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Moth developing inside pupal shell (stereo)

 
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rjlittlefield
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Joined: 01 Aug 2006
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PostPosted: Tue Jul 24, 2018 10:33 pm    Post subject: Moth developing inside pupal shell (stereo) Reply with quote

This project had more than the usual number of interesting aspects.

First, here is the subject. It's a moth pupa with a well developed adult forming inside. You can easily see the antenna inside its case, a couple of legs inside theirs, patterns forming on the scaled wings, and so on. This is a stereo pair, crossed-eye.



Here's a closer crop, showing more detail around that claw in the middle of the image. (The claw is located at the joint between tibia and tarsus, several segments above the foot. As far as I know, nobody is quite sure what the claw is good for. There are suspicions that it's used to help the critter drag itself out of the pupal shell and surrounding debris, to get somewhere that it can expand its wings.)



Direct lllumination for all these shots was provided by a single undiffused Jansjo lamp, cross-polarized. I had to kill surface reflections in order to get a good view of the subsurface detail. The subject is lying on a gray card which provides non-polarized back light. You can see how bad the surface reflections are, on the sides of the pupa. So that's interesting aspect #1, having to use cross-polarization just to see what I cared about.

Interesting aspect #2 is that I had to shoot the focus stack by hand, turning the fine focus knob of my focus block system. I tried to shoot it automatically, with stepper motor control. But this is a live specimen, and apparently there's something about the noise of a stepper motor that agitates the critter, because every couple of frames that leg with the claw would jump to a new location. I was worried that the camera's shutter action might be causing the problem, but fortunately it was not. Still, I'm thinking that a setup providing dead quiet operation and much faster acquisition would have been more robust.

Interesting aspect #3 is that this subject is extraordinarily subject to color shift due to metamerism. The camera sees light that comes from the inside of the pupa as much more red than it appears to my human eye. To produce a rendition that is anything close to what I saw by eye, I had to desaturate the colors across the board by a Photoshop Hue/Saturation layer with Saturation of -43. Here's a comparison of the images as adjusted and as shot, with the ridiculous red cast to the pupa even though the background is neutral gray as it should be.



Interesting aspect #4 may be seen in the second image, the stereo crop. If your stereo perception is good enough, you'll notice that the band of bright reflections just below the eye appears to be depressed below the rest of the surface. It looks almost like the pupa is dented in that area. But in fact the pupa is not dented, it's just that the reflections behave quite oddly in that area. Because the light source is very small, and the pupa is very shiny, and the lens aperture is pretty wide (NA 0.14), those reflections are highly vulnerable to the "utilized aperture" effect. Reflections from one side of the band use one part of the lens, while reflections from the other side of the band use another part of the lens, so as the lens is moved to focus, the reflections appear to move around in interesting ways that cause the stereo rendering to look dented. Here's a simple animation of the source frames. (Note: these reflections would be extremely bright and blown out if it were not for the cross-polarization. What you're seeing here is just the remnant light that got through the crossed polarizers. In the original images that remnant is quite a bit more blue, again due to the polarizers. But here the blueness has been diminished by desaturation as described above.



Interesting aspect #5 is back to the subject itself, in particular how its patterns change over time. Here are three views of the subject that were shot about 1 day apart. The last view is from tonight, just a few minutes ago. In addition to being very dark, the wing covers are starting to soften and dimple, so I'm expecting that very soon now I'll have an adult moth to look at. This seems like a pretty normal sequence for moth development, but I've never followed it this closely before. I'm very interested to note how the basic geometry of the wing patterns are laid down early, and are quite obvious, with the pattern then filling in with increasingly pigmented scales.



And finally, interesting aspect #6 was a reminder of just how confusing things can be for a new user. I do most of my work on Windows. But I shot and processed these stacks on an iMac with 5K display, partly because that machine was conveniently located and partly as another end-to-end test to check out the latest build of Zerene Stacker on a high dpi Mac display with Apple's current O/S. Everything was going along just fine until I decided to press the little green "maximize" button so as to use the whole screen. To my surprise and horror, the main Zerene Stacker window suddenly became so large that the app's top-of-screen menu bar disappeared, along with the resizing buttons. Argghh! Hung! I could not figure any way to do a controlled exit. I finally figured out how to bring up a macOS Force Quit menu, which allowed me to kill Zerene Stacker and regain control of my computer. Clearly some horrendous bug had been introduced with JRE 10 (Java Runtime Environment), and I had to figure out how to work around it. But after some web searching to see if anybody else had reported this, I gradually realized that in fact the observed behavior was actually a feature, not a bug. Several years ago Apple changed their user interface spec so that the green button that used to mean "maximize" was now recommended to mean "full screen" by default. The JRE had finally gotten around to implementing that recommendation. It's completely normal for all Mac applications to lose their menu bars and resizing buttons when they go to full screen mode, but those can be easily retrieved by just hovering the mouse at the top of the screen for 1/2 second or so. That part is implemented by the JRE also, so in the end, everything was fine except that I had not known the proper gesture. I suppose I should be embarrassed to tell this story, but I'm not. The world is a very complicated place, and I only know a little bit of it. Lessons learned, reminders heeded.

