1997
The Journal of Experimental Biology 214, 1997-2004 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jeb.056531

RESEARCH ARTICLE Some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket Gryllus texensis
Shelley A. Adamo* and Maggie M. E. Lovett
Department of Psychology, Neuroscience Institute, Dalhousie University, 1355 Oxford Street, Halifax, NS, Canada, B3H 3X5
*Author for correspondence (sadamo@dal.ca)

Accepted 3 March 2011

SUMMARY In many parts of the world, climate change is increasing the frequency and severity of heat waves. How do heat waves impact short-lived poikilotherms such as insects? In the cricket, Gryllus texensis, 6days of elevated temperatures (i.e. 7°C above the average field temperature and 5°C above their preferred temperature) resulted in increased egg laying, faster egg development and greater mass gain. The increased temperature also increased activity of phenoloxidase and lysozyme-like enzymes, two immune-related enzymes, and enhanced resistance to the Gram-negative bacterium Serratia marcescens. When given a sublethal S. marcescens infection, G. texensis maintained increased reproductive output at the elevated temperature (33°C). These data suggest that heat waves could result in more numerous, disease resistant, crickets. However, resistance to the Gram-positive bacterium, Bacillus cereus was lower at temperatures above or below the average field temperature (26°C). A sublethal infection with B. cereus reduced egg laying at all temperatures and suppressed the increase in egg laying induced by higher temperatures. These results suggest that for some species–pathogen interactions, increased temperatures can induce trade-offs between reproduction and disease resistance. This result may partly explain why G. texensis prefers temperatures lower than those that produce maximal reproductive output and enhanced immune function.
Key words: global warming, insect, Orthoptera, phenoloxidase, oviposition.

INTRODUCTION

Global warming is producing increases in both average temperatures and in the frequency and severity of heat waves (Stone et al., 2010). The magnitude of these changes varies across the globe. For example, central Texas, USA, has not experienced any significant warming over the 20th century; however, temperatures are expected to rise gradually during this century (Nielsen-Gammon, 2011). Nevertheless, Austin, TX, has experienced an increase in the frequency and severity of heat waves over the last 50years (Stone et al., 2010), and for this region, the increase in heat waves remains the largest present-day impact of global warming. For poikilotherms such as insects, ambient temperature profoundly affects the rate of biochemical reactions critical to their physiology (Chown and Nicolson, 2004). For shortlived animals such as insects, even one extended heat wave may have significant impacts. Unfortunately, the effects of heat waves on insect biology and behaviour are hard to predict. Increased temperature should increase the rate of enzymatic reactions within individuals (Angilletta et al., 2010); however, the effect of temperature on enzymatic pathways does not necessarily predict what will happen to the whole organism (Chaui-Berlinck et al., 2004). Nonetheless, it is the effect of warmer temperatures on whole organisms that will have important ramifications for ecosystems. Within physiological limits, increased enzymatic rates in insects should lead to higher metabolic rates, more rapid growth and increased reproduction (Angilletta et al., 2010). Such effects have been demonstrated for a number of insect species, usually under laboratory conditions (Frazier et al., 2006). In many temperate-zone insects, the optimal temperature (i.e. temperature that generates the greatest number of offspring) is higher than the environmental

