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eukaryotic cell division cycle? Trends Cell Biol. 11, 366371. Hetzer, M., Gruss, O.J., and Mattaj, I.W. (2002). The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly. Nat. Cell Biol. 4, E177E184. Dasso, M. (2002). The Ran GTPase: theme and variations. Curr. Biol. 12, R502R508. Gruss, O.J., Carazo-Salas, R.E., Schatz, C.A., Guarguaglini, G., Kast, J., Wilm, M., Le Bot, N., Vernos, I., Karsenti, E., and Mattaj, I.W. (2001). Ran induces spindle assembly by reversing the inhibitory effect of importin a on TPX2 activity. Cell 104, 8393. Koffa, M.D., Casanova, C.M., Santarella, R., Kocher, T., Wilm, M., and Mattaj, I.W. (2006). HURP (Hepatocarcinoma-Upregulated) is part of a Ran-dependent complex involved in spindle. Curr. Biol. 16, 743754. Sillje, H.H.W., Nagel, S., Korner, R., and Nigg, E.A. (2006). HURP is a Ran-importin beta regulated protein that stabilizes kinetochore microtubules in the chromosomes. Curr. Biol. 16, 731742. Fleig, U., Salus, S.S., Karig, I., and Sazer, S. (2000). The ssion yeast Ran GTPase is required for microtubule integrity. J. Cell Biol. 151, 11011112. Salus, S.S., Demeter, J., and Sazer, S. (2002). The Ran GTPase system in ssion yeast affects microtubules and cytokinesis in cells that are competent for nucleocytoplasmic protein transport. Mol. Cell Biol. 22, 84918505. Hirose, E., Mukai, M., Shimada, A., Nishitani, H., Shibata, Y., and Nishimoto, T. (2006). Loss of RanGEF/

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Pim1 activity abolishes the orchestration of Ran-mediated mitotic cellular events in S. pombe. Genes Cells 11, 2946. Tanaka, K., Mukae, N., Dewar, H., van Breugel, M., James, E.K., Prescott, A.R., Antony, C., and Tanaka, T.U. (2005). Molecular mechanisms of kinetochore capture by spindle microtubules. Nature 434, 987994. Sato, M., and Toda, T. (2007). Alp7/TACC is a crucial target in Ran-GTPasedependent spindle formation in ssion yeast. Nature 447, 334337. Oliferenko, S., and Balasubramanian, M.K. (2002). Astral microtubules monitor metaphase spindle alignment in ssion yeast. Nat. Cell Biol. 4, 816820. Sato, M., Vardy, L., Garcia, M.A., Koonrugsa, N., and Toda, T. (2004). Interdependency of ssion yeast Alp14/ TOG and coiled coil protein Alp7 in microtubule localization and bipolar spindle formation. Mol. Biol. Cell 15, 16091622. Sato, M., Koonrugsa, N., Toda, T., Vardy, L., Tournier, S., and Millar, J.B. (2003). Deletion of Mia1/Alp7 activates Mad2-dependent spindle assembly checkpoint in ssion yeast. Nat. Cell Biol. 5, 764766. Zheng, L., Schwartz, C., Wee, L., and Oliferenko, S. (2006). The yeast transforming acidic coiled coil-related protein Mia1/Alp7 is required for formation and maintenance of persistent microtubule-organizing centers at the nuclear envelope. Mol. Biol. Cell 17, 22122222. Caudron, M., Bunt, G., Bastiaens, P., and Karsenti, E. (2005). Spatial coordination of

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Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK. 2Department of Biochemistry, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA. E-mail: p.r.clarke@dundee.ac.uk