All images shot with Canon T1i camera, Mitutoyo M Plan Apo 5X NA 0.14 objective with Canon 100 mm f/2.8 L IS USM macro lens used as tube lens to give 2.5X magnification on sensor. 40 micron focus step, stack lengths about 80 frames each. Synthetic stereo, +-3%. All images PMax, retouched from DMap to avoid starbursts around bright debris.

I hope you find some part of this interesting also!

--Rik
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Beatsy



Joined: 05 Jul 2013
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PostPosted: Wed Jul 25, 2018 12:09 am    Post subject: Reply with quote

Fascinating narrative and pics.

Interesting aspect #7: with my recently-learnt ability to see stereos properly, I found yours with shifts of +/- 3 much easier/quicker to "lock" than mine (and others) with +/- 4. Not sure, but fairly confident it's the shift rather than the subject, so I'll try that setting in future stereos. Ta.
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Smokedaddy



Joined: 07 Oct 2006
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PostPosted: Wed Jul 25, 2018 8:35 am    Post subject: Reply with quote

I thought the stereo images turned out very nice. Looks like a lot of work overall too.
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Roel Wijtmans



Joined: 23 Nov 2016
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PostPosted: Wed Jul 25, 2018 8:58 am    Post subject: Reply with quote

Not just some really beautiful photos, but also some very interesting information! Thanks for this post!
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rolsen



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PostPosted: Wed Jul 25, 2018 9:01 am    Post subject: Reply with quote

Very nice shots and detailed information.
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mawyatt



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PostPosted: Wed Jul 25, 2018 10:55 am    Post subject: Reply with quote

Rik,

Very nice images and great narrative!! Alway enjoy when folks take the time to describe what's actually going on, so hat's off to you sir Very Happy

What did you use for the polarization of the Ikea and the lens setup?

Best,
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Lou Jost



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PostPosted: Wed Jul 25, 2018 11:04 am    Post subject: Reply with quote

Fascinating story. I am surprised by the ending, though. I don't recall pupae going black just before the moth emerges. On the contrary I thought they normally become transparent. Did it in fact emerge successfully? I'm worried now!
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rjlittlefield
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PostPosted: Wed Jul 25, 2018 2:14 pm    Post subject: Reply with quote

Thanks for all the comments/questions!

Beatsy wrote:
I found yours with shifts of +/- 3 much easier/quicker to "lock" than mine (and others) with +/- 4.

Determining the optimum shift is often a matter for some experimentation. Too much shift and things get weird; too little shift and there's not enough depth.

See "What is a good amount of stereo separation?" for a variety of people's opinions.

There are formulas that relate the shift percentage and stack properties to viewing angle.

Working backward for this stack, assuming a sensor size of 14.9 mm across the subject, magnification of 2.5, and 87 frames at 0.040 mm spacing, I calculate off-axis angles of +-2.94 degrees, for a total viewing angle of just under 5.9 degrees.

It's probably no surprise that what I used is very close to the community consensus that 6 degrees is the largest value everybody liked. But I would be lying if I said I calculated it that way. In reality I just know from experience that +-3% often works out well for the stacks I usually shoot, so I usually start there, visually evaluate the result, and adjust accordingly. That approach might change if I had a calculator that was really convenient to use, but such a beast is still on the future-features list.

Smokedaddy wrote:
Looks like a lot of work overall too.

Yes, quite a few hours. It takes a certain amount of time to actually do the work, and then writing it up takes a lot more. But I'm usually in the game for knowledge more than pretty pictures, and if I don't take the time to document what I did, it's kind of like the other time got wasted because I'll soon forget.

mawyatt wrote:
What did you use for the polarization of the Ikea and the lens setup?

I used a couple of 52mm lens-filter type polarizers, one just behind the objective and one fastened in front of the Ikea with a bit of gaffer's tape. These were circular polarizers, so I took care that both of the polarizers were positioned with the "outside" linear polarizing surface facing the subject.

Here is the full setup, with the Ikea turned off, the room lights turned on, and a black machine screw standing in for the pupa. (The polarizer on the Ikea has been removed from its rotating housing with filter threads.)



Note that it's a vertical setup with a horizontal positioning stack consisting of linear rails for XY shift, plus one goniometer and one rotary stage for adjusting tilt with respect to the lens. It's hard to tell in this photo, but I have a steel and balsa "shelf" that fastens to the rotary stage with magnets. Normally I use a vertical setup as shown here, because it's a better match to my subjects. But everything is fastened together with Arca-Swiss parts, so when I want to shoot horizontal, that's just a matter of swapping the focus rail and the subject positioning stack, and typically moving that column off to the side so that it's not in any of the sight lines.

Lou Jost wrote:
I don't recall pupae going black just before the moth emerges. On the contrary I thought they normally become transparent.

