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Cherry and friend (fruit fly -- Rhagoletis indifferens)
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rjlittlefield
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Joined: 01 Aug 2006
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Location: Richland, Washington State, USA

PostPosted: Fri May 29, 2015 12:21 am    Post subject: Cherry and friend (fruit fly -- Rhagoletis indifferens) Reply with quote



This is, of course, a cherry. But as you've probably already guessed, I'm not very interested in the cherry.

No, this time I'm interested in that small lesion on the side of the cherry, about half way up the left side.

That's actually a hole, extending from the outside of the cherry clear into a void that has formed next to the pit.

It's the access portal, if you will, into the world of the Western Cherry Fruit Fly, Rhagoletis indifferens.

You might want to stop at this point and review my 2008 posting about this beast. There, I looked closely at the ovipositor of the adult.

This time, it's the larvae that have captured my attention.

More precisely, the front end of the larvae, the mouthparts mostly. What prompted my interest is that while sorting and cleaning some cherries, one of the maggots happened to get on my finger and make a run for safety. The sensation was quite strange: "Say what?! Maggots are scratchy??"

On closer inspection, I figured out what was going on.

Turns out, fruit fly maggots have fangs! Well, officially they're called "mouth hooks", but they could just as well be fangs.

Here, see what I mean:



As far as I can tell, these mouth hooks are multifunction tools, good both for locomotion and for shredding the flesh of the cherry.

When the larvae want to move fast, they stop squirming and start lunging, repeatedly thrusting the head forward, sinking the mouth hooks into whatever's handy, then pulling their head back to pull the rest of the body forward. It's very effective.

I wanted to shoot a movie of the action, but that would have required far more skill than I have. So instead I decided to "make do" by just shooting a bunch of stills.

Here's a montage of the few acceptable shots I got. You can tell from the image numbers that there were a lot of duds too.



As usual, I find that the structure of the beast is more richly conveyed in stereo. This is crossed eye, and I've tried to avoid window violations for those of you who are sensitive to that.



The fan-shaped structure at the top of the image is one of the two anterior spiracles. They feed large tracheae that run back along the body of the larva, with smaller tracheae branching off those to provide oxygen to the rest of the body. You can see many of these under the skin, in the nearly transparent outer portions of the body.

Here's one more stereo, a different specimen showing the same structures. This one gives a little better view of how deep inside the head the sclerotized portions extend.



The high mag images are shot with Mitutoyo 10X on Raynox 150 tube lens, Canon T1i camera, illumination with diffused Jansjo LED lamps, mostly directed at a piece of white card under the specimen, with a small black card held in place as a partial darkfield stop. Vertical setup, shot through a thin layer of water with the specimen stuck into a drop of sticky water-soluble transparent "personal lubricant". I'll post pictures of the setup separately (HERE).

I hope you find this interesting!

--Rik

Edit: add link to pictures of the setup


Last edited by rjlittlefield on Sun Aug 02, 2015 8:56 am; edited 1 time in total
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BugEZ



Joined: 26 Mar 2011
Posts: 592
Location: Loves Park Illinois

PostPosted: Fri May 29, 2015 4:44 pm    Post subject: Reply with quote

Very Nice Rik! I look forward to the setup photos. I presume vertical?

I am anxious to photograph some fly larvae and you have set the bar quite high...

edit...Oh, I see vertical...

Keith Short
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RobertOToole



Joined: 17 Jan 2013
Posts: 155
Location: United States

PostPosted: Fri May 29, 2015 11:32 pm    Post subject: Reply with quote

Excellent topic and images Rik.

Very interesting details.

It reminds me a little of some kind of mutated walrus from hell. Very Happy

Nice to see your Mitutoyo 10X and Raynox combo working well.

Robert
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Carmen



Joined: 10 Feb 2015
Posts: 273
Location: Buenos Aires

PostPosted: Sat May 30, 2015 8:06 pm    Post subject: Reply with quote

Thank you Rik for the interesting foto essay. The bug resembles our local fat walrus (I think that's the correct english) because of its fat body and tusk like fangs.

Question please: what treatment was given to the specimen? If a stacked photo, then I imagine it must have been dead, yet it looks alive!
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rjlittlefield
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PostPosted: Sat May 30, 2015 8:50 pm    Post subject: Reply with quote

Carmen wrote:
Question please: what treatment was given to the specimen? If a stacked photo, then I imagine it must have been dead, yet it looks alive!