DOI: 10.1016/j.cub.2007.06.023

Respiratory Biology: Why Insects Evolved Discontinuous Gas Exchange

Many, but not all, insects breathe in a discontinuous gas-exchange cycle. A recent study has evaluated rival hypotheses for the evolution of this trait, concluding that the most likely is the one invoking minimization of respiratory water loss. John R.B. Lighton Drama resides in unexpected places, and the way insects breathe is no exception. Rather than rely on passive diffusion, many taxa of tracheate arthropods including insects the most numerous animals on the planet express an extraordinary discontinuity in external gas exchange. It veers from almost nothing, to normal oxygen uptake but minimal CO2 output, to a burst of CO2 and an incoming ood of oxygen. Meet the tripartite discontinuous gas-exchange cycle, or DGC, which has invited much speculation. A recent paper by White et al. [1] examines the competing hypotheses to explain the origins of the DGC, which are that it evolved to: reduce respiratory water loss (the hygric hypothesis [2]); generate concentration gradients that allow breathing in underground environments (the chthonic hypothesis [3,4]); or reduce exposure to oxygen (the oxidative damage hypothesis [5]). To make sense of the controversy, consider the story of the DGC (reviewed in [4,6,7]). The DGC begins with the spiracles, the gateways to the

tracheal system, tightly closed (the closed-spiracle or C phase). Negligible external gas exchange occurs, so oxygen concentration within the trachea falls and CO2 concentration rises. At a critical oxygen concentration of about 45% the spiracles start to utter (the F phase begins) and oxygen enters through the spiracles at a rate equivalent to mitochondrial respiration. The primary entry mechanism is diffusion [7], but bulk ow down a pressure gradient, generated by removal of oxygen from the tracheal space, may also play a role in minimizing water loss during the F phase [6]. This is all well and good. The mitochondria stoke their res while the animal conserves its scarce water reserves. Yet the mitochondria also generate CO2. Some CO2 escapes by diffusion and is measurable with sensitive instrumentation, but this amounts to only about 1525% of the insects mitochondrial rate of CO2 production.

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The curious reader might wonder why the insect cannot release CO2 at its mitochondrial production rate, seeing that it is obtaining sufcient oxygen. The answer lies in the difference between the oxygen and CO2 concentration gradients across the spiracles. Tracheal oxygen concentration holds steady at about 45% during the F phase [4,6,7], generating a formidable trans-spiracular concentration gradient of 1617% in normal air which contains 21% oxygen. On the other hand, tracheal CO2 levels hover around 4% [5], a fourfold weaker concentration gradient against the outside air which for present purposes contains zero CO2. This handily explains the approximately four-fold mismatch between CO2 production (by the mitochondria) and CO2 emission (through the spiracles) during the F phase. Thus, CO2 accumulates during the F phase; the F phase is not sustainable. Eventually CO2 accumulation prevails over the water-conserving F phase, and the spiracles open up (the O phase). At this point, by denition, CO2 has built up to its maximum tolerable concentration and it oods down the strong concentration gradient, encouraged in larger insects by active ventilation. With it goes precious water vapor. If you have a sensitive enough balance, you can watch the digits ratchet downward faster during each O phase [8]. And oxygen comes ooding in [5]. This is the oxidative damage hypothesiss pivotal weakness. The F phase allows oxygen to be regulated at a low, constant level, but with every cycle the DGC brings a tsunami of oxygen. Also, insects such as termites accommodate obligate symbionts with a known hypersensitivity to oxygen. If the oxidative damage hypothesis is correct, surely termites will boast champion DGCs? But instead, they are continuous exchangers [9], at least in normoxia (what they do in their hypoxic citadels is unknown). The y in the ointment is that, although many insects express a DGC, many others, including those that you would predict would

express it, such as desert-dwellers, do not. What to make of this? Like me, most scientists are happy to be higher primates preadapted for no good reason to making fabulously complicated twigs with which to sh out delicious and entertaining nuggets from the termite mound of consensual reality. Some nugget collections form patterns that jibe with accepted theory, itself a product of induction based on observation. The DGCs role in reducing water loss is just one of millions of examples. My early work described DGCs in some beetles and ants. Wherever I looked, I found more examples. Yet I also found some beetles and ants ignorant of the literature; they exchanged respiratory gases continuously [4]. Heres the thing: Many successful inhabitants of dry areas turned out to be continuous breathers, including workers of a hyper-xeric ant yet in this species the queen, who spends her early life sealed in an underground chamber and later always stays in the nest, expresses the DGC [3]. Looking at published data, it became evident that insects and other tracheate arthropods such as solphugids generally displayed a DGC if they spent at least some of their time underground, where oxygen concentrations were low and CO2 concentrations high. The above account of the DGC shows that it generates spectacular concentration gradients that should allow successful oxygen uptake and CO2 emission, even in seriously challenging underground or chthonic environments [3,4]. Water conservation is a given. Any spiracular control regimen will be evolutionarily selected to accomplish it, and evidence is mounting steadily that merely expressing the DGC confers no special hygric blessings [10]. White et al.s [1] study is the rst informed, rigorous and phylogenetically correct broad-scale attempt to evaluate the hygric, oxidative damage and chthonic hypotheses. Apart from phylogeny their analysis includes environmental temperature, water availability and likely microhabitat oxygen availability, all tested in various combinations against DGC

