Why insects aren't huge: a new challenge to a decades-old idea

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1:56Welcome back to The Nature Podcast. This week, working out why insects aren't giant. And cutting the cost of CAR T therapy. I'm Nick Patrick-Chow. And I'm Maren Hansberger.

2:16Here's a question for you. Why are insects small? For decades, researchers have thought they've had a pretty good idea of what's limiting insect sizes. But now a new paper is challenging that idea. Because while there might be some insects living today that look pretty big, they don't even come close to the giants that lived 300 million or so years ago. Griffinflies, for example, were ancient dragonfly-like insects that could have wingspans up to 70 centimeters, which is about the same as a crow has today. Now, if that fills you with dread like me, don't worry, because these giant insects are extinct, and you won't be seeing others this size anytime soon.

2:58But why is that? Well, insects' ability to use oxygen might be the key. You see, insects have relatively simple respiratory systems that rely in part on oxygen diffusing into body tissue. This process isn't the most efficient, so it's been thought that it wouldn't be able to provide enough oxygen to tissues if insects became too big. And this could be holding back the resurgence of enormous insects. But 300 million years ago, atmospheric oxygen levels were a lot higher than they are today, potentially allowing this diffusion process to work more effectively, letting insect sizes increase. This theory was crystallized in the pages of Nature in 1995, and since then has become something of a dogma.

3:44Fast forward to today, though, and there's a new paper in Nature challenging this idea, suggesting that oxygen might not be the limiting factor after all. The team behind this work have looked at the smallest sections of an insect's respiratory system, known as tracheals, very fine structures a bit like capillaries that deliver oxygen into tissue. One of the authors of this paper is comparative physiologist Ned Snelling, and reporter Benjamin Thompson called him up to hear about this work and asked Ned why he wanted to investigate this decades-old idea. Well, I did it because I didn't believe it.

4:21The reason I didn't think it was true was because part of my PhD work required me to build little flight chambers and fly locusts in order to measure their flight metabolic rate. One thing I noticed when I was doing this was that once the locusts finish flying, they recovered very quickly. And even though locusts aren't the largest insects, it kind of suggested to me that oxygen supply is not really a big challenge for insects. and when I examined locust flight muscle under a transmission electron microscope, I noticed that the tracheoles occupied a very small fraction of the locust flight muscle.

5:08So despite this being a tissue that needs a lot of oxygen, you weren't seeing a lot of the final oxygen delivery system, the tracheoles. Exactly. So your work then was suggesting that insects are very good at getting oxygen where it needs to be and so you don't necessarily need more of it in the atmosphere to get enough supply. And so potentially that's not the barrier preventing insects getting bigger. That's what I thought. And so how did you go about testing that in this new work? If oxygen supply is a challenge in larger and larger insects, then we would expect that these tracheoles that supply oxygen to the flight muscle, where oxygen demand is extreme, their investment should increase in larger and larger insects.

5:55Perhaps if oxygen really did constrain body size, the tracheole investment would reach a level that could possibly constrain flight in some way because they were occupying so much flight muscle that there wasn't room for anything else. And so you might expect this relationship then between the amount of tracheoles and insect size. Is that what you saw then? Well, we did see some evidence for that. So what we did was we took little samples and we could quantify the percent volume of the flight muscle that is dedicated to these tracheole structures. and we notice that the investment does increase with body size but despite that increase it remains trivial.

6:55In a little half milligram insect the investment of tracheoles is only about 0.5 % of the flight muscle volume. And that increases in a five gram insect up to about 0.8%. So this is four orders of magnitude. It's a 10 ,000 fold body mass range. And it increases from about half a percent to about 0.8%. So there isn't a one-to-one ratio between size and tracheal need. There is an increase, but relatively slight. So you put forward evidence that you don't need a lot more tracheals to get the amount of oxygen to be bigger, which is at odds with the previous ideas around for 30 odd years that the way insects got so big was that there was just more oxygen.

7:46I mean, how come no one's come up with this idea before, do you think? I think the main reason people haven't looked at this before, is because I work across different animal groups, I've been able to look at tissue and particularly capillary investment in birds and mammals. And the capillary investment of birds and mammals, particularly the flight muscle and the cardiac muscle, it can be about 10%. So from looking at that tissue and then working with insect flight muscle, I noticed that the investment of tracheoles, which are effectively analogous structures to the capillaries, they occupy only about 1%.

8:29So I was able to use that comparative aspect to think it might be worth having a closer look at how the tracheole density varies as a function of body size in insects. Obviously, this previous idea about the oxygen concentration has been a popular one and does make a lot of sense based on the evidence that folk have known. And of course, extraordinary claims require extraordinary evidence. I mean, what do you think folk will make of your results when they see it? And what questions do you think they'll have? So for a long time, we thought that the tracheal system was this entirely diffusion dominated system from the outside air all the way to the cells.

