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Comparative Biochemistry and Physiology, Part A

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Anoxia tolerance of the adult Australian Plague Locust (Chortoicetesterminifera)R. Meldrum Robertsona,b,⁎, Arianne J. Ceasea,1, Stephen J. Simpsonaa School of Biological Sciences, University of Sydney, A08 - Heydon-Laurence Building, NSW 2006, AustraliabDepartment of Biology, Queen's University, Kingston, ON K7L 3N6, Canada

A R T I C L E I N F O

Keywords:InsectSuffocationAnoxic comaSolitariousGregarious

A B S T R A C T

Optimal breeding conditions for locust swarms often include heavy rainfall and flooding, exposing individuals tothe risk of immersion and anoxia. We investigated anoxia tolerance in solitarious and gregarious adults of theAustralian Plague Locust, Chortoicetes terminifera, by measuring the time to enter an anoxic coma after sub-mersion in water, the time for recovery of ventilation and the ability to stand on return to air. We found a longertime to succumb in immature adults that we attribute to a larger tracheal volume. Time to succumb was alsolonger after autotomizing the hindlegs to reduce the energetic cost of muscular activity. Time to recover waslonger in gregarious males and this developed during maturation, suggesting an increase in the cost of neuralprocessing associated with social interactions under crowded conditions. Short-term changes in rearing condi-tions had effects that we interpret as stress responses, potentially mediated by octopamine.

1. Introduction

Under stressful environmental conditions many insects enter acoma, which is a state characterized by immobility and a lack of re-sponse to stimulation. It is associated with a reduction in energy con-sumption (e.g. anoxia (Campbell et al., 2018); hypothermia (Robertsonet al., 2017)), suggesting that it is an adaptive strategy to cope withextreme conditions. An important contributor to the paralysis is ashutdown of neural operations in the CNS via a spreading depolariza-tion (SD) of neurons and glia (Rodgers et al., 2010; Spong et al., 2016).The collapse of CNS ion gradients results from a failure of Na+/K+-ATPase activity and, given the high energetic cost of running these ionpumps to maintain neural function (Laughlin et al., 1998; Niven, 2016),this is a highly effective means of reducing energy consumption. TheCNS response to anoxia demonstrates positive feedback in that thetrigger for pump failure is itself due to a critical reduction in ATP supplywhen oxidative phosphorylation is compromised. The threshold forentering a coma varies with development (e.g. for anoxia (Campbellet al., 2018)), and in the context of species adaptation and acclimation(e.g. for chill coma (Andersen et al., 2018)). Thus, a complete under-standing of how insects tolerate harsh environments requires knowl-edge of what (e.g. prior exposure, developmental stage, activity level)determines the threshold for SD and subsequent coma, and the rate ofrecovery upon return to normoxia. We investigated the effect of rearing

conditions that result in density-dependent phase differences (gregar-ious and solitarious phenotypes) on anoxic coma and recovery in theAustralian Plague Locust (Chortoicetes terminifera).

Whereas entry into an anoxic coma appears primarily determinedby SD in neural integrative centres, full recovery has at least twophases. In Drosophila melanogaster, ionic concentration gradients in theCNS are restored rapidly on return to normoxia, even after 90min ofanoxia in a minimally dissected preparation (Rodriguez and Robertson,2012). However, recovery of locomotor activity takes considerablylonger than recovery of the CNS ionic disturbance (Evans et al., 2017).In mammalian systems, full recovery of neural circuit function dependson the clearance of the metabolic by-product, adenosine, which acts asa presynaptic inhibitory transmitter at A1 receptors (Lindquist andShuttleworth, 2012, 2017). However, clearance of accumulated K+ inthe haemolymph may determine recovery time in D. melanogaster(Campbell et al., 2018). It is worth stressing that extracellular K+

concentrations ([K+]o) in the CNS can change rapidly due to the small,restricted extracellular volume, but [K+]o in the haemolymph takesmuch longer to increase and decrease (e.g. for Locusta migratoria chillcoma: CNS (Robertson et al., 2017) and haemolymph (MacMillan et al.,2014)). Whatever the mechanism, recovery time depends on theduration of the exposure to anoxia (Krishnan et al., 1997; Lighton andSchilman, 2007) and metabolic rate (Schilman et al., 2011). The surgeof CNS [K+]o appears unaffected by the means of inducing anoxia (L.

https://doi.org/10.1016/j.cbpa.2018.12.005Received 23 October 2018; Received in revised form 7 December 2018; Accepted 9 December 2018

⁎ Corresponding author at: Department of Biology, Queen's University, 3118 Biosciences Complex, Kingston, ON K7L 3N6, Canada.E-mail address: [email protected] (R.M. Robertson).

1 current address of AJC: School of Sustainability, Arizona State University, PO Box 875,502, Tempe, AZ 85287–5502, USA.

Comparative Biochemistry and Physiology, Part A 229 (2019) 81–92

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migratoria: exposure to nitrogen gas (Rodgers et al., 2007); suffocationby immersion in water (Hou et al., 2014)). However, water immersionis more ecologically relevant and less damaging than gas exposure inflies, perhaps by limiting desiccation when the spiracles are openduring the coma (Benasayag-Meszaros et al., 2015).

Phase polyphenism in locusts is a dramatic example of phenotypicplasticity induced by variation in population density (Pener andSimpson, 2009). Most notably, solitarious phase locusts avoid con-specifics and gregarious phase locusts aggregate, have increased ac-tivity levels, and can migrate long distances en masse. However, there isa suite of species-specific traits that are associated with these twophases (reviewed in (Cullen et al., 2017)). While the major evolutionarydrivers of this plasticity are not understood, it is likely an adaptation toliving in an unstable and unpredictable environment. Behaviouralpolyphenism (aggregating versus avoiding conspecifics) indicates thatthe neural circuits underlying behaviour have been modified (Burrowset al., 2011). This is notably evident in Schistocerca gregaria for whichthe concentrations of neurochemicals are substantially different in so-litarious and gregarious animals (Rogers et al., 2004) and the brain is30% larger in the gregarious phase (Ott and Rogers, 2010), suggestingan increased metabolic cost of operation. Indeed, resting metabolic ratetends to be proportionally higher in gregarious locusts (Applebaum andHeifetz, 1999; Butler and Innes, 1936; Cease et al., 2010), potentiallydue to increased cost of maintaining larger brains and/or flight muscles(Nespolo et al., 2008). These differences imply that anoxia tolerancemay depend on phase of locusts.

Although the Australian Plague Locust (APL; Chortoicetes termini-fera) does not exhibit the striking coloration differences of solitariousand gregarious S. gregaria or L. migratoria, it does show a behaviouralpolyphenism that contributes to the formation of marching bands andswarms (Cullen et al., 2012; Gray et al., 2009). Behavioural changesassociated with crowding (increased activity and attraction to con-specifics), or isolation, are evident within 72 h and complete within aweek. High rainfall has a strong effect on population growth of APL(Veran et al., 2015) and other locusts (Pedgley, 1979; Rainey, 1951) byproviding optimal breeding conditions (Clark, 1974; Hemming et al.,1979). Thus, flooding of the breeding areas is a consistent hazard forAPL and their eggs can survive at least 14 days of water immersion,longer at cooler temperatures (Woodman, 2015). First instar APLnymphs enter a coma within ~2 mins of water immersion and showhigh survival rates (~80%) for at least 6 h of immersion, with the re-covery time being dependent on immersion time and temperature(Woodman, 2013). No information is currently available on anoxiatolerance of adult APL, either solitarious or gregarious.

We investigated tolerance to anoxia by water immersion in adultAPL to determine the effects of behavioural polyphenism on entry to,and recovery from, an anoxic coma. We predicted that, in gregariousAPL, increased neural processing associated with the social demands ofcrowding would increase neural metabolic rate and promote a shortertime to succumb under water and a longer time to recover upon returnto air. In addition, we characterized the effects on anoxia tolerance ofmaturation, tracheal volume, struggling activity, nutrition, and priorexposure.

