rebeccmeister: (cricket)
Figuring out how to count cricket growth rings has been an interesting endeavor. Friday morning, after I'd posted about my quandary, I brainstormed with [personal profile] slydevil and [personal profile] sytharin about further methods for cutting thin sections of cricket legs. I mentioned the slice of potato method, which really didn't work, and L came up with the idea of trying some other sort of vegetable that is firmer than a potato, like a radish, perhaps.

Armed with this notion, I headed in to work. After some further failures with the potato (I didn't happen to have radishes sitting around at work), I decided to try cutting thin slices without any extra support, but working under the dissecting scope instead.

And, success. The scope made it much easier to position the razor blade at just the right spot to get multiple beautiful sections. I know it worked well because I was then able to compare growth rings for Day 0 adult crickets (hint: zero growth rings) versus Day 6 adults. Here's a photo of the Day 6 adult:

Gryllus lineaticeps growth rings

I also did a bit more reading about methods for preserving stuff on microscope slides. Apparently I'm not the only one who has wondered about this. Our lab storeroom had little bottles of clear nail polish for sale next to the various flavors of slides and coverslips, so I figured I'd give that a try at first.

As of today, it looks like it works well! This is really good because it means I can work in batches and don't have to do each cricket one at a time. So now the rest will be fairly routine: prepare and count rings for crickets of known adult ages to make a calibration curve, then prepare and count rings from the field crickets.

I'm really enjoying the chance to play with the compound microscope we're borrowing from another lab.
rebeccmeister: (cricket)
We have five nights remaining here, so I've been switching things into high gear, to the degree possible. That means two circadian experiments a day, one at noon, and one at about midnight, with whatever crickets we can catch. Even if we can't fill out our long-winged sample sizes, we can at least get information about the short-winged crickets, to compare with data from the lab. We've also managed to pinpoint one location that seems to have a slightly higher proportion of long-winged crickets, so I was able to run 3 long-winged females last night. L is also starting to have slightly better success with her pitfall traps: she managed to get another long-winged female this morning. Progress! We actually have a bare minimum sample size complete for the first tracer!

The blacklight hasn't really attracted much in the way of crickets, but there have been a lot of interesting and beautiful moths.

The exhaustion is cumulative. I am struggling to verbalize late at night, when I'm tired. I'm screwing up numbers. At least I'm catching myself...I think. Sample schedule: wake up, drink coffee, check the last-instar crickets for new adults. By 10 am, start prepping for the noon timepoint, which runs from 10:55-1:30. Eat lunch. Collapse in a puddle, or go to town for groceries. Prep for the evening timepoint. Eat dinner early, so we can get out to the cricket hunting grounds by dusk. Hunt crickets until 10 pm. Bring crickets back, run more experiments, wrap up by 1 am, try to unwind for 20 minutes, try to sleep as much as possible. Repeat.

I'm adding tons of photos to the album. I want to run around more at night, to try and get pictures of the tarantulas out here (big, black, hairy, fast!). There are also some amazingly huge wolf spiders with abdomens the size of a quarter, that hang out in the same cracks where the crickets hide. They are so cool, but also shy, so it's hard to get pictures of them. We've gotten to watch the wolf spiders and the deer mice eat the crickets. There are lots of toads out, too. The nights are busy out there.
rebeccmeister: (cricket)
Projects with the big crew:

Monday night: thorough coverage of our mark-recapture areas. The weather stayed reasonably warm into the night, so teams were able to gather up a lot of crickets. I stayed behind to run a set of 9 pm crickets.

On Tuesday, we concluded that one of the paint types we'd been using for mark-recapture was not good for the crickets. Crickets painted with Testor's enamel paint were noticeably more sluggish than unpainted crickets or crickets painted with the acrylic paint we'd been using in the lab up until then. We also concluded that we were reaching a point where we weren't learning a whole lot more from repeatedly re-surveying our mark-recapture area, so we decided to switch gears for our evening plans. Oh, we also went to the beach in Santa Barbara, which was a much nicer trip than our trip to Pismo Beach last year. Not only was the beach less crowded, almost everyone actually went in the water. I should have gone for a fuller swim, but oh well.

So then, Tuesday evening, we formed four small teams and headed out in four different directions to get a better sense of the broader population structure out here. I also wanted to encourage everyone to help me collect up as many long-winged crickets as possible, because they're much more rare than the short-winged crickets and are a huge limiting factor for the circadian experiments. In order to give everyone extra incentive, we decided there needed to be a prize for the team that collected the most long-winged crickets. Casting about for ideas, I settled on a prize of a cake of that team's choosing.

It worked! Mk and I wound up hiking up a road in a valley along the southwestern part of the reserve. C and P were supposed to hike along the corresponding eastern edge, but when they reached the gate for that road, C observed two sets of predator eyes shining back at her: either coyotes or mountain lions. So they wisely stuck closer to home. B and CH headed north, and L, G, and Ms headed south.

The cake bribe worked. C and P won, and we came in second, but I still feel like a winner given that I wound up with 6 long-winged females with pink flight muscle. We set them up for a noontime circadian experiment.

