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Beneficial nematodes -- Heterorhabditis bacteriophora

 
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rjlittlefield
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Joined: 01 Aug 2006
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Location: Richland, Washington State, USA

PostPosted: Mon Apr 16, 2018 12:33 am    Post subject: Beneficial nematodes -- Heterorhabditis bacteriophora Reply with quote

"Say what?! Nematodes don't have prolegs -- do they??"

OK, it was a momentary reaction, but that's actually what I thought for a moment when I saw this structure:



The background here is that my strawberry patch is infested with strawberry root weevils, which I'm attempting to treat by applying a spray of "beneficial nematodes".

More precisely, these are Heterorhabditis bacteriophora, which BioLogic Company (https://biologicco.com/) apparently cultures in enormous quantities and ships out to people like myself in little envelopes containing a fluffy white powder described as follows:
Quote:

ACTIVE INGREDIENTS
Heterorhabditis bacteriophora............35.4%
INACTIVE INGREDIENTS:
Water.............................................14.6%
Diatomaceous earth (OMRI listed)......49.9%
Potassium sorbate (GRAS)..................0.1%
TOTAL..........................................100.0%


The instructions are to disperse the contents in water, spray the affected area, and water thoroughly. Then over the next few weeks the nematodes are supposed to parasitize the weevil grubs and kill them.

Of course I could not stand to do this without taking a closer look.

So, midway through spraying, I diverted the stream into a small plastic container and captured 100 ml or so. After a quick look under stereo scope to confirm that it contained little wigglies, I let the liquid settle overnight, then put a drop of the thick stuff from the bottom onto a slide.

With 10X objective, this is what I saw:



At the scale shown here, there's nothing particularly interesting.

But at full resolution through the eyepieces, even with 10X objective and 10X eyepiece there was some interesting detail that immediately attracted my attention: the critters seemed to have a multitude of little projections that reminded me ever so much of prolegs on a caterpillar.

Here's a crop of the image above:



Cranking up the magnification to a 40X objective produced the image I showed first:



Now, the odd thing was that while most of the nematodes showed the ripple pattern wherever they bent very much, there were also a few individuals who never did. Those also tended to be a bit smaller:



Of course I started searching for more information. It did not take long, mostly with Google image search, to find a plausible explanation.

According to THIS SOURCE, the infective stage of Heterorhabditis bacteriophora is actually a third-stage juvenile, described more fully like this:
Quote:
3. What is an infective juvenile?
A third-stage juvenile of an entomopathogenic nematode is called as an infective juvenile because it initiates the infection in its host. Infective juvenile is the only non-feeding and free-living stage found in the soil but all other stages including fourth and fifth (adult) and egg stages are completed inside the host.

4. What is a dauer juvenile?
The infective juveniles are actually third-stage juvenile that also called as dauer juveniles because they are enclosed in a second-stage cuticle, which arrests their further development (Fig.3; adopted from http://www.nematodeinformation.com) and helps to survive outside the host i.e. in the soil environment. Furthermore, these developmentally arrested dauer juveniles are physiologically adapted to remain in the environment (i.e. soil) without feeding until a perspective host is located. These dauer juveniles recover and resume their development only when they enter the perspective insect host’s body cavity via natural openings and shed their second stage cuticle. The dauer juveniles are also well known to tolerate harsh environmental conditions including extreme hot and cold temperatures, and desiccation (Jagdale and Gordon, 1997; Jagdale and Grewal, 2003; 2007; Jagdale et a., 2005).

Matching against their picture, I'm reasonably sure that most of what I'm looking at are dauer juveniles, with the ripples due to that retained second stage cuticle. And then I'm guessing that the slightly smaller individuals that never develop the ripples are just one stage earlier, probably about ready to become dauers, but still with just a single cuticle.

Interesting!

Other interesting snippets...

1. It's not actually the nematodes that kill the grubs. No, the nematodes just deliver a dose of specialized bacteria that live symbiotically with the nematodes. It's the bacteria that kill the grubs, and the nematodes make their living by feeding on the nutritious goo that's left behind.