2. Materials and methods

2.1. Animals

Locusts were obtained from breeding colonies that have beenmaintained in the School of Biological Sciences at the University ofSydney since their collection from outbreaks in New South Wales andWestern Australia in 2005. Gregarious stocks were reared undercrowded conditions with a 14 L:10D photoperiod in a room held at32 °C. During lights on, an incandescent 60W bulb behind each cageprovided an extra source of heat enabling locusts to thermoregulate topreferred body temperatures. They were fed daily with an ad libitum

supply of wheat grass and wheat germ.To obtain locusts in the solitarious phase, hatchlings were in-

dividually reared in small, plastic containers each of which had its ownsupply of pumped filtered air and food (wheat grass and wheat germ)and was separated from its neighbours by opaque barriers. Thus, visualand olfactory cues that could induce gregarization were eliminated.This culture method has been adapted from those used for Schistocercagregaria (Simpson et al., 1999) and it is effective at inducing beha-vioural phase polyphenism in Chortoicetes terminifera (Gray et al.,2009).

2.2. Maturation

To characterize the maturation of anoxia tolerance, ages and de-velopmental stages were standardized by taking all locusts from a singlecage initially populated with first instar hoppers that hatched on thesame day. Males and females were collected as fifth instars and then ondays numbered relative to the day of the moult to the adult. Anoxiatolerance was measured on 10 non-consecutive days, from the 5th in-star to the 15th day after the final moult, not including D4, D5, D10,D11, D12 and D13 (D=day). Data were collected from a total of 52females and 45 males of different ages.

2.3. Suffocation by immersion

Locusts were submerged in de-chlorinated tap water at room tem-perature (~25 °C) by placing them within an inverted 500mL glassbeaker that held them underwater. To speed data collection, they weretested in pairs that could be distinguished either by sex (1 male and 1female tested together) or by cutting short the wings of one of a pair ofmales. For some experiments, to test the contribution of muscular ac-tivity, both jumping hindlegs were induced to autotomize by pinchingthe distal end of the femur and twisting the leg. The time to enter ananoxic coma was measured from immersion to cessation of movement,which was usually preceded by convulsions and leg kicking. After30min immersion, locusts were removed from the water, patted drywith a paper towel, weighed and placed on their sides to recover. Thetime to recover ventilation and the ability to stand were measured fromthe time they were removed from the water to, respectively, the time ofthe first visible signs of abdominal ventilatory movements and the timethey abruptly resumed a normal standing posture with the thoraxsupported evenly and off the surface.

2.4. Estimation of tracheal volume

At the end of some experiments, we estimated tracheal volumeusing a water displacement method (Bartholomew and Barnhart, 1984).Each locust was rendered comatose by immersion, removed from thewater, dried with a paper towel and weighed. Then it was placed in a50mL syringe filled with water to which had been added a smallamount of detergent (~1mL of detergent in 250mL of tap water).Tracheal air was replaced with water by expelling air in the syringe,sealing it and applying negative pressure. This was repeated 3 timesbefore drying the locust as before and re-weighing it. The increase inweight was used as an estimate of tracheal volume assuming a waterdensity of ~1 g/mL.

2.5. Diet manipulation

Dry, artificial diets were made as described previously (Cease et al.,2012) and had equal total macronutrients (42% of dry weight) butthree different protein:carbohydrate ratios (35:7, 21:21 and 7:35). Forthese experiments, 13 males and 13 females (adults ~4 days afterfledging) for each diet treatment were housed in small(16×9×11 cm) plastic cages with the food freely available in dishesand a separate supply of water from an inverted test-tube stoppered

R.M. Robertson et al. Comparative Biochemistry and Physiology, Part A 229 (2019) 81–92

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with cotton wool. The supply of food and water was checked daily but,in spite of this, there was high mortality and cannibalism under theseconditions. After 7 days of confinement and diet treatment, mortalitywas highest in the protein-deprived group (54%), intermediate in thelow carbohydrate group (50%) and lowest in the group with a balancedprotein:carbohydrate diet (38%). Mortality was higher for females(54%; 62% of the protein-deprived females) than males (41%; 46% ofthe protein-deprived males). On the eighth day of treatment we testedanoxia tolerance of the most active 5 males and 5 females from eachdiet treatment and compared them with 10 male and 10 female age-matched locusts collected from the breeding colony and fed with theirnormal diet of wheat grass and wheat germ.

2.6. Phase manipulation

Adult locusts in the solitarious and gregarious phases were reared asdescribed above. We also examined the effects of acute isolation ofgregarious locusts and acute crowding of solitarious locusts. For theformer, gregarious locusts were isolated at 21 days post-hatching(~3 days after the imaginal moult) and, after 6 and 7 days of isolation,were tested alongside age-matched gregarious locusts from the originalcage. For the latter, solitarious locusts were crowded at 30 days post-hatching (solitarious locusts developed more slowly and thus the de-velopmental stage was approximately the same: ~3 days after theimaginal moult) by transferring them into a cage of gregarious locusts.They were identified by colouring their pronota and forewings withpink, waterproof chalk so that they could be removed after 6 and 7 daysof crowding for testing alongside age-matched solitarious locusts. Westarted with 40 solitarious and 40 gregarious with the intent of com-paring 20 individuals in each condition (with equal numbers of malesand females), however, sample sizes were affected by mortality, pri-marily of solitarious locusts. In addition, we increased the sample sizeof the gregarious locusts by 10 males and 10 females to get a betterestimate of results from un-manipulated locusts under their normalrearing conditions. Sample sizes for this experiment are provided inTable 3.

2.7. Statistics

We used Sigmaplot 13 (Systat Software Inc.) for statistical analysisand to prepare figures. Data were tested for normality and equal var-iance using the Shapiro-Wilk and Levene median tests respectively.Parametric data are reported as mean ± standard error and werecompared using ANOVA and Holm-Sidak tests for multiple compar-isons. Nonparametric data are reported as median and interquartilerange (IQR) and were compared using Kruskal-Wallis ANOVA on ranksand Dunn's test for multiple comparisons. There has been much recentdiscussion about the validity of P value thresholds (Benjamin et al.,2018; Lakens et al., 2018) and the misinterpretation of P values(Colquhoun, 2017). We recognize that P < 0.05 is an arbitrarythreshold for determining whether the results indicate real effects.Nevertheless, as a conventional guide, differences with P values< 0.05are indicated in the figures using either an asterisk (one comparison) orletters (multiple comparisons) as described in the text.

3. Results

For convenience, we refer to locusts reared in crowded conditions asgregs and locusts reared isolated as sols. In preliminary experimentscomparing 26 gregs with 32 sols we found that, upon immersion, gregstook longer to succumb (2.1 ± 0.14min) than sols (1.3 ± 0.12min;Holm-Sidak P < 0.001). On return to air, there was no effect of phaseon the time taken to resume ventilation (gregs – 11.8 ± 0.6min; sols –11.3 ± 0.6min; P=0.5) but gregs took longer to stand(17.2 ± 1.0min) than sols (12.5 ± 0.9min, Holm-Sidak P < .001).Sex had no obvious effect on the time to succumb (P=0.96) or the time

to stand (P= 0.76) but there was weak evidence that females recoveredventilation faster than males (female – 10.7 ± 0.6min; male –12.4 ± 0.6min; P=0.05). For most animals, ventilation resumedaround 5min before they righted themselves. However, a striking ob-servation was that 12 of 13 male sols stood abruptly before ventilationresumed, suggesting that the recovery of the neuromuscular coordina-tion and strength required to stand was possible in smaller animals(males are about half the size of females) with simple diffusion of airthrough the tracheae. This time difference between the recoveries ofventilation and standing was dependent on phase (P < 0.001) and sex(P= 0.016) with no interaction (P= .9) (male greg – 4.4 ± 0.9min;male sol – 0.08 ± 0.9min; female greg – 6.4 ± 0.9min; female sol –2.4 ± 0.7min). A problem with this initial dataset was that there wasno strict control over the age of the locusts, particularly for gregs.However, the results suggested effects of phase and sex on measures ofanoxia tolerance in C. terminifera and encouraged us to examine thismore closely.

