Responses of terrestrial insects to hypoxia or hyperoxia

Respiratory Physiology & Neurobiology 154 (2006) 4–17

Responses of terrestrial insects to hypoxia or hyperoxia夽 Jon Harrison a,∗ , Melanie R. Frazier b,1 , Joanna R. Henry a,1 , Alexander Kaiser c,1 , C.J. Klok a,1 , Brenda Rasc´on a,1 a

Section of Organismal, Integrative and Systems Biology, School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, United States b Department of Biology, Box 351800, University of Washington, Seattle, WA 98115-1800, United States c Department Basic Sciences, Midwestern University, Glendale, AZ 85308, United States Accepted 10 February 2006

Abstract Oxygen is critically important for catabolic ATP generation but is also a dangerous source of reactive oxygen species. Insects respond to short-term exposure to hypoxia or hyperoxia with compensatory changes in spiracular opening and ventilation that reduce variation in internal PO2 . Below critical PO2 values (Pc), nitric oxide and hypoxia inducible factor (HIF)-mediated pathways induce long-term responses such as compensatory tracheal growth, suppressed development, and acclimation of ventilation. Pc values are strongly affected by activity and ontogeny, due to changes in the ratio of tracheal conductance to metabolic rate. Although growth rates and development are suppressed by significant hypoxia in all species studied to date, adult body size is only affected in some species. Severe hyperoxia causes major oxidative stress and reduces survival, while moderate hyperoxia increases development times and body sizes in some species by unknown mechanisms. © 2006 Elsevier B.V. All rights reserved. Keywords: Oxygen; Insects; Critical PO2 ; Hyperoxia; Hypoxia

夽 This paper is part of a special issue entitled “Frontiers in Comparative Physiology II: Respiratory Rhythm, Pattern and Responses to Environmental Change”, guest edited by W.K. Milsom, F.L. Powell and G.S. Mitchell. ∗ Corresponding author. Tel.: +1 480 965 9459. E-mail addresses: [email protected] (J. Harrison), [email protected] (M.R. Frazier), [email protected] (J.R. Henry), [email protected] (A. Kaiser), [email protected] (C.J. Klok), [email protected] (B. Rasc´on). 1 These authors contributed equally.

1569-9048/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2006.02.008

1. Introduction For terrestrial insects, like all aerobic organisms, O2 is critically important in catabolic ATP generation but is also a dangerous source of reactive oxygen species (ROS) that cause damage to biomolecules. Insects must regulate internal PO2 within a fairly narrow range to maintain aerobic metabolism while avoiding oxygen toxicity. The regulation of internal PO2 is challenging

J. Harrison et al. / Respiratory Physiology & Neurobiology 154 (2006) 4–17

for all insects, even those living in open, low-elevation habitats due to scope of their metabolic rates. Studies of the effects of ambient PO2 (APO2 ) on function are central to understanding basic and applied questions in insect respiratory physiology including: (1) mechanisms of respiratory control, (2) acclimation and adaptation to hypoxic environments such as altitude or burrows, (3) the effects of oxidative stress, (4) the influence of APO2 on body size, and (5) use of controlled atmospheres as pest control. Here we review the literature, add new data, and suggest frontiers for study of the responses of terrestrial insects to hypoxia and hyperoxia. In Part I, we consider central nervous systemmediated, homeostatic responses of the respiratory system that reduce effects of APO2 on internal PO2 . These mechanisms provide rapid compensatory responses to variation in APO2 by altering resistance to gas exchange at each of the major steps in oxygen delivery from atmosphere to cell. The molecular mechanisms of oxygen-sensing by the insect nervous system are poorly known, but may involve haem-containing proteins and/or O2 -sensitive K+ channels as in mammals (Prabhakar, 2006). The CO2 - and O2 -sensitive central nervous system controls both the muscles of the spiracular valves and the muscles that drive convective air flow through tracheae and spiracles (Fig. 1). Resistance to oxygen flux through the terminal tracheoles is altered by changes in tracheolar fluid levels in response to local PO2 (Wigglesworth, 1983). Despite the many mechanisms for modulating tracheal conductance, changes in tissue PO2 do occur, and these lead to a variety of longer-term responses, which are covered in Part II. Longer-term tissue hypoxia activates secretion of nitric oxide (NO) from specific cells and expression of hypoxia inducible factor (HIF) in others (Fig. 1). These lead to a variety of downstream effects such as secretion of a fibroblast growth factor (FGF) homologue from hypoxic tissues to stimulate tracheal growth and branching (Jarecki et al., 1999). Changes in internal PO2 can have other direct effects on long-term physiological function by altering mitochondrial ATP or ROS production (Fig. 1). The short- and long-term homeostatic responses to hypoxia or hyperoxia are imperfect and have limited capacities. In Part III, we address physiological, behavioral and ecological factors that determine when oxygen becomes limiting or excessive. The critical

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Fig. 1. Schematic of the insect respiratory system and a generalized insect cell illustrating data-supported homeostatic pathways of responses to variation in ambient PO2 . Locations of putative oxygen sensors are indicated by a star; details of these pathways are in the text. The diagram illustrates responses to hypoxia, but for most links, there is evidence that converse responses occur during hyperoxia. For explanation of acronyms, see text.