duration. Unfortunately the oxygen availability metric is guesswork, thanks to an almost complete lack of relevant microhabitat measurements in the literature. Oxygen availability turns out not to have a signicant effect, but including it in the model causes only a modest reduction of the models explanatory power. Thus, the chthonic and oxidative damage hypotheses are on the ropes but by no means out of the game. New measurements might energize either, and White and his collaborators have provided an excellent broad-scale analytical framework for future work. The caveat is that intercomparing DGCers, as they did, may not be as enlightening as comparing DGCers with non-DGCers. Experimental manipulation continues to be important, because broad-scale approaches [1] usually address what is now the case, not the mechanistic basis underlying how that came to be. Given that all of the insects in the study expressed the DGC, it is not surprising that the DGC was strongly affected by habitat-temperature and water availability, thus favoring the hygric hypothesis. But what were the selective pressures behind the evolution of the DGC in these groups in the rst place, versus those groups that did not evolve this dramatic way of breathing, or have lost it secondarily? This question is at the heart of the controversy, and remains tantalizingly unresolved.
References
1. White, C.R., Blackburn, T.M., Terblanche, J.S., Marais, E., Gibernau, M., and Chown, S.L. (2007). Evolutionary responses of discontinuous gas exchange in insects. Proc. Natl. Acad. Sci. USA 104, 83578361. 2. Lighton, J.R.B. (1996). Discontinuous gas exchange in insects. Annu. Rev. Entomol. 41, 309324. 3. Lighton, J.R.B., and Berrigan, D. (1995). Questioning paradigms: Caste-specic ventilation in harvester ants, Messor pergandei and M. julianus (Hymenoptera: Formicidae). J. Exp. Biol. 198, 521530. 4. Lighton, J.R.B. (1998). Notes from underground: Towards ultimate hypotheses of cyclic, discontinuous gas exchange in tracheate arthropods. Amer. Zool. 38, 483491. 5. Hetz, S.K., and Bradley, T.J. (2005). Insects breathe discontinuously to avoid oxygen toxicity. Nature 433, 516519. 6. Kestler, P. (1985). Respiration and respiratory water loss. In Environmental Physiology and Biochemistry of Insects,

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K.H. Hoffmann, ed. (Springer: Berlin), pp. 137186. 7. Chown, S.L., Gibbs, A.G., Hetz, S.K., Klok, C.J., Lighton, J.R.B., and Marais, E. (2005). Discontinuous gas exchange in insects: A clarication of hypotheses and approaches. Physiol. Biochem. Zool. 75, 345349. 8. Lighton, J.R.B. (1992). Simultaneous measurement of CO2 emission and mass loss in two species of ants. J. Exp. Biol. 173, 289293.

9. Lighton, J.R.B., and Ottesen, E.A. (2005). To DGC or not to DGC: Oxygen guarding in the termite Zootermopsis nevadensis (Isoptera: Termopsidae). J. Exp. Biol. 208, 46714678. 10. Lighton, J.R.B., Schilman, P.E., and Holway, D.A. (2004). The hyperoxic switch: Assessing respiratory water loss rates in tracheate arthropods with continuous gas exchange. J. Exp. Biol. 207, 44634471.

School of Life Sciences, University of Nevada at Las Vegas, 4505 Maryland Parkway, Las Vegas, Nevada 89154-4004, USA, and Sable Systems International, 6340 S. Sandhill Rd., Suite 4, Las Vegas, Nevada 89120, USA. E-mail: Jrlighton@aol.com

DOI: 10.1016/j.cub.2007.06.007