3.1. Maturation

To characterize the effects of age on measures of anoxia tolerancewe focused on changes taking place between the late stage 5th instarand reproductively competent adults. During this period the orthop-teran CNS increases in volume (S. gregaria: (Sbrenna, 1971)) as neuronsgrow and form new connections (L. migratoria: (Gee and Robertson,1994; Gray and Robertson, 1996)). Neural operation also changes and,by the end, adult behaviours are fully developed (e.g. thoracic neuronalcircuits operate fast enough to enable sustained flight (Gray andRobertson, 1994; Kutsch, 1989)).

Maturation affected the time to succumb depending on the sex ofthe locust (Two Way ANOVA: Page < 0.001; Psex= 0.012; P sex x

age= 0.003). Females showed a large increase for at least 3 days afterthe imaginal moult (most P < .001) and this returned to pre-moultlevels by D6, whereas, for males, there was a similar trend and weakevidence for a difference between D1 and D14 (P=0.04) (Fig. 1A).

The time to ventilate was also affected by maturation in a sex-de-pendent fashion (Two Way ANOVA: Page < 0.001; Psex < 0.001; Page xsex < 0.001) with no effect in females but an increase in males from D6onwards (Fig. 1B). Time to stand was similar, and it should be notedthat the time to stand measure includes the time to ventilate so anychange in the latter would affect the former (Two Way ANOVA:Page < 0.001; Psex= 0.147; Psex x age= 0.31). Although the effect ofsex was not significant and there was no interaction between sex andage, the effect of age appears most prominent in males (Fig. 1C).

In summary, the most striking effects of maturation were: 1. a largeincrease in time to succumb (~four-fold in females) for several daysafter the imaginal moult; and 2. a maintained increase in time to ven-tilate (~1.5 times longer) only in males around the time that they be-came sexually mature and started to mate.

3.2. Tracheal volume

A simple explanation for the transient increase in time to succumbafter the imaginal moult is that it reflects a change in tracheal capacityassociated with moulting, which would be expected to be larger in fe-males (> twice the size of males). In S. americana, tracheal volumeincreases between instars but decreases within an instar as mass in-creases within constrained body dimensions (Lease et al., 2006). Also, achange in tracheal capacity can account for an effect of starvation onincreasing time to succumb in L. migratoria (Rodgers-Garlick et al.,2011).

Tracheal volume was larger in females (median 48 μL, IQR33–61 μL, n=15) compared to males (median 35 μL, IQR 28–46.5 μL,n=14) (Kruskal-Wallis One Way ANOVA: P=0.047). However, mass-specific tracheal volume was smaller in females (58 ± 7.2 μL/g)compared to males (113 ± 13.3 μL/g) (One Way ANOVA: P= 0.001)


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indicating that the increased size of females was not accompanied by aproportionate increase in tracheal volume. For both males and femalesthere were negative linear relationships between tracheal volume andbody mass (Fig. 2A; male P= 0.034; female P=0.006). However,there was no difference in time to succumb between males and females(One Way ANOVA: P=0.7) and only in males was there a negativerelationship between time to succumb and body mass (Fig. 2B; maleP=0.004; female P=0.36). Nevertheless, for both males and femalesthere were positive linear relationships between time to succumb andtracheal volume (Fig. 2C; male P=0.015; female P=0.031).

3.3. Autotomy

Another factor that would affect the time to succumb is the rate ofoxygen consumption due to struggling during immersion. Most animalskicked repetitively using both hindlegs prior to entering a coma (someanimals beat their wings immediately they entered the water, but thiswas rare, 1–2% of individuals). To determine the contribution of vig-orous muscular activity to measures of anoxia tolerance we comparedintact locusts (12 male and 12 female) with locusts whose metathoraciclegs had been autotomized (12 male and 11 female) (Fig. 3).

Not surprisingly there was an effect of hindleg autotomy on mass inboth males and females (Fig. 3A, Two Way ANOVA: Psex < 0.001;

Plegs < 0.001; Psex x legs= 0.03). Female mass dropped from810 ± 16 g to 663 ± 17 g (Holm-Sidak P < 0.001) whereas malemass dropped from 330 ± 16 g to 255 ± 16 g (Holm-SidakP= 0.002). This was associated with an increase in the time to suc-cumb (Fig. 3B, Two Way ANOVA: Psex= 0.5; Plegs= 0.019; Psex x

legs= 0.62). Female time to succumb increased from 0.99 ± 0.15minto 1.29 ± 0.16min, whereas male time to succumb increased from1.0 ± 0.15min to 1.46 ± 0.15min. Autotomy had no effect on thetime to ventilate although, because the locusts were>6 days old therewas a difference between sexes (Fig. 3C, Two Way ANOVA:Psex < 0.001; Plegs= 0.77; Psex x legs= 0.29). Finally, there was evi-dence for an effect of autotomy on the time to stand (Fig. 3D, Two WayANOVA: Psex= 0.5; Plegs= 0.02; Psex x legs= 0.62). Although there wasno main effect of sex and no interaction, the effect of autotomy on timeto stand was driven by a 2.87min increase in females (One WayANOVA P= .01) compared with a 0.42min increase in males (One WayANOVA P=0.74).


Nutrition plays important roles in determining organisms' phy-siology and behaviour, including social interactions (Lihoreau et al.,2015). In particular, investigation of locust feeding choices and their

Fig. 1. Anoxia tolerance during maturation of gregarious, female and male C. terminifera. A. Time to succumb to anoxia after water immersion. B. Time to startventilation and C. Time to stand after return to normoxia. Note that not all days between the 5th instar and D15 are represented (days 4–5 and 10–13 are missing).Box plots are 25th to 75th percentile, indicating median, with whiskers to 10th and 90th percentile. Number in parentheses under the abscissa indicate sample sizes(no repeated individuals). Statistical comparisons are described in the main text.


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consequences has been instrumental in establishing the NutritionalGeometry framework for understanding the regulation of macro-nutrient consumption (Raubenheimer and Simpson, 1993, 2003). Thesechoices and consequences differ between solitarious and gregarious S.gregaria 5th instar nymphs (Simpson et al., 2002). In addition, in-creasing metabolic rate to compensate for overfeeding carbohydrate is astrategy for regulating nutrition in 5th instar L. migratoria (Zanottoet al., 1997; Zanotto et al., 1993). In cases where locusts are fed dietstoo imbalanced to maintain body composition homeostasis, locustseating carbohydrate-biased diets lay on more lipids and less proteinrelative to locusts fed protein-biased diets (Simpson et al., 2002). Thesestudies suggest that diet composition would affect responses to anoxia.We examined the effect of diet manipulation on anoxia tolerance ingregarious adults. As detailed in the Methods, we started with 13 malesand 13 females in each of 3 diet treatments (low protein:carbohydrate,balanced, high protein:carbohydrate) but, after mortality over 7 days,we ended up comparing 5 males and 5 females from each of the arti-ficial diets against 10 males and 10 females, unconfined and fed theirregular diet of wheat grass and bran (Fig. 4).

Generally, the effects were similar in males and females, but morepronounced in females (see Table 1 for values). Two Way ANOVAsrevealed main effects of sex for all measures, though this was marginalfor time to succumb and time to stand. All the artificial diets reduced

mass (Two Way ANOVA: Psex < 0.001; Pdiet < 0.001; Psex x diet <0.001), with concomitant increases in tracheal volume (Two wayANOVA: Psex < 0.001; Pdiet < 0.001; Psex x diet < 0.001), times tosuccumb (Two Way ANOVA: Psex= 0.044; Pdiet < 0.001; Psex x

diet = 0.24), times to ventilate (Two Way ANOVA: Psex, 0.001;Pdiet < 0.001; Psex x diet = 0.34), and times to stand (Two Way ANOVA:Psex= 0.33; Pdiet = 0.027; Psex x diet = 0.48). Within the artificial diets,for which the locusts were reared in small cages, there was a tendencyfor the high protein:carbohydrate diet to have greater effects. Thisreached P < 0.05 for the tracheal volume of females.

