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Keywords:

  • Danaus plexippus;
  • feeding;
  • moulting;
  • thermoregulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Body temperature in insects is affected by interactions between environmental variables, behavioural choices and the physiological state of the animal. Insect larvae display a number of tactics for thermoregulation which allow their body temperatures to deviate several degrees from ambient. Thermoregulatory adaptations can reduce generation time and allow higher population growth rates. However, few investigations have examined thermoregulation of moulting larvae. This study measured whether moulting Monarch butterfly (Danaus plexippus) larvae thermoregulate, and, if so, whether they thermoregulate similarly to feeding larvae. Body temperatures of moulting and feeding larvae were compared to their microhabitat temperatures. In three of four environments, feeding larvae were able to thermoregulate in such a way that their body temperature was higher than ambient at low ambient temperature, and lower than ambient at higher ambient temperature. However, moulting larvae appear not to thermoregulate because their body temperature did not differ from ambient in three of four environments. These results pose the question of whether larvae are behaviourally and/or physiologically constrained from thermoregulating during the moulting process. Additional studies are needed to address such questions and would help to elucidate the impact of thermoregulation of moulting larvae on the ecology and evolution of insects.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Body temperature in insects is affected by interactions between environmental variables, behavioural choices and the physiological state of the animal (e.g. Gilbert & Raworth 1996; Chown & Nicolson 2004). Body temperature is strongly influenced by changes in the organism's physical environment (Kingsolver 2000) and the small size of insects allows them to utilise small-scale variations in microclimate that are not available to larger animals (Kingsolver 2000). Consequently, insects can potentially experience a great deal of diversity in microclimatic conditions; hence, correct identification and characterisation of the microclimate at the proper scale experienced by an insect is necessary to quantify and interpret the physiological ecology of an insect species.

Two general adaptive mechanisms used by insects to cope with the wide range of ambient temperatures they experience are behavioural thermoregulation (e.g. Heinrich 1979; Casey 1993) and physiological thermoregulation (e.g. Ruf & Fiedler 2000). These two adaptive mechanisms are often used together (e.g. Casey 1992, 1993) to respond to diurnal and seasonal temperature changes. Adult insects display a number of tactics for thermoregulation (Nice & Fordyce 2006) that allow their body temperatures to deviate several degrees from ambient (Willmer 1986). To a certain extent, some insects can utilise endothermic processes to generate their own internal heat (James 1986); however, most insects, with their small body size and weak endothermic capacity, largely rely on heat acquired from the environment (Heinrich 1981).

Sun-radiated heat is often the most efficient and most readily available thermal source used for insect thermoregulation (Heinrich 1981). In particular, many insect larvae use basking, aggregation, tent making, colour, setae and behavioural postures and movements to help regulate their body temperatures in variable thermal conditions (Stamp & Casey 1993). For example, colour plasticity in monarch (James 1986) and swallowtail larvae (Hazel 2002), cryptic basking within apples in codling moth larvae (Kuhrt et al. 2005) and aggregation in sawfly larvae (Fletcher 2009) all demonstrate how larval body temperatures can be altered significantly from ambient temperatures. Larvae can also thermoregulate by utilising heat avoidance mechanisms to lower body temperatures during the hottest times of the day (Rawlins & Lederhouse 1981). These thermoregulatory adaptations facilitate more rapid growth during the larval stage, reduce mortality rates by reducing the amount of time larvae remain in their most vulnerable state as prey and increase the probability of reaching the minimum body mass limit for proper pupation, all of which reduce generation time and allow higher population growth rates (Rawlins & Lederhouse 1981; Nice & Fordyce 2006). For monarch butterflies, this also provides the potential for expanding into a greater range of temperature environments (Rawlins & Lederhouse 1981; Davis et al. 2005).

