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The effects of CO2 and chronic cold exposure on fecundity of female Drosophila melanogaster
Journal of Insect Physiology 57 (2011) 35–37
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
The effects of CO2 and chronic cold exposure on fecundity of female Drosophila melanogaster Jessie L.P. MacAlpine a, Katie E. Marshall b, Brent J. Sinclair b,* a b
Huron Park Secondary School, Woodstock, Ontario, Canada Department of Biology, The University of Western Ontario, London, Ontario, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 July 2010 Received in revised form 14 September 2010 Accepted 15 September 2010
Carbon dioxide and chilling are sometimes used to immobilise insects for laboratory research. Both of these methods are known to have short-term effects on behaviour and physiology in Drosophila, but their long-term impacts are unknown. We exposed female D. melanogaster adults to high CO2 concentrations (4 h at 18,000 ppm) and chronic cold (72 h at 4 8C). The carbon dioxide exposure increased chill coma recovery time, but did not result in changes in offspring number, sex ratio, or size. By contrast, the cold exposure resulted in fewer, smaller offspring, and resulted in a male-biased sex ratio compared to controls. There was no significant interaction between CO2 and cold. We conclude that although caution must be used in choosing an immobilisation method, CO2 appears to have less long-term impact than cold. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Carbon dioxide Anaesthesia Offspring sex ratio Parental investment Chilling
1. Introduction Exposure to high concentrations of carbon dioxide is frequently used to immobilise Drosophila melanogaster and other insects in the laboratory (Nicolas and Sillans, 1989). The mechanisms of this immobilisation seem to be associated with antagonising glutamate receptors in the neuromuscular junction (Badre et al., 2005). However, in D. melanogaster, CO2 exposure has been shown to affect survival of newly eclosed adults (Perron et al., 1972), mating behaviour (Barron, 2000), the rapid cold-hardening response (Nilson et al., 2006), heat hardening (Milton and Partridge, 2008) and, in D. simulans, male fecundity (Champion de Crespigny and Wedell, 2008). Cold exposure, inducing chill coma, is often used as an alternative means of immobilisation, but has its own effects on behaviour (e.g. Phelan et al., 2001), fecundity (e.g. through sperm dumping; Ashburner et al., 2005) and (in D. simulans), survival (Champion de Crespigny and Wedell, 2008). D. melanogaster is a common model in biology, and is becoming the primary model for understanding the effects of cold on chill susceptible insects (Hoffmann et al., 2003). The scale of many of these experiments reduces the practicality of handling flies without anaesthesia (e.g. the experiments described by Marshall and Sinclair (2010) required handling over 100,000 flies). Nilson et al. (2006) and David et al. (1998) found that the interaction between acute CO2 exposure and immediate response to cold
* Corresponding author. Tel.: +1 519 661 2111x83138, fax: +1 519 661 3935. E-mail address: [email protected] (B.J. Sinclair). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.09.003
became undetectable after a few hours’ recovery, and suggested that a recovery period may be sufficient to allow CO2 anaesthesia to be used prior to cold tolerance investigations. However, Nilson et al. (2006) examined only acute cold tolerance, and David et al. (1998) observed time to recover from chill coma, whereas the fitness effects of cold and other stressors are often sub-lethal, and play over a less immediate timescale (e.g. Marshall and Sinclair, 2010). Thus, to ensure the integrity of experiments on the response of D. melanogaster to cold, there is a need to determine whether CO2 exposure interacts with the response to cold in the long term. Here, we examine the sub-lethal effects of chronic cold and acute CO2 exposure, and their interactions in insects with a view to determining the long-term implications of CO2 and cold anaesthesia. We expose virgin female D. melanogaster to CO2 and then chronic cold. We measure the effects of CO2 on recovery from chill coma, and the effects of CO2 and chronic cold exposure on longterm reproductive output. 2. Methods D. melanogaster (collected from the vicinity of London, Ontario in 2007) were mass-reared on cornmeal-agar medium at 22 8C and 14:10 L:D and 70% RH, as described in detail elsewhere (Rajamohan and Sinclair, 2008). Eighty adult, virgin female D. melanogaster were collected after eclosion by aspiration without anaesthesia and placed individually into 30 mL vials containing food. The vials were divided into four treatment groups of twenty flies each. The first was housed in ambient temperature and CO2 levels (Control group), the second received ambient CO2 but
