Calliphora vicina, proboscis and labella.

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Greenfields
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Calliphora vicina, proboscis and labella.

Post by Greenfields »

Calliphora vicina, proboscis and labella
230410 Run_24Cd.JPG
These are the labella or lobes of the oral sucker of a bluebottle blowfly: Calliphora vicina.

Under the name Musca/Calliphora erythrocephala it was probably the most intensively studied of all diptera in Europe in the early twentieth century, later to be overtaken by the similar Black Blowfly: Phormia regina in North America, then the much smaller and ubiquitous Drosophila which has many anatomical features in common with the two blowflies.

The labella is surrounded by chemosensory ["taste"] hairs springing from its aboral [back] surface. Each hair is hollow with a pore at the tip which allows tastants to enter and make contact with the chemosensory neurons inside. All labellar taste hairs house a single mechanosensory neuron, but the number of receptor sites, each of which responds to a different chemical structure, varies from two to four..

The hairs can be divided into four groups by length, and it turns out that these groups are related to the chemical structures to which the hairs respond. This is also true of similar hairs on the feet. There have been many experiments in which the electrical activity of individual hairs has been recorded in response to a variety of chemical stimulants.

The arrangement of the chemosensory hairs around the labella is roughly symmetrical. Maps of the positions of the base of the hairs on the aboral surfaces of the labellar lobes show that they are almost - but not quite perfectly, mirror images. There are also variations between individuals so the arrangement can't be used to identify species.

Gustatory neurons on the leading edges of the wings and chemosensory cells on blowly's palps and antennae are used for foraging and can detect traces of odours over long distances [maybe more than a kilometer].

On landing on potential food the hairs on the feet, which curve downwards, test the surface for attractive compounds [e.g. water and sugars] or noxious [e.g. alcohols] chemicals. An attractive response stimulates extension of the proboscis to explore the substrate. With the labella closed the hairs on the aboral surface point forwards. If the response continues to be attractive the labella open to sample the surface.

The response is sophisticated. If the sensilla detect water, the proboscis will only extend if the fly is thirsty. If sugar or other nutrient is detected, the proboscis will only extend if the fly is hungry. It is possible for only a single neuron to induce a response because each hair contains only one water detector and one salt detector [plus up to two others].

The surfaces of the labella are covered by lines of pseudotrachea, which are tubes formed around a skeleton of thousands of forked, chitinous rings, resembling curved pitchforks, which can collect fluid and conduct it by capillary action towards the oral opening leading to the food canal. There are more chemosensors in small pegs (papillae) between the pseudotrachea, and cells inside the alimentary tract to keep testing the fluid as it is ingested.

Surrounding the oral channel in the centre of the labella are the "prestomal teeth". These vary between species, but where they exist they can rasp the surface of some foods to expose liquids for ingestion. [The scrape marks have been recorded after flies have been feeding on sugar or baits laced with harmless dyes.] In C. vicina there are three rows of prestomal teeth on each side of the oral opening but the outermost row is usually difficult to see.

In C. vicina the pseudotrachea are covered by an almost transparent and presumably semi-permeable membrane.

As a specimen dries out the well-known collapse of delicate tissues due to surface tension effects means that the labella becomes dark and shrivelled. The membrane "shrink wraps" the crumpled tissue making it difficult to photograph because of the specular reflections: as if wrapped in cling film.

This shape of the labella are similar, but it not identical to the appearance fully distended labella have in life. The main difference is that the living fly extends the proboscis and labella using a set of muscles [not by inflation as was once thought], whilst in these images different post-mortem methods have been used to get the labella to distend and remain stable.

This specimen has been macerated, of course. 3 hours in 9% aqueous KOH. I think that this may be close to the limit at which the structure becomes too fragile to be handled.


Fluorescence induced by staining with citrazinic acid.
The first image is one tenth of its original scale, and the second a detail of the same image.
230514 Citrazinic_Whole_1k.jpg
230514_Citrazinic_Crop_1K.jpg
It is difficult to reproduce the result of photographing the fluorescence of a dye stain on large subjects because of the variables, some of which are hard to control.