As is typical for field experiments, we're making a ton of decisions on-the-fly out here, so part of the reason for trying to thoroughly blog about everything is to try and remember why those decisions were made (and also try to retain shreds of sanity because there is major Thought Tragedy of the Commons* out here).

After the noontime data collection, I became concerned that catching crickets, holding them overnight and through the next day, and then running them, was affecting their metabolic rates. The noontime crickets had higher respiration rates that are comparable to the respiration rates I've observed so far with laboratory crickets, whereas the 9 pm crickets tended to have about half to two-thirds the respiration. We aren't really aiming to study metabolism under starvation conditions, so that was a problem.

Thus, last night, we changed things up again. At 9 pm, teams set off to the two locations out of the four that had been the most fruitful on Tuesday night. Larger groups seemed prudent after the creepy nighttime predator eyes (and a note: nighttime fieldwork is a whole different ballgame than daytime fieldwork!!). I stayed back at the ranch house to prep supplies for another circadian experiment. At 10 pm, teams returned with their haul up to that point, and I wound up setting up 1 long-winged female, 6 short-winged females, and 6 long-winged males for another run starting at 10:24 pm.

At noon, I trained Mk how to help me run the experiment, so she also helped me with the evening timepoint and we turned that crank as best we could. [Interesting tidbit: a large proportion of the crickets have some sort of mite hanging out under their wing covers. I need to photograph them.] We wrapped up by around 1 am. Teams went back out at 10 pm for a second search shift, but temperatures dropped substantially last night, so the crickets weren't all that active anymore.

It's tough out here, when all our best efforts just can't quite net the numbers we need for this kind of experiment. I sort of expected that, and figure we're learning a TON out here anyway, so I'm still optimistic we'll be able to get some good papers for our efforts, even if they aren't quite what we set out to do. We shall see.

I am still feeling grumbly about that rejected manuscript, although this morning as I revisit the comments I'm coming to terms with it all. Gotta get up, dust off my fragile, bruised ego, and keep going.

*Thought Tragedy of the Commons is [personal profile] scrottie's term for what happens when one person tries to hold onto their thoughts about what they're going to be doing next, by saying that thing out loud. When they do, they disrupt the peaceful thoughts of the other people around them, who often respond in turn by voicing all their own thoughts out loud. The net effect can be complete disintegration of one's internal monologue and will for doing things, especially if one is a rather sensitive introvert.
rebeccmeister: (cricket)
Yesterday was warm, so it seemed like there were more crickets out and about during our evening surveys.

I was going to run circadian crickets last night, but in the evening B came into the house with a worried look on his face. When we went out to the table on the porch, the deli cups that had held the crickets for the experiment had been knocked over, and a lid was missing. Out of the four crickets I'd set aside, the long-winged one was the only one that was totally gone.

Later in the evening, when I went out to put out crickets from the day's haul, I startled a rat, who raced off the table into a big, open grating at the edge of the porch.

We are now keeping the pint cups underneath a larger bin, with a rock on top, and I'm now being more careful to seal the lids closed.

Other wildlife: lots of very large wolf spiders (abdomen the size of a dollar coin), a handful of tiny mice (one of which was spotted eating a cricket), lots of toads and tree frogs, rattlesnakes, and an alligator lizard.

It looks like the Whittier wildfire is drawing to a close, to judge by the decrease in smoke and bright spots that we can see at night.
rebeccmeister: (cricket)
After Friday's long hike (losing track of days of the week already, here...), it seemed prudent to make Saturday a low-key day. Besides, our conclusion from the hike was that we're stationed in the part of the reserve that is the most crickety, so we don't really need to go too far.

So after morning pitfall trap checks and miscellanea, it was time for another trip into town. On our first trip, L pointed out a sign for U-Pick berries right as we crossed highway 154 to take Edison Street into town. We had enough to do that time that we didn't have the capacity to stop. This time, however, we did.

And now I'm having some strongly conflicting emotions. On the one hand, I'm utterly ecstatic to get to eat fresh raspberries and apricots and blackberries, and to buy farm-direct vegetables. YAY! On the other hand, it's tremendously difficult to quell the urge to Pick All The Berries and Jam All The Things. This has bumped Sedgwick from, "Ehh, this field site is fine" to "OMG OMG I LOVE THIS PLACE." The woman running sales said that with the highway closures for the fire, traffic has been dead and she hasn't had customers, but it isn't a big deal because she has a lot of other things to do anyway.

I am trying to work on a horribly overdue manuscript review, but the exhaustion from fieldwork and dealing with logistics and lots of other human beings is making it very challenging to concentrate. In the evening, two students from a collaborating lab showed up, so there's been a lot of "Getting to know you" and such. Also a lot of intense mentoring of the current undergraduate and post-bac who used to work with me. Those are important, but draining. We're currently at 7 people. On Monday there will be a partial changing of the guard, and we'll be up to 10 people until that Thursday. The good news is that I think we're reaching a point where we'll have fairly quiet mornings and a mid-day break, which I desperately need.