PO2 (Pc) can be measured as the minimum or maximum PO2 that can sustain a rate process (such as metabolic or growth rate). Measurement of Pc across and within species, and for different processes can provide insight into many aspects of tracheal system design. In each section, we attempt to synthesize results of past research and point to important directions for future study. Throughout, we integrate previously unpublished data on responses of grasshoppers to hypoxia and hyperoxia. Most of these new data are from a study of the response of the American locust, Schistocerca americana to long-term rearing in different APO2 . Animals were reared from the 1st instar to adulthood, in 5 l chambers perfused with APO2 of 5, 10, 21, or 40 kPa (balance N2 ) at 30 ◦ C. We used two rearing chambers for each oxygen treatment, each containing 40 grasshoppers (N = 80 grasshoppers per rearing oxygen). Growth, tracheal dimensions, and ventilatory responses of these animals were measured as described below.

↓ ↓ ↑ ↑ ↓ = ↑

40–59 35–100 25–50

= 31–61 ↑ ↑

=

↓ =

=

↓ =

4–14.4 0–15 6.4–13

↑

↓ ↓

↑ ↑

=

↓ = ↓ ↑ ↓ ↓ ↓ ↑ ↓ = 5–15 6–15 4–8 11

Hyalophora cecropia Hyalophora cecropia Attacus atlas

Dung beetle Carpenter ant Longhorn beetle Horse lubber grasshopper Cecropia moth Cecropia moth Atlas moth Aphodius fossor Camponotus vicinus Orthosoma brunneum Taeniopoda eques

7 7 7.3

References tF tC tcycl Hyperoxia

APO2 (kPa) tC tcycl

tF

tO

O(VCO2 )

O(MCO2 )

F(MCO2 ) Hypoxia

APO2 (kPa)

Mass (g)

Effects of APO2 on the spiracles of insects exhibiting cyclic or discontinuous gas exchange (DGC) are complex and must be analyzed for each part of the cycle. During DGC, the burst-like CO2 release in the open phase (O-phase) is followed by a phase of spiracular constriction (C-phase) and a succeeding flutter phase (F-phase), during which quick openings of single spiracles allow release of some CO2 until the next Ophase. (see Gibbs and Quinlan, this volume; Bradley, this volume). The affects of APO2 on some parameters of DGC depend on the insect species; for example, the duration of the cycle can be increased, decreased, or unaffected by APO2 (Table 1). This observation suggests that there is important among-species variation in parameters such as PO2 and PCO2 trigger points, buffering capacity, relative tracheal volumes, and the ability to generate convective ventilation. Insects using DGC show two consistent responses to APO2 ; as APO2 decreases (a), the C-phase becomes shorter in duration, and (b) during the F-phase, the rate of CO2 emission increases (Table 1). The positive correlation between APO2 and C phase duration supports the hypothesis that depletion of oxygen inside the tracheal system triggers the end of the C-phase at a certain setpoint determined by PO2 rather than by subatmo-

Common name

2.2. Ambient PO2 effects on discontinuous gas exchange cycle (DGC) patterns

Genus species

The spiracles control oxygen delivery across the body wall and are the major barrier to oxygen transport for insects at low metabolic rates. Spiracles open more frequently, for longer duration, or wider when external PO2 is reduced (Wigglesworth, 1935; Case, 1956). The spiracular valves are closed for longer periods when the ambient air is hyperoxic (Burkett and Schneiderman, 1974). Thus, hypoxia usually causes a temporary increase in CO2 and respiratory H2 O emission (Greenlee and Harrison, 1998, 2004a), while hyperoxia causes the converse (Lighton et al., 2004). These effects are mediated by sensors and integratory centers in the central nervous system (Burkett and Schneiderman, 1974).

Table 1 Observed effects (↑: increase; =: no consistent change; ↓: decrease) of ambient hypoxia and hyperoxia on DGC pattern and CO2 release rates of resting insects

2.1. Ambient PO2 effects on spiracles

Effects on durations of the entire cycle (tcycl ), closed phase (tC ), flutter phase (tF ), and open phase (tO ) are given, as well as effects on volume of CO2 released during the O phase (VCO2 ), and rates of CO2 release during the open (O(MCO2 )) or flutter phases (F(MCO2 )). See text for further explanation. * For O. brunneum, no mass was given so body length is presented.

Chown and Holter (2000) Lighton and Garrigan (1995) Paim and Beckel (1963) Harrison et al. (1995)

2. Short-term, homeostatic responses of the respiratory system to hypoxia/hyperoxia

Levy and Schneiderman (1966) Schneiderman (1960) Hetz and Bradley (2005), Grabbert et al. (2003)

