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Testing and commissioning the dual-choice olfactometer
With the build completed I moved the olfactometer into the insectary for testing and commissioning. This was quite an exciting time as I was getting closer to the moment of truth. Would the equipment, that had thus far lived in my head and in computer software, be fit for purpose. Would the careful design, modelling and building pay off and furnish me with kit that would be central to my study of mosquito olfactory mediated behaviour, or would it leave me scratching my head on the way back to the drawing board.
Hurrying the development of the soon to be experimental candidate mosquitoes. Preconditioning for temperature and light cycle.
Once all the connections has been made to the newly purchased air compressor, carbon filter, inline heater and humidifier it was time to check that it was capable of delivering steady air flow at the right temperature and humidity.
Air flow was a simple matter of setting the litres per minute to match the modeled input. The humidity and temperature required a little more finesse, as it required a balancing of the water volume and temperature in the humidifier; once set, however, it remained steady at 25 degrees centigrade and 75 % relative humidity.
Importantly, I was unable to find any airflow bias between the trap chambers and variable delivery chambers, meaning that I could move on to the next step – a live run with mosquitoes.
Building the olfactometer!
After a long digital design and modelling process I was delighted to finally have all the component parts in hand and ready to assemble. In the interest of working uninterrupted, I chose to carry out the construction at home rather than in the lab. Hence the dining table, and occasional clutter you’ll see in these images.
Due to the late design changes after the computational fluid dynamic modelling of the previous iteration I had enough acrylic sheet to build 2 main flight chambers rather than one. Since the build this has proven fortuitous as it means I can carry out twice as many behavioural assays at the same time. This is particularly important as my study mosquitoes require overnight assays which makes time a real limiting factor.
If you could buy a flat-pack dual choice olfactometer this is what it might look like. (N.B. the syringe is for applying the acrylic cement)
Acrylic is quite nice to work with as the “glue” is very watery and so provide you can get a tight bond between surfaces you can apply the glue in one place and allow capillary action to draw it in and fill the void.
Insulating tape was used to hold the pieces in place whilst the glue was applied and set. Because
Insulating tape is slightly elastic it is great for this as it can be pulled very tight and allowed to relax again; once relaxed the acrylic sheet is held securely but not deformed.
Also, because the edges were very nearly square, after the laser cutting, forming the box was easier than I feared it might be.
Once the insulating tape was removed I was left with something remarkably similar to the design!
The gallery below shows the stages of construction before moving the units back to the laboratory.
In the next blog post I’ll look back over the installation and first test assays using this dual choice olfactometer.
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- If you could buy a flat-pack dual choice olfactometer this is what it might look like
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- The first side of the flight chamber being attached to the base
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- Insulating tape is slightly elastic it is great for this as it can be pulled very tight and allowed to relax again; once relaxed the acrylic sheet is held securely but not deformed.
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- Coming together nicely and the edges are all square
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- Once the insulating tape was removed I was left with something remarkably similar to the design!
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- The view from the other end showing the choice apertures
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- Why build 1 when you make 2
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- With first prototype sample chamber and trap chamber attached.
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- In this image it can be seen that the trap/release chambers are interchangeable.
Up close and personal with Culex pipiens
Recently I have been able to spend a little time Staffordshire University’s environmental scanning electron microscope (ESEM). I’d like to share some of the images that were captured.
Click any of the images for the full sized version!
These first 3 images show the complexity, and beauty, of the antennae of this male mosquito. I particularly like the way that new detail is revealed at each new magnification level. From an entirely non-scientific point of view, I find these hugely satisfying aesthetically
Male Culex pipiens antennae. Zoom level 1
Male Culex pipiens antennae. Zoom level 2
Male Culex pipiens antennae. Zoom level 3
The next 3 images focus on the maxillary palps of the same male Culex pipiens mosquito. Again, starting at low magnification and progressively increasing.
The end of a Male Culex pipiens’ maxillary palp. x100 magnification.
Male Culex pipiens maxillary palp x250 magnification
Male Culex pipiens maxillary palp x500 magnification. (specimen is getting quite charged now- hence the “glowing” hairs)
And last but not least, a 3 image series of the end of the proboscis of the same Culex pipiens male.
Male Culex pipiens proboscis x130 magnfication
Male Culex pipiens proboscis x250 magnfication
Male Culex pipiens proboscis x500 magnfication
Experienced microscopists please excuse the specimen charging throughout, and the missing scale data on the first 3 images.
A trip to the laser table.
Having never worked with acrylic before it has been an interesting challenge to accurately, and tidily cut my materials. The cylinder sections were quite easy to do, requiring only a junior hacksaw and some finishing with sandpaper to get a nice square edge. The flat sheets with the need for true edges suitable for gluing provided more of a challenge.
I tried using a hacksaw which in my hands was too inaccurate and resulted in a wavy line, entirely unsuitable for bonding to the face of another sheet.
I next tried to score and snap the sheets, but again this gave a poor finish, and sometimes causes a “step” profile in the edge. My final DIY attempt was to use a fine toothed bandsaw; this gave a truer edge but caused too much chipping of the edges, even when I tried protecting them with tape.