We're starting to converge on a pitfall trap method. The first night, we netted a total of 1 male cricket across 5 traps. The second night, we got 5 crickets, all of which were in the 3 traps that held recorders that played back male cricket chirps and not the 2 empty traps. Last night B had the idea to take some of the males that we collected during surveys and put them out in the traps that don't have recorders in them. A team also went out and set up additional pitfall traps in another location, so we're gradually extending our reach.

Long-winged crickets with pink flight muscle are still incredibly scarce. I want to try and walk around right at dusk, but I won't get to do that tonight because I need to run the cricket oxidation procedure at 9 pm again. I'm still optimistic about working with our ongoing collection of last-instar crickets.
rebeccmeister: (cricket)
4 reasons why it's easier to run the circadian experiment here than in the lab at Berkeley:

1. Kitchen's right here, so I don't have to prepare 3-5 meals in advance and cart them in with me.
2. Bed's right here, so I can sleep in a real bed without an added commute.
3. We are collecting the crickets in the evening and then running the experiment the following day. While we hold them, we're giving them water, but no food. So I don't have to arrange to take away food several hours before the experiment. This means I won't have to get up early to take away food on any days where I run the noon timepoint.
4. Our schedules are consistently night-shifted, so sleep is much more consistent.

Main reason why it's harder: Very, very few long-winged crickets with pink flight muscle so far. But L pointed out that it's still worthwhile to run the oodles of short-winged crickets to get at least some sense as to metabolism in lab vs. field. And while we're getting some exercise, it's in smoke-filled air (Whittier fire), and it isn't rowing. Also, it's unsurprisingly hard to find any quiet space whatsoever to gather my thoughts.

Cool things observed during last night's circadian trial (9 pm timepoint): one female with a spermatophore, two short-winged females with underdeveloped ovaries, which means they're on the younger side, which is good. The 5x Granny Lamp works decently well for field dissections, which is great.

Yesterday during the day: hiked 11 miles, north up the main road to Gate 2 along Figueroa Mountain Road, then back down along Lisque Valley road, pausing to listen for daytime crickets every so often. Figueroa Mountain Road gave us better views of the Whittier Fire, which grew a lot bigger yesterday. It appears to be a favorite among local cyclists (argh should have brought my bike for multiple reasons). The others learned about how stupid it is to hike in the middle of the day in the summer in a climate like this (I knew but participated anyway because I'm stupid and was curious about the terrain). Lots of spots along Lisque Valley Road looked like decent cricket habitat, but we didn't hear anything. It appears that even the most desperate of males stop chirping during the middle of the day, from around 11 am to 3 pm, perhaps due to the heat. We confirmed this by listening near the fields where we've been doing our nighttime surveys towards the end of the day. Still, I feel like we learned a lot, and it also looks like our trip could add a lot of info to Open StreetMaps.

Speaking of which - at the last minute we got a GPS at REI, right as we were heading out of town. Based on [personal profile] scrottie's recommendation, we got a Garmin etrex 20x hiking GPS. (also, I've used his GPS before, so I had some familiarity with how to operate it). I am SO GLAD we got it. Totally worthwhile, and now I'm tempted to keep it instead of passing it over to the lab and getting a reimbursement for it.

Double nighttime surveys last night. Nighttime temperatures have been on the low side, compared to what I remember from last year. Yesterday I finally had the presence of mind to put an iButton outside near the crickets we're holding, so we'll have more information about daytime and nighttime temperatures in the shade, at least. We have been able to collect around 80 crickets within an hour, among 4 people. We saw much less activity during our second survey, from 11 pm - midnight, but part of that may have to do with the fact that we still had all the crickets from the earlier survey in captivity. We're having reasonably good luck with mark-recapture within our two survey plots so far - "classroom" and "garden." They're maybe 1/8 of a mile apart, and there isn't any evidence of movement across that scale yet. Collections are female-biased, because the females are running around, looking for males, while the males establish territories at the openings to burrows, to amplify their songs. When we get too close, they will abruptly stop chirping and dive into their holes.

As we add more people to the team, we'll keep adding to our survey areas, which will hopefully help with getting more crickets for my experiments, too. We're also keeping all of the last-instar crickets we collect, so we can wait until they emerge as adults and see what ratio of long-winged to short-winged crickets we get. That should tell us more about why we're getting an extreme short-winged bias again (same pattern as last year) - whether it's because the long-winged crickets are off flying somewhere where we aren't catching them, or whether there just aren't all that many long-winged crickets out there under the current conditions.

Other interesting wildlife: one rattlesnake (of course B pestered it more so he could get video of it rattling), a couple of big frogs, a tree frog, a mouse (eating a cricket!!), lots of large spiders (maybe tarantulas, but not all that hairy??), lots of black widows, lots of darkling beetles. We hear coyotes a lot at night.

Pitfall traps have been highly unsuccessful so far (1 male out of 5 traps). I think they're going to require a bunch of tweaking.

A lot of the long-winged males I've checked for flight muscle status also have a whole bunch of parasites glommed on to their armpits.