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0.1 0.1 4 cm* 2.3

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spheric pressure (Hetz and Bradley, 2005). As APO2 increases, more oxygen enters the tracheal system during the O-phase, extending the time it takes for internal PO2 to become low enough to trigger cessation of the C-phase. The negative correlation between APO2 and F-phase CO2 emission may be due reduced suction ventilation at lower APO2 . Suction ventilation occurs during the Cand F-phases due to negative internal pressures created when oxygen is consumed from the tracheal system and not replaced by CO2 , much of which dissolves in body fluids (Levy and Schneiderman, 1966; Hetz and Bradley, 2005). During the F-phase, net CO2 emission will result from the balance between diffusive efflux and suction ventilation influx. At lower APO2 , there will be less oxygen in the tracheae to be consumed and therefore less of a negative internal pressure will be created. Hypoxia may also reduce internal negative pressures by increasing spiracular opening. In many insects, the DGC pattern is replaced by a continuous CO2 release pattern at a critical APO2 (Lighton and Garrigan, 1995; Chown and Holter, 2000). The most obvious explanation (as yet, not rigorously tested) is that tracheal PO2 falls below a threshold, stimulating the central ganglia to continuously open the spiracles. 2.3. Ambient PO2 effects on ventilation To support and facilitate gas exchange, insects can compress regions of their body to create convective gas flow. These observable ventilatory movements are stimulated by hypoxia and suppressed by hyperoxia (Fig. 2). Oxygen-sensitivity of ventilation appears to be driven by central nervous system sensors (Bustami et al., 2002; Bustami, this volume). The increase in ventilation during hypoxia is sufficient to account for the increase in spiracular conductance in grasshoppers (Greenlee and Harrison, 1998). Ventilation is strongly stimulated by ambient hypoxia, but only weakly suppressed by ambient hyperoxia (Fig. 2). Suppression of ventilation by hyperoxia must cause CO2 to accumulate, providing a counterbalancing drive to increase ventilation, perhaps explaining the less-than-expected suppression of ventilation by hyperoxia. An additional factor may be stimulation of ventilation by reactive oxygen species produced by hyperoxia, as has been hypothesized in ver-

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Fig. 2. Ventilatory frequency as a function of APO2 (open triangles, dashed line, T. eques, Bustami et al., 2002; closed circles, Schistocerca americana, Greenlee and Harrison, 1998; closed diamonds, Schistocerca americana, Harrison, unpublished data), or as a function of PO2 in perfused trachea (filled triangles, Schistocerca americana, Gulinson and Harrison, 1996), or PO2 in perfused buffer solutions (open circles, dotted line, N. cinerea, Snyder et al., 1980).

tebrates (Dean et al., 2004). Likewise, hypoxia’s stimulatory effects on ventilation are likely to be mitigated by a reduction in internal PCO2 . In support of this idea, hyperoxia or hypoxia induced by tracheal (Gulinson and Harrison, 1996) or hemolymph (Snyder et al., 1980) perfusions that maintain a constant PCO2 affect ventilation more strongly than changes in APO2 (Fig. 2). In contrast to the strong effect of hemolymph PO2 on ventilation rate in cockroaches (Snyder et al., 1980; Fig. 2), spiracular behavior is not affected by varying PO2 in the buffer solution surrounding the controlling ganglion in silkmoth pupa (Burkett and Schneiderman, 1974). Either ventilation and spiracular activity are controlled by different mechanisms in cockroaches and moth pupae, or the hemolymph–tissue barriers possess different permeabilities for respiratory gases in these two species. 3. Longer-term responses to variation in ambient PO2 3.1. Molecular pathways As in vertebrates (Moncada and Higgs, 1993), hypoxia activates both the nitric oxide (NO)/cyclic

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J. Harrison et al. / Respiratory Physiology & Neurobiology 154 (2006) 4–17

GMP pathway and the hypoxia-inducible factor (HIF) pathway in insects (Fig. 1). Local hypoxia causes a rise in NO production in certain tissues of Drosophila larvae, especially in pouches of tissues near the spiracles and in the imaginal disks (Wingrove and O’Farell, 1999). NO diffuses to other tissues and activates guanylyl cyclase, stimulating a downstream cascade that includes activation of protein kinase G (PKG) and a variety of effects including escape behavior, tracheal proliferation, and developmental arrest (Wingrove and O’Farell, 1999). Over-expression of nitric oxide synthase (NOS) causes a greater hypoxia response (more developmental arrest and tracheal proliferation), whereas knocking out the PKG gene or inhibiting NOS reduces such responses. Hypoxia also activates HIF pathways, at least in Drosophila embryos and larvae (Ma and Haddad, 1999). Many of the structural and functional properties of HIF in insects appear to be very similar to those of vertebrate homologues (Lavista-Llanos et al., 2002). As in vertebrates (Maxwell, 2004), HIF in insects is a heterodimer of basic-helix-loophelix-PAS proteins that bind to hypoxia response elements (HREs) during hypoxia. Concentrations of the HIF-␣ subunit depend on PO2 , whereas the HIF␤ subunit is expressed regardless of PO2 . HIF-␣ is broken down at a high rate in normoxia by an oxygen-dependent ubiquination process and by prolyl hydroxylation (Lavista-Llanos et al., 2002; Maxwell, 2004). The downstream consequences of HIF induction are poorly understood in insects. However, there is evidence that HIF leads to induction of a homologue of fibroblast growth factor (FGF), a product of Drosophila gene branchless. The FGF is secreted by hypoxic tissues, stimulating local tracheoles to form new branches and move towards hypoxic tissues (Jarecki et al., 1999). In addition, hypoxia and HIF homologue induction stimulates scylla and charybdis (homologues of mammalian genes with roles in apoptosis, Reiling and Hafen, 2004). Overexpression of scylla inhibits growth, and flies lacking functional scylla and charybdis experience higher mortality in hypoxia. HIF levels are highest during late embryogenesis (Ma and Haddad, 1999; Lavista-Llanos et al., 2002), which may indicate that embryos tend to be hypoxic before hatching or that HIF has other, yet-to-be explored functions.