3.5. Triple dip

To investigate the effect of prior exposure on anoxia tolerance wesubjected locusts to repeated immersions: an initial immersion, another1–2 h after the first immersion and another 24 h later. We used 14 males(~10 days post imaginal ecdysis) with autotomized hindlegs to reducevariation associated with sex and struggling activity. Individuals wereidentified by separating and testing them as numbered pairs (wingslong and wings cut short in each pair). During the overnight periodeach pair was kept in a separate container with ad libitum access towater and their regular diet. To control for this overnight treatment, wealso tested 10 autotomized males taken from the main colony and heldin containers under the same conditions overnight in the testing room.

There was no change in mass or time to succumb after prior ex-posure 1–2 h previously (Fig. 5; see Table 2 for values) but they re-covered from the coma in a shorter time. After 24 h the pre-exposedlocusts had lost mass but had retained a shorter time to recover fromthe coma. None of these changes could be attributed to the overnighttreatment because the control animals were not different in any wayfrom the experimental animals' first immersion.


As for previous data, sex had an effect on mass and time to ventilate(Two Way ANOVAs; Psex < 0.001 for both measures) with the smallermales exhibiting the longer time to ventilate that developed duringmaturation. Phase (original or manipulated) affected all measures (TwoWay ANOVAs; Pphase < 0.001 for all), with significant interactionswith sex for time to ventilate (Psex x phase= 0.002) and time to stand(Psex x phase= 0.03) (Fig. 6 and Table 3).

The main difference between gregs and sols was that male sols werefaster to recover from a coma (both for ventilation and the ability tostand) than male gregs, which is in accord with our preliminary data.Short-term isolation of locusts that had been reared under crowdedconditions reduced the mass of both males and females and increasedthe time to ventilate of females. Short-term crowding of locusts that hadbeen reared under solitary conditions reduced the time to succumb ofboth males and females, increased the time to ventilate in males (si-milar trend in females) and increased the time to stand in females (si-milar trend in males). There is no clear evidence that crowding a solmade it similar to a greg, in fact in females the opposite is true; short-term crowding of sols made them less like gregs. The same is true forshort-term isolation of gregs, which did not make them like sols (exceptperhaps for female time to ventilate).

In summary, there was an effect of the original phase on time torecover from anoxia in males, but this was not replicated by an acuteisolation of gregs. Moreover, changing rearing conditions to manipulatephase had unpredicted effects that lasted for at least 7 days.

4. Discussion

Our goal was to determine the effect of behavioural polyphenism onanoxia tolerance in the APL. For locusts that were reared isolated orcrowded starting from the 1st instar, we found that the only clear dif-ference was in males: male gregarious adults took longer to recover

Fig. 2. Tracheal volume of gregarious, male and female C. terminifera. A.Tracheal volume negatively correlates with mass in males and females. B. Timeto succumb to anoxia negatively correlates with mass in males. C. Time tosuccumb positively correlates with tracheal volume in males and females.Statistics for linear regressions are described in the main text.


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from anoxia than male solitarious adults. This difference could also beinduced by ~7 days of crowding male and female adults that had beenreared solitarious. On the other hand, ~7 days of isolating adults thathad been reared gregarious had no effect on males but increased thetime to ventilate of females. An interesting observation was that the sexdifference in recovery time arose during maturation when the time toventilate for males increased permanently about 6 days after the ima-ginal moult, whereas maturation had no effect on time to ventilate infemales. In the discussion below we consider entry to and recovery fromanoxic coma separately.

4.1. Entry to anoxic coma

The time to succumb to anoxia was not different in 5th instar andfully mature adults, however it was striking that immature adults tooklonger to succumb, particularly for females. The simplest explanationfor this is that immature adults would have had a larger tracheal ca-pacity, and hence a greater reservoir of oxygen, because linear di-mensions increase immediately at the time of the imaginal moult (moreso for females, which are more than twice the size of males) whereasmass takes several days to increase. These data are consistent with ourfinding that time to succumb was positively correlated with tracheal

capacity in males and females. Also, starvation of L. migratoria, whichincreases tracheal capacity, increases time to succumb (Rodgers-Garlicket al., 2011) and in S. americana tracheal capacity diminishes within aninstar as animals grow (Lease et al., 2006). The longer time to succumbof immature adults can explain our preliminary observations withgregarious animals of mixed ages (some immature) compared withsolitarious animals that were all mature.

The other major factor affecting time to succumb would be the rateat which the locusts consumed oxygen, including a resting metabolicrate and increased metabolic rate associated with vigorous muscularactivity. Reducing the latter, by removing the hindlegs, reduced mass(in a way that would not change tracheal capacity) and increased thetime to succumb for both males and females. Much of the resting me-tabolic rate can be attributed to the activity of energy-consumingmembrane pumps in the CNS. Prior exposure to anoxia in L. migratoriareduces neural performance and resting metabolic rate suggesting atrade-off between performance and the metabolic cost of signalling(Money et al., 2014). Consistent with this pattern we found that priorexperience of anoxia increased the time to succumb in male APL thathad limited ability for vigorous muscular activity (i.e. hindlegs auto-tomized).

Entry to an anoxic coma is thus dependent on the reservoir of

Fig. 3. Anoxia tolerance of gregarious, female and male C. terminifera with and without hindlegs. A. Mass. B. Time to succumb. C. Time to ventilate. D. Time to stand.Vertical bars indicate mean ± standard error. Asterisks indicate P < 0.05 for effects of hindleg autotomy. Details in text.


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Fig. 4. Anoxia tolerance of gregarious, female and male C. terminifera fed different diets. A. Mass. B. Tracheal volume. C. Time to succumb. D. Time to ventilate. E.Time to stand. Regular – normal diet of wheat grass and wheat germ; Low protein – prepared diet with low proportion of protein (7:35%); Balanced – prepared dietwith equal proportions of carbohydrate:protein (21:21% of dry weight); Low carb – prepared diet with low proportion of carbohydrate (35:7%);. Different letters onbars indicate P < 0.05. Details in text.

Table 1Mass, tracheal volume and anoxia tolerance measures after dietary manipulation.

N Mass /g Volume /μL Succumb /min Ventilate /min Stand /min

Female Regular 10 833 ± 23 52 ± 6 1.0 ± 0.1 8.1 ± 0.3 14.1; 12.2–16.4Balanced 5 * 559 ± 32 * 122 ± 22 * 3.3 ± 0.2 * 10.8 ± 0.5 17.3; 15.9–20.6Low carb 5 * 543 ± 32 * # 188 ± 19 * 4.1 ± 0.6 * 12.7 ± 0.8 * 17.3; 16.2–28.9Low protein 5 * 579 ± 32 * 129 ± 22 * 3.5 ± 0.6 * 12.1 ± 0.9 16.5; 13.1–20.6

Male Regular 10 363 ± 23 37 ± 3 0.9 ± 0.09 11.2 ± 0.4 13.8; 12.5–16.3Balanced 5 328 ± 32 34 ± 10 1.7 ± 0.4 11.8 ± 0.4 13.4; 13.1–15.0Low carb 5 * 285 ± 32 53 ± 6 * 3.6 ± 0.5 * 14.6 ± 0.8 16.3; 11.6–16.4Low protein 5 * 286 ± 32 71 ± 21 * 3.2 ± 0.8 13.4 ± 1.3 16.0; 12.5–16.3

Values are presented as mean ± SE or median; IQR. Asterisks and bold font indicate P < 0.05 when compared with the regular diet; # indicates P < 0.05 whencompared with balanced and low protein diets. Within sex One Way ANOVA and Kruskal-Wallis One Way ANOVA.