Today we'll take it easier than yesterday, probably with another trip into town for groceries and such.
rebeccmeister: (cricket)
The 3 of us make for a really nice fieldwork Dream Team.

The drive down took about 5.5 hours, hitting periodic random traffic congestion, as one does in California. With an extra 1h delay at the car rental, load-up at the lab plus 3 houses, and a stop at REI for a GPS plus lunch, we didn't get on the road until 12:30 pm. Still not too bad.

I am SO GLAD I got to pack all the lab supplies. Last year was a nightmarish giant pile of stuff, whereas this year I know exactly where everything is / is supposed to be. Everything got packed very neatly into the 12-passenger van, with ample room to spare, and didn't feel like a hellish mad scramble. I have a certain hatred of stuff-piles stacked so high that things slide all over.

So far I think the cricket population density is on par with last summer. C and A got here a couple of days before we did, and in one evening were able to finish collecting what they needed, so they offered to help us. In an hour, the 5 of us collected ~80 crickets, heavily biased towards short-winged males and females. All of the 10 long-winged crickets we found had histolyzed (white) flight muscle. So we'll have to keep easter-egg hunting.

Today has involved a debriefing with one of the reserve managers, picking up some additional supplies in town, getting meals and groceries squared away for the next couple of days, setting up the full respirometry rig, and beginning to test out pitfall traps. Tonight we'll repeat our population survey (mark-recapture with last night's crickets) and will hopefully work towards gathering up crickets for some initial metabolic experiment test runs tomorrow.


Last year we had to stay in tent cabins because the ranchhouse was undergoing renovations and refurbishment. The cabins had healthy black widow populations (though no one was bitten), and we cooked in an outdoor kitchen adjacent to a classroom space where we worked. Showers and toilets were in a freestanding, rustic structure. It was pretty good for a fieldwork setup, all things considered.

This year, we're the first research group staying in the ranchhouse as the renovations wrap up. And OMG it is POSH. Apparently the UCSB donor who funded the project will be staying in the master bedroom on occasion. It has a panoramic view up the Reserve's central valley. It's fully air-conditioned. There were some interesting decisions during the renovation, such that certain bathroom fixtures are still adorably historic, and all of the new windows are still single-pane. Sad to see so much energy loss.

Still, there's countertop space in the kitchen that is PERFECT for the respirometry rig, and we're using the dining room for staging other projects and plotting and scheming about how to take over the world. The living room contains the most enormous television I have ever seen in my life.
rebeccmeister: (cricket)
Several photos from around the lab.

First, my lab succulent collection, which makes it nice to gaze toward the windows:
My succulent collection in the lab

This Granny Lamp arrived today. I was going to buy a dissecting scope for fieldwork, but got to thinking that one of these lamps would probably be just fine instead, and it might be nicer than the scope for teaching students how to dissect crickets.
A granny lamp's an entomologist's best friend

Before I headed in to work today, I paid a visit to the Albany Aquarium to see what they had in the way of air pumps. Here's the challenge: when I do nighttime cricket experiments, I need to keep the crickets in the dark / under red light, up until the very end of the procedure. We have a small room that doesn't have too much light pollution, but it's too small for the whole benchtop respirometry rig. See, it's full even when I just have injection/blood collection supplies laid out:

Daytime circadian setup

(nighttime view):
Nighttime circadian setup

Here's the scale of the benchtop respirometry setup, for comparison. Too much stuff to fit in that small, multi-use room.
Li-Cor / Oxilla stop-flow respirometry setup

Previously, I was able to use a second respirometry rig that's built into a Pelican case and designed for field measurements, but a grad student in the lab is going to take that rig out into the field this summer, so it's off-limits now.

The people working at the Albany Aquarium Store were knowledgeable and helpful, and helped me find an adjustable aquarium pump that provides enough air flow, but not too much, for the purpose of flushing dry, CO2-free air through the syringes that house the crickets during the circadian procedures (at ~800 mL/min, in case you wondered - lowest setting on the pump). Perfect!
Aquarium air pump CO2 flush setup

Okay. To round things out, here's a sophisticated gadget I designed myself. It's a cardboard tube, modified to help keep the cricket syringe upright in a hands-free manner, so I can do other things while the syringe is being flushed:
Sophisticated syringe holder

Time to design something similar for the aquarium pump setup.
rebeccmeister: (1x)
I managed to get myself out of bed to go rowing this morning. As usual, I'm glad I did. The wind was blowing up the BAP out of the south, as it often does, but the water was still nice enough for a good steady-state piece.

I'm continuing to work on my posture in the boat. A long while back, Iz had some useful feedback on how to think about it, and I've finally reached a stage where I can feel the feedback from my muscles when I'm doing things correctly vs. being sloppy. Good postural control at the finish/release makes for much smoother rowing and better preparation at the catch. It should also help smooth things between M's rowing style and mine.

The challenge is that I have to keep rowing with at least a minimal amount of consistency to hold onto improvements like this one. Days like Monday through Wednesday this week made that challenging, however, so I have to be satisfied taking what I can get.