3.2. Compensatory responses of tracheal morphology to ambient PO2 While the spiracles are open, the number, length, diameter and wall thickness of the tracheae and tracheoles will determine the diffusive capabilities of the insect respiratory system. Dimensions of the major tracheae will also determine the pressures required for convective transport. Oxygen movement through the tracheal system can be conceptualized as a longitudinal movement down the length of the tracheae, followed by lateral movement across the walls of the finest tracheae and tracheoles, and finally through the hemolymph, plasma membrane and cytosol to the mitochondria. Lateral movement of oxygen across the walls of the larger tracheae is inconsequential due to the thickness of these walls and the relatively low surface area of these tracheae (Schmitz and Perry, 1999; Hartung et al., 2004). As yet, no studies have provided an unbiased statistical evaluation of the effect of hypoxia or hyperoxia on the diffusing capacity of the tracheal system of an entire insect (e.g. as done by Schmitz and Perry, 1999). However, a compensatory change in tracheal number (branching) in response to APO2 has been demonstrated in all species for which this parameter has been measured (Table 2). Compensatory changes in tracheal diameter or length in response to changing APO2 also occur in insects, but this response varies among insect species (Table 2). In the mealworm, Tenebrio molitor, changes in tracheal diameter nearly perfectly accomodate changes in APO2 (assuming diffusive gas exchange, Loudon, 1989). However, the butterfly, Calpodes ethlius, and the sucking bug, Rhodnius prolixus, reared under the same hypoxic conditions as Tenebrio, show much weaker or minimal changes in tracheal dimensions (Locke, 1958). In our study of S. americana, we measured the lengths and diameters of the 6th dorsal transverse tracheae (for methods description, see: Harrison et al., 2005) at each instar of grasshoppers reared at 5, 10, 21, and 40 kPa APO2 . A regression analysis including both instar and oxygen treatment as factors revealed that rearing oxygen had no significant effect, or apparent trend, on either tracheal length (t = −0.579, P = 0.566, N = 41) or diameter (t = 1.275, P = 0.21, N = 41; 1/trachea width was analyzed to make this variable normally distributed). The explanation for this among species variation is

J. Harrison et al. / Respiratory Physiology & Neurobiology 154 (2006) 4–17

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Table 2 Developmental plasticity of insect tracheal systems in response to hypoxia or hyperoxia Genus species

Common name

APO2

Drosophila melanogaster

Fruitfly

Drosophila melanogaster Drosophila melanogaster

Fruitfly Fruitfly

Tenebrio molitor Tenebrio molitor

Mealworm Mealworm

Calpodes ethlius Rhodnius prolixus Rhodnius prolixus Schistocerca americana

Brazilian skipper butterfly Sucking bug Sucking bug American locust

Hyperoxia Hypoxia Hypoxia Hyperoxia Hypoxia Hypoxia Hypoxia Hyperoxia Hypoxia Hypoxia Hypoxia Hypoxia Hyperoxia

unclear. One interpretation is that multiple mechanisms can achieve the same phenotype (compensation for APO2 ). In support of this hypothesis, at least one insect species, R. prolixus appears to compensate for hypoxia by increasing the number, rather than the size, of tracheae (Table 2). Compensatory changes in tracheal dimension also appear to vary among populations within an insect species. Beitel and Krasnow (2000) found that length of the main dorsal tracheae decreased without a change in diameter in response to hypoxia in Drosophila melanogaster, whereas Henry and Harrison (2004) found that hypoxia increased diameters of the same tracheae without a change in length (length data were collected and analyzed but unpublished by Henry and Harrison). The fact that both changes increase tracheal diffusive capacities, but by different mechanisms, supports the hypothesis that identical functional outcomes can occur through different pathways, depending on the genetic background of the populations being used. Thus far, there have been no comparative studies of the tracheal systems of insect species that live in hypoxic environments such as high altitudes or burrows. However, the intraspecific developmental plasticity in tracheal number and dimensions certainly suggest that insects living in hypoxic conditions would exhibit compensatory increases in tracheal system conductance. Laboratory selection for fitness in hypoxic or hyperoxic environments produces heritable, compensatory changes in tracheal morphology of fruitflies (Henry and Harrison, 2004).

Length

↓

↑

Diameter

= ↓ ↑ ↑ ↑

Branching

References

↓ ↑

Jarecki et al. (1999) Beitel and Krasnow (2000) Henry and Harrison (2004)

↑

↑ = ↑

= =

Loudon (1989) Locke (1958)

= =

Wigglesworth (1954) This study

3.3. Developmental plasticity of ventilatory response to ambient PO2 Many terrestrial insects modulate ventilation rate in response to acute changes in APO2 , however, it is unknown how ventilation changes in insects exposed to chronic hypoxia or hyperoxia. Mammals exposed to hypoxic conditions for hours to days exhibit enhanced ventilation at a given APO2 (Huey et al., 2000). However, very long-term exposures (e.g. rearing in hypoxic conditions) causes a blunting of the ventilatory response to hypoxia in vertebrates (Ward et al., 2000). We measured the ventilation rate of adult grasshoppers that had been reared, from the 1st instar, in 5, 10, 21, or 40 kPa APO2 . These animals had statistically identical ventilation rates (counted visually, rates measured with a stopwatch) when measured at rest in their cages, despite the dramatically different APO2 values (ANOVA, F1,40 = 0.19, P = 0.66; Fig. 3). However, when we measured the ventilation rates of these same grasshoppers at 21 kPa APO2 , animals reared in hypoxic conditions exhibited dramatically reduced ventilation rates (ANOVA, F1,40 = 12.14, P = 0.0012; Fig. 3). The degree of abdominal compression at 21 kPa APO2 (measured using micro-videography as described in Greenlee and Harrison, 2004a) did not vary among treatment groups (ANOVA, F1,14 = 0.79, P = 0.38) suggesting that the hypoxic-reared grasshoppers strongly reduced convective ventilation in 21 kPa PO2 . One explanation for this pattern is sensory adaptation of the PO2 sensors that drive ventilation.