temperature (Frazier et al., 2006; Deutsch et al., 2008). For some temperate-zone insects, temperatures remain below the optimal temperature for population growth, even in summer (Deutsch et al., 2008). In part this is because insect enzymes often show their highest activity at temperatures 10 or 20°C above the environmental temperature [e.g. phenoloxidase in Heliothis virescens (Lockey and Ourth, 1992) and Musca domestica (Hara et al., 1993)]. These data suggest that many temperate-zone insects should show enhanced performance, including increased reproduction, at higher temperatures. Although insects lack the complex neural and physiological mechanisms required to maintain a constant body temperature against changes in external temperature, some insects can control their internal temperature behaviourally (Chown and Nicolson, 2004). For example, crickets thermoregulate by moving to microenvironments that allow them to maintain their preferred body temperature (Adamo, 1998). Behavioural thermoregulation can produce body temperatures considerably above ambient temperature (Chown and Nicolson, 2004). Given the expected benefits of elevated temperatures to insect reproduction, and presumably fitness (Deutsch et al., 2008), it might be expected that most temperate-zone insects would have temperature preferences far above their typical environmental temperature. However, many insects that behaviourally thermoregulate prefer temperatures not too far above the environmental temperature [e.g. crickets (Louis et al., 1986; Adamo, 1998)]. This puzzling issue has not been specifically addressed. One possible reason why many temperate-zone insects do not prefer extremely warm temperatures, despite the thermodynamic advantage they provide, is that warmer temperatures induce physiological trade-offs that may decrease an individual’s fitness

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1998 S. A. Adamo and M. M. E. Lovett
(Angiletta et al., 2003). For example, reproduction is an extremely expensive activity in terms of time, energy and resources for female insects (Harshman and Zera, 2007). Enhanced reproduction may take energy and resources away from other metabolically expensive systems [e.g. immune function (Freitak et al., 2003)]. Many studies have shown that reproduction occurs at the expense of immune function and disease resistance, even when food is abundant (Harshman and Zera, 2007; Lawniczak et al., 2007). Therefore, at higher temperatures, insects may increase their rate of reproduction, but may become more susceptible to disease as a result, leading to reduced lifespan and possibly reduced fitness in the field. Trade-offs between immune function and reproduction could be offset by the positive effects of elevated temperatures on insect immune systems. Individual enzymatic reactions should become faster, leading to an enhanced immune response. Empirical evidence suggests that warmer temperatures do increase a variety of immune responses (e.g. Ouedraogo et al., 2002; Ouedraogo et al., 2003). However, other studies demonstrate that warmer temperatures lead to a decline in some immune functions [e.g. melanisation (Suwanchaichanda and Paskewitz, 1998)]. Determining how increased temperature will influence reproduction and how this will influence immune function and disease resistance requires empirical study. Few studies have examined these interactions (Guinnee and Moore, 2004), yet they may play a crucial role in the response of an insect to climate change. If both immune function and reproduction are simultaneously enhanced, then, at least for some species, climate change will result in the production of more insects, and these insects will be more disease resistant. If higher temperatures induce or exacerbate trade-offs between reproduction and immune function, then this might be a partial explanation as to why species appear to prefer ‘suboptimal’ temperatures. The Texan cricket Gryllus texensis (Cade and Otte, 2000) is a good model organism for studying this question. Temperature is known to influence cricket physiology [e.g. metabolic rate is highly correlated with temperature (Nespolo et al., 2003)] and behaviour (e.g. Martin et al., 2000). Moreover, crickets behaviourally thermoregulate [G. texensis (Adamo, 1998), Gryllus integer (Hedrick et al., 2002), Gryllus bimaculatus (Vanwyk and Ferguson, 1995)]. Gryllus texensis can be maintained at different temperatures, and female reproductive output is easy to quantify (e.g. Shoemaker et al., 2006a; Shoemaker et al., 2006b). It has been used for ecoimmunological studies, and some immune functions can be quantified (e.g. Adamo, 2004a). There is also evidence that immune system activation is costly in this cricket (Shoemaker and Adamo, 2007). Immune challenge usually reduces female reproduction in insects (Reaney and Knell, 2010 and references therein), however, under normal temperatures, there is little evidence for trade-offs between reproduction and immune function in female G. texensis (Shoemaker et al., 2006a; Shoemaker et al., 2006b; Shoemaker and Adamo, 2007). Therefore, it is possible to use this species to test whether such trade-offs are induced at higher temperatures. In this study, we examined whether raised temperatures, mimicking a typical heat wave, increase reproduction in G. texensis. We then determined whether increased temperature also enhances immune function as well as disease resistance to two different pathogens. Finally, we examined the effects of infection on reproduction at different temperatures. These experiments will help us to determine whether increased temperatures are likely to result in trade-offs between immune function and reproduction, even if temperature enhances each trait individually. Such trade-offs will influence how this species responds to climate change.
MATERIALS AND METHODS Animals