Wednesday through today I have been dissecting the crickets from this week's inulin experiments. My primary goal is to determine the flight muscle status for the long-winged crickets (pink, active flight muscle or white, histolyzed flight muscle). My main focus is on the long-winged pink-muscled crickets because they are metabolically distinct from the short-winged white-muscled crickets.

But on any given day, the ratio of long-winged pink to long-winged white varies. For the 11 pm - 1 am timepoint, I had 7 pink and 1 white, so that timepoint is set. However, for the 11 am - 1 pm timepoint, I only had 2 pink and 8 white, and for the 8 pm - 10 pm timepoint I only have 1 pink and 7 white.

So I will need to redo two out of the three timepoints. Based on the current results, I'll set up long-winged to short-winged crickets at a ratio of 2:1.

These dissections have been very tedious and labor-intensive because in addition to checking the flight muscle status, I'm also dissecting out and weighing it, and also the crickets' ovaries and fat body. The ovaries are straightforward, but the fat body is a diffuse and sticky tissue that has to be gathered up.
rebeccmeister: (cricket)
Things went smoothly with last night's and this morning's timepoints, overall. So now that's a wrap and I can focus more fully on other projects, like data analysis, woo!

It's both interesting and frustrating to recognize common effects of sleep deprivation. I lay down on the couch at 2:30 am, but didn't really sleep properly because I was pretty wired from the experiment. I got up at 4 am to take food away from a box of crickets, and then got up again at around 6:30 because I wasn't really sleeping anyway. The morning's experiment ran from 7:30-10:30.

I would have liked to have dug in more on some data analysis projects last night, and I did manage to get some stuff done, but I'm still doing too much task-switching these days. Add on tiredness and/or sleep deprivation, and trying to get work done is like trying to sludge my way through molasses.

That said, here are some interesting things I've been learning:

In conjunction with our measurements of nutrient oxidation rates, we've been working on quantifying the activities of some cherry-picked key metabolic enzymes. For example, way back in the day when you learned about glycolysis in high school biology, you might remember learning the names of the enzymes that catalyze the steps of glycolysis. In crickets where we see an increase in glucose oxidation rates, we'd like to know how increase oxidation is accomplished, under the idea that an increase in glucose oxidation indicates an increase in rates of flux through glycolysis. One method used by many organisms is to increase the activity of enzymes associated with that metabolic pathway, but in theory that end point could be accomplished in no small number of ways. Still, the first enzyme of glycolysis, hexokinase, is a fairly straightforward place to start.

So, how do you quantify an enzyme's activity? It's not particularly simple. You need to figure out a method that lets you quantify either the disappearance of a substrate or the appearance of a product, ideally over time. Fortunately, for common enzymes like hexokinase, lots of people have figured out clever ways to do this, and it's even possible to buy kits that take the guesswork out of figuring out the ideal reaction parameters.

Even with that out of the way, you still need to think about ways to standardize your measurements - hexokinase activity relative to what? The generally accepted standard method involves quantifying the total protein in your tissue sample. Dividing your rate by the protein concentration then gives you what's known as the enzyme's "specific activity." Note that you might get a very different result if you standardized based on your tissue sample's mass - more on that below.

"Specific activity" only gives you enzyme activity relative to the protein concentration. The next level is to multiply the specific activity by the total mass of the tissue being assayed, to quantify the total enzyme activity within that tissue.

Anyway, I've learned some interesting things in the process of quantifying hexokinase activity in cricket fatbody. Interesting thing 1: the protein concentration of reproductive (short-winged) crickets' fatbody is much higher than the protein concentration of the dispersal (long-winged) crickets' fatbody. If you think about this for a minute, it can make sense when you realize that the dispersal morph uses its fatbody as a storage depot for the lipids that are accumulated for endurance flight. If there are more lipids present per unit of fatbody, there will be a lower protein concentration.

But what about total activity? Yesterday, I finished collecting data for the second piece of the enzyme analysis: quantification of the total mass of the fatbody in each morph. That led to interesting thing 2: The dispersal morph has a larger total fatbody mass than the reproductive morph. Okay, maybe that's not too surprising if it's doing double-duty with its fatbody, using it for metabolic purposes AND using it to store lipids. Something else to think about, though, is that unlike mammals, insects have limited storage capacity within their non-stretchy exoskeletons. So this is where the rubber hits the road in terms of a flight-reproduction trade-off: if your fatbody is bigger (and the flight muscles are, too), something else is probably smaller. And that something else is the ovaries.

Anyway, with regards to hexokinase, there's a trend towards higher hexokinase specific activity in the dispersal morph. So given that the dispersal morph also has a larger fatbody, I expect the difference between the morphs to be exaggerated when considering the total hexokinase activity in the fatbody. That aligns nicely with our glucose tracer oxidation results, where there's a trend towards generally higher rates of glucose oxidation in the dispersal morph and a larger-amplitude change in oxidation rates across the circadian cycle.

It's not quite a smoking gun, however, because the specific activity of hexokinase doesn't appear to change much across the circadian cycle. So having constitutively higher hexokinase specific activity may help facilitate big changes in glucose flux, but it isn't modulating rates of flux.