These include the instability of many of the reagents used when they are in solution, pH [which always seems to be close to an inflection in the titration curve making it difficult to repeat accurately], the purity of the dye [often not that good], the concentration [planar dyes form aggregates at increasing concentrations which have different properties] and the fact that the concentrations used may be very low [of the order of 5 to low hundreds of parts per million] which makes them vulnerable to accidental contamination.

One of the biggest problems is photodegradation. These images were made with Mitutoyo 10x and 20x objectives on a full frame DSLR. For the fluorescent signal to be bright enough to be captured at a low ISO to avoid shadow noise, the intensity of the excitation causes rapid photodegradation within a single run. A succession of runs may produce quite different results - and the first is not necessarily the best. Adding an oxygen scavenger helps and does not fluoresce so allows deep background shadows. An anti-fade reagent helps even more - but commercial products are egregiously costly and my improvised formula fluoresces blue. That does not affect the foreground of opaque subject but does produce a blue background [not always bad].

Illumination was a Nichia 6W 365 nm. LED driven by benchtop PSU in current-limiting mode, through a pulse-width modulator to control the output. A pair of N-BK7 glass collecting lenses projected a 7 mm square image of the dye onto the subject, slightly defocused to blur the image of the die electrodes. No diffuser was used because the images are of light emitted by the subject, not reflected by it.


And finally, at least for now here are two images of the prestomal teeth.
These subjects have not been macerated.
One was made with white light, and the other captures autofluorescence in 365 nm light.

Prestonal_White.jpg
Prestonal_Autofluorescence.jpg
Henry
Last edited by Greenfields on Fri Aug 11, 2023 12:01 pm, edited 1 time in total.
Feel free to edit my images.


Adalbert
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Re: Calliphora Vvcina. Proboscis and labella.

Post by Adalbert »

very nice :smt041

Greenfields
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Re: Calliphora vicina. proboscis and labella.

Post by Greenfields »

THank you for your coments.

The next two images, of a labella which has not been stained or macerated, is the closest I can come to show the labella in its naturals state
Prestonal_White.jpg
200417_Run_58_Not_Macerated.jpg
It liife the oral surface of the labells is pressed against the substrate so will not be convex. Watching a blowlfy feeding shows that the proboscis and labella move faster than the temporal resolution of human vision, so only time-laps video will show exactly how it behaves.

Henry
Feel free to edit my images.

Pau
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Re: Calliphora Vicina. Proboscis and labella.

Post by Pau »

Great work!

I most like the fluorescence images. Any idea about the cause of the different emission colours of different parts? (I assume that all are made of chitin)
Pau

rjlittlefield
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Re: Calliphora Vicina. Proboscis and labella.

Post by rjlittlefield »

Very nice!

I especially like image #3 (THIS ONE) because it shows the prestomal teeth from a vantage point I have never seen before.

Image #7 (HERE) shows the prestomal teeth very well also, but in essentially the same way that is shown in my stereo pair at https://www.photomacrography.net/forum/viewtopic.php?p=103825#p103825 .
Greenfields wrote:
Fri Aug 11, 2023 8:27 am
Surrounding the oral channel in the centre of the labella are the "prestomal teeth". These vary between species, but where they exist they can rasp the surface of some foods to expose liquids for ingestion. [the scrape marks have been recorded after flies have been feeding on sugar or baits laced with harmless dyes.]
Thank you for the info about scrape marks. In earlier discussions about my images, now over 11 years ago, there was some debate about the function of the prestomal teeth. I'm glad to hear that in at least some cases a scraping function has been documented.

--Rik

Lou Jost
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Re: Calliphora Vicina. Proboscis and labella.

Post by Lou Jost »

Really interesting photography and equally interesting biology! Thanks for taking the time to make such an interesting write-up.

Greenfields
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Re: Calliphora Vicina. Proboscis and labella.

Post by Greenfields »

Pau asked:

"Any idea about the cause of the different emission colours of different parts? (I assume that all are made of chitin)"

Thus is an interesting and complicated question. I don't have an answer but clues are starting to emerge from the most recent professional research.

Chitin is not a homogenous substance, of course. It it only one part of the exoskeleton and its composition varies accoding to its purpose.