36
[()TD$FIG]
J.L.P. MacAlpine et al. / Journal of Insect Physiology 57 (2011) 35–37
chronic cold (Cold group), the third received extreme CO2 but ambient temperature (CO2 group) and the fourth was treated with extreme CO2 followed by chronic cold (CO2 + cold). Two days post-eclosion, vials containing flies (loosely sealed with a porous foam stopper) were placed in one of two 6 L airtight plastic containers containing a beaker of c. 250 mL of water. The next day, CO2 in one of the containers was elevated to 18,000 ppm for four hours by adding 12 alka-seltzer tablets (each containing 1000 mg citric acid and 1916 mg NaHCO3; Bayer, Morristown, NJ, USA) to the beaker of water, and measured with a Data-Harvest Easy Sense Q5 sensor (Data Harvest, Buffalo, NY, USA). Half the flies in each of the control and CO2-exposed groups were then exposed to chronic cold (72 h at 4 8C). After the cold treatment, flies were returned to room temperature, and observed at 5 min intervals to measure the time taken to recover from chill coma, determined when the individual could fly in response to a gentle tap to the vial. After all flies had recovered from chill coma, two virgin males of identical age to the female were added to each vial. Adults were removed after ten days, and all offspring from those adults removed and frozen fifteen days later (the latest date at which we could be sure to avoid inclusion of F2 offspring). The offspring from each vial were dried over silica gel for two days, sexed, and biomass of male and female offspring for each vial measured separately using a UMX5 microbalance (Mettler-Toledo, Mississauga, ON, Canada). Chill coma recovery was compared between CO2-treated and control flies using Kaplan–Meier survival analysis in Sigmaplot 11.2 (Systat software, San Jose, CA, USA). The overall number of offspring was compared among treatments using a generalised linear model with a negative binomial error distribution and a log link function on SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA). The sex ratio of the offspring was compared among treatments using a generalised linear model with binomial error and logit link, also using SPSS. Finally, the mass of the offspring was compared among sexes and treatments using an analysis of variance on SPSS. 3. Results There was no mortality in any of the CO2 or chronic cold treatments. The shape of the chill coma recovery curve was significantly changed by CO2 exposure. Even though the fastest initial recovery from chill coma was observed in flies exposed to CO2, most CO2-exposed flies took significantly longer to recover
[()TD$FIG]
Fig. 2. (A) Mean (s.e.m.) number of male (closed bars) and female (open bars) offspring produced by adult female D. melanogaster exposed to 4 h 18000 ppm CO2 (CO2), 72 h 4 8C (Cold) or both (CO2 + Cold). (B) Mean (s.e.m.) dry mass of male (closed bars) and female (open bars) exposed to CO2 or cold (treatments as above).
than their control counterparts (Fig. 1; Log-rank statistic: 5.535, df = 1, p = 0.019). Chronic cold exposure significantly reduced the number of offspring produced (Fig. 2a; Wald x2 = 15.588, df = 1, p < 0.001), but exposure to CO2 did not alter the total number of offspring produced (Wald x2 = 0.504, df = 1, p = 0.478), nor was there a CO2 cold interaction (Wald x2 = 0.002, df = 1, p = 0.961). Exposure to chronic cold significantly reduced the proportion of female offspring produced (Fig. 2a; Wald x2 = 16.807, df = 1, p < 0.001), and exposure to both CO2 and cold resulted in a slight shift to a male-biased offspring ratio (Fig. 2a; Wald x2 = 14.718, df = 1, p < 0.001). Female offspring were larger than male offspring (Fig. 2b; Table 1). CO2 exposure did not affect dry mass of offspring
Table 1 Results of a general linear model comparing mean size of offspring of female D. melanogaster exposed to chronic cold or high CO2 (or both), to the size of offspring of their relevant controls. N = 16–18 female parents per treatment combination. p Values in bold indicate statistically significant terms.
