway knocked out.  Then they measured the amount of food eaten by the different strains of mutant larvae as well as their resultant growth. By using immunohistochemical labelling, they also were able to find that reduced insulin signalling in the mushroom body on reduced the total number of neurons produced in the brains of these larvae.

Taken together, Dr. Campos and Xiao Li suggest their results mean that the mushroom body could be the brain region responsible for collecting signals about nutritional status in insects, and helps regulate feeding behaviour.  More broadly, this contributes to the knowledge about how insulin signalling impacts brain function.

Zhao, X. L. and Campos, A.R. (2012) Insulin signalling in mushroom body neurons regulates feeding behaviour in Drosophila larvae. J. Exp. Biol. http://www.ncbi.nlm.nih.gov/pubmed/22786647

Keywords: Physiology Fridays, Mushroom body, Insulin, Drosophila melanogaster, research blogging

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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