So we'll proceed on to our next candidate.
rebeccmeister: (cricket)
State of the science:

-I am making progress on two manuscripts. My PhD advisor is largely giving the next leafcutter manuscript the go-ahead for submission, finally. The final stages always take way longer than one would like, but they're concrete. The significance statement will still need some thorough work, but I suspect we can ask someone else for solid help with it. Meanwhile, I've gotten caught up on the most recent literature in the field of nutrition and ageing, and am making progress on couching our related project in the appropriate context. Rewarding stuff to think about.

-I am running a couple more circadian trials with the current species, Gryllus firmus, the Florida sand cricket. Specifically, I feel like we need to finish exploring a final hypothesis about lipid metabolism. After we determined that our original lipid tracer was too high in concentration to really be a tracer, most of our efforts have focused on quantifying lipid metabolism by injecting a low-concentration tracer. As I read and think more about lipid metabolism, however, I'm coming to think that there could be interesting differences between the crickets in their capacity to oxidize lipids across the circadian cycle even if there aren't constitutive differences in lipid oxidation rates. Plus we are only missing data from 2 timepoints, one of which has the potential to be especially informative and interesting, based on our other data. I'll admit I'm glad the missing data aren't from the 9 am or 1 am timepoints.

-We also have questions about interactions between feeding and nutrient oxidation rates. For all of the Florida cricket experiments, I withheld food for 4 hours prior to the oxidation trial. With our video setup, we should soon have data about feeding patterns, but I'll also do some experiments comparing fed and starved crickets so we can get a better handle on feeding influences.

-After all that, I am going to transition over to the California cricket species, Gryllus lineaticeps. My experiments in Nebraska with lab vs. field G. firmus have suggested lab evolution of several aspects of metabolism, which makes it impossible to relate lab findings back to wild crickets. Related to the previous point, we haven't determined what we'll do for feeding status for G. lineaticeps, so there are a number of logistical elements to work out before we take this work to the field in early July.

-For some reason, I volunteered to give a talk on Friday, which I need to write. So of course there are too many interruptions in the lab today to be able to focus on anything. Sigh-ugh.
rebeccmeister: (cricket)
You can probably ascertain, by relative quiet, that I've been busy.

I've been working on some enzyme assays with our Chinese Visiting Scholar, B. Mostly, he's the one who's working on the assay logistics, while I'm the one who knows where the equipment and samples are, and how to work the equipment. This leads to a lot of back-and-forth and a bit of babysitting on my part, but it is important work.

Wednesday morning, he ran some preliminary experiments. At around 3 pm, we needed to make a decision: forge ahead with the actual assay, or wait? For forty logistical reasons, we decided to forge ahead. It took me around an hour to dissect out and prep the tissue samples. Due to some miscommunication, I then wound up having to wait for a very long time before I could set up protein assays, which have to be completed on the same samples as the enzyme assays. If I'd known in advance, I would have brought in dinner. Unfortunately, I get really grumpy when I miss dinner. But it was satisfying to get that batch of work out of the way, and we should be able to make good progress next week.

Meanwhile, I'm working with three different undergrads these days. One is learning the ropes for analyzing cricket activity across the day and night, and on Thursday we finally hit the stage where he commented that things finally feel like real science. Working out that pipeline became something of an ordeal because the open-source software we're using isn't being maintained anymore, so we had to figure out a machine where we could install Ubuntu 12.04. By itself, that wouldn't be a big deal, but there's something weird about the Dell we were trying to work with, where we couldn't simply boot Ubuntu from a flash drive or DVD. Finally, we installed Ubuntu on a separate hard drive and stuck it in the Dell, and it works. So C got to do his first test-tracking on Thursday, which worked well, all things considered.

The second undergrad is new to the lab, but not new to research. I'm teaching her how to do various basic biochemical assays, so we can assay the composition of cricket hemolymph in the California species we work with. The early stages of mentoring a new student take the most time and effort, but she's a quick study, so that's good.

The third undergrad is cooking along on her honors thesis project, which involves feeding different diets to crickets and then tethering them and flying them in front of a fan. One day, she had a conflict and wasn't able to fly her cricket, so I got to do it instead:

Tethered cricket flying

I'm generally excited about her project, which could turn into a nice publication.

But I'm feeling antsy about making progress on academic writing, because working with a lot of people sucks up a lot of time and mental energy. Time to get more strategic about scheduling.
rebeccmeister: (cricket)
[As posted to other social media]:

As you undoubtedly know by now, one of my favorite George Pocock quotations is: "No one will ask you how long it took to build; they will only ask who built it."

Nonetheless, at the end of a long day of data quality-control and analysis I find it satisfying to tally up some numbers.

So far, this circadian experiment has involved gearing up to run time points 45 times, across a 5-month period. I've poked and dissected 393 crickets, and we'll wind up using data from 211 of them. In some respects this is a far cry from the 6 different radiotracer experiments from 2015 (averaged ~250 crickets/experiment), but of course there are some major qualitative differences between experiment types. Overall I would say the radiotracer experiments were less grueling.