Fig. 1. Cumulative chill coma recovery in female Drosophila melanogaster adults after 72 h at 4 8C. Control flies (solid line) recovered faster than flies that had been exposed to 18,000 ppm CO2 for 4 h (dashed line).
Source
df
MS
F
p
CO2 Cold Sex CO2 Cold CO2 Sex Cold Sex CO2 Cold Sex Error
1 1 1 1 1 1 1 98
0.0009 0.0030 0.0459 0.0017 0.0007 0.0148 0.0018 0.0033
0.282 0.901 13.947 0.528 0.200 4.500 0.531
0.596 0.345 <0.001 0.469 0.655 0.036 0.468
J.L.P. MacAlpine et al. / Journal of Insect Physiology 57 (2011) 35–37
(Fig. 2b; Table 1). However, there was a treatment sex interaction such that cold-treated flies produced male offspring that were significantly smaller than their control or CO2-treated counterparts, but chronic cold exposure did not alter the size of female offspring (Fig. 2b; Table 1). There were no significant interactions between CO2 and cold or CO2 and sex, and the threeway interaction was also non-significant (Table 1). 4. Discussion Here, we show that CO2 exposure alters chill coma recovery, but has no impact on fecundity or maternal investment in female D. melanogaster. This concurs with previous work showing that CO2 modifies chill coma recovery time (Milton and Partridge, 2008; Nilson et al., 2006). The mechanisms underlying both chill coma recovery and CO2 anaesthesia are poorly understood, but it appears that both act upon the nervous system (Badre et al., 2005; Hosler et al., 2000), and that this is why the two treatments interact. Chronic cold exposure (3 days at 4 8C) had a substantial impact on fecundity of female flies, as well as on the size and sex ratio of their offspring. Thus, CO2 exposure (as might be used in anaesthesia) does not appear to have long-term sub-lethal consequences for female D. melanogaster, at least in terms of offspring quantity, size or sex ratio. This suggests that CO2 exposure may induce fewer overall physiological responses (or less damage) than cold, or that the pathways targeted are primarily short-term, compared to the longer-term impacts of chronic cold exposure. Low temperature exposure may affect reproduction of D. melanogaster by several routes. Cold can be used to induce spermdumping by mated females (Ashburner et al., 2005), but our females were unfertilised. Stress can also have effects on receptivity of females, and although Shreve et al. (2004) have previously shown that a cold exposure that induces RCH preserves mating behaviour, the impact of cold on longer-term behavioural responses has not been investigated (but see Champion de Crespigny and Wedell, 2008). We did not count eggs laid by females in our study, and it is possible that there may have been increased (and differential) mortality of embryos after cold exposure. Marshall and Sinclair (2010) showed a change in sex ratio and number of offspring in female flies repeatedly exposed to cold. They interpreted this to be a result of a physiological response to cold, possibly as a result of reallocation of resources to support repair of cold-induced damage. Although the mechanisms of chronic cold damage are likely different from those underlying acute cold injury (see Sinclair and Roberts, 2005), it is possible that a similar process is taking place: that female flies either have depleted energy reserves after the chronic cold exposure, or that chilling injuries are accrued during the three day exposure to 4 8C and flies must invest energy in repair rather than reproduction. However, flies have full access to food during this time, so it seems that persistent physiological damage is a more likely explanation for reduced fecundity than depleted energy reserves. Additional experiments will be necessary to determine whether the impact is life-long (implying physiological damage) or whether our method is biased towards reproductive output in the first few days postexposure, when recovery and repair are still occurring. The carbon dioxide exposure (4 h) was likely an extreme case – few researchers would use such long exposures to immobilise flies for experiments. Assuming that the impact of a lesser exposure is not worse (which seems reasonable); we therefore suggest that