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J. Harrison et al. / Respiratory Physiology & Neurobiology 154 (2006) 4–17

Fig. 3. Ventilation rates of adult Schistocerca americana grasshoppers reared at various ambient PO2 values. Ventilation rates did not differ among the APO2 treatment groups (P = 0.54, F = 0.7322, df = 3, 38) for animals measured in their cages, at their rearing PO2 . When animals were removed from the cages and tested at 21 kPa, there was a strong effect of rearing APO2 on ventilation rate (P = 0.007, F = 4.68, df = 3, 38).

Alternatively, hypoxic-rearing could have increased the number of tracheae and tracheoles, and grasshoppers achieved a target tissue PO2 using a lower ventilation rate. 3.4. Effects of hypoxia and hyperoxia on insect development In all terrestrial insects studied to date, APO2 values of 10 kPa or lower reduce growth rate and increase developmental times (Table 3). This trend was also observed in S. americana grasshoppers studied here (Fig. 4 left, Table 4). Some mechanisms responsible for the suppression of growth rates by hypoxia are emerging. Cell cycle and division are strongly affected by hypoxia in a dose-dependent manner in Drosophila embryos and larvae (Wingrove and O’Farell, 1999; Douglas et al., 2001, 2005). Cells arrest during interphase, suggesting oxygen-sensitivity of DNA replication, or during metaphase, which may be due to improper assembly of the spindle apparatus. Fruitflies reared under hypoxic conditions have fewer and smaller cells (at least in the wing), consistent with suppression of the cell cycle and cell growth by hypoxia (Peck and Maddrell, 2005). One of the reg-

ulatory pathways controlling this oxygen-dependence of cell division and size is the nitric oxide/PKG pathway (Fig. 1; Wingrove and O’Farell, 1999); another is the HIF/Scylla/S6K pathway (Reiling and Hafen, 2004). The effect of hypoxia on body size varies among species (Table 3). For D. melanogaster, there is a linear, positive correlation between APO2 and pupal and adult sizes below 21 kPa (Peck and Maddrell, 2005). Similarly, for T. molitor, APO2 values of 10 kPa or below reduce size. However, for S. americana, body size at each developmental instar is unaffected by APO2 (Fig. 4 right; Table 4). What explains the variable effect of hypoxia on body size? One possibility is that hypoxia suppresses size in holo- but not hemi-metabolous insects. In support of this hypothesis, the effect of oxygen on size is reversible during embryonic or larval, but not pupal stages of fruitflies (Peck and Maddrell, 2005). Alternatively, hypoxia may cause reduced body size in organisms living in environments where hypoxia signals deteriorating environmental conditions that require rapid maturity at small body sizes for ecological success. Both D. melanogaster and T. molitor seem likely to experience hypoxia in nature, whereas, this scenario seems unlikely for S. americana, which lives above ground at low elevations. Extreme hyperoxia (100 kPa) kills flies in a few days, while exposure to 49% oxygen cuts longevity approximately in half (Kloek et al., 1976; Sohal et al., 1993). Interestingly, there is no evidence for up-regulation of the enzymes involved in detoxifying oxygen radicals (e.g. catalase, superoxide dismutase) when flies are exposed to 100 kPa APO2 (Sohal et al., 1993), perhaps because this treatment is so severe that compensatory protein production is impossible. At most temperatures, moderate hyperoxic rearing conditions (between 21 and 40 kPa) slightly increase development time and body size but not growth rate in fruitflies (Frazier et al., 2001; Table 3). Similarly, hyperoxia increases the time between molts in mealworms, although pupal and adult size is unaffected (Loudon, 1988; Greenberg and Ar, 1996; Table 3). The extension of development time by hyperoxia suggests that decline in internal PO2 to a trigger point could be involved in the initiation of molting in these insects. Internal PO2 may decrease as insects approach

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Table 3 Developmental responses to long-term exposure to hypoxia or hyperoxia in terrestrial insects Species

APO2 (kPa)

Calliphora vomitoria, blowfly Tenebrio. molitor, mealworm beetles

5 10 10

Drosophila melanogaster, fruitfly

Schistocerca americana, grasshopper

Manduca sexta, caterpillar eggs

Growth rate

Development time

↓1,2

↑ ↑ ↑1,2

15 40 7.5

=1 =2

=1 ↑2

10 15 30 40 5 10 40 11

↓1

↑1,2

=1 ↓ ↓ =

=2 ↑1 ↑ ↑ = ↑

Number of molts

Body size

Survival

References Houlihan (1974)

↑1,2

↓1,2

↓ ↓ ↓1,2

↓2

=1 =2 ↓2

=1 =2 ↓2

↓1 ↓2

↓1,2

↑1 = = =

=

Loudon (1988), Greenberg and Ar (1996)

Frazier et al. (2001), Peck and Maddrell (2005)

This study

↓

Woods and Hill (2004)

1: Loudon (1988) and Frazier et al. (2001); 2: Greenberg and Ar (1996) and Peck and Maddrell (2005).

a molt, due to the growth of metabolic tissue while the dimensions of the spiracles and major tracheae remain constant. The rise in Pc as insects approach a molt supports this suggestion (see Section 4.3 below). Internal PO2 does decline during embryonic development of Manduca sexta eggs (Woods and Hill, 2004). Hyper-

oxia (40 kPa) does not affect growth rate or body size in grasshoppers (Fig. 4; Table 4), suggesting that these grasshoppers either prevented a change in internal PO2 by some homeostatic mechanism, or that reaching a critical size is more important to molt initiation in these insects than internal PO2 .