9:10But we now know that the larger air sacs that are more laterally located in the insect help tremendously with oxygen supply by acting as bellows. Now, some of the criticism could be that we know in four species of running beetle that the space occupied by these air sacs within the body increases in larger beetles. And if a similar relationship occurred in insects more generally, then that might suggest that actually these air sacs might begin to constrain body size. Effectively, the air sacs might take up so much room that there's no more space in the body for larger air sacs to support this convection.

10:09Which brings us back to this idea that you'd need elevated atmospheric oxygen levels to overcome this. Now, my response to that criticism would be that, well, even if there is a hypothetical constraint in these air sacs, stands to reason, because the tracheal system is quite a responsive system, that any constraint could be compensated by increasing tracheal density because there's so much room. And one question that struck me, and I'm sure others listening as well, is that the evidence you're putting forward is that oxygen concentration shouldn't be a limit for insect size, right? Why are there no truly massive insects alive today?

10:50There are a few ideas out there. Probably the one that I think might have most support is the observation that large insects are very easy to catch. I had a very large black emperor dragonfly enter my tent in Zambia a few months ago and I was able to catch it with my hand. now we have birds and bats and i would say that predation pressure which didn't occur 300 million years ago would stop insects evolving into large body size because there would be easy to catch and eat another idea is that there might be a mechanical constraint so when an insect moults its exoskeleton it becomes soft and pliable so it can shed its old exoskeleton and emerge as an adult insect and when you're a large adult with a soft exoskeleton it makes you susceptible to collapsing and of course if your exoskeleton is soft then you can't move the insect would damage its limbs.

12:09So that makes you a little bit susceptible to predation as well. I mean, if you look at it in the round, there's now a couple of ideas as to what may or may not have limited insects' size. What do you think it would take to put it to bed other than a time machine, I suppose? Yeah, I think we really need to do research on the effect of body size on those factors, particularly the biomechanics of the exoskeleton. We know crustaceans that live in water where their body weight is supported, they can become quite large. That's some circumstantial evidence to suggest that there are limitations on the biomechanical loading of very large insects and that that might be a factor.

12:57That was Ned Snelling from the University of Pretoria in South Africa there. To read his paper, look out for a link in the show notes. Coming up, how to engineer supercharged immune cells inside the body. Right now though, it's time for the research highlights with Dan Fox.

13:16Tracking how quickly a person's biological clock ticks could be a powerful tool for predicting when they might die. Biological age takes a person's health into consideration by looking at how DNA and cells are affected by a person's lifestyle and genetics. Therefore, this age can be higher or lower than their actual chronological age. The pace at which this biological age changes can speed up or slow down, but scientists have debated whether such changes matter. To answer this, researchers evaluated DNA collected from almost 700 older adults over a 24-year span. People whose biological clocks accelerated more rapidly faced a higher risk of death over the course of the study independent of their initial biological age than did people whose clocks ticked at a slower pace.

14:09The findings suggest that the speed of biological ageing shifts as a person's health changes, and the authors say researchers could eventually monitor these shifts to test interventions for extending lifespans. Read that research in Nature Aging.

14:29Physicists have made an ultra-powerful magnet which fits in the palm of a hand. Nuclear magnetic resonance is a technique that places objects inside powerful magnets to determine their molecular structures. But powerful magnets are usually large and energy-hungry. So researchers set out to build a small, ultra-strong magnet that would still be able to fit samples inside its core. To do this, they made the magnet's coils from metal tape coated with a superconducting ceramic, which when cooled to around minus 269 degrees Celsius can carry enormous electric currents that generate a magnetic field.

15:09A stack of four coils produced a magnetic field that was more than 800 ,000 times stronger than the Earth's and consumed less than one watt of power. The team hopes that magnets like these could make high-sensitivity studies of molecular structures more widely accessible. If you're feeling attracted to that research, you can find it in Science Advances.

15:37CAR T therapies have shown impressive results in tackling cancer but it is a slow and expensive process. Now researchers have shown a way to make it easier and quicker by creating CAR T cells inside the body. In the fight against cancer or other diseases one treatment method involves engineering someone's own immune cells to root out things like tumours or things that could be damaging the body. That's what CAR T therapy is. In this case, T cells are made to express chimeric antigen receptors, or CAR. These receptors help the T cells find rogue cells like those in tumours. But typically, this requires taking blood from people, filtering it out, engineering the T in a lab before re-injecting the CAR T-cells back into the person being treated.

16:28This takes time and money, so researchers have been experimenting with trying to get this process to work within the body. They've used things like viruses, particularly lentiviruses, that can deliver the DNA to the T-cells and get them to express the CAR. This DNA insertion can be a bit random though, and it could be introduced into the wrong place in the T-cell's genome. Also, cells other than the target immune cells may be affected. In rare cases, this could even lead to secondary cancers. Recently though, researchers published a study in Nature about a more targeted approach that can integrate the relevant CAR gene into a section of the T-cell's genome known as the T-cell alpha constant or TRAC locus by combining a few different delivery mechanisms or vectors.