37
CO2 may be used as an immobilising agent for D. melanogaster with little or no long-term sub-lethal effect on reproduction. However, we note that CO2 does interact with several short-term responses, especially cold acclimation and hardening and heat hardening (Milton and Partridge, 2008; Nilson et al., 2006), and suggest that care should be taken to determine appropriate recovery periods, and allow for them between anaesthesia and stress experiments. Finally, we note that the reproductive output assay we conducted is relatively straightforward, and we suggest that it would be a good adjunct to other rapid assays (for example, chill coma; Gibert et al., 2001) for determining the impacts of physiological stress on performance of Drosophila. Acknowledgements JLPM thanks Kelly and Brock MacAlpine for help and support, Roger Hall for the loan of equipment and the Sanofi-Aventis Biotalent Challenge for facilitating this project. This work was supported in part by NSERC Discovery and Canadian Foundation for Innovation grants to BJS. We are grateful to two anonymous referees for their constructive suggestions, which improved the clarity of the manuscript. References Ashburner, M., Golic, K., Hawley, S.H., 2005. Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, New York. Badre, N.H., Martin, M.E., Cooper, R.L., 2005. The physiological and behavioral effects of carbon dioxide on Drosophila melanogaster larvae. Comparative Biochemistry and Physiology A 140, 363–376. Barron, A.B., 2000. Anaesthetising Drosophila for behavioural studies. Journal of Insect Physiology 46, 439–442. Champion de Crespigny, F.E., Wedell, N., 2008. The impact of anaesthetic technique on survival and fertility in Drosophila. Physiological Entomology 33, 310–315. David, R.J., Gibert, P., Pla, E., Petavy, G., Karan, D., Moreteau, B., 1998. Cold stress tolerance in Drosophila: Analysis of chill coma recovery in D. melanogaster. Journal of Thermal Biology 23, 291–299. Gibert, P., Moreteau, B., Petavy, G., Karan, D., David, J.R., 2001. Chill-coma tolerance, a major climatic adaptation among Drosophila species. Evolution 55, 1063– 1068. Hoffmann, A.A., Sorensen, J.G., Loeschcke, V., 2003. Adaptation of Drosophila to temperature extremes: bringing together quantitative and molecular approaches. Journal of Thermal Biology 28, 175–216. Hosler, J.S., Burns, J.E., Esch, H.E., 2000. Flight muscle resting potential and speciesspecific differences in chill-coma. Journal of Insect Physiology 46, 621–627. Marshall, K.E., Sinclair, B.J., 2010. Repeated stress exposure results in a survivalreproduction trade-off in Drosophila melanogaster. Proceedings of the Royal Society B 277, 963–969. Milton, C.C., Partridge, L., 2008. Brief carbon dioxide exposure blocks heat hardening but not cold acclimation in Drosophila melanogaster. Journal of Insect Physiology 54, 32–40. Nicolas, G., Sillans, D., 1989. Immediate and latent effects of carbon dioxide on insects. Annual Review of Entomology 34, 97–116. Nilson, T.N., Sinclair, B.J., Roberts, S.P., 2006. The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster. Journal of Insect Physiology 52, 1027–1033. Perron, J.M., Huot, L., Corrivault, G.-W., Chawla, S.S., 1972. Effects of carbon dioxide anaesthesia on Drosophila melanogaster. Journal of Insect Physiology 18, 1869– 1874. Phelan, L.L., Rodd, Z.A., Hirsch, H.V.B., Rosellini, R.A., 2001. Exposure to cold: aversive Pavlovian conditioning in individual Drosophila melanogaster. Physiological Entomology 26, 219–224. Rajamohan, A., Sinclair, B.J., 2008. Short-term hardening effects on survival of acute and chronic cold exposure by Drosophila melanogaster larvae. Journal of Insect Physiology 54, 708–718. Shreve, S.M., Kelty, J.D., Lee, R.E., 2004. Preservation of reproductive behaviors during modest cooling: rapid cold-hardening fine-tunes organismal response. Journal of Experimental Biology 207, 1797–1802. Sinclair, B.J., Roberts, S.P., 2005. Acclimation, shock and hardening in the cold. Journal of Thermal Biology 30, 557–562.