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oxygen at the time of immersion and the rate at which it is consumed.Nevertheless, it is likely that the trigger for SD, which initiates coma,can be controlled independently of metabolic rate. Under anoxia, larvaland adult Drosophila both decrease metabolic rate to around 3% ofnormal, however adults rapidly enter a coma whereas larvae continueto locomote strongly for many minutes (Callier et al., 2015; Campbellet al., 2018). Also, the time to CNS shutdown under anoxia is affectedby acclimation temperature in D. melanogaster and differs in tropicaland temperate drosophilid species in parallel with differing chill comatemperatures (Andersen et al., 2018). In L. migratoria entry to an anoxiccoma can be manipulated with the neuromodulator, octopamine(Money et al., 2016). These observations suggest that neural mechan-isms exist that could control entry to an anoxic coma in adaptive re-sponses to environmental variation.

4.2. Recovery from anoxic coma

Recovery from an anoxic coma requires the restoration of ion gra-dients in the CNS, which are necessary for neural signalling. However,this occurs rapidly on return to normoxia and can be discounted in ourconsideration of the timing of neuromuscular recovery. This timingpositively correlates with the duration of the anoxia (Krishnan et al.,1997; Lighton and Schilman, 2007) due to an increasing build-up ofmetabolic disturbance during the coma (Hochachka et al., 1993; Weyel

and Wegener, 1996) that takes increasing time to clear. Within the CNS,metabolites such as adenosine might inhibit neural circuits even whenion gradients have been restored (as for mammals, (Lindquist andShuttleworth, 2012)). In addition, restoring ion balance in the hae-molymph takes longer than in the CNS and a build-up of haemolymph[K+]o would delay neuromuscular recovery (Campbell et al., 2018).The metabolic disturbance during the coma and the rate of recovery onreturn to normoxia are both increased by raising the temperature andthereby increasing metabolic rate (Schilman et al., 2011). Our experi-ments did not address the mechanisms underlying recovery, but weinterpret our results with reference to accumulation and clearance of ametabolic disturbance.

A robust finding was that time to ventilate was longer in adultgregarious males than in equivalent females, in spite of the much largermass of females. Hindleg autotomy did not affect time to ventilate or itsdifference between males and females indicating that time to ventilatewas not dependent on any increase in metabolic rate brought on byvigorous activity. The time to stand was marginally longer in auto-tomized females relative to intact females but this might be attributedto the lack of hindlegs making it more difficult for a larger locust tostand. It is particularly interesting that the increase in male time toventilate developed during maturation, around the time of sexual ma-turity. For these reasons, we suggest that it is due to increased neuralactivity (increased CNS metabolic rate) associated with competition formates in crowded rearing conditions. Consistent with this are our re-sults showing that, in males without hindlegs, prior experience of ananoxic coma, which reduces neural performance and metabolic rate inL. migratoria (Money et al., 2014), reduces the time to ventilate and thetime to stand for at least 24 h.

For most locusts, ventilatory movements recovered before theability to stand. This likely reflects the differential complexity of theneuromuscular mechanisms for ventilation and standing. The first signsof ventilation are controlled by a single central pattern generator in themetathoracic ganglion (Bustami and Hustert, 2000) supplying smallmuscles in the abdomen relatively close to the thorax. Standing, on theother hand, requires correct neuromuscular coordination of multipleperipheral muscles in all six (or four) legs controlled by multiple neuralpattern generators in at least three thoracic ganglia. It is perhaps to beexpected that a more complex, dispersed control system (standing)

Fig. 5. Anoxia tolerance of gregarious, male C. ter-minifera without hindlegs after repeated pre-ex-posure to anoxia. A. Mass. B. Time to succumb. C.Time to ventilate. D. Time to stand. Suffocationepisodes were repeated one hour (1 h) and one day(24 h) after the first immersion (1st) and comparedwith a control group (C). Different letters on bars orbox-plots indicate P < 0.05. Details in text.

Table 2Mass and anoxia tolerance measures after immersion pre-treatments in maleswith autotomized hindlegs.

N Mass / g Succumb /min Ventilate/min Stand /min

First dip 14 269 ± 8 1.1; 1.0–1.3 10.5 ± 0.4 14.8; 14.3–16.91 h dip 14 265 ± 7 1.4; 1.0–1.8 * 8.7 ± 0.3 * 11.8; 10.8–12.924 h dip 14 * 254 ± 7 * 1.6; 1.3–2.0 * 9.0 ± 0.2 * 10.9; 10.3–11.5Control 10 273 ± 8 1.6; 1.3–1.9 10.5 ± 0.3 15.9; 15.3–19.4

Values are presented as mean ± SE or median; IQR. Asterisks and bold fontindicate P < 0.05 when compared with the first immersion (One WayRepeated Measures ANOVAs, excluding Control). Subsequent One WayANOVAs including Control showed no difference of Control from first immer-sion values.


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Fig. 6. Anoxia tolerance of female and male C. terminifera reared under different conditions of crowding. A. Mass. B. Time to succumb. C. Time to ventilate. D. Timeto stand. GREGARIOUS – reared under normal crowded colony conditions; GREG-isolated – reared in a crowded colony and subsequently isolated for 6–7 days; SOL-crowded – reared in isolation and subsequently crowded for 6–7 days; SOLITARIOUS – reared under isolated conditions. Different letters on box-plots indicateP < 0.05. Details in text.

Table 3Mass and anoxia tolerance measures of gregarious and solitarious locusts before and after manipulation of phase.

N Mass / g Succumb /min Ventilate /min Stand /min

Female GREG 30 799 ± 21 1.2 ± 0.1 8.3; 7.7–8.7 14.8 ± 0.4GREG-isolated 14 * 656 ± 30 1.4 ± 0.1 * 9.5; 8.8–10.3 14.9 ± 0.5SOL-crowded 9 861 ± 54 * 0.6 ± 0.8 * 9.6; 9.1–10.6 * 18.0 ± 1.3SOL 8 794 ± 32 1.4 ± 0.1 8.8; 8.0–9.3 12.7 ± 0.6

Male GREG 29 340 ± 6 1.1; 0.9–1.3 11.5 ± 0.3 15.9; 13.9–17.7GREG-isolated 19 * 298 ± 8 1.3; 1.1–1.7 11.1 ± 0.3 13.0; 11.9–19.0SOL-crowded 7 327 ± 9 * 0.9; 0.5–1.2 11.8 ± 0.8 14.8; 12.3–17.3SOL 7 326 ± 8 1.5; 1.3–2.0 * 9.6 ± 0.4 * 10.8; 8.8–12.3

Values are presented as mean ± SE or median; IQR. Asterisks and bold font indicate P < 0.05 when compared with GREG within a sex but see Fig. 6 for results of allpair-wise comparisons (One Way ANOVAs or Kruskal-Wallis One Way ANOVAs).


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would take longer to recover than a simpler one (ventilation). However,we found that there was little difference between time to ventilate andtime to stand in solitarious males. For example, in the preliminarydataset, 12 of 13 mature solitarious males stood abruptly before wenoted the start of ventilation. The most obvious distinguishing featureof solitarious males is that they have the smallest linear dimensions,though there is no clear difference of mass compared with gregariousmales. One explanation for the lack of a delay between ventilation andstanding is that, on return to air, simple diffusion through the tracheaeprovides sufficient oxygen to enable a more compact control system torecover without ventilation. A contributing factor may be a reducedhaemolymph volume, which would promote a more rapid clearance ofhigh haemolymph [K+]o.


We expected to see more of an effect between the artificial diets(e.g. between carbohydrate-biased and protein-biased diets), howeverthe data were compromised by high mortality due to cannibalism.Cannibalism is common in orthopterans, particularly when they aredeprived of protein (Simpson et al., 2006), and avoiding it can drivemass migratory behaviour (Bazazi et al., 2008). Cannibalism mayprovide an adaptive advantage by increasing maximum survival timeand maximum distance travelled for groups of marching locusts, but notindividuals (Hansen et al., 2011). Protein-satiated (i.e. previously fed a35:7 protein:carbohydrate ratio) C. terminifera will cannibalize im-mobile but live locusts within two hours and the level of cannibalism isincreased with protein-deprivation (i.e. previously fed a 7:35 pro-tein:carbohydrate ratio). Moreover, in L. migratoria, artificial diets, evenwhen appropriately balanced, are not sufficient to support growthduring adult maturation and mortality is increased with low proteinconcentrations (Chyb and Simpson, 1990). Our general observationswere consistent with these previous results in that we found the highestmortality for adults fed the low protein:carbohydrate diet.

