Tracheae and spiracles of grape leaffolder caterpillar

Images taken in a controlled environment or with a posed subject. All subject types.

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rjlittlefield
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Tracheae and spiracles of grape leaffolder caterpillar

Post by rjlittlefield »

This is the larva of Desmia funeralis, one of the grape leaffolder moths. Working from large to small...

This is a crop from a 1X image that I intended to shoot just for overview. But when I looked closer, I saw that the light and focus had accidentally been just right to capture the tracheae, visible behind the transparent cuticle. The view is especially good just forward of that first abdominal spiracle.

Image

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Of course that got me curious: what might I see if I looked closer?

This is a dead but fresh specimen, photographed at 5X and rendered in stereo. There are some distracting bits of debris on the surface, but perhaps this at least confirms the structure of the tracheae.

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At the bottom of the preceding image, again appears that first abdominal spiracle. Here is a closer look, shot at 20X, rotated clockwise, and seriously cropped:

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In the above 20X view, we can see pretty clearly that the spiracle is mostly filled up with some things that look remarkably like Q-tips. I have seen such structures before, in other caterpillar spiracles, but I have never been able to see convincingly what the structure is.

So this time I decided to push harder.

First, I spent many hours in what turned out to be a fruitless effort to photograph this spiracle with a 40X NA 0.80 apochromat. The problem is lighting -- the Q-tips are so far down inside the lip of the spiracle that by the time the available angles are limited by the high-NA objective, there's no light hitting them directly. Under those conditions, the whole scene loses detail and effectively blurs into a mass of shapes that shows no more than the 20X NA 0.42 did. Despite preparing a permanent specimen by drying in acetone, and trying several lighting schemes, I got nothing useful.

So then I switched strategies to more or less classic microscopy. I harvested another specimen (my grape vines have lots of them), killed it in boiling water, dissected the fresh specimen to extract a small piece of cuticle holding the first abdominal spiracle and essentially nothing else. Then I dried that small piece of cuticle in acetone and mounted it under a cover slip using --- wait for it --- Bondic. Yeah, Bondic. It turns out that Bondic loves chitin. It flowed nicely around and into the piece of dry cuticle, displacing essentially all the air with only a bit of manipulation. After all the air bubbles had separated from the cuticle and flowed away, I dropped a cover slip over the specimen, waited for capillary force to suck the cover slip down tight, and cured the Bondic with blue light.

That gave me something very much like a permanent slide mount, which was simple to image with a 40X NA 0.65 bio objective with condenser illumination.

There was even enough depth left in the mount to make a decent stereo rendering. Here we have 54 frames at 0.001 mm focus step, rendered at +-6 degrees for total 12 degrees of stereo separation.

Cropped to show the whole spiracle. and very heavily sharpened to pull out as much detail as possible:

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Cropped even tighter to show more structure in the hairs:

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So, now I'm pretty comfortable that I understand the structure. Those "Q-tip "structures are tufts of hairs, mounted on the inner faces of the lips of the spiracle. I expect that in operation, the lips can be closed to reduce water loss, or opened to allow air exchange, but then with the air being filtered by the tufts of hairs.

It would, of course, be even more satisfying to image these things with SEM, something like HERE. But that will have to wait for some bigger budget: either time, or money, or space, or more likely all three. I am not expecting to move in that direction any time soon.

In any case, I hope you find this interesting!

--Rik

Rikisub
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by Rikisub »

Amazing work Rik, absolutely impressive, congratulations!!

wwheeler48
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by wwheeler48 »

Rik-that is incredible. I will have to look up some of the physiology of how these structures work. I just had a simplistic view that air just kind of flow into tubes. It is obviously much more complicated. Biology is fascinating. Thanks for sharing!

Sym P. le
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by Sym P. le »

... the light and focus had accidentally been just right ...
Sure, RIk, sure. But absolutely well done, even if you did have to struggle a bit. =D>

leonardturner
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by leonardturner »

Strikingly excellent work on multiple levels of magnification. Can't wait till you get that SEM!

Adalbert
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by Adalbert »

Hello Rik,
very interesting story, great pictures!
What WD's have the two objectives 40x/0.80 and 40x/0.65 ?
Best, ADi

pierre
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by pierre »

Impressive work & results Rik.
Thanks for sharing.
Regards

Pierre

rjlittlefield
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by rjlittlefield »

Everyone, thank you for the kind words. This has been a very challenging subject, which has somehow gotten its hooks into me so that it's difficult to let go.

I tried again at higher resolution. The technique was to prepare a dry whole specimen by dehydrating in acetone and washing in HMDS before air-drying, then I physically split the spiracle down its middle by using a #10 scalpel while working under a 45X stereo scope. That opened up the structure so that I could shoot the cross section, with some illumination shining directly on the tufts of hair.

Using a 40X NA 0.80 plan apochromat objective, plus a 1.4X teleconverter, with very aggressive sharpening and curves (levels) adjustment to bring out as much structure as I could, here is the result:

Image

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These are close crops from a single stack, 87 frames at 0.7 micron focus step. Canon R7 with full electronic shutter, nominal 1 second exposure, flashed in the middle using an add-on flash sync controller that I made about 10 years ago for use with StackShot and EFSC. With the total absence of camera vibration, and using a microscope focus block for stepping, I was able to entirely skip computational alignment and get a clean result with no added alignment issues.

ADi, the 40x/0.80 is about 0.7 mm WD. I do not know a number for the 40x/0.65, but it hardly matters because that one is designed to image material mounted flat under cover glass.

--Rik

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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by rjlittlefield »

Here is the overall setup for the preceding shot. Illumination is very simple, just a single flash unit sitting a few inches behind a piece of thick vellum tracing paper. I figured that with the structure and orientation of this subject, there was only one path for light to get into the spiracle anyway.

Image

--Rik

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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by rjlittlefield »

For context, here is a 5X view of the entire cross-section. The spiracle being imaged is at the left side, just above center.

Image

This is a single frame, not cropped. At this web-sized scale the entire depth of the spiracle fits comfortably inside the effective DOF of the 5X NA 0.14 objective.

--Rik

BugEZ
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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by BugEZ »

Amazing!

Keith

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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by rjlittlefield »

Today I tripped across an SEM image that I suspect pretty closely represents the spiracle of my grape leaffolder caterpillar.

Image

This is from https://www.researchgate.net/publicatio ... utterflies . The species illustrated is Bicyclus anynana, a Southeast Asian butterfly not closely related to my moth. But the spiracle structure sure looks remarkably similar.

--Rik

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Re: Tracheae and spiracles of grape leaffolder caterpillar

Post by MarkSturtevant »

An exceptional study!
Mark Sturtevant
Dept. of Still Waters

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