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
The effects of CO2 and chronic cold exposure on fecundity of female Drosophila melanogaster Jessie L.P. MacAlpine a, Katie E. Marshall b, Brent J. Sinclair b,* a b
Huron Park Secondary School, Woodstock, Ontario, Canada Department of Biology, The University of Western Ontario, London, Ontario, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 July 2010 Received in revised form 14 September 2010 Accepted 15 September 2010
Carbon dioxide and chilling are sometimes used to immobilise insects for laboratory research. Both of these methods are known to have short-term effects on behaviour and physiology in Drosophila, but their long-term impacts are unknown. We exposed female D. melanogaster adults to high CO2 concentrations (4 h at 18,000 ppm) and chronic cold (72 h at 4 8C). The carbon dioxide exposure increased chill coma recovery time, but did not result in changes in offspring number, sex ratio, or size. By contrast, the cold exposure resulted in fewer, smaller offspring, and resulted in a male-biased sex ratio compared to controls. There was no significant interaction between CO2 and cold. We conclude that although caution must be used in choosing an immobilisation method, CO2 appears to have less long-term impact than cold. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Carbon dioxide Anaesthesia Offspring sex ratio Parental investment Chilling
1. Introduction Exposure to high concentrations of carbon dioxide is frequently used to immobilise Drosophila melanogaster and other insects in the laboratory (Nicolas and Sillans, 1989). The mechanisms of this immobilisation seem to be associated with antagonising glutamate receptors in the neuromuscular junction (Badre et al., 2005). However, in D. melanogaster, CO2 exposure has been shown to affect survival of newly eclosed adults (Perron et al., 1972), mating behaviour (Barron, 2000), the rapid cold-hardening response (Nilson et al., 2006), heat hardening (Milton and Partridge, 2008) and, in D. simulans, male fecundity (Champion de Crespigny and Wedell, 2008). Cold exposure, inducing chill coma, is often used as an alternative means of immobilisation, but has its own effects on behaviour (e.g. Phelan et al., 2001), fecundity (e.g. through sperm dumping; Ashburner et al., 2005) and (in D. simulans), survival (Champion de Crespigny and Wedell, 2008). D. melanogaster is a common model in biology, and is becoming the primary model for understanding the effects of cold on chill susceptible insects (Hoffmann et al., 2003). The scale of many of these experiments reduces the practicality of handling flies without anaesthesia (e.g. the experiments described by Marshall and Sinclair (2010) required handling over 100,000 flies). Nilson et al. (2006) and David et al. (1998) found that the interaction between acute CO2 exposure and immediate response to cold
* Corresponding author. Tel.: +1 519 661 2111x83138, fax: +1 519 661 3935. E-mail address: [email protected] (B.J. Sinclair). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.09.003
became undetectable after a few hours’ recovery, and suggested that a recovery period may be sufficient to allow CO2 anaesthesia to be used prior to cold tolerance investigations. However, Nilson et al. (2006) examined only acute cold tolerance, and David et al. (1998) observed time to recover from chill coma, whereas the fitness effects of cold and other stressors are often sub-lethal, and play over a less immediate timescale (e.g. Marshall and Sinclair, 2010). Thus, to ensure the integrity of experiments on the response of D. melanogaster to cold, there is a need to determine whether CO2 exposure interacts with the response to cold in the long term. Here, we examine the sub-lethal effects of chronic cold and acute CO2 exposure, and their interactions in insects with a view to determining the long-term implications of CO2 and cold anaesthesia. We expose virgin female D. melanogaster to CO2 and then chronic cold. We measure the effects of CO2 on recovery from chill coma, and the effects of CO2 and chronic cold exposure on longterm reproductive output. 2. Methods D. melanogaster (collected from the vicinity of London, Ontario in 2007) were mass-reared on cornmeal-agar medium at 22 8C and 14:10 L:D and 70% RH, as described in detail elsewhere (Rajamohan and Sinclair, 2008). Eighty adult, virgin female D. melanogaster were collected after eclosion by aspiration without anaesthesia and placed individually into 30 mL vials containing food. The vials were divided into four treatment groups of twenty flies each. The first was housed in ambient temperature and CO2 levels (Control group), the second received ambient CO2 but