I can say that in high resolution images, part the distribution of fluorescence correlates with visible features which suggests that the physical and chemical propertiies of the different features play a role indetermining whether and to what degree a fluorophore will dissolve.

This open access paper on the machanical properties of Prestomal published a year ago last February:

https://www.researchgate.net/publicatio ... Brachycera

Includes come confoval images of a labell with a blue background which the authors attributed to the elastomeri protein resilin, so may be that is responsible for the blue fluorescence where maceration has rendered the exoskeleton translucent.

Another image and detail of the preszxtomal teeth.

The fluorophore was Blankophor REU-P. This was used in place of Calcofluor [which I don't have] in the hope that it was sufficiently simular to Calcofluor to adsorb to chitin.

Interpreting the image is complex. The fluoropohre is practically insoluble in water so a solubilising agent [triethanolamine] is needed to prepare a concentrated stock solution. This will determine the final pH and may affect the emission. The subject was previously stored in "10%" phosphate buffered formalin [mainly to prevent fungal growth which otherwise appears rapidly] so there may also be contributions from formalin induced fluorescence and there is also some autofluorescence from the resilin


Final.jpg

Final_Sel.jpg


Henry
Feel free to edit my images.

Pau
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Re: Calliphora Vicina. Proboscis and labella.

Post by Pau »

Henry,
Thank you for the further info, liked paper and new images, most interesting stuff.
Pau

Hatchetj0e
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Re: Calliphora Vicina. Proboscis and labella.

Post by Hatchetj0e »

Spectacular, especially given all the challenges of using fluorescence! I hate to think of how much time you had to spend on this!

Greenfields
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Re: Calliphora Vicina. Proboscis and labella.

Post by Greenfields »

Thank you, Hatchetj0e

It did take a while to learn how to get the proboscis to extend and labella inflate [fortunately they are rubbery, so survive handling well but tend to elastically retract], build a suitable obervation cell in which the sides can easily be replaced because they have to be too thin to clean, and keep the UV light stable [I originally tried aluminium flashlights, but they all quickly became unreliable : i suspect due to the difficulty of manufacturing a reliable electrical connection between the aliminium body which is used a current path [and forms an insulating oxide layer almost immediately on exposure to air] and the brass and copper components.

Then comes the question of getting colours "right". There have been discussions on this forum about how to get the colours of self-luminous subjects "accurate" and many more on forums covering astrophotography. There is no consensus. I just record the visual appearance of key features and try to match that record in post by adjusting the colour balance and contrast.

Among many unexpected things I found is that the published absorbance and fluorescence data for compounds are not a reliable guide to their behaviour when adsorbed. For example, the dye I have most experience with is acridine orange and that has an absorbance minimum at 365 nm. and fluoresces very weakly [if at all] at that excitation wavelenght in solution. When adsorbed if fluoresces strongly. [Acridine orange jumped in price by a factor of six this spring.]

At the risk of becoming tiresome, here is another image:

This time the subject was stained by acridine orange, which usually produces a red-orange fluorescence but photodegrades very rapidly on exposure to NUV light. In visible light the sub ject looked orange, just as it should, but for this experiment thr subject was immersed in deionised water containing 0.56 g./l of potassium sulphite with its pH adjusted to 4.25 with dilute HCl as an oxygen scavenging reage to see whether this would retard the photogadation. I don't know: The fluorescence was blue. I wonder if the sulphite completely quenched the AO fluorescence leaving only the autofluorescence.


01_1Kr.jpg

Crtop_01.jpg


Henry
Feel free to edit my images.

rjlittlefield
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Re: Calliphora Vicina. Proboscis and labella.

Post by rjlittlefield »

Greenfields wrote:
Fri Aug 18, 2023 4:29 am
At the risk of becoming tiresome...
I suspect that risk is quite small. Speaking only for myself, I'm loving this thread.

--Rik

quenoteam
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Re: Calliphora Vicina. Proboscis and labella.

Post by quenoteam »

Really brilliant job!!

Sippyjug
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Re: Calliphora Vicina. Proboscis and labella.

Post by Sippyjug »

Stunning series. Absolutely stunning. =D>

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