2. The recommended application rate of these things is not as high as I imagined. BioLogic quotes a rate of 10 million per 400 square feet, which works about to be about 1 nematode for every 4 square mm of ground surface. Bugsforgrowers.com says "the optimal rate of 1 billion infective juvenile nematodes in 100 to 260 gallons of water per acre is generally recommended". One acre is 4.047 billion square mm, so their number ends up being almost the same. Given that these things have to disperse through several inches of soil column, I'm pretty surprised that such a low dose rate is considered effective.

3. This species of nematode is an active hunter, moving through the soil with guidance from chemical cues. Some other species are ambush predators, basically just waiting in place for some insect to come ingest them or at least get close enough to be pounced on.

4. This species is fully hermaphroditic. Only one individual needs to infect a victim, to end up essentially mating with itself to produce the next generation. Other species require simultaneous infection by male and female nematodes.

I hope you find this as interesting as I did!

--Rik

Photo details: shot with Canon T1i camera (APS-C sensor size) using 10X and 40X objectives, direct projection through a 1.4X teleconverter for net 14X and 56X on sensor. Auto exposure times at ISO 800 are showing as 1/40 second for 56X and 1/1300 second for 14X.
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micro_pix



Joined: 11 May 2012
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PostPosted: Mon Apr 16, 2018 6:16 am    Post subject: Reply with quote

Very interesting Rik.



I did post an image a while ago of a L3 stage larvae of a “redworm” species that infects deer.

While it has a very different effect on the host than the nematode in your photos, it also has the protective sheath at the L3 stage to allow it to get to the right place in the host. I understand that the protective sheath is something that helps distinguish parasitic nematodes from free living nematodes.

http://www.photomacrography.net/forum/viewtopic.php?p=218982&highlight=#218982

David
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Beatsy



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PostPosted: Mon Apr 16, 2018 7:52 am    Post subject: Reply with quote

Superb illustration of how the microscope, plus a little curiosity and effort, can make many things far more interesting. Thoroughly enjoyed the write up. Thanks Rik.
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Jacek



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PostPosted: Mon Apr 16, 2018 8:03 am    Post subject: Reply with quote

Very interesting. Good job.
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Roel Wijtmans



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PostPosted: Mon Apr 16, 2018 10:08 am    Post subject: Reply with quote

Interesting! Thanks for the write-up.
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rjlittlefield
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PostPosted: Wed Apr 18, 2018 10:17 pm    Post subject: Reply with quote

Thanks for the feedback, everybody!

micro_pix, thanks especially for the link to that posting of yours. In my brief reading, I got the same impression as you, that the protective sheath is a distinguishing feature.

Perhaps I would have been quicker to recognize the sheath in mine, except that in mine, the wrinkling was so smooth and regular that it gives a completely different impression from a flexible sheath with a liquid layer underneath it.

Did you see the deer redworms live and wiggling? If so, what did their sheaths look like in motion?

--Rik
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vasselle



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PostPosted: Thu Apr 19, 2018 3:26 pm    Post subject: Reply with quote

Très bon travail
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Walter Piorkowski



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PostPosted: Sat Apr 21, 2018 12:13 pm    Post subject: Reply with quote

Rik. Liked the sharp images and the information provided. Walt
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micro_pix



Joined: 11 May 2012
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PostPosted: Sat Apr 21, 2018 11:48 pm    Post subject: Reply with quote

rjlittlefield wrote:
Thanks for the feedback, everybody!

Did you see the deer redworms live and wiggling? If so, what did their sheaths look like in motion?

--Rik


Hi Rick,

I did watch them in motion, it was only really when seeing the head and tail occasionally retracting a little from the outer sheath that I realised the whole larvae was encased and that it was an L3 stage larvae. In future I’ll look for that “concertina” effect on the inside of the bends.

David
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Sumguy01



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PostPosted: Sun Apr 22, 2018 10:37 am    Post subject: Reply with quote

Very Happy Very interesting.
Thanks for sharing.
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