If adult C. terminifera were regulating nutrition by increasing re-spiration after overfeeding on carbohydrate (Zanotto et al., 1997) wewould expect locusts eating the low protein:carbohydrate diet to haveincreased tracheal volume or to succumb more quickly to anoxia.However, in contrast, female locusts eating the protein-biased diet hadthe largest tracheal volume (no effect for males) and there was nodifference between carbohydrate- and protein-biased diet treatments ontime to succumb. In spite of minimal differences among adults fed ar-tificial diets, we compared these with the control group fed the normaldiet of wheatgrass and bran. Males and females were generally similarin their responses though the effects were larger in females, which weattribute to the fact that females are twice the size of males, are pre-paring for oogenesis, and may be more vulnerable to a nutritional de-ficiency. Notwithstanding the ad libitum provision of food, and thecannibalism, the locusts lost mass indicating insufficient nutrition. Asexpected, this mass loss was associated with an increase in trachealvolume and a longer time to succumb, particularly for females fed theprotein-biased diet. The time to recover was also longer, which isconsistent with an effect of starvation in L. migratoria, mediated by thecellular energy sensor, AMP kinase (AMPK) (Hardie et al., 2012;Rodgers-Garlick et al., 2011). Moreover, food deprivation increaseslevels of phosphorylated AMPK in fat body, muscle and CNS of L. mi-gratoria and this is associated with increased neural performance thatwould be expected to increase CNS metabolic rate (opposite to the ef-fect of a prior anoxic coma) (Cross et al., 2017). Another possiblecontributor to the effects of nutritional stress is the neuromodulatoroctopamine, which increases in the haemolymph after food deprivationin S. gregaria (Davenport and Evans, 1984) and modulates the recoveryfrom a water immersion coma in L. migratoria (Money et al., 2016).Octopamine could also be involved in mediating the effects of crowdingsolitarious locusts (see below).


Male solitarious APL recovered from an immersion coma faster thanmale gregarious APL. Their times to ventilation were similar to those offemale APL, which were lower overall across treatments compared tomales. One explanation for this is that isolating males during matura-tion prevented the increase in the time to ventilate that we argue isassociated with reproductive competence and the stress of matingcompetition, the latter of which would increase CNS metabolic raterelative to isolated males. We did not characterize maturation of anoxiatolerance in solitarious APL but predict that isolated males would notshow an increase in time to ventilate during maturation.

Short-term isolation of gregarious APL caused a loss of mass in bothmales and females. However, this body mass loss was not sufficient toincrease the time to succumb, as we found in response to the muchlarger body mass loss after diet manipulation. There was a modest in-crease in the time to ventilate for females, suggesting that isolation mayhave been stressful for locusts used to crowded conditions. On the otherhand, short-term crowding of solitarious locusts more clearly indicatedthat changing rearing conditions was stressful, potentially increasingCNS metabolic rate. Time to succumb was shorter and time to recoverwas longer in males and females. There is an interesting parallel withCNS levels of octopamine, which is recognized as a stress hormone(Adamo, 2017). Whereas baseline octopamine levels in the CNS of S.gregaria are not different in solitarious and gregarious adults (in markedcontrast with other bioactive chemicals), short term crowding induces alarge octopamine increase in solitarious adults during the process ofgregarization (Rogers et al., 2004). Hence, we interpret the above re-sults as a stress response to the dramatic change in rearing conditions.

5. Conclusion

The simple measures that we used to characterize anoxia tolerancein the APL are clearly limited in their ability to discover the cellularmechanisms underlying anoxic coma and recovery. Nevertheless, as welearn more about these mechanisms in other species and use more so-phisticated techniques, it becomes possible to make inferences abouthow animals cope with environmental stress and how different factorscontribute to resilience. Here we found a clear sex difference in theability to recover after suffocation under water, a difference that is alsophase-dependent and that develops in males during maturation. Wespeculate that the slower time to recover of gregarious males is due to ahigher cost of neural processing and consequent increase in the accu-mulation of metabolites during the coma. It is worth noting that all ourresults were obtained with 30min of anoxic coma and survival is notaffected until after about 6 h of immersion (seven species of rangelandgrasshoppers (Brust et al. 2007); 1st instar APL nymphs (Woodman,2013); adult L. migratoria (Wu et al., 2002)). Hence, with longer im-mersion times the differences we found are likely only to be ex-acerbated.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

We thank Steve Rogers for helpful discussion, Tim Dodgson, TamaraPulpitel and Ximonie Clark for their help setting up and maintainingcolonies of C. terminifera in gregarious and solitarious phases andRachel Van Dusen for comments on the manuscript.

Funded by a Discovery Grant from the Natural Sciences andEngineering Research Council of Canada to RMR and a LaureateFellowship from the Australian Research Council to SJS.


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References

Adamo, S.A., 2017. The stress response and immune system share, borrow, and re-configure their physiological network elements: evidence from the insects. Horm.Behav. 88, 25–30.

Andersen, M.K., Jensen, N.J.S., Robertson, R.M., Overgaard, J., 2018. Central nervousshutdown underlies acute cold tolerance in tropical and temperate Drosophila spe-cies. J. Exp. Biol. 221.

Applebaum, S.W., Heifetz, Y., 1999. Density-dependent physiological phase in insects.Annu. Rev. Entomol. 44, 317–341.

Bartholomew, G.A., Barnhart, M.C., 1984. Tracheal gases, respiratory gas-exchange,body-temperature and flight in some tropical Cicadas. J. Exp. Biol. 111, 131–144.

Bazazi, S., Buhl, J., Hale, J.J., Anstey, M.L., Sword, G.A., Simpson, S.J., Couzin, I.D.,2008. Collective motion and cannibalism in locust migratory bands. Curr. Biol. 18,735–739.

Benasayag-Meszaros, R., Risley, M.G., Hernandez, P., Fendrich, M., Dawson-Scully, K.,2015. Pushing the Limit: Examining Factors that Affect Anoxia Tolerance in a SingleGenotype of Adult D. melanogaster. (Scientific reports 5).

Benjamin, D.J., Berger, J.O., Johannesson, M., Nosek, B.A., Wagenmakers, E.J., Berk, R.,Bollen, K.A., Brembs, B., Brown, L., Camerer, C., Cesarini, D., Chambers, C.D., Clyde,M., Cook, T.D., De Boeck, P., Dienes, Z., Dreber, A., Easwaran, K., Efferson, C., Fehr,E., Fidler, F., Field, A.P., Forster, M., George, E.I., Gonzalez, R., Goodman, S., Green,E., Green, D.P., Greenwald, A.G., Hadfield, J.D., Hedges, L.V., Held, L., Hua Ho, T.,Hoijtink, H., Hruschka, D.J., Imai, K., Imbens, G., Ioannidis, J.P.A., Jeon, M., Jones,J.H., Kirchler, M., Laibson, D., List, J., Little, R., Lupia, A., Machery, E., Maxwell, S.E.,McCarthy, M., Moore, D.A., Morgan, S.L., Munafó, M., Nakagawa, S., Nyhan, B.,Parker, T.H., Pericchi, L., Perugini, M., Rouder, J., Rousseau, J., Savalei, V.,Schönbrodt, F.D., Sellke, T., Sinclair, B., Tingley, D., Van Zandt, T., Vazire, S., Watts,D.J., Winship, C., Wolpert, R.L., Xie, Y., Young, C., Zinman, J., Johnson, V.E., 2018.Redefine statistical significance. Nat. Hum. Behav. 2, 6–10.

