Week 3 Quiz

Respiratory water loss in insects is a controversial topic. While earlier studies considered respiratory water loss a significant component of overall water loss, later work suggested that respiratory water loss was unlikely to contribute significantly to overall water loss. This prompted Dr. Steven Chown (from Monash University in Australia) to investigate respiratory water loss in five species of dung beetles and to ascertain to what degree respiratory water loss contributed to overall water loss in these insects.

a) Why does knowing the significance of respiratory water loss to an insect's overall water budget matter, especially in the context of our understanding of the evolution of discontinuous gas exchange?

At present, we do not clearly understand why some insects exhibit discontinuous gas exchange (DGE). One hypothesis proposed to explain the occurrence of DGE in insects is the hygric hypothesis, which proposes that DGE evolved in order to minimize respiratory water loss. However, if respiratory water loss in insects does not significantly contribute to their overall water loss, then the hygric hypothesis would be invalid. That is, why would insects evolve a respiratory pattern to minimize respiratory water loss if such water loss represents only a small fraction of their overall water loss?

b) All five dung beetle species that Dr. Chown studied are known to exhibit discontinuous gas exchange? How does this choice of study organisms impact the generalizability of Dr. Chown's results?

The hygric hypothesis proposes that DGE evolved in order to minimize respiratory water loss. On this basis, the dung beetles studied by Dr. Chown should have low respiratory water loss. Therefore, if Dr. Chown were to show that respiratory water loss was only a small proportion of the overall water loss in these insects, which would lead him to conclude that respiratory water loss is insignificant in insects, this conclusion may not actually apply to the vast majority of insects that do not exhibit DGE and, consequently, should have much higher respiratory water loss.

In a 2010 study published in Journal of Insect Physiology, researchers examined gas exchange patterns in silkmoths with unblocked (a,b) or blocked (c,d) spiracles at two different ambient temperatures. As you can see, silkmoths with unblocked spiracles exhibited discontinuous gas exchange (DGE) at both 6 and 14 degrees C, whereas silkmoths with blocked spiracles exhibited DGE at 6 degrees C, but transitioned to continuous gas exchange at 14 degrees C. Provide a reasonable explanation for these observations. [NOTE: For moths with blocked spiracles, all spiracles were blocked except one.]

Because insects are ectothermic poikilotherms, as ambient temperature increases, so, too, does their body temperature, leading to a higher metabolic rate. To sustain a higher metabolic rate, insects must bring more air into their tracheal system. In the silkmoths with unblocked spiracles, this is accomplished by retaining DGE but shortening the length of the closed periods, as clearly evident in the figure provided. By contrast, in silkmoths with blocked spiracles, in order to bring a sufficient volume of air into their tracheal system, they must keep their one unblocked spiracle open continuously. The blockage of all their other spiracles greatly limits the maximum rate at which air can enter their tracheal system, and, consequently, they cannot afford for their one blocked spiracle to close when their oxygen demands are elevated by increased ambient temperature.

In class, I mentioned that insects rarely have respiratory pigments. Truthfully, while insects rarely have respiratory pigments in their hemolymph (i.e., insect blood), many insects do have respiratory pigments inside their cells (e.g., Drosophila). What role might these intracellular respiratory pigments play in insect physiology?

There are two conceivable roles that intracellular respiratory pigments might play in insects:

i) they may serve to bind up oxygen during periods of rest in order to minimize how much oxygen reaches the mitochondria, where it would get turned into superoxide/ROS, thereby causing biomolecular damage that contributes to aging; and/or,

ii) they may serve to bind up oxygen during periods of normoxia so that oxygen can be made available during periods of hypoxia.

A 2016 study published in Insect Biochemistry and Molecular Biology showed that chitin is a component of the midgut in Rhodnius prolixus, the bug species that transmits the Chagas parasite. What insight does this observation provide us with regards to the ontological development of the insect gut?

Chitin is a component of the insect exoskeleton, and, as we discussed in class, its presence inside the insect body is considered to reflect that structures have developed by invagination of the body wall (e.g., the trachea). On this basis, it would seem that the entire insect gut, including the midgut, most likely develops via invagination of the body wall, rather than developing from the organization of some internal tissues.

In a 2017 study published in Journal of Physiology, Drs. Graham Scott and Grant McClelland (both from McMaster University) found that, in muscle cells from high-altitude deer mice, mitochondria are largely clustered near the plasma membrane, whereas, in muscles cells from low-altitude deer mice, they are more uniformly distributed throughout the cell. Provide a reasonable explanation to explain this difference.