Yes, well, um . . . that's an excellent question. Does somebody have a simpler one? Rolling Eyes

Perhaps the best explanation is if I just quote from some email that I wrote to a colleague a few days ago...

Quote:
Subject: How can I kill a cherry maggot and still have it look good??

I have a goal that so far I've failed miserably to achieve. I'm hoping that you can give me some guidance about how to proceed.

The goal seems simple enough: I just want to kill a cherry maggot and have it retain a lifelike appearance.

By "lifelike appearance", I mean in an extended posture with mouth hooks and anterior spiracles sticking out, and with body tissues retaining their normal transparency.

So far, nothing I've tried has worked.

1. Drowning. After full submersion in water for several days, they still wiggle. Not much, just enough to mess up a stack.
2. Heating. Heated to about 140 degrees, they die, but the tissues go milky like a cooked egg yoke.
3. Freezing. They ball up into something about the shape of a puparium. When thawed and left to soak, they don't expand.
4. Isopropyl alcohol, in various concentrations. Either they shrug it off or (in high concentration) the tissues go milky.
5. Acetone, similar.
6. Soaking in a spinetoram solution. Dow "Delegate" to be precise. They still wiggle. Not much, etc.
7. Ethyl acetate, assorted failures.

I'm thinking of gearing up to drop them live and wiggling into acetone and dry ice. Never have played with that combo, might be an interesting experience.

What else would you suggest?

--Rik


Alas, I did not have the proper materials to follow any of my colleague's suggestions. But I did finally luck into an acceptable alternative...

Quote:

Thanks for the suggestions. Chlorotone sounds interesting. Can you suggest a place that I might buy some?

I was finally able to get specimens prepared, as shown at http://www.photomacrography.net/forum/viewtopic.php?t=27702 , by the simple(?) expedient of trying to drown them for a couple of days, then finishing the dispatch by blotting them dry on a piece of Kleenex tissue and finally treating them to ethyl acetate by dropping it onto the tissue. The attempted drowning of active maggots left quite a few of them with mouth parts suitably visible.

Adding to the list of things that failed, by the way, was continued submersion in a saturated salt solution at room temperature. Those larvae are a lot tougher than I would have guessed!

--Rik

Today I tried one more of the possibilities, thinning some 7.5% benzocaine teething gel about 1:2 with water, and letting a few maggots bathe in that solution for an hour or so. At the end of that time I just gave up and photographed a critter live and wiggling. Repeat refrain: those larvae are a lot tougher than I would have guessed!

The one bright side of all this is that these maggots, unlike the adult flies, don't seem to elicit much sympathy from the anti-cruelty folks.

My defense in any case is that I am bound by local law to kill these things, using any non-prohibited methods and materials. The ones who got photographed at least got immortalized.

--Rik
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rjlittlefield
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PostPosted: Sun May 31, 2015 12:29 am    Post subject: Reply with quote

I had another specimen mounted up tonight for other purposes, but it looked so good I couldn't resist pushing the envelope a little farther.

This stack is shot with the Mitutoyo 20X NA 0.42 , 166 frames at 3 microns focus step, rendered in stereo at +-6%. Canon T1i, ISO 100, 0.1 second exposure, 6.5 seconds per frame including 5 seconds settling time. Shot as RAW+JPEG with RAW recording only to the memory card for faster operation. Developed in Lightroom from the RAW, with sharpening and noise reduction to give the best-looking individual frames.

Stereo output rendered with pre-sizing to 50%, shifts at +-6%, saved images then aggressively sharpened with Photoshop USM at 1 pixel radius, 100-200% depending on taste, so as to give the best stereo lockup (for me).

All are crossed-eye.

This first one is almost the whole frame.



These others are tighter crops of the same output pair.







--Rik
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NikonUser



Joined: 04 Sep 2008
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Location: southern New Brunswick, Canada

PostPosted: Sun May 31, 2015 6:31 am    Post subject: Reply with quote

As in Rik's fly maggot the maggots of the house flies and blow flies have similar hooked-mouthparts.

Here are a couple of shots from a young maggot of one of theses flies. Dissected out to show the entire structure known as a "cephalopharyngeal skeleton", all the bits have names including mandibles (mouth hooks).

http://www.photomacrography.net/forum/viewtopic.php?t=14675&highlight=nu11117
_________________
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Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

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rjlittlefield
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PostPosted: Sun May 31, 2015 11:32 am    Post subject: Reply with quote

Tony, thanks for the link -- I had forgotten that one.