Brust, M.L., Hoback, W.W., Wright, R.J., 2007. Immersion tolerance in rangeland grass-hoppers (Orthoptera: Acrididae). J. Orthoptera Res.arch 16, 135–138.

Burrows, M., Rogers, S.M., Ott, S.R., 2011. Epigenetic remodelling of brain, body andbehaviour during phase change in locusts. Neural Syst. Circuits 1, 11.

Bustami, H.P., Hustert, R., 2000. Typical ventilatory pattern of the intact locust is pro-duced by the isolated CNS. J. Insect Physiol. 46, 1285–1293.

Butler, C.G., Innes, J.M., 1936. A comparison of the rate of metabolic activity in thesolitary and migratory phases of Locusta migratoria. Proc. R. Soc. Ser. B Biol. 119,296–304.

Callier, V., Hand, S.C., Campbell, J.B., Biddulph, T., Harrison, J.F., 2015. Developmentalchanges in hypoxic exposure and responses to anoxia in Drosophila melanogaster. J.Exp. Biol. 218, 2927–2934.

Campbell, J.B., Andersen, M.K., Overgaard, J., Harrison, J.F., 2018. Paralytic hypo-en-ergetic state facilitates anoxia tolerance despite ionic imbalance in adult Drosophilamelanogaster. J. Exp. Biol. 221.

Cease, A.J., Hao, S.G., Kang, L., Elser, J.J., Harrison, J.F., 2010. Are color or high rearingdensity related to migratory polyphenism in the band-winged grasshopper, Oedaleusasiaticus. J. Insect Physiol. 56, 926–936.

Cease, A.J., Elser, J.J., Ford, C.F., Hao, S.G., Kang, L., Harrison, J.F., 2012. Heavy live-stock grazing promotes locust outbreaks by lowering plant nitrogen content. Science335, 467–469.

Chyb, S., Simpson, S.J., 1990. Dietary selection in adult Locusta migratoria. Entomol Exp.App. 56, 47–60.

Clark, D.P., 1974. Influence of rainfall on densities of adult Chortoicetes-Terminifera(Walker) in Central Western New-South-Wales, 1965-73. Aust. J. Zool. 22, 365–386.

Colquhoun, D., 2017. The reproducibility of research and the misinterpretation of p-va-lues. R. Soc. Open Sci. 4, 171085.

Cross, K.P., Britton, S., Mangulins, R., Money, T.G.A., Robertson, R.M., 2017. Food de-privation and prior anoxic coma have opposite effects on the activity of a visualinterneuron in the locust. J. Insect Physiol. 98, 336–346.

Cullen, D.A., Sword, G.A., Simpson, S.J., 2012. Optimizing multivariate behaviouralsyndrome models in locusts using automated video tracking. Anim. Behav. 84,771–784.

Cullen, D.A., Cease, A.J., Latchininsky, A.V., Ayali, A., Berry, K., Buhl, J., De Keyser, R.,Foquet, B., Hadrich, J.C., Matheson, T., Ott, S.R., Poot-Pech, M.A., Robinson, B.E.,Smith, J.M., Song, H., Sword, G.A., Vanden Broeck, J., Verdonck, R., Verlinden, H.,Rogers, S.M., 2017. From molecules to management: mechanisms and consequencesof locust phase polyphenism. Insect Epigenetics 53, 167–285.

Davenport, A.P., Evans, P.D., 1984. Changes in hemolymph octopamine levels associatedwith food-deprivation in the locust, Schistocerca gregaria. Physiol. Entomol. 9,269–274.

Evans, J.J., Xiao, C., Robertson, R.M., 2017. AMP-activated protein kinase protectsagainst anoxia in Drosophila melanogaster. Comp. Biochem. Physiol. A Mol. Integr.Physiol. 214, 30–39.

Gee, C.E., Robertson, R.M., 1994. Effects of maturation on synaptic potentials in the lo-cust flight system. J. Comp. Physiol. A. 175, 437–447.

Gray, J., Robertson, R., 1994. Activity of the forewing stretch-receptor in immature andmature adult locusts. J. Comp. Physiol. A. 175, 425–435.

Gray, J.R., Robertson, R.M., 1996. Structure of the forewing stretch receptor axon inimmature and mature adult locusts. J. Comp. Neurol. 365, 268–277.

Gray, L.J., Sword, G.A., Anstey, M.L., Clissold, F.J., Simpson, S.J., 2009. Behaviouralphase polyphenism in the Australian plague locust (Chortoicetes terminifera). Biol.Lett. 5, 306–309.

Hansen, M.J., Buhl, J., Bazazi, S., Simpson, S.J., Sword, G.A., 2011. Cannibalism in thelifeboat - collective movement in Australian plague locusts. Behav. Ecol. Sociobiol.

65, 1715–1720.Hardie, D.G., Ross, F.A., Hawley, S.A., 2012. AMPK: a nutrient and energy sensor that

maintains energy homeostasis. Nat. Rev. Mol. Cell Biol. 13, 251–262.Hemming, C.F., Popov, G.B., Roffey, J., Waloff, Z., 1979. Characteristics of desert locust

plague upsurges. Philos. Trans. R. Soc. London Series B 287, 375–386.Hochachka, P.W., Nener, J.C., Hoar, J., Saurez, R.K., Hand, S.C., 1993. Disconnecting

metabolism from adenylate control during extreme oxygen limitation. Can. J. Zool.Revue Canadienne De Zoologie 71, 1267–1270.

Hou, N., Armstrong, G.A., Chakraborty-Chatterjee, M., Sokolowski, M.B., Robertson,R.M., 2014. Na+-K+-ATPase trafficking induced by heat shock pretreatment cor-relates with increased resistance to anoxia in locusts. J. Neurophysiol. 112, 814–823.

Krishnan, S.N., Sun, Y.A., Mohsenin, A., Wyman, R.J., Haddad, G.G., 1997. Behavioraland electrophysiologic responses of Drosophila melanogaster to prolonged periods ofanoxia. J. Insect Physiol. 43, 203–210.

Kutsch, W., 1989. Development of the flight motor pattern. In: Goldsworthy, G.I.,Wheeler, C.H. (Eds.), Insect Flight. CRC Press, Boca Raton, FL, pp. 51–73.

Lakens, D., Adolfi, F.G., Albers, C.J., Anvari, F., Apps, M.A.J., Argamon, S.E., Baguley, T.,Becker, R.B., Benning, S.D., Bradford, D.E., Buchanan, E.M., Caldwell, A.R., VanCalster, B., Carlsson, R., Chen, S.-C., Chung, B., Colling, L.J., Collins, G.S., Crook, Z.,Cross, E.S., Daniels, S., Danielsson, H., Debruine, L., Dunleavy, D.J., Earp, B.D., Feist,M.I., Ferrell, J.D., Field, J.G., Fox, N.W., Friesen, A., Gomes, C., Gonzalez-Marquez,M., Grange, J.A., Grieve, A.P., Guggenberger, R., Grist, J., van Harmelen, A.-L.,Hasselman, F., Hochard, K.D., Hoffarth, M.R., Holmes, N.P., Ingre, M., Isager, P.M.,Isotalus, H.K., Johansson, C., Juszczyk, K., Kenny, D.A., Khalil, A.A., Konat, B., Lao,J., Larsen, E.G., Lodder, G.M.A., Lukavský, J., Madan, C.R., Manheim, D., Martin,S.R., Martin, A.E., Mayo, D.G., McCarthy, R.J., McConway, K., McFarland, C., Nio,A.Q.X., Nilsonne, G., de Oliveira, C.L., de Xivry, J.-J.O., Parsons, S., Pfuhl, G., Quinn,K.A., Sakon, J.J., Saribay, S.A., Schneider, I.K., Selvaraju, M., Sjoerds, Z., Smith, S.G.,Smits, T., Spies, J.R., Sreekumar, V., Steltenpohl, C.N., Stenhouse, N., Świątkowski,W., Vadillo, M.A., Van Assen, M.A.L.M., Williams, M.N., Williams, S.E., Williams,D.R., Yarkoni, T., Ziano, I., Zwaan, R.A., 2018. Justify your alpha. Nat. Hum. Behav.2, 168–171.