Fig. 4. Effects of APO2 on growth rate (left) or size at a given developmental stage (right) for Schistocerca americana grasshoppers. Hypoxia reduced growth rates, but animals reached the same size at any given instar (see Table 4 for statistical analysis). Open gray circles and dotted line=5 kPa O2 ; gray closed circles and dashed line=10 kPa O2 ; black open circles and dashed line=21 kPa O2 ; closed black circles and solid line=40 kPa O2 . Points on right graph are slightly staggered for visibility.

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Table 4 Analyses of rearing APO2 influences on growth rate and body size of Schistocerca americana APO2 (kPa)

Intercept (95 kPa CI)

Slope (95 kPa CI)

N

1.65 (1.56–1.74) A 1.76 (1.65–1.87) A 1.79 (1.70–1.87) A 1.79 (1.67–1.90) A

0.025 (0.020–0.031) A 0.039 (0.034–0.045) AB 0.047 (0.041–0.052) B 0.049 (0.040–0.057) B

34 47 41 38

(2) ln femur length ∼ instarb 5 10 21 40

1.33 (1.24–1.43) A 1.33 (1.26–1.39) A 1.30 (1.22–1.38) A 1.38 (1.28–1.47) A

0.272 (0.24–0.27) A 0.260 (0.25–0.27) A 0.274 (0.26–0.29) A 0.260 (0.24–0.28) A

34 47 41 38

(3) ln mass ∼ ln daya 5 10 21 40

Forced through 0 Forced through 0 Forced through 0 Forced through 0

0.567 (0.450–0.683) A 0.995 (0.915–1.075) AB 0.107 (0.972–1.161) B 0.106 (0.964–1.163) B

10 12 10 10

(4) ln mass ∼ instarb 5 10 21 40

Forced through 0 Forced through 0 Forced through 0 Forced through 0

0.481 (0.413–0.550) A 0.540 (0.496–0.584) A 0.567 (0.529–0.606) A 0.584 (0.516–0.653) A

10 12 10 10

(1) ln femur 5 10 21 40

length ∼ daya

Reduced oxygen slowed growth rate (analyses 1 and 3), but did not reduce final body size at each instar (analyses 2 and 4). The letters after the intercept and slope estimates denote statistically significant treatment differences. a Regression analysis including both development day and rearing oxygen (ln femur length or mass (day) + oxygen) showed that femur length and mass increased with oxygen (P-values < 0.0001). A comparison of the regression slopes at each oxygen level suggests this result was due only to the 5 kPa rearing treatment. However, the oxygen effects in regression analyses on growth rates for femur length or mass were still significant with the 5 kPa data removed, supporting a significant depressive effect of 10 kPa PO2 on growth rate. b Regression analysis including both instar and rearing oxygen (ln femur length or mass (instar) + oxygen) showed that oxygen did not affect size at each developmental instar (P-values > 0.1).

3.5. Toxic effects of hypoxia and hyperoxia on insect tissues 3.5.1. Severe hypoxia/anoxia Unlike vertebrates, most insects can recover from hours to days of complete anoxia with little damage. The mechanisms responsible for this anoxia tolerance are poorly understood, and potentially of great biomedical relevance (Farahani and Haddad, 2003). During anoxia, ATP levels and metabolic rates fall to very low levels, as in vertebrates (Weyel and Wegener, 1996). In many vertebrates, anoxia causes a loss of ATP catabolites from the cell, impeding recovery during reoxygenation (Weyel and Wegener, 1996). In locust flight muscle, anoxia causes ATP to be catabolized to AMP, with little further conversion to hypoxanthine, xanthine, or uric acid (Weyel and Wegener, 1996), aiding recovery during reoxygenation. Another metabolic

response that enhances anoxia stress-resistance is the induction of trehalose by anoxia, which reduces protein denaturation (Chen, 2002). As previously noted, insects respond to anoxia or severe hypoxia by cessation of cell cycle activity. Amazingly, embryos are able to maintain an arrested state of development for up to 2 h of anoxia exposure, and then resume cell cycle activity within 20 min of reoxygenation (Douglas et al., 2001). Stabilized levels of the proteins cyclin A and E2F1, and increased levels of cyclin B are suggested to function as intermediaries of developmental arrest induction during metaphase and pre-S phase (Douglas et al., 2001). 3.5.2. Hyperoxia and oxidative stress Oxygen can be toxic in large amounts and hyperoxia is known to reduce the life span of many organisms, including insects (Sohal et al., 1993). The mechanisms