My final solution was to reach out to the Faculty of Arts and Creative Technologies to see what solutions they had. To my delight they have 2 laser cutting tables, 1 of which was suitable for my needs, available to hire. This was the perfect solution, guaranteeing straight cuts with a great finish – ready to glue.
Flight chamber design is now finalised.
Following a number of different design versions I am pleased to have settled on a final version that is going forward to prototyping.
The biggest, most obvious, change is the size of the flight chamber. This is significantly reduced in cross sectional area in response to the computational fluid dynamic work that has been carried-out. The reduced area leads to a reduction of required air volume needed to generate the flow rate and means that I can use the same unit for both static air testing and dynamic wind tunnel like tests.
In the static air tests the mosquitoes are loaded as normal, as are the variables ( or variable and control). However, no air supply will be attached. Testing with no air flow will allow me to test whether the odours from the variables is enough to cause the mosquitoes to investigate them without other ‘activators’. These tests will typically run overnight, and require a slight, removable, modification to the mosquito traps to prevent exit once a ‘decision’ has been made.
The dynamic, or wind tunnel, tests are typically much quicker, and can occur in a few seconds to minutes. There are different assumptions made of these types of tests than in the static air test, as a method it is equally applicable to my study. It is my plan to use both methods in conjunction to develop as complete a representation of behaviour as possible.
Reader contributed content. Animation of gate mechanisms
The animations seen in this video were contributed by regular blog reader David, and I would like to thank you for your input. They show quite nicely how each release/trap gate rotates on its axis to open or close the chamber. They also indicate the fact that these chambers will be removable to allow easier handling of the mosquitoes at the start and end of each experimental iteration.
David I hope you don’t mind my adding the title and captions.
David was instrumental in helping develop the model in Autodesk Inventor 2015.
He has also convinced me to test the design using computational fluid dynamics, which is yielding interesting results and may result in some design changes to the main body of the olfactometer.
Thanks again David!
First drawings of the experimental chamber for my olfactometer
I am delighted to be able to share some of the imagery showing the design of the olfactometer as it currently stands. Hopefully these pictures are worth a thousand words, as today’s blog post is intended to be quite short…
The material being used for the panels and tubing which make the parts illustrated is transparent, colourless, acrylic. The pin which for the hinge and handle for the rotating doors in the mosquito trap/release chambers is stainless steel, as is the mesh which covers the ends and surface of the gate.
3 dimensional drawing of the proposed olfactometer experimental chamber.
With reference to the 3 dimensional diagram above, the air will be blown into the ports at the rear of the unit, where they will pass through the 2 variable chambers. Here the air will flow over the variable/s being tested (or the control), where it will become ‘loaded’ with chemicals which may or may not cause a behavioural change in the mosquitoes. The air will then flow through the main flight chamber before exiting through the mosquito release chamber at the front of the unit.
Plan view of the experimental chamber. 3 dimensional drawings of the proposed olfactometer mosquito release/traps chambers.
There are a few design points which are currently still under discussion; such as the design of the rotating gates in the mosquito trap/release chambers. Currently they are designed as an acrylic ring which has a stainless steel mesh across it, there is some suggestion that it may be easier to simply use an acrylic disk and drill this through with many small holes. This is the potential drawback that the drilled holes will not provide a visual barrier to the stimulus being offered, where the stainless steel mesh is opaque and will therefore help control against the mosquitoes being able to see rather that smell the stimuli.
Side view of the experimental chamber, showing plane for sectional diagram to the right.
The airflow inlets shown in sectional view in the 3rd drawing are slightly misrepresented in the drawing. The internal portion, that which is inside the chamber, is actually shorter than shown. This will be adjusted in the next round of design reviews.
As always comments and questions are greatly appreciated!
CAD drawings soon, and parts on order. Video update.
Meet Ochlerotatus punctor, the puddle dweller!
Today’s blog post is not directly linked to the olfactometer design and build process but instead introduces a little detail around one of the UK’s native mosquitoes.
Both of these are Ochlerotatus punctor and were collected as larvae from the same temporary puddle in the middle of a path through a forest. They were then reared on to adult emergence in the university insectary. It is worth noting that the puddle only lasted about 10 days, yet it yielded many larvae; I do not know however whether the development cycle completed before the puddle dried up. I do know that I did not collect any 4th instars (the final larval stage of mosquitoes) or pupae from the puddle before it dried up.
The images below are included to help highlight some of the physical differences between male and female mosquitoes. The lighting is not great as the photos are taken through the eyepiece of the dissecting microscope in the insectary. These are quite small mosquitoes, about 2/3 the size of the Culiseta species which forms the title image of this blog site.
Ochlerotatus punctor are widely distributed in Britain dependent upon the availability of temporary woodland pools. They females feed on a range of mammals and will readily bite humans, particularly at dusk near aquatic sites (Snow, 1990).
Female Ochlerotatus punctor head with annotations showing pilose antennae and short maxillary palps.
Male Ochlerotatus punctor head with annotations showing plumose antennae and elongate maxillary palps.
References:
Snow, K.R. (1990). Mosquitoes. Slough: The Richmond Publishing Co. Ltd.