Laughlin, S.B., de Ruyter Van Steveninck, R.R., Anderson, J.C., 1998. The metabolic costof neural information. Nat. Neurosci. 1, 36–41.

Lease, H.M., Wolf, B.O., Harrison, J.F., 2006. Intraspecific variation in tracheal volume inthe American locust, Schistocerca americana, measured by a new inert gas method. J.Exp. Biol. 209, 3476–3483.

Lighton, J.R.B., Schilman, P.E., 2007. Oxygen Reperfusion damage in an Insect. PLoSOne 2.

Lihoreau, M., Buhl, J., Charleston, M.A., Sword, G.A., Raubenheimer, D., Simpson, S.J.,2015. Nutritional ecology beyond the individual: a conceptual framework for in-tegrating nutrition and social interactions. Ecol. Lett. 18, 273–286.

Lindquist, B.E., Shuttleworth, C.W., 2012. Adenosine receptor activation is responsible forprolonged depression of synaptic transmission after spreading depolarization in brainslices. Neuroscience 223, 365–376.

Lindquist, B.E., Shuttleworth, C.W., 2017. Evidence that adenosine contributes to Leao'sspreading depression in vivo. J. Cereb. Blood Flow Metab. 37, 1656–1669.

MacMillan, H.A., Findsen, A., Pedersen, T.H., Overgaard, J., 2014. Cold-induced depo-larization of insect muscle: differing roles of extracellular K+ during acute andchronic chilling. J. Exp. Biol. 217, 2930–2938.

Money, T.G., Sproule, M.K., Hamour, A.F., Robertson, R.M., 2014. Reduction in neuralperformance following recovery from anoxic stress is mimicked by AMPK pathwayactivation. PLoS One 9, e88570.

Money, T.G., Sproule, M.K., Cross, K.P., Robertson, R.M., 2016. Octopamine stabilizesconduction reliability of an unmyelinated axon during hypoxic stress. J.Neurophysiol. 116, 949–959.

Nespolo, R.F., Roff, D.A., Fairbairn, D.J., 2008. Energetic trade-off between maintenancecosts and flight capacity in the sand cricket (Gryllus firmus). Funct. Ecol. 22,624–631.

Niven, J.E., 2016. Neuronal energy consumption: biophysics, efficiency and evolution.Curr. Opin. Neurobiol. 41, 129–135.

Ott, S.R., Rogers, S.M., 2010. Gregarious desert locusts have substantially larger brainswith altered proportions compared with the solitarious phase. Proc. Biol. Sci. 277,3087–3096.

Pedgley, D.E., 1979. Weather during Desert Locust Plague Upsurges. Philos. Trans. R. Soc.London Series B 287, 387–391.

Pener, M., Simpson, S., 2009. Locust phase polyphenism: An update. In: Simpson, S.J.,Pener, M.P. (Eds.), Advances in Insect Physiology. Academic Press, pp. 1–272.

Rainey, R.C., 1951. Weather and the movements of locust swarms - a new hypothesis.Nature 168, 1057–1060.

Raubenheimer, D., Simpson, S.J., 1993. The geometry of compensatory feeding in thelocust. Anim. Behav. 45, 953–964.

Raubenheimer, D., Simpson, S.J., 2003. Nutrient balancing in grasshoppers: behaviouraland physiological correlates of dietary breadth. J. Exp. Biol. 206, 1669–1681.

Robertson, R.M., Spong, K.E., Srithiphaphirom, P., 2017. Chill coma in the locust, Locustamigratoria, is initiated by spreading depolarization in the central nervous system. Sci.Rep. 7, 10297.

Rodgers, C.I., Armstrong, G.A.B., Shoemaker, K.L., Labrie, J.D., Moyes, C.D., Robertson,R.M., 2007. Stress preconditioning of spreading depression in the locust CNS. PLoSOne 2.

Rodgers, C.I., Armstrong, G.A., Robertson, R.M., 2010. Coma in response to environ-mental stress in the locust: a model for cortical spreading depression. J. InsectPhysiol. 56, 980–990.

Rodgers-Garlick, C.I., Armstrong, G.A.B., Robertson, R.M., 2011. Metabolic stress mod-ulates motor patterning via AMP-activated protein kinase. J. Neurosci. 31,3207–3216.


91

http://refhub.elsevier.com/S1095-6433(18)30283-6/rf0005









































































































































































Rodriguez, E.C., Robertson, R.M., 2012. Protective effect of hypothermia on brain po-tassium homeostasis during repetitive anoxia in Drosophila melanogaster. J. Exp.Biol. 215, 4157–4165.

Rogers, S.M., Matheson, T., Sasaki, K., Kendrick, K., Simpson, S.J., Burrows, M., 2004.Substantial changes in central nervous system neurotransmitters and neuromodula-tors accompany phase change in the locust. J. Exp. Biol. 207, 3603–3617.

Sbrenna, G., 1971. Postembryonic growth of the ventral nerve cord in Schistocerca gregariaforsk. (Orthoptera:Acrididiae). Ital. J. Zool. 38, 48–74.

Schilman, P.E., Waters, J.S., Harrison, J.F., Lighton, J.R.B., 2011. Effects of temperatureon responses to anoxia and oxygen reperfusion in Drosophila melanogaster. J. Exp.Biol. 214, 1271–1275.

Simpson, S.J., McCaffery, A.R., Hagele, B.F., 1999. A behavioural analysis of phasechange in the desert locust. Biol. Rev. Camb. Philos. Soc. 74, 461–480.

Simpson, S.J., Raubenheimer, D., Behmer, S.T., Whitworth, A., Wright, G.A., 2002. Acomparison of nutritional regulation in solitarious- and gregarious-phase nymphs ofthe desert locust Schistocerca gregaria. J. Exp. Biol. 205, 121–129.

Simpson, S.J., Sword, G.A., Lorch, P.D., Couzin, I.D., 2006. Cannibal crickets on a forcedmarch for protein and salt. Proc. Natl. Acad. Sci. USA 103, 4152–4156.

Spong, K.E., Andrew, R.D., Robertson, R.M., 2016. Mechanisms of spreading depolar-ization in vertebrate and insect central nervous systems. J. Neurophysiol. 116,

1117–1127.Veran, S., Simpson, S.J., Sword, G.A., Deveson, E., Piry, S., Hines, J.E., Berthier, K., 2015.

Modeling spatiotemporal dynamics of outbreaking species: influence of environmentand migration in a locust. Ecology 96, 737–748.

Weyel, W., Wegener, G., 1996. Adenine nucleotide metabolism during anoxia and post-anoxic recovery in insects. Experientia 52, 474–480.

Woodman, J.D., 2013. Temperature affects immersion tolerance of first-instar nymphs ofthe Australian plague locust, Chortoicetes terminifera. Aust. J. Zool. 61, 328–331.

Woodman, J.D., 2015. Surviving a flood: effects of inundation period, temperature andembryonic development stage in locust eggs. B Entomol Res 105, 441–447.

Wu, B.S., Lee, J.K., Thompson, K.M., Walker, V.K., Moyes, C.D., Robertson, R.M., 2002.Anoxia induces thermotolerance in the locust flight system. J. Exp. Biol. 205,815–827.

Zanotto, F.P., Simpson, S.J., Raubenheimer, D., 1993. The regulation of growth by locuststhrough postingestive compensation for variation in the levels of dietary-protein andcarbohydrate. Physiol. Entomol. 18, 425–434.

Zanotto, F.P., Gouveia, S.M., Simpson, S.J., Raubenheimer, D., Calder, P.C., 1997.Nutritional homeostasis in locusts: is there a mechanism for increased energy ex-penditure during carbohydrate overfeeding. J. Exp. Biol. 200, 2437–2448.


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