17:18I spoke to one of the authors, Justin Aikim, and he laid out for me the initial idea behind their approach. First, we demonstrated about nine years ago, also in nature, that you can use genome editing, and in that paper it was CRISPR-Cas9, to deliver specifically the car into one single locus in the genome. And we showed that one specific locus called the TRAC locus was generating really potent CAR T cells. And so we since spent a lot of time optimizing that type of engineering and moving it to the clinic. And that technology has been then adopted by many people because it creates CAR T cells that are more predictable.

17:57It really standardized the manufacturing, making it safer. But we also showed that the CAR T cells engineered this way were also more potent than those engineered with randomly integrated vital vectors. When we were interested in moving in vivo, we wanted to move in vivo without losing the precision that we optimized the last 10 years. And so the greater challenge was, well, can we go in vivo and achieve the same level of precision and place the car into one single locus? So what did you try to try and get this position? To do that, then we need to deliver both something that was going to cut the DNA, the CRISPR-Cas9 molecules, and then we need to provide a DNA template to repair the cuts and then integrates the CAR transgene.

18:39And so the first part of the equations was actually already solved by the laboratory of Jennifer Darna, who is one of the pioneering CRISPR used for genome editing. And she already optimized some particles that can deliver or fully form Cas9 and a gadRNA to cut the tract locus, which is the locus that we already optimize for CAR T cells. And so I've seen Jenny Hamilton from her lab presenting data before it was published with that particle delivering Cas9. And we were already excited and had developing tools to deliver DNA, the other part of the equations. And so when I saw these first presentations, immediately I discussed with Jenny and then Jennifer.

19:25Could we combine our two particles and deliver together the Caesars and the DNA to create the site-specific engineering in vivo? And what was remarkable is that the very first time we tested it in a humanized mouse, it works the very first time, suggesting that it was radiatoral. Wow. Okay. So the very first time you tried this out, it worked. And it's really just about combining these approaches, this delivery of the DNA and also the scissors to cut the specific target that you wanted to have on the T cells. Yeah. I mean, I would say the reason why it worked so well the first time is because all those parts that were optimized for the past 10 years individually when put together made it work so well from the first time.

20:08So you said you tried this out in humanized mice. These are mice that have human cells and other tissues. And into these mice you sent your vectors. These were adeno-associated viruses that carried the DNA and what are called enveloped delivery vehicles for the CRISPR-Cas9 machinery. What diseases or conditions did you try out in the mice to see how effective this was. So we use those humanized model in which we are engrafting a human immune cells to create a humanized immune system. And in those, there are mostly T cells and B cells. And so the first thing we tested, can we engineer the T cells to eliminate the B cells?

20:46And this is actually really relevant in the context of autoimmune disorders. There are recently really exciting data suggesting that you can use CAR T cells targeting B cells and completely deplete the B cells and remove what we call autoreactive B cells that are at the origin of those autoimmune disorders, such as lupus. And we did it completely depleting all the B cells. We were not able to detect a single one after a single injection of our vectors. And then we did three different tumor models. The first one in lymphoma, where we were trying to deplete some B cell malignancies, a second one in myeloma, where those are plasma cells that turn into cancer that we were also able to deplete.

21:27And then the greater challenge was to do that in the context of a solid tumor, because here we are generating the T cells in the blood. It's considered a higher challenge to be able to engineer cells in the blood and then for them to go into the solid tumors, identify the targets, and then start doing their job proliferating and curing those mice. And we were able to achieve really, really good responses in those challenges models, which was pretty remarkable. And while these mice have sort of humanized immune cells, how much do you think needs to be done in order to actually translate this to work in humans?

22:04We are now moving from what was originally CAR T cell considered a cell therapy to now what's called a gene therapy. We're not delivering cells, we're delivering genes. And the type of experiments the FDA is requiring for us to initiate a clinical trial is based on gene therapy. And it usually has to be done in non-human primates, so monkeys usually, that can be as close as it gets to a human immune system and test vectors in that context. And so that would be the next step. And I want to put you in the shoes of Reviewer 2 for a second. If you were to criticise your own work, what would you say are the sort of limitations of it?

22:45I think the main limitations that we are working around is the fact that now we have a two-vector system. In manufacturing, clinical-grade vectors come at a certain cost. And so manufacturing two vectors adds some complexity. And so I think in the future, the field will try to move to a single vector system. And here we're working with viral vectors also that are a little bit more complex to manufacture. And so there is a push in the field to try to achieve that with non-viral vector. And so I think that we did the first demonstrations that site-specific engineering is possible and efficiency in immune cells.

23:26and I think now the field will collectively try to make the system simpler and even cheaper, that is. That was Justin A. Kim from the University of California, San Francisco in the US. For more on that story, check out the show notes for some links. And that's all for this time. We'll be back later in the week with The Briefing Podcast. But until then, if you've enjoyed this show, do let us know. You can leave a review on your podcast app of choice or you can reach out to us on social media. We're at Nature Podcast. And of course, we're on email too, podcast at nature.com. I'm Maren Hansberger.

23:58And I'm Mick Petrie-Chow. Thanks for listening.

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