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of oxygen’s toxic effects on tissues are not well understood, although hyperoxia clearly increases ROS production and oxidative damage. Exposure to 100% APO2 causes mortality in houseflies in 4–5 days, associated with accumulation of oxidative damage as measured by protein carbonyl content (Sohal et al., 1993). Oxidative damage causes conformational changes in cytochrome oxidase, which then cause deformations of the mitochondrial cristae and mitochondrial apoptosis that leads to widespread cell death (Walker and Benzer, 2004). As in mammals (Dean et al., 2004), the central nervous system of Drosophila is quite vulnerable to the toxic effects of hyperoxia. Flies exposed to 100 kPa oxygen exhibited rapid brain degeneration and textural changes, revealing a “spongy” appearance, a typical characteristic of aged flies raised in normoxia (Philpott et al., 1974). The forebrain bears most of the damage during oxygen toxicity (Kloek et al., 1978). Flies exposed to 55 kPa oxygen show extensive vacuolation, regional shrinkage of the forebrain, and a loss of cephalic adipose tissue that normally populates the space between the brain and compound eye. Surprisingly, we were unable to find any direct measures of tissue damage at levels of PO2 other than 100 and 21 kPa. Fig. 1 suggests that variation in tissue PO2 will be correlated with variation in ROS production and oxidative damage. However, an alternative hypothesis is that tissue antioxidants and detoxification enzymes prevent significant oxidative damage over a wide range of tissue PO2 values, but that these defenses are overwhelmed at some very high tissue PO2 level. 4. What determines the critical PO2 for oxygen delivery? 4.1. Body size Based on historical correlations between high atmospheric O2 and the appearance of gigantic fossil animals, several high-profile publications have hypothesized that elevated atmospheric O2 enabled evolution of giant animals including terrestrial insects in the late Paleozoic, late Cretaceous and early Tertiary periods (reviewed by Dudley, 2000). Although the historical correlations between APO2 and maximal insect size are very intriguing, there are many alternative hypotheses

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for gigantism. For example, historical changes in atmospheric O2 level were driven partly by alterations in primary producer abundance and community composition (reviewed by Berner et al., 2003) that may have affected animal size by changing food quality or quantity. One problem with the hypothesis that O2 limits maximal insect size is that we currently lack a reasonable physiological explanation for such an effect. The most obvious explanation is that insects rely, at least partially, on diffusion through their tracheal system to maintain internal PO2 , and longer diffusion distances of larger insects challenge oxygen delivery (Dudley, 2000). However, our recent data provide strong evidence that oxygen delivery does not become more challenging for larger insects. During ontogeny, there is no evidence that larger body size is associated with smaller safety margins for oxygen delivery in resting grasshoppers (Greenlee and Harrison, 2004a), jumping grasshoppers (Kirkton et al., 2005), or feeding caterpillars (Greenlee and Harrison, 2005). Changes in tracheal morphology and increasing use of convection allow larger insects to overcome diffusion limitations posed by increasing size (Greenlee and Harrison, 2004a; Hartung et al., 2004). However, additional across-species studies are required, especially for large flying insects and insects that do not use convective ventilation, to fully eliminate the possibility that Pc rises with size in insects. Potentially, oxygen effects on size could be determined by mechanisms that are independent of the size of the insect, such as oxygen effects on the cell cycle or cell size (Peck and Maddrell, 2005), or on the hormonal cascade for molting (see Sections 3.4 and 4.3). 4.2. Metabolic rate Active insects with higher metabolic rates tend to have higher Pc values than inactive insects (Fig. 5). During tethered flight, the Pc for steady-state metabolic rate of S. americana grasshoppers rises dramatically compared to resting animals, despite a 10fold rise in tracheal conductance and convection (Rasc´on and Harrison, 2005, Fig. 5, point 10). Pooling species, Pc values are significantly lower for inactive than active insects (Fig. 5, t-test, active mean Pc = 12.2 kPa, S.D. = 7.6, inactive mean Pc = 5.9 kPa, S.D. = 4.9, P = 0.03, df = 1,22). The highest Pc values

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mental responses to PO2 at higher temperatures, but minimal responses at cooler temperatures (Frazier et al., 2001; Woods and Hill, 2004). These studies suggest that even tiny insects such as Manduca eggs and Drosophila larvae can be oxygen-limited under natural conditions, and indicate a need for understanding the trade-offs between investment in respiratory structures versus other tissues that might explain these minimal safety margins. 4.3. Ontogeny

Fig. 5. Pc values for acute exposure to various APO2 levels for inactive or active insects. Numerical data points are from studies conducted at normobaric pressure; lower-case letters refer to different species within the same study. Capital letters refer to studies that utilized hypobaric hypoxia. Asterisks (* ) indicate the same species from different studies, within a category. Dotted lines between data points indicate variation in Pc within a species, due to ontogenic effects. To match a data point to it’s reference, see superscripts in text and reference section. Datapoints labeled 5 are grasshoppers from Waclawski and Harrison, unpublished data—5a: Schistocerca americana, 5b: Teaniopoda eques, 5c: Melanoplus differentialis, 5d: Trimerotropic pallidipennis. MR = metabolic rate. Point 1: Chappell and Rogowitz (2000); Point 7: Holter and Spangenberg (1997); Point 9: Paim and Beckel (1964); Point 11: Timmins et al. (1999); Point 12: Wegener and Moratzky (1995); Point 13: Zhou et al. (2000).

yet measured occur during very high activity (dragonfly flight metabolic rate, Harrison and Lighton, 1998, Fig. 5, point 6; and adult grasshopper jumping rate, Kirkton et al., 2005, Fig. 5, point 14). However, the amount of interspecific variation in Pc is striking, with some resting insects having Pc values higher than other active species (Fig. 5). Higher temperature increases metabolic rates much more dramatically than diffusion rates, causing a rise in Pc. This effect has been shown most dramatically for Manduca eggs (Woods and Hill, 2004). These eggs have a Pc for metabolic rate below 9 kPa at 22 ◦ C, and a Pc of 40 kPa at 37 ◦ C! Temperature also strongly affects developmental and growth responses to APO2 . Both fruitflies and caterpillar eggs exhibit strong develop-