36
[()TD$FIG]
J.L.P. MacAlpine et al. / Journal of Insect Physiology 57 (2011) 35–37
chronic cold (Cold group), the third received extreme CO2 but ambient temperature (CO2 group) and the fourth was treated with extreme CO2 followed by chronic cold (CO2 + cold). Two days post-eclosion, vials containing flies (loosely sealed with a porous foam stopper) were placed in one of two 6 L airtight plastic containers containing a beaker of c. 250 mL of water. The next day, CO2 in one of the containers was elevated to 18,000 ppm for four hours by adding 12 alka-seltzer tablets (each containing 1000 mg citric acid and 1916 mg NaHCO3; Bayer, Morristown, NJ, USA) to the beaker of water, and measured with a Data-Harvest Easy Sense Q5 sensor (Data Harvest, Buffalo, NY, USA). Half the flies in each of the control and CO2-exposed groups were then exposed to chronic cold (72 h at 4 8C). After the cold treatment, flies were returned to room temperature, and observed at 5 min intervals to measure the time taken to recover from chill coma, determined when the individual could fly in response to a gentle tap to the vial. After all flies had recovered from chill coma, two virgin males of identical age to the female were added to each vial. Adults were removed after ten days, and all offspring from those adults removed and frozen fifteen days later (the latest date at which we could be sure to avoid inclusion of F2 offspring). The offspring from each vial were dried over silica gel for two days, sexed, and biomass of male and female offspring for each vial measured separately using a UMX5 microbalance (Mettler-Toledo, Mississauga, ON, Canada). Chill coma recovery was compared between CO2-treated and control flies using Kaplan–Meier survival analysis in Sigmaplot 11.2 (Systat software, San Jose, CA, USA). The overall number of offspring was compared among treatments using a generalised linear model with a negative binomial error distribution and a log link function on SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA). The sex ratio of the offspring was compared among treatments using a generalised linear model with binomial error and logit link, also using SPSS. Finally, the mass of the offspring was compared among sexes and treatments using an analysis of variance on SPSS. 3. Results There was no mortality in any of the CO2 or chronic cold treatments. The shape of the chill coma recovery curve was significantly changed by CO2 exposure. Even though the fastest initial recovery from chill coma was observed in flies exposed to CO2, most CO2-exposed flies took significantly longer to recover
[()TD$FIG]
Fig. 2. (A) Mean (s.e.m.) number of male (closed bars) and female (open bars) offspring produced by adult female D. melanogaster exposed to 4 h 18000 ppm CO2 (CO2), 72 h 4 8C (Cold) or both (CO2 + Cold). (B) Mean (s.e.m.) dry mass of male (closed bars) and female (open bars) exposed to CO2 or cold (treatments as above).
than their control counterparts (Fig. 1; Log-rank statistic: 5.535, df = 1, p = 0.019). Chronic cold exposure significantly reduced the number of offspring produced (Fig. 2a; Wald x2 = 15.588, df = 1, p < 0.001), but exposure to CO2 did not alter the total number of offspring produced (Wald x2 = 0.504, df = 1, p = 0.478), nor was there a CO2 cold interaction (Wald x2 = 0.002, df = 1, p = 0.961). Exposure to chronic cold significantly reduced the proportion of female offspring produced (Fig. 2a; Wald x2 = 16.807, df = 1, p < 0.001), and exposure to both CO2 and cold resulted in a slight shift to a male-biased offspring ratio (Fig. 2a; Wald x2 = 14.718, df = 1, p < 0.001). Female offspring were larger than male offspring (Fig. 2b; Table 1). CO2 exposure did not affect dry mass of offspring
Table 1 Results of a general linear model comparing mean size of offspring of female D. melanogaster exposed to chronic cold or high CO2 (or both), to the size of offspring of their relevant controls. N = 16–18 female parents per treatment combination. p Values in bold indicate statistically significant terms.