My feeling is that these brown naked "cutworm" pupae don't change transparency very much, but as the moth develops, its increasingly well defined features become more visible through the same shell. In this case I was not surprised to see that the adult moth was quite dark.

Quote:
Did it in fact emerge successfully? I'm worried now!

No worries. The moth emerged just before bedtime, a couple of hours after my post. I went to bed before it was expanded, but in the morning it was fully expanded and inclined to fly at the slightest provocation.

Here's a quick snapshot of the moth and the pupal shell, fresh from the refrigerator where I'm keeping the moth placid. It's a very handsome beast, well worth a stacked portrait, but I'm not quite sure yet how to do that, given how quickly it gets agitated when I shine a light on it.



--Rik
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Lou Jost



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PostPosted: Wed Jul 25, 2018 2:19 pm    Post subject: Reply with quote

That's good news!
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rjlittlefield
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PostPosted: Wed Jul 25, 2018 3:05 pm    Post subject: Reply with quote

Yes, it would have been a shame to not be able to ID the beast.

But now that it's out, I'm pretty confident identifying this critter as Mamestra configurata, for example as described at Pacific Northwest Moths, Moth Photographers Group, and BugGuide.

This species is also known as the "Bertha armyworm", if that gives you any ideas. The Pacific Northwest Moths site describes its habits like this:
Quote:
Habitat
This species is widely distributed in moist open habitats throughout much of western North America. It is usually very rare and sporadic in more natural habitats, but often builds into epidemic outbreaks in agricultural crops and other disturbed habitats at low elevations. In the Pacific Northwest, it is a major pest in crops of peppermint and alfalfa both east and west of the Cascades.

My specimen came home as a late instar larva, apparently in a bundle of beets purchased at the local farmers' market. When the beet leaves started wilting too much, I tried switching it to broccoli, on which it fed eagerly. The caterpillar was attractive also, but I did not have enough time and interest to photograph that.

--Rik
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BugEZ



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PostPosted: Thu Jul 26, 2018 8:53 pm    Post subject: Reply with quote

A very interesting post, thanks for sharing!

Rik wrote
Quote:
l’m very interested to note how the basic geometry of the wing scale patterns are laid down early...


This is similar to the eye patterns on some flies. The biologists that study this talk about the progression of the” morphogenetic furrow” across the stem cell membranes in the pupa that determine the eye patterning. As the morphogenetic furrow progresses, one cell identifies what type of ommaridium will develop and the rest of the eye cells (7-8 sensor cells lens cells, corneal filters, lenses, downstream nerves etc) are regulated by the initial fate selection. Perhaps this is similar in moth and butterfly wings? The scientists at NYU were quite interested in the alternating stripes in some Dolichopodidae ommatidia. Were they easier to rear, no doubt they would have done experiments, but rearing difficulties made detailed study impractical.

Anyway, the pattern is laid down and then the embellishments are added.

Keith
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rjlittlefield
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PostPosted: Thu Jul 26, 2018 9:24 pm    Post subject: Reply with quote

OK, here's a better shot of the adult:



The above is not a stack. It was supposed to be, but a couple of things went bad in the process.

As I mentioned earlier, this moth is inclined to get agitated when exposed to light. So I kept the critter in the refrigerator for a few hours to chill out, while I prepared my stacking setup. I had everything set up perfect and ready to go: framing, exposure and aperture, step size, number of frames, all giving an excellent result on the dead moth I tested it with.

I took away the dead test moth and replaced it with this live one, freshly removed from the refrigerator. It ruffled around while being transferred, then settled down, completely motionless, while I shot the stack.

At least, I thought it was completely motionless. That's the way it looked in real time as I shot the stack.

But when I later processed the stack, I learned that in fact the critter had been slowly and continuously adjusting its position, with the result that the stacked images looked, well, a little degraded.

Here's the PMax. The DMap wasn't quite this bad, but still nowhere acceptable.



Actually, it's a bit of miracle that the stack ended up looking even this good. At least it's complete. What was supposed to have been 30 frames, with some spare in the background, turned out to be 25 frames with none at all to spare. That's because frames 26 and 27 showed only blank gray board, after the moth suddenly sprang to life, scampered to the edge of the board, and swung itself over the edge where it was darker. I decided that was a good time to stop. Brick wall

The moth has earned its reward. It's been released to go play outside now.

--Rik
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rjlittlefield
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PostPosted: Thu Jul 26, 2018 9:38 pm    Post subject: Reply with quote

Keith, thanks for the information about fly eyes.

The general theme of laying down a gross pattern and then embellishing it, seems to be universal. Some of the most easily seen examples that I know are with insect pupae where the appendages remain distinctly separated from the body, rather than all fusing into a mostly smooth surface as in this moth pupa. There's a good example at Metamorphosis: pupation of the wasp Polistes diminula. I'm watching a couple of click beetle pupae go through the same process right now.

--Rik
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