And regardless, that's a lot of crickets to have poked. Thank you, crickets.


Also, I suspect I'm not quite finished with it all. I'll take my current analyses and will run with them for now because I'm giving a conference presentation in one and a half weeks, but I am thinking that I'll have more confidence in these results if I boost all my sample sizes to closer to 9 crickets per morph per tracer per timepoint (so 9 crickets x 2 morphs x 3 tracers x 5 timepoints = 270 total). The good news is that if I decide to proceed, I at least have my cricket-rearing protocol in good shape, so completion should be straightforward. In addition, I'm feeling fairly confident about my method for characterizing lipid oxidation.

It has been quite a ride.
rebeccmeister: (cricket)
Yesterday morning left me feeling sufficiently terrible that I left work at around 1:30, went to Berkeley Bowl to pick up some groceries and cricket food, then went home and curled up on the couch with the cat. Cat therapy.

This is one of those head colds that starts with a scratchy back of the throat. I am pretty sure that the only cure is rum.

[ profile] scrottie and L are both sufficiently civilized human beings that they came home with candy to give out for Halloween. I was feeling sufficiently antisocial that I didn't want to repeatedly answer the door, so we set out one of those plastic pumpkin buckets and periodically refilled it. Seemed like a reasonable compromise. I feel bad about missing out on an opportunity to get to know more of the neighbors and see kids in cute costumes, but at least we contributed to the collective sugar-high. And I didn't pass on my cold.

This evening we'll do another 9 pm timepoint, and then we'll ship out a box of samples in the morning. Then we'll take a couple of days off while we wait to find out the results and whether we need to continue re-tooling things for the glutamate and oleic acid tracers. But just for fun, we'll probably stay up until 1 am on another night this week anyway to finish collecting data for a different project (characterizing cricket hemolymph across the circadian cycle). Given that we finally have good cricket numbers, we might as well put the crickets to use.
rebeccmeister: (cricket)
Here is a grid of sample sizes for the circadian experiment:

Sample sizes.jpg

We haven't been able to decide which is worse, the 1 am timepoint or the 9 am timepoint. I'm leaning towards the 9 am timepoint. You'd think 9 am doesn't sound all that bad, right? But we have to make sure the crickets are in a postingestive state, so someone has to take away their food 4 hours before the timepoint. And the 9 am timepoint actually starts at 8 am, so that means getting to campus by 4 am.

Even morning people have limits, and my limit is having to wake up before 4 am.

Also, when the timepoint starts at 8 am, that means we have to prepare certain things right before the experiment itself starts, so the work actually begins closer to 7 am.

To top things off, the person who has been helping on this project, through thick and thin, lost track of things and slept in this morning. As a result, I learned that I *can* run the whole experiment by myself. That said, I really, really, really hope I never, ever have to do that again.

Those glucose samples are crossed out because of a snafu with our respirometry setup. I have to manually set the range for our high-precision carbon dioxide detector, but early on I set the range too low and so our measurements of the amount of carbon dioxide exhaled are underestimates. We might be able to get that info from our collaborator who measures the C-13 sample enrichment, but I'm not certain of that yet.

Also, my Halloween costume is made of polyester, and polyester smells awful, especially when one is sweating from all the extra anxiety and running around.
rebeccmeister: (cricket)
We're back on deck with the circadian experiment.

One of the most challenging aspects, for me, is when I have these long evenings that I can't really put to effective use. I tried to work on some statistical analyses last night, but I really needed to look stuff up in Zar, which was at home. So I just ate too many biscotti instead.

I have this other massive stats book at work called Applied Linear Statistical Models (which I will call NKNW after its authors), but it's ever-so-slightly more mathematical and less pragmatic than Biostatistical Analysis (aka Zar). So when I need to look up something like how to calculate statistical power, NKNW doesn't actually help.

Nor does the internet, because as we all know the internet isn't a vetted source, and you'll find about 12 different sources with 12 different styles of calculations. In addition, for a lot of stats stuff it's helpful to learn a consistent set of variables and calculations because there's a hell of a lot of sloppy work out there and it's easy to get stuck in a swamp of incomprehension because someone defines things in a slightly different fashion. And that's how "Data Science" got invented.

Today, however, I have Zar again, so maybe I can fwump myself over this hurdle and on to the next thing (job applications again).
rebeccmeister: (cricket)
1. K overheard me saying: "Stop jumping on my hand! You have no head" (said to a freshly decapitated cricket)

2. Last night a feisty cricket managed to jump straight out of its box at my face and onto my glasses. If you've ever worked with crickets, you're already acquainted with the Scrambling-After-the-Escaped-Cricket Dance. But the glasses took things to a whole new level. However, I still won.
rebeccmeister: (cricket)
I totally didn't make it to the boathouse this morning. I'm pretty sure that I needed those extra 2.5 hours of sleep on C's office floor instead. Especially given how physically painful it felt to get up again at 7 am (more painful than the 3:30 wake-up to ride over to the lab).