Crickets (long-winged G. texensis) were collected near Austin, TX, USA, and have been maintained as a laboratory colony for many generations with occasional additions of fresh animals from the field. Crickets were reared at 26±3°C on a 12h:12h light:dark cycle. This temperature represents the average temperature crickets experience during their adult life (National Weather Service Climate Report; http://www.weather.gov/climate/index.php?wfoewx). G. texensis become sexually mature in Austin, Texas in the spring (May) or fall (September–October) (Adamo et al., 1995; Bertram, 2002). Pellets of dry cat food and water were provided ad libitum. Crickets were used 2weeks (±3days) after the moult to adulthood. At this age female G. texensis have mated (Solymar and Cade, 1990) and are within their lifespan in the field (Murray and Cade, 1995). It is also well before the immune system begins to decline as a result of senescence (~4weeks) (Adamo et al., 2001). No cricket was used in more than one experiment. All experiments included at least three independent replicates spanning multiple generations within the insect colony. All studies were approved by the Animal Care Committee of Dalhousie University (#I9-026) and are in accordance with the Canadian Council on Animal Care. Chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA) unless otherwise noted.
Temperature preference of G. texensis

Crickets were placed individually in opaque tubs (10 10cm; diameter depth) for 1day prior to trials. A temperature gradient was created by placing one end of a glass aquarium (26 48 28cm) onto a hot plate. The glass aquarium was divided into five lanes, each 5cm wide. Crickets were unable to see their neighbours through the metal section dividers. The floor of the aquarium was covered with 2cm of sand. Once the aquarium temperature had reached equilibrium (~3h), one cricket was placed in the centre of each lane, males and females in alternate lanes. The temperature ranged from 26°C at the cool end to 38°C at the warm end. Crickets were given 30min to acclimatize to the aquarium, and then the temperature at their position was measured with an electronic thermometer at 30 and 60min and the average of the two temperatures was calculated. A high proportion of the crickets (38/45, 84%) were at the same temperature for both time points. An earlier study (Adamo, 1998) showed that these time points give an accurate estimate of an individual’s temperature preference. A cricket’s internal body temperature is identical to the external temperature when crickets are in a temperature gradient (Louis et al., 1986). The aquarium was rotated between trials so that the heated end of the aquarium was changed each trial. The sand was also mixed thoroughly between trials.
Effects of temperature on reproduction

Females were weighed and isolated in containers (15 17 9.5cm) with food and water ad libitum. They were assigned to one of three temperature groups: 18, 26 or 33°C for 6days, which is 28% of an adult’s lifespan (Murray and Cade, 1995). Heat wave temperatures were represented by the 33°C treatment, as experienced in Austin, TX (National Weather Service; http://www.weather.gov/climate/ index.php?wfoewx; e.g. 14–24 July 2010, average daily temperature, 33°C). A cold snap during the breeding season (e.g. 9–12 October 2009) was represented by the 18°C treatment. Average temperatures during the times G. texensis are actively seeking mates (morning and evening) in the field are typically in the range of 17–23°C (Souroukis et al., 1992).