Unlike tracheoles, capillaries, which bring oxygen and nutrients to cells, do not penetrate into cells; therefore, oxygen and nutrients must diffuse from capillaries, across the cell membrane, and into the mitochondria where they are used to create ATP to power cellular activity (e.g., muscle contraction). At high altitude, there is very little oxygen in the air, which slows down the rate at which oxygen can diffuse from capillaries to mitochondria, thereby limiting the rate of ATP production. By moving the mitochondria close to the cell membrane, high altitude deer mice are trying to minimize the distance over which oxygen diffusion takes place, which speeds up the diffusion rate and thereby restores faster rates of ATP production despite limited oxygen availability.

Would you expect discontinuous gas exchange to be more common in small insects or large insects? Explain.

In class, we noted that metabolic rate is the primary determinant of whether discontinuous gas exchange (DGE) is expressed in insects. On this basis, I would expect that discontinuous gas exchange would be more common in large insects, as, due to negative allometric scaling, which we learned about in BIOB34, large insects have relatively slower metabolic rates than small insects.

In 2018, Dr. Do-Hyoung Kim and colleagues presented a paper titled "Suffocating insects: hormonal regulation of tracheal air-filling". In the paper, Dr. Kim noted that the administration of kinins (a group of hormones found in the majority of insects) to the epithelial cells lining the tracheoles caused them to mobilize calcium ions from intracellular stores to the cytosol. On the basis of this finding, do you suppose that kinin causes or prevents suffocation in insects? Justify your answer.

Since kinins bring about the mobilization of calcium ions to the cytosol in the tracheolar epithelial cells, they should also cause the osmolarity of these cells to increase. Consequently, water will be attracted to flow into these cells by osmosis from the tracheolar lumen, thereby reducing the amount of the fluid present in the lumen. Since this will simultaneously increase the amount of air present in the lumen, which makes it easier for oxygen to diffusion throughout the tracheal system, kinins likely prevent suffocation in insects.

Why might animal physiologists studying the evolution of discontinuous gas exchange in insects consider forging collaborations with plant physiologists studying gas exchange in leaves?

Discontinuous gas exchange (DGE) in insects involves closing, fluttering, and opening the spiracles that guard the entrance to the tracheal system for reasons that are hotly debated (e.g., minimize water loss, maximize gas exchange in hypoxia/hypercapnia, etc.). Gas exchange in leaves involves closing and opening the stomata, which connect the leaf tissue to the outside atmosphere. Given the similarities in how gas exchange in regulated (at least mechanistically) in these two groups of organisms, perhaps collaborations between animal and plant physiologists could help expedite our understanding of the evolution of DGE or yield novel insights about gas exchange in leaves.

In class, I mentioned how insect physiologists typically measure gas exchange via CO₂ release rate because insects, given their small size, do not consume adequate amounts of O₂ to be reliably detected. In a study published in October 2024, researchers from Université de Strasbourg (France) used an Oroboros Respirometry System, which is typically used to measure O₂ consumption rate of organelles suspended in a liquid solution, to measure O₂ consumption rate of carpenter ants in air. A representative data trace is shown below. The researchers stated that their measurement of O₂ consumption rate in these ants demonstrated that they exhibit discontinuous gas exchange, and they highlighted the data corresponding to each of the three phases. If you had been asked to review this paper prior to its publication, would you have had any concerns about the researchers' claim that carpenter ants exhibit discontinuous gas exchange? Explain.

I would have two major concerns with their claim. First, the closed phase has an oxygen consumption rate that is distinctly non-zero, suggesting that some degree of oxygen consumption is occurring during this phase when all the spiracles are supposed to be closed. The fact that oxygen consumption rate is reduced but still occurs is more reminiscent of cyclic gas exchange. Second, during the fluttering phase, while oxygen consumption rate is higher than during the closed phase, it is still quite low. As we discussed in class, during the fluttering phase, air is able to enter into the tracheal system due to a favourable pressure gradient between the atmosphere and the tracheal system, so one would expect that oxygen consumption rates would be much higher, closer to what is observed when the spiracles are open. Thus, overall, I propose that the researchers are observing cyclic gas exchange, not discontinuous gas exchange in their carpenter ants.

In a 2011 study published in Journal of Experimental Biology, Dr. Heidy Contreras showed that common water striders (pictured below) exhibited discontinuous gas exchange. Why do you suppose that this observation merited publication in such a prestigious scientific journal?