--Rik
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Carmen



Joined: 10 Feb 2015
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Location: Buenos Aires

PostPosted: Sun May 31, 2015 6:24 pm    Post subject: Reply with quote

thank you tony for the interesting link, impressive work once again!

Rik, I still don't understand how you acheive the life-like appearance using the focus stacking technique. I infer that the specimen must be nearly dead, am I close? or perhaps very intoxicated? In any event, it must remain inmovilized.

I naïvely supposed that the cold freezer temperature would preserve bugs. But I noticed that after a few weeks in the freezer, they become dry and dead looking, colors fade, etc. I consider that the healthy anímate appearance has more visual impact than a desiccated appearance; thus the motivation of my inquiry.
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Walter Piorkowski



Joined: 14 Aug 2006
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PostPosted: Sun May 31, 2015 7:04 pm    Post subject: Reply with quote

Rik. Remarkable effort and images. You don't seem to need a microscope.
Walt
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rjlittlefield
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PostPosted: Sun May 31, 2015 8:45 pm    Post subject: Reply with quote

Carmen, sorry, I did not mean to be unclear. Yes, the subjects were dead or very close to it. For the 10X stacks, the subjects were submerged in water for a couple of days, then blotted dry, treated with ethyl acetate fumes for several minutes, and then put back in water until they could be photographed. There was no opportunity for them to dry out to any significant extent, hence the lifelike appearance. For the 20X stack, I did not leave the subject in water for so long, because I wanted the tracheae to remain filled with air as much as possible. I treated it with submersion in a benzocaine solution for close to an hour, which still left it quite wiggly, so I gave up and used it anyway for the two pictures shown at http://www.photomacrography.net/forum/viewtopic.php?p=171003#171003. But then several hours later I found that it had stopped moving, so I used the opportunity to shoot the 20X stack.

The problem with your bugs in the freezer is what home cooks call "freezer burn" when it occurs to frozen meat. Even at freezer temperature, ice still sublimates quite a bit, and unfortunately the molecules are happier to migrate and form pure water crystals on the inside of the container than to stay distributed throughout the specimen. With large chunks of meat, the problem can be avoided by packaging in coated paper or plastic that fits so tightly around the meat there is no place for ice crystals to accumulate. But that won't work with something like a fly, and I have no idea how to prevent dessication with those.

Walter, thanks for the compliment. There's some question what to call setups like this. Here in the forums at photomacrography.net, the images clearly go into the Macro category because they're shot outside the frame of a conventional microscope and thus can have more flexibility for illumination. But if I were submitting to Nikon Small World, I would have no hesitation about describing the setup as a "home-made microscope".

--Rik
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lothman



Joined: 14 Feb 2009
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PostPosted: Mon Jun 08, 2015 6:11 am    Post subject: Reply with quote

Hi Rik,

these stereo pairs of the translucent body comes out excellent. Did you have any problems by detecting the sharp areas?

Regards
Lothar
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rjlittlefield
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PostPosted: Mon Jun 08, 2015 8:48 am    Post subject: Reply with quote

Lothar, thanks. I was pleased by how these came out. The transparency of the body tissues makes at least some of the internal structures quite visible, especially the tracheal tubes and the sclerotized mouthparts.

No problems with these except for specimen prep and positioning, as described above and in the setup description. For transparent structures like these PMax is the only realistic option. It doesn't have any controls so there's no tuning to be done. It's also relentless about detecting whatever is sharp. That includes pixel noise, so I shoot at lowest ISO and apply noise reduction during development in Lightroom. Then after the stereo pairs are generated, I also apply aggressive sharpening because for me that makes the stereo lock up better and brings out the detail that was the whole point in the first place.

--Rik
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NikonUser



Joined: 04 Sep 2008
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PostPosted: Tue Jun 09, 2015 1:12 pm    Post subject: Reply with quote

rjlittlefield wrote:

Today I tried one more of the possibilities, thinning some 7.5% benzocaine teething gel about 1:2 with water, and letting a few maggots bathe in that solution for an hour or so. At the end of that time I just gave up and photographed a critter live and wiggling. Repeat refrain: those larvae are a lot tougher than I would have guessed!
--Rik


I reckon that just annoyed them.

I just bought some "Anbesol", it's 20% benzocaine
_________________
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives
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esotericman



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PostPosted: Tue Jun 09, 2015 2:28 pm    Post subject: Reply with quote

Rik,

I very much appreciate you taking the time to explain this. The anterior spiracles are great.

Christian
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