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Today in Cricket Hemolymph News

I'm at one of those frustrating stages with a project. The short-term goal is to quantify the total volume of cricket hemolymph across the circadian cycle (light-dark cycle) in conjunction with quantifying the concentration of protein, carbohydrate, and lipid in the hemolymph. This work is largely just backdrop for a larger project to characterize protein, carbohydrate, and lipid metabolism across the circadian cycle - an ambitious, but impactful, project.

Anyway. In lieu of using radiolabeled tracer compounds, we're using stable isotopes for this project because we currently don't have institutional approvals set up for radioisotope work. I suspect that, in the long term, radioisotopes will be preferable for a whole host of reasons, but for now, it is what it is. One potential issue is that due to logistical limitations we could be using amounts of the stable isotopes that are well above "trace" amounts, which means they could have effects on cricket metabolism independent of anything that might be going on across the circadian cycle. So we need to have good baseline information about what's present in the hemolymph relative to what we're injecting into the crickets.

The standard method for estimating the total volume of the hemolymph in an animal goes something like this: inject some sort of compound into the animal that is biologically inert but that can be tracked and quantified in some fashion. Wait for some period of time for the compound to diffuse throughout the animal, then sample a small amount of hemolymph and assay it for the amount of compound. The degree of dilution in the assay sample can be used to determine the total volume of hemolymph. In my case, I'm using fluorescently-labeled inulin to do the job. We have this awesome fancy microplate reader that can detect trace amounts of fluorescence in samples.

This procedure raises a question: how long is "some period of time"? Clearly it will be different for an elephant vs. a shrew. I suppose I could simply cite an older paper that used a 15-minute time period, but overall it's better to directly validate things. So I spent some quality time with just four crickets today. I also needed to test out a method to inject them in a place that won't leak as much as the abdomen. This method basically involves trapping their feet in modeling clay, lifting their wing covers, applying a dab of vacuum grease to the base of the wing, and then injecting through the vacuum grease into the thorax.

Then I tried to do as many repeated samples of their hemolymph as I could manage, which was the really hard part. I just don't like having to disfigure crickets. I started out by snipping off the ends of the cerci, but in several cases the snipped ends didn't produce a sufficient amount of hemolymph. So then I had to do other things, like snipping off part of a leg or jabbing the cricket with a fat needle. This is also kind of hard, because it means that I'm draining the cricket of the substance I'm trying to measure. Plus it was really time-consuming for each individual cricket, and the crickets really don't like having their feet stuck in the modeling clay. It's abusive to the point where they can easily lose a leg or two. Ugh.

I got through four crickets today, and actually managed to get pretty good-looking timecourse data for those four. I should try and do 2-4 more on Monday. And then I hope I never have to do this again.



( 1 remark — Remark )
May. 8th, 2016 09:02 pm (UTC)
; )
Happy Mother Earth Day!

MoM turned upside down is WoW ! feeling..

have a lovely day bella!
; )
( 1 remark — Remark )

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