As the image illustrates, common water striders spend a considerable amount of time on the surface of the water; consequently, they inhabit a relatively humid environment where water is readily available for consumption and where the air directly above the water, where they are found, should be fairly saturated with water. The fact that common water striders exhibit discontinuous gas exchange (DGE) seems to refute the hygric hypothesis: this hypothesis states that DGE evolved in order to minimize respiratory water loss, but this would seem to be unimportant for common water striders given the abundance of water in their habitat. Given that the hygric hypothesis is one of the widely-accepted explanations for DGE evolution in insects, data that refute the hypothesis would be noteworthy to the biological community, justifying their publication in prestigious journals. In a similar vein, because water striders spend a considerable amount of time on the surface of the water, they should not be routinely subjected to hypoxia and/or hypercapnia, which would mean that their exhibition of DGE cannot be explained by the chthotic hypothesis, either. Thus, the occurrence of DGE in water striders likely challenged the accepted hypotheses for DGE and, therefore, was a a noteworthy observation for the scientific community. 

The "eye and ruler" method for measuring hematocrit from centrifuged blood samples within a capillary tube--as we did in this week's lab exercise--has been the "gold standard" for hematocrit determination for decades. In a 2022 paper, researchers from University of Zurich (Switzerland) proposed an alternative method for hematocrit determination that involves using Image J to analyze photographs of centrifuged blood samples with a capillary tube. Specifically, the researchers proposed using Image J to draw lines from the top to the bottom of the packed red blood cells (as shown) and from the top to the bottom of the total blood, then having Image J determine the length of these two lines. To validate their proposed method, the researchers measured hematocrit for the same blood samples using both methods. Their findings are shown below. As you can see, there was a very strong correlation between the two methods, but the Image J method always gave higher hematocrit values compared to the eye and ruler method.

a) Why do you Image J method generally yielded higher hematocrit values compared to the eye and ruler method?

Because there are spaces between even the smallest markings on the rulers, there is always going to be some level of estimation when measuring the packed red blood cells or total blood with a ruler, which doesn't exist with Image J. What is most interesting here is that, with the ruler, we must either i) always overestimate the length of the total blood or ii) always underestimate the length of the packed red blood cells. That's the only way to get consistently higher hematocrit values. Perhaps the difference in the colour of these two components has a role here? 

b) Do the findings of this study invalidate any conclusions that you made in this week's experiment regarding hematocrit? Explain.

No. This week's experiment was mostly a comparative study; that is, we were investigating how hematocrit varies among different mammal and bird species. On this basis, the absolute values of hematocrit are not important per se: only the relative values of hematocrit, i.e., how hematocrit of one species compares to that of the other species, is important. Therefore, although the Image J method yields higher absolute values for hematocrit, given the strong correlation between the two methods, the relative hematocrit values are the same for both methods. So, either method could be used in a comparative context, but, if we were doing a study where the absolute value of hematocrit was important, then we would have to carefully consider which method is giving a more accurate result. 

A diagnosis of spontaneous bacterial peritonitis (SBP) requires the detection of the presence of elevated numbers of white blood cells (WBCs) in the peritoneal fluid (i.e., > 1000 cells/mm³). In a 2013 study published in Clinical Biochemistry, researchers collected peritoneal fluid from 100 hospitalized patients and used two methods to count WBCs within this fluid: manual counting with hemacytometer and microscope (as in our laboratory exercise) and automatic counting using flow cytometry. 

a) The researchers found that the WBC count was consistently higher with one method compared to the other. Which method do you suppose yielded higher WBC counts? Justify your answer.

Probably the flow cytometer. Having had the experience of conducting a manual count of RBCs in this week's lab exercise, I know that it can be difficult to ensure that every single cell has been counted, especially if one is not very systematic in their approach to counting. Also, one may quickly fatigue from counting cells, such that, while the first few squares counted may be accurate, the last few squares may be less accurate. Even having two people conduct each count and taking an average still means that, for the reasons above, the manual count is likely to be lower than an automatic count because if at least one of the two people "undercounts" the cells, then the average will be lower than it should be.

b) In light of your answer to part a) above, do you think that the Biological Sciences Department at UTSC should invest in a flow cytometer for the BIOB32 labs? Justify your answer.

b) The objective of this lab experiment was to compare RBC counts among different mammals and birds. Even if the students consistently undercount the number of RBCs in the blood sample, as long as they do so for all species, then the relative differences in RBC counts among species would be same with manual vs. automatic counting. However, given the comment above about the potential for students to fatigue of counting over time, it would be imperative that the order in which blood samples are counted is not the same for all groups (i.e., some groups should do bovine first, but some groups should do bovine last) to minimize any bias resulting from counting fatigue over the course of the experiment. Given the significant cost of a flow cytometry system, it would not likely provide much better information than can be achieved by manual counting. In the case of SBP diagnosis described in the question, an accurate count is more important--and hence flow cytometry may be worthwhile--because it's the absolute WBC number that determines the diagnosis.