A considerable amount of research has focused on the effects of temperature on larval growth and development in Lepidoptera in general (e.g. Kingsolver 2000; Hazel 2002; Irlich et al. 2009) and monarchs in particular (Rawlins & Lederhouse 1981; Zalucki 1982). However, the direct relationship between thermoregulation and feeding has not been adequately addressed and that of temperature and moulting is poorly understood and rarely studied. Evidence from previous studies on thermal behaviour suggests that optimal body temperature is important during moulting to minimise the duration of, and the risks associated with, moulting as larvae are anchored to a silk mat and cannot readily respond to predators (Malcolm & Zalucki 1993; Solensky & Larkin 2003). On the other hand, the increased risks of predation and moisture loss because of overheating, as well as the physiological changes and possible constraints that occur during the moulting process, may also influence selection of sites microclimatically suited to moulting (Notter-Hausmann & Dorn 2010), although this has not been much studied. Hence, we ask here whether moulting larvae thermoregulate and, if so, whether they thermoregulate similarly to feeding larvae.

To examine these questions, the thermoregulatory patterns of monarch larvae, Danaus plexippus (Linnaeus), were measured. Monarch butterflies are well known for their extensive migratory patterns from breeding to over-wintering sites throughout North America (Zalucki & Clarke 2004; Davis et al. 2005) and Australia (Clarke & Zalucki 2004). As a result of their expansion into more temperate climates following the spread of their food source, the common milkweed (Asclepias spp.), they have evolved extensive thermoregulation capabilities and are known to utilise both basking and colour plasticity throughout the larval and adult life stages (James 1986; Davis et al. 2005). Individuals with increased melanism (darker pigmentation with less yellow and white colouring) absorb thermal energy at a faster rate and larval development rates are increased and adults can fly at lower temperature (Davis et al. 2005).

In this study, the body temperature of moulting and feeding larvae were compared to the microhabitat ambient temperature (i.e. leaf surface temperature) in which they were located. We hypothesised that although feeding larvae would thermoregulate to maintain optimal body temperature, as has been described previously (Harrison & Fewell 1995; Kingsolver 2000), moulting larvae would not because of their immobility (although they may be selective as to where they moult). These hypotheses were tested by statistically comparing the slope of body temperature vs. leaf surface temperature to 1; a slope not different from 1 is indicative of conforming to the thermal environment, whereas a slope different from 1 is indicative of thermoregulation (Willmer 1986; Willmer et al. 2004). Hence, we predict that moulting larvae will have a slope that does not differ from 1, whereas feeding larvae will have a slope that differs from 1.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Under natural conditions of temperature and radiation, monarch larvae were periodically monitored for 5 days in November and 5 days in May at an open range field site (Pinjarra Hills −27° 32′S; 152° 54′E) in Brisbane, Australia. Larvae, ranging from first to fifth instars, were either found and observed in situ or taken from another site as eggs and positioned on randomly selected milkweed after hatching. For each observed larva, body temperature and ambient temperature of the adjacent leaf surface in which they were located were measured using an infrared temperature gun (Raytek® PM Plus), which was calibrated within 0.1°C of known temperatures. Larval location within the sun or shade was also recorded.

Simple linear regression analyses were conducted on body temperature and leaf surface temperature using Proc Reg in SAS separately for each behaviour in each environment in both November and May. For each regression analysis, the slope of body temperature on leaf temperature, and its 95% confidence interval, was calculated. The slope was considered to be different from 1 if 1 was outside the 95% confidence interval; the slope was considered to be different from 0 if the R2 value was statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Diurnal fluctuations in mean daily temperature across all sampling days in the sun and shade for both November and May are presented in Figure 1; times were binned to the nearest hour or half-past the hour. Not surprisingly, temperature fluctuations and daily maxima were higher in the austral summer month of November, as was the variation between sun and shade temperatures.

figure

Figure 1. Individual leaf temperature as a function of time of day in both the sun and the shade in November and May. Within each month, the sun and shade temperatures were taken on the same day on multiple leaves within each habitat type (sun vs. shade). (image) sun; (image) shade.

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Larval body temperature vs. leaf surface temperature for all individuals on all measurement days are presented in Figures 2 and 3. Figure 2 shows the results from November, and Figure 3 shows the results from May; in both figures, panel a presents the results from the sun and panel b presents the results from the shade. In each month in each habitat (sun or shade), there is substantial overlap in the temperatures experienced by feeders and moulters. In moulting larvae in both the sun and the shade in November, the relationship between leaf temperature and body temperature has slopes that explain a statistically significant proportion of the variation (i.e. the slopes are significantly different from 0; R2 = 0.61; P = 0.0047 and R2 = 0.93; P = 0.0012, respectively) and do not differ significantly from 1 (Table 1).

figure

Figure 2. Body temperature as a function of leaf temperature in November for both moulting and feeding larvae in sun (panel a) and shade (panel b) environments. (image) feeding sun; (image) moulting sun; (image) feeding shade; (image) moulting shade.