Fig. 1. Cumulative chill coma recovery in female Drosophila melanogaster adults after 72 h at 4 8C. Control flies (solid line) recovered faster than flies that had been exposed to 18,000 ppm CO2 for 4 h (dashed line).
Source
df
MS
F
p
CO2 Cold Sex CO2 Cold CO2 Sex Cold Sex CO2 Cold Sex Error
1 1 1 1 1 1 1 98
0.0009 0.0030 0.0459 0.0017 0.0007 0.0148 0.0018 0.0033
0.282 0.901 13.947 0.528 0.200 4.500 0.531
0.596 0.345 <0.001 0.469 0.655 0.036 0.468
J.L.P. MacAlpine et al. / Journal of Insect Physiology 57 (2011) 35–37
(Fig. 2b; Table 1). However, there was a treatment sex interaction such that cold-treated flies produced male offspring that were significantly smaller than their control or CO2-treated counterparts, but chronic cold exposure did not alter the size of female offspring (Fig. 2b; Table 1). There were no significant interactions between CO2 and cold or CO2 and sex, and the threeway interaction was also non-significant (Table 1). 4. Discussion Here, we show that CO2 exposure alters chill coma recovery, but has no impact on fecundity or maternal investment in female D. melanogaster. This concurs with previous work showing that CO2 modifies chill coma recovery time (Milton and Partridge, 2008; Nilson et al., 2006). The mechanisms underlying both chill coma recovery and CO2 anaesthesia are poorly understood, but it appears that both act upon the nervous system (Badre et al., 2005; Hosler et al., 2000), and that this is why the two treatments interact. Chronic cold exposure (3 days at 4 8C) had a substantial impact on fecundity of female flies, as well as on the size and sex ratio of their offspring. Thus, CO2 exposure (as might be used in anaesthesia) does not appear to have long-term sub-lethal consequences for female D. melanogaster, at least in terms of offspring quantity, size or sex ratio. This suggests that CO2 exposure may induce fewer overall physiological responses (or less damage) than cold, or that the pathways targeted are primarily short-term, compared to the longer-term impacts of chronic cold exposure. Low temperature exposure may affect reproduction of D. melanogaster by several routes. Cold can be used to induce spermdumping by mated females (Ashburner et al., 2005), but our females were unfertilised. Stress can also have effects on receptivity of females, and although Shreve et al. (2004) have previously shown that a cold exposure that induces RCH preserves mating behaviour, the impact of cold on longer-term behavioural responses has not been investigated (but see Champion de Crespigny and Wedell, 2008). We did not count eggs laid by females in our study, and it is possible that there may have been increased (and differential) mortality of embryos after cold exposure. Marshall and Sinclair (2010) showed a change in sex ratio and number of offspring in female flies repeatedly exposed to cold. They interpreted this to be a result of a physiological response to cold, possibly as a result of reallocation of resources to support repair of cold-induced damage. Although the mechanisms of chronic cold damage are likely different from those underlying acute cold injury (see Sinclair and Roberts, 2005), it is possible that a similar process is taking place: that female flies either have depleted energy reserves after the chronic cold exposure, or that chilling injuries are accrued during the three day exposure to 4 8C and flies must invest energy in repair rather than reproduction. However, flies have full access to food during this time, so it seems that persistent physiological damage is a more likely explanation for reduced fecundity than depleted energy reserves. Additional experiments will be necessary to determine whether the impact is life-long (implying physiological damage) or whether our method is biased towards reproductive output in the first few days postexposure, when recovery and repair are still occurring. The carbon dioxide exposure (4 h) was likely an extreme case – few researchers would use such long exposures to immobilise flies for experiments. Assuming that the impact of a lesser exposure is not worse (which seems reasonable); we therefore suggest that
