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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

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