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* These authors contributed equally
This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.
Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.
Maintenance of salt and water levels in insects allows them to succeed in many ecological and environmental niches, utilizing a variety of feeding strategies1. Most insects have evolved mechanisms to regulate the composition of their haemolymph within narrow limits in order to withstand the different challenges associated with their particular environment2. Terrestrial insects are often faced with the challenge of conserving water and the excretory system undergoes anti-diuresis to prevent loss of water and some essential salts, therefore, avoiding desiccation. In contrast, diuresis occurs when the insect feeds and is ch....
1. Making silicone-lined dishes
NOTE: This step should be done prior to the experiments. These dishes will be made to prepare the assay dish for dissections, and for contraction assay experiments.
Application of DH31 against unstimulated MTs results in a significant increase in fluid secretion rate, confirming its role as a diuretic hormone in Aedes mosquitoes (Figure 1A). When tubules are treated with AedaeCAPA-1, a reduction in secretion rate is observed in DH31-stimulated MTs. Figure 1B demonstrates the use of ion-selective electrodes to measure Na+ concentrations in the secreted droplets. Treatment of.......
When ingesting a blood meal, haematophagus insects face the challenge of excess solutes and water in their haemolymph2. To cope with this, they have a specialized excretory system, which is tightly controlled by hormonal factors, allowing the insects to rapidly initiate post-prandial diuresis. The Ramsay assay and use of ion-selective microelectrodes allows for measurement of fluid secretion rates along with ion concentrations and transport rates in isolated insect MTs. Critical steps within these.......
This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to AD and J-PP. AL and FS received NSERC CGS-M awards in support of their graduate research.
....Name | Company | Catalog Number | Comments |
1 mL syringes | Fisher Scientific | 14955456 | |
35 mm Petri dishes | Corning Falcon (Fisher Scientific) | C351008 | |
Borosillicate glass capillary filamented tubes (OD 1 mm, ID 0.58 mm, length 100 mm) | World Precision Instruments | 1B100F-4 | used for ISME reference electrodes |
Borosillicate glass capillary filamented tubes (OD 2 mm, ID 1.12 mm, length 102 mm) | World Precision Instruments | 1B200F-4 | used for SIET reference electrodes |
Borosillicate glass capillary unfilamented tubes (OD 1.5 mm, ID 1.12 mm, length 100 mm) | World Precision Instruments | TW150-4 | used for ISME and SIET electrodes |
CO2 pad | Diamed | GEN59-114 | |
Dimethyltrimethylsilylamine solution | Sigma-Aldrich | 41716 | |
Faraday cage | Custom | Can be fabricated by local machine shop | |
Ferric chloride | Sigma-Aldrich | 157740 | |
Forceps (Dumont #5) | Fine Science Tools | 91150-20 | |
Glass Petri dish | Fisher Scientific | 08-748A | |
Hydrated mineral oil | Fisher Scientific | 8042-47-5 | Specific brand is not important |
INFINITY1-2CB video camera | Luminera | INFINITY1-2CB | |
Micromanipulators (left and right handed) | World Precision Instruments | MMJL and MMJR | Specific brand is not important so long as high quality manipulator |
Mineral Oil, Light | Fisher Scientific | 0121-4 | |
Minutien pins (0.1 mm stainless steel) | Fine Science Tools | 26002-10 | |
Non-hardening modeling clay | Sargent Art | Specific brand is not important | |
Olympus light microscope (FOR SIET) | Olympus | customized system | |
Plastic Pasteur (transfer) pipette | Fisher Scientific | 13-711-7M | |
Poly-L-lysine solution (0.1 mg/mL) | Sigma-Aldrich | A-005-M | 84 kDa |
Polyvinyl chloride (PVC) | Sigma-Aldrich | 81395 | |
Scalpel Blade | Fine Science Tools | 10050-00 | |
Scalpel Handle | Fine Science Tools | 10053-09 | |
Schneider's Drosophila medium | Sigma-Aldrich | S0146 | |
SIET system | Applicable Electronics | customized system | Details available at: http://www.applicableelectronics.com/overview |
Silver wire | World Precision Instruments | AGW1010 | |
Sodium ionophore II cocktail A | Fluka | 99357 | |
Standard polystyrene Petri (culture) dishes | Fisherbrand | FB012921 | Any size would work, but 60 mm dishes are good for both dissections and assay |
Stereomicroscope with ocular micrometer | Nikon | SMZ800 | |
Sutter P-97 Flaming Brown Pipette puller | Sutter Instruments | FGPN7 | |
Sylgard 184 Silicone Elastomer Kit | Dow Chemical Company | NC9285739 | |
Tetrahydrofuran | Sigma-Aldrich | 401757 | |
VWR advanced hotplate stirrer - aluminum | VWR | 9578 | Specific brand is not important |
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