37
CO2 may be used as an immobilising agent for D. melanogaster with little or no long-term sub-lethal effect on reproduction. However, we note that CO2 does interact with several short-term responses, especially cold acclimation and hardening and heat hardening (Milton and Partridge, 2008; Nilson et al., 2006), and suggest that care should be taken to determine appropriate recovery periods, and allow for them between anaesthesia and stress experiments. Finally, we note that the reproductive output assay we conducted is relatively straightforward, and we suggest that it would be a good adjunct to other rapid assays (for example, chill coma; Gibert et al., 2001) for determining the impacts of physiological stress on performance of Drosophila. Acknowledgements JLPM thanks Kelly and Brock MacAlpine for help and support, Roger Hall for the loan of equipment and the Sanofi-Aventis Biotalent Challenge for facilitating this project. This work was supported in part by NSERC Discovery and Canadian Foundation for Innovation grants to BJS. We are grateful to two anonymous referees for their constructive suggestions, which improved the clarity of the manuscript. References Ashburner, M., Golic, K., Hawley, S.H., 2005. Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, New York. Badre, N.H., Martin, M.E., Cooper, R.L., 2005. The physiological and behavioral effects of carbon dioxide on Drosophila melanogaster larvae. Comparative Biochemistry and Physiology A 140, 363–376. Barron, A.B., 2000. Anaesthetising Drosophila for behavioural studies. Journal of Insect Physiology 46, 439–442. Champion de Crespigny, F.E., Wedell, N., 2008. The impact of anaesthetic technique on survival and fertility in Drosophila. Physiological Entomology 33, 310–315. David, R.J., Gibert, P., Pla, E., Petavy, G., Karan, D., Moreteau, B., 1998. Cold stress tolerance in Drosophila: Analysis of chill coma recovery in D. melanogaster. Journal of Thermal Biology 23, 291–299. Gibert, P., Moreteau, B., Petavy, G., Karan, D., David, J.R., 2001. Chill-coma tolerance, a major climatic adaptation among Drosophila species. Evolution 55, 1063– 1068. Hoffmann, A.A., Sorensen, J.G., Loeschcke, V., 2003. Adaptation of Drosophila to temperature extremes: bringing together quantitative and molecular approaches. Journal of Thermal Biology 28, 175–216. Hosler, J.S., Burns, J.E., Esch, H.E., 2000. Flight muscle resting potential and speciesspecific differences in chill-coma. Journal of Insect Physiology 46, 621–627. Marshall, K.E., Sinclair, B.J., 2010. Repeated stress exposure results in a survivalreproduction trade-off in Drosophila melanogaster. Proceedings of the Royal Society B 277, 963–969. Milton, C.C., Partridge, L., 2008. Brief carbon dioxide exposure blocks heat hardening but not cold acclimation in Drosophila melanogaster. Journal of Insect Physiology 54, 32–40. Nicolas, G., Sillans, D., 1989. Immediate and latent effects of carbon dioxide on insects. Annual Review of Entomology 34, 97–116. Nilson, T.N., Sinclair, B.J., Roberts, S.P., 2006. The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster. Journal of Insect Physiology 52, 1027–1033. Perron, J.M., Huot, L., Corrivault, G.-W., Chawla, S.S., 1972. Effects of carbon dioxide anaesthesia on Drosophila melanogaster. Journal of Insect Physiology 18, 1869– 1874. Phelan, L.L., Rodd, Z.A., Hirsch, H.V.B., Rosellini, R.A., 2001. Exposure to cold: aversive Pavlovian conditioning in individual Drosophila melanogaster. Physiological Entomology 26, 219–224. Rajamohan, A., Sinclair, B.J., 2008. Short-term hardening effects on survival of acute and chronic cold exposure by Drosophila melanogaster larvae. Journal of Insect Physiology 54, 708–718. Shreve, S.M., Kelty, J.D., Lee, R.E., 2004. Preservation of reproductive behaviors during modest cooling: rapid cold-hardening fine-tunes organismal response. Journal of Experimental Biology 207, 1797–1802. Sinclair, B.J., Roberts, S.P., 2005. Acclimation, shock and hardening in the cold. Journal of Thermal Biology 30, 557–562.