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Climate change effects on insects
Females were placed in one of two incubators at 18 or 33°C, with a 12h:12h light:dark cycle, or left in the rearing room at 26°C. An oxygen monitor placed in the incubators demonstrated that oxygen levels remained normal throughout the incubation period. After 6days, crickets were removed and the number of eggs laid was counted and females were re-weighed. A random subset of eggs (five eggs from each female) was set aside. These eggs were returned to their previous temperature regime to determine hatching success. Eggs were left in cotton and buried in vermiculite. Hatchlings were collected and counted every day for 50days. Eight eggs randomly chosen from eight females per temperature were tested for their total protein content using a Bradford total protein assay (Bradford, 1976). Eggs were sonicated in distilled water and the supernatant was added to the Bradford reagent. The change in absorbance at 595nm was measured 10min later and compared with values obtained using a protein standard (bovine albumen) run on the same day.
Effect of temperature on immune function

1999

was measured at 600nm. Cuvettes were incubated at 18, 26 and 33°C and the absorption was read prior to incubation and at 6, 12 and 24h. Similarly, 1ml of nutrient broth was added to 100l of B. cereus (~1 105cellsl–1) in a 1.5ml cuvette. Absorption was measured at 600nm. Cuvettes were incubated at 18, 26 and 33°C and the absorption was read prior to incubation and at 8, 18 and 24h. To test resistance, females were isolated and assigned to the three different temperature regimes as previously described. After 6days, females were injected with the LD50 dose for one of the two bacteria [S. marcescens, ~1 104 cells (Adamo et al., 2010); B. cereus ~6 04 cells (S.A.A., unpublished data)]. Mortality was recorded for the next 6days. At room temperature, mortality as a result of these pathogens occurs within 4days with S. marcescens (Adamo et al., 2001) or 5days with B. cereus (S.A.A., unpublished data).
Effect of infection on egg laying

Females were isolated as previously described. After 6days at one of the three temperatures (18, 26 or 33°C), 2l of haemolymph was removed to assess either phenoloxidase activity or lysozyme-like activity at different temperatures. Phenoloxidase activity was measured using a method modified from that of Bidochka et al. (Bidochka et al., 1989). Haemolymph (2l) was removed through the membrane below the pronotum using a chilled Hamilton syringe. The haemolymph was added to 50l of phosphate-buffered saline (PBS) and immediately vortexed for 5s. The PBS contained bovine pancreas -chymotrypsin (2mg1.5ml–1PBS), and was left to incubate for 20min at room temperature. The mixture was then transferred to a cuvette containing 0.9ml of 0.02moll–1 L-DOPA and the change in absorbance was measured for 60min at 490nm (Novaspec II spectrophotometer; Pharmacia, Peapack, NJ, USA). During this 60·min period, the cuvettes were maintained at the cricket’s incubation temperature (i.e. 18, 26 or 33°C). Lysozyme-like enzymatic activity was estimated using a turbidity assay (Adamo, 2004a). Haemolymph (2l) was removed from crickets after 6days at 18, 26 or 33°C and added to 100l of PBS and vortexed. The haemolymph–PBS mixture was added to 0.9ml of a Micrococcus luteus cell wall suspension (12.5mg30ml–1double distilled water). The change in absorbance at 450nm was measured over 45min. Lysozyme standards were run daily at room temperature and were used to normalize results across days. During the assay, cuvettes were maintained at the crickets’ incubation temperature.
Effect of temperature on disease resistance

Females were isolated and assigned to one of the three temperature regimes as described above. Crickets were then injected with an LD01 dose (i.e. only 1 cricket in 100 kept at 26°C was expected to die from the treatment) of either S. marcescens or B. cereus, or left uninjected as controls. After 4days (S. marcescens) or 5days (B. cereus), the number of eggs laid by surviving crickets was counted.
Statistics

Data were tested for normality. Nonparametric data were analyzed following the method of Meddis (Meddis, 1984). In cases where multiple tests were performed on the same data set the alpha criterion was adjusted accordingly (i.e. post hoc tests). Data are given as means ± 1standard deviation unless otherwise noted.
RESULTS Temperature preference of G. texensis

There was no significant difference between males and females in their temperature preference (t-test: t0.044, d.f.43, P0.97; 22 males, 23 females). Crickets preferred 28.1±1.1°C (N45), a significantly warmer temperature than the rearing temperature of 26°C (one-sample t-test: t4413, P