Ontogeny can have dramatic, but diverse effects on Pc, producing variation similar in magnitude to that induced by metabolic rate (Fig. 5). For example, during ontogeny of grasshoppers, Pc values for resting metabolic rate fall dramatically in older instars (Greenlee and Harrison, 2004a, Fig. 5, point 3), due to increased tracheal development and use of convection (Hartung et al., 2004). Despite an increase in tracheal diffusing and aerobic capacities, older jumping grasshoppers have increasing Pc and lactate production, and reduced endurance (Kirkton et al., 2005, Fig. 5, point 14). Finally, in recently molted Manduca caterpillars, there is little change in Pc across instars, but as animals approach a molt their Pc increases strongly (Greenlee and Harrison, 2005, Fig. 5, point 4). Changes in Pc through ontogeny are due to changes in metabolic rates and tracheal conductances (Greenlee and Harrison, 2004a, 2005). Within an instar, oxygen consumption rises due to the growth of aerobic tissue while dimensions of spiracles and major tracheae remain constant. Tracheal conductance may be further compromised by compression of flexible tracheae and air sacs due to tissue growth (Greenlee and Harrison, 2004b, 2005). At least in 5th instar caterpillars, Pc for metabolic rate rises to values approaching 21 kPa prior to molting. At present, it is unknown whether the rise in Pc for animals nearing a molt is accompanied by a fall in tissue PO2 that might serve as a trigger in the hormonal pathways controlling molting. However, the strong rise in Pc as animals near the molt suggests that requirements for a larger tracheal system provide an ultimate need, if not a proximate cue for molting. The strong ontogenic effects on Pc suggest that at least some of the scatter in the data shown in Fig. 5 could be due to ontogenetic effects. Even in

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adults, many developmental processes may influence Pc. These include factors that affect oxygen consumption, such as growth of reproductive tissue or muscle, and factors that influence power requirements during activity such as mass and cuticular elasticity.

reserves, high-energy phosphates, or anaerobic lactate production (Rasc´on and Harrison, 2005).

4.4. Hypo- versus normobaric hypoxia To our knowledge, no study to date has directly compared Pc in hypobaric and normobaric hypoxia for a terrestrial insect. Theoretically, if a major barrier to oxygen delivery is aerial diffusion within the trachea, then hypobaric Pc values should be substantially higher than equivalently hypoxic normobaric Pc, because diffusion rates in air increase proportionally as pressure decreases. However, the small amount of data available do not support this hypothesis, as the hypobaric Pc values appear similar to those measured under normobaric conditions (Tenney, 1985, Fig. 5, point T; and compare Withers, 1981, Fig. 5, point W with Joos et al., 1997, Fig. 5, point w). Direct tests of the effects of normo- versus hypobaric hypoxia on Pc would be useful as an index of the relative importance of diffusive gas exchange, and to allow us determine whether results from normobaric hypoxia studies are relevant to high altitude insects.

These reviewed studies suggest that internal PO2 is a critical physiological parameter involved in many processes, and that the morphology and physiology of the insect tracheal system is modulated in a compensatory manner by APO2 . Many frontiers remain in this field. Here are a few key unanswered questions: what role do tracheolar fluids play in regulating tracheal conductance, and what mechanisms regulate tracheole fluid movements? Where are the major sites of resistance to gas exchange in insects with open spiracles? What mechanisms allow acclimation of the ventilatory rates to rearing PO2 ? Can a drop in internal PO2 to some trigger point contribute to initiation of molting? Why does APO2 affect body size in some insects but not others? How is investment in the tracheal system influenced by body size? How does APO2 affect oxidative damage, and how do insects prevent oxidative damage? How do insects survive hours of anoxia? Clearly many fundamental unanswered questions remain concerning the responses of insects to hypoxia and hyperoxia.

4.5. Comparison of the oxygen-sensitivity of metabolic rates and behavioral functions

Acknowledgements

Whether the Pc for metabolic rate is a good predictor of the Pc for behaviors appears to depend partly on behavior duration. For long-duration behaviors, such as feeding in Manduca caterpillars, whole body CO2 emission and feeding Pc values were very similar (Greenlee and Harrison, 2005, Fig. 5, point 4). The Pc for short-duration behaviors appears to be significantly lower than for metabolic rates. For example, the Pc for metabolic rate was above 21 kPa in a flying dragonfly, but most animals could remain in flight in APO2 values as low as 10 kPa (Harrison and Lighton, 1998, Fig. 5, point 6). Similarly, for grasshoppers, the Pc for peak force production during tethered flight was about 5 kPa, well below the 16 kPa found for steady-state gas exchange (Rasc´on and Harrison, 2005, Fig. 5, point 10). Short-duration, high-power-output behaviors are relatively insensitive to hypoxia because they can be accomplished using quickly depleted air sac oxygen

5. Summary and a few frontiers

This work was partially supported by NSF IOB 0419704 to JFH. We thank Phillip Waclawski for collecting the Pc data for resting and jumping grasshoppers (points labeled 5 in Fig. 5) and TimaSue Cantu for collecting much of the growth, tracheal dimension, and ventilation rate data for S. americana reared at different oxygen levels.

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