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figure

Figure 3. Body temperature as a function of leaf temperature in May for both moulting and feeding larvae in sun (panel a) and shade (panel b) environments. (image) feeding sun; (image) moulting sun; (image) feeding shade; (image) moulting shade.

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Table 1. Results from linear regression analyses of body temperature on leaf temperature for feeding and moulting larvae in sun and shade in both November and May
 Slope95% CISlope diff from 1aR2Sig of R2
  1. a

    Bold answer matches hypothesis.

November     
Feeding sun0.620.287Yes0.440.0004
Moulting sun0.700.353No0.610.0047
Feeding shade0.560.392Yes0.390.0233
Moulting shade1.130.274No0.930.0012
May     
Feeding sun0.800.588No0.380.0251
Moulting sun0.580.706NO0.280.2086
Feeding shade0.670.157Yes0.87<0.0001
Moulting shade1.020.157No0.88<0.0001

In contrast, feeding larvae in November in both environments have slopes that are significantly less than 1; in both the sun and shade, leaf temperature explains a significant proportion of the variation (R2 = 0.44; P = 0.0004 and R2 = 0.39; P = 0.0233, respectively, Table 1). Slopes less than 1 suggest thermoregulation, and suggest that, at lower temperatures, the feeding larvae are able to elevate their body temperature above ambient, and at warmer leaf temperatures, feeding larvae are able to reduce their body temperatures below ambient. In moulting larvae, slopes are not significantly different from 1, suggesting a lack of thermoregulation, because the larval body temperature does not differ significantly from ambient temperature.

This same pattern supporting our hypotheses was seen during May in the shade environment: moulting larvae have a slope that does not differ from 1, and feeding larvae have a slope that is significantly less than 1; in both cases, leaf temperature explains a significant fraction of the variance in body temperature (R2 = 0.87 for feeding and 0.88 for moulting, with P < 0.0001 in both instances). However, in the sun environment in May, the slope for the feeding larvae does not differ from 1, and the R2 value of 0.38 is only marginally significant (P = 0.0251, Table 1); the slope for the moulting larvae does not differ from either 1 or 0 (R2 = 0.28; P = 0.2086, Table 1). Hence, the results for the sun environment in May do not support the hypotheses, although it is important to note that sample sizes for both feeding and moulting larvae are low (n = 11 and 5, respectively), and the range of leaf temperatures is quite restricted (approximately only a 6 degree temperature range).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The present data largely support the idea that monarch larvae thermoregulate during normal feeding activities. In three of four cases, feeding larvae were able to thermoregulate in such a way that they kept their body temperature higher than ambient at lower temperatures and lower than ambient at higher temperatures (Figs 2, 3). This suggests that feeding larvae are able to adjust to diurnal fluctuations in temperature as they attempt to maintain a steady internal body temperature close to the optimum (about 29°C; Zalucki 1982). Although minimal research has been done on the direct relationship between larval feeding and thermoregulation, some evidence suggests that lepidopteran larvae and grasshoppers must thermoregulate to reach a minimum body temperature threshold for activation of feeding mechanisms and that increases in body temperature increase rates of feeding (Harrison & Fewell 1995; Kingsolver 2000). Codling moth larvae developing in apple fruits are known to thermoregulate via cryptic basking and microhabitat selection, choosing to build feeding tunnels in the warmer apple hemispheres (Kuhrt et al. 2005), while sawfly larvae (Fletcher 2009) have the ability to thermoregulate under shaded conditions, in the absence of direct solar radiation. The present results on monarch larvae fit these patterns of thermoregulation in feeding larvae of other insect species.

On the other hand, moulting larvae appear not to thermoregulate because their body temperature did not differ from ambient leaf surface temperature in three of four cases (In the fourth case, the sun environment in May, the slope of the regression line for moulting larvae did not differ from either 0 or 1; the low sample size (n = 5) undoubtedly contributed to this result). They do not alter their body temