Even in Kyoto

I ran my first circadian trial in California back on July 5, 2016. Around a month before that, a collaborator had declared a need for one of our sensitive respirometry instruments (a Li-Cor CO2 analyzer) to conduct field measurements, so the early stages of the first circadian experiment involved some scrambling to get our less sensitive respirometry setup up and running (a FoxBox). This shouldn't be a big problem, given the nature of the circadian experiments (crickets use enough oxygen and exhale enough carbon dioxide to allow relatively easy measurement with the FoxBox). But then two months later the more sensitive Li-Cor was returned to us and I was encouraged to switch back over to that setup. In case that wasn't enough fun, I had to switch back to the FoxBox again two months later, I think because someone else in the lab once again needed the more sensitive setup.

Something has plagued me ever since then: the oxygen measurements from the more sensitive setup (an OxZilla) just never seemed quite right. We'd initially dismissed this, until it became so blindingly obvious that we couldn't ignore it anymore. Unfortunately, that was at around the same time as I was trying to wrap things up in California, so I never had an opportunity to conduct a series of tests to try and figure out what was wrong.

Thankfully, here in Arizona I'm working with a respirometry guru, who has been giving me access to the PC-based software I used for the initial data collection. Conversations with J convinced me to contact the instrument manufacturers, which led to some in-depth conversation with one of their excellent technical support people. I then convinced my advisor in California to conduct a small hypothesis test, which confirmed my suspicion that something was wrong with the voltage transmission from the Oxzilla to the computer.

The only annoying thing about all of this is that it would have been tremendously helpful if only I'd recorded all three of the measurements from the OxZilla instead of just the oxygen delta (reference oxygen, sample oxygen, and oxygen delta [sample minus reference]). That would have quickly made it obvious where the problem lay.

And yet - we can do a simple algebraic correction for the voltage transmission problem. After applying this correction, as noted in the subject line, I feel SO MUCH BETTER while looking through the data. The corrected data are now way easier to work with.

And now all I need to do is re-run a whole bunch of analyses. But this is a comparatively trivial step that will just take a known amount of time, and then it will be done - hours in a day, as compared to days, weeks, months, years of my life - which is what it took to collect the data in the first place.

The transition back to more data analysis and writing is always a bit jarring, and I need to remind myself of this. I can't sit at a desk and think/write for as long as I can work on labwork or things like cricket care. And I need to be okay with that, and remember that it's okay to build in mental breaks (take a walk on this beautiful campus!).

And then, on the other hand, it's really fun to get to do all this thinking. I wake up thinking about all the data I've collected and all the experiments I've done, and I have all these ideas and I can't wait to get back to poring through everything.

Wednesdays are when I finally start to get a sense as to the number of potential crickets available for experiments in the upcoming 2 weeks. I mostly work with crickets that are at Day 5 of adulthood, which means that I can start to sort through the bins of last-instar crickets on Wednesdays and pull out new adults that will reach Day 5 by the following Monday. L also sorts through the California crickets on Wednesdays, and sorts out the last-instar crickets from the younger juveniles, so it's when we can assess the size of the last-instar stockpile and get a feel for how many crickets are going to reach adulthood over the next two weeks.

It was disappointing but unsurprising to note that our last-instar stockpiles still aren't looking all that great yet. There will be enough to perpetuate our lab stocks, but probably not enough to spare for experiments. In this case, this isn't actually a product of the food issues I've been blogging about recently. It's about the quality of cricket care while L and I were both away doing fieldwork at Sedgwick. When we returned, we discovered that the hatchling bins were full of mold and that they were out of water. It takes about 2.5 months for hatchlings to reach the last instar, so the current last-instar crickets are what remains from those poor, traumatized hatchlings.

And this is why, as much as it sounds nice to have undergraduates do all of our cricket care, we really can't. I think back to my first year as a graduate student, when I was also extremely terrible about taking care of seed-harvester ant colonies, and seed-harvesters are really easy compared to the crickets. It isn't the undergraduates' fault, really. Cricket care requires people who can keep a consistent and reliable schedule, and who can stay late when there are emergencies or when there's a shortage of help. It's difficult to do that when your student lifestyle involves running all over the place to participate in all sorts of activities, like classes and student groups and sports and studying.

And so, while in some respects crickets are a phenomenal study organism, the theory of being able to accrue huge sample sizes doesn't match up with the amount and nature of the work that must go into ensuring those sample sizes. I mean, we're still way better off than anybody studying vertebrates, but we have our fair share of hiccups, and crickets are a non-trivial amount of work. And this has been quite a year for those hiccups.

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.

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.

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 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.

Several photos from around the lab.

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


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.


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:



(nighttime view):


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


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!


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:


Time to design something similar for the aquarium pump setup.

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.

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:



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.

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.

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.

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).