Our current stable isotope tracer for characterizing lipid metabolism is not ideal. Apparently L started out trying palmitic acid in the beginning. That's the same lipid tracer as I used in Nebraska, though I used a radiotracer, not a stable isotope tracer. He was never able to obtain a reproducible signal with palmitic acid, so eventually they triangulated on oleic acid instead, injecting a bolus of pure oleic acid with a single C-13 label on the carbon that's easiest to label. A quick check of my notes about my palmitic acid experiences confirm why L never got any signal - only around 1% of the injected radiolabeled palmitate ever got oxidized over a 3-hour time period.

There are a couple of problems with the 1-13C oleic acid. The biggest one is that the amount of oleic acid we're injecting is equivalent to the total amount of lipid present in the entire cricket hemolymph pool. Hardly a tracer, unless there's incredibly high hemolymph lipid turnover. Along with that, as I'm doing the injections, I've observed a couple of crickets that have apparently bled heavily sometime during the 60-minute incubation period, and I'm not entirely sure about what's going on there. I haven't seen nearly as much bleeding during the injections themselves as compared to the glucose injections, but it still seems like there's something strange going on.

Uniformly-labeled C-13 oleic acid should generate a stronger signal, but it's also expensive. I would also need to think about the best injection vehicle for to dilute out this nonpolar compound.

We shall see what the preliminary samples reveal. They have just reached our collaborator today.
rebeccmeister: (cricket)
Yesterday was a long day. After tending to a handful of miscellaneous errands in the morning, I headed in to work. During our lab meeting, we hashed out plans for a field expedition this upcoming weekend. Originally we were going to leave on Friday, but the circadian crickets made things complicated, so the new plan is for us to finish out the circadian experiment Friday night/Saturday morning, then sleep, then head out to the field site. We aim to return by midday on Monday so our intrepid undergrads can make it back in time for their summer classes. Then I will turn around and head out to the Midwest via Amtrak on Tuesday morning for some Midwestern bicycling adventures.

I'm reminded of that one 400k brevet that [ profile] scrottie and I completed last September, where work was super hectic and we barely got any sleep before the brevet. The early hours of that brevet were such a merciful relief from being in the lab, but by the end of the ride we were so exhausted that we had to keep flopping down on the ground for little naps.

I don't know quite what to bring along for the Amtrak segment of the trip yet. Probably the cat bed quilting project and a book or two.

Anyway, after hashing out fieldwork plans, I had to do a somewhat hastily tacked-on project to start repeating the procedures we've worked out for the sand crickets (Gryllus firmus) in the variable field crickets (Gryllus lineaticeps, the species we'll go hunt for over the weekend). That made yesterday a triple-procedure kind of day, oof, where I had to roll straight from one task into the next.

The thing that hits me the hardest is the carryover aspect of things. I'm feeling so unmotivated today because task-switching takes extra energy that I just don't have right now, so then I wind up with awkward gaps of time where I can't seem to get anything done. It's not easy to be at peace with the gaps, even though that's probably the best answer for things.
rebeccmeister: (cricket)
The science jet lag isn't so horribly wretched at the moment, although I'm expecting some carryover through tomorrow.

We did the first run of the 1 am timepoint last night. That meant hanging out in the lab until 8 pm, when it was time to remove food from the cage, then hanging out until 11:30 when it was time for our final preparations, then running the show from 12-2 am.

To fool the crickets into thinking it's nighttime, we worked under red light. K took some photos at my request:

Nighttime tracer injection setup

Nighttime tracer injection setup

Nighttime tracer injection setup

Nighttime tracer injection setup

We made several mistakes, which could mean having to do a three-peat of this timepoint. At some point, the light to the left of my head fell down and the bulb burned out. While plugging in a replacement light, K must have accidentally jostled the adapter for the fan pump we were using to flush those 60-mL syringes with dry, CO2-free air. Unfortunately she didn't notice quite when that happened, so we had to re-flush all of the syringes and start the stop-flow incubation 30 minutes late. We'll still be able to compare long-winged versus short-winged crickets, but since we don't know the full timecourse for our injected tracer we don't know if these results will be comparable to the rest of our results.

At one point, I also opened the door and exposed the crickets to some incandescent light. Argh.

Then I slept on C's office floor in a sleeping bag until 4 am, when I got up to remove food from another set of crickets for the 9 am timepoint. This system wound up being less hellish than just running one of the two timepoints. Once the crickets were squared away at 4 am, I slept until 7:10 and then walked over to Yali's for a latte and scone. We wrapped up the 9 am timepoint by 10, and I headed home for a couple of hours.

The downside of this kind of arrangement is trying to figure out what to do from 10 am - 2 pm. I'm not very good at taking naps, nor do I want to completely throw my sleep schedule out the window. So I rested and worked on a couple of household chores.

Tomorrow we only have a couple of crickets that will be ready, so we'll take things easy and just run the 5 pm timepoint. We'll be doing another 1 am stint on Friday night. We're actually making very good progress overall, although things can get a little frustrating because we lose data left and right due to various small mishaps. It's just a difficult experiment to complete smoothly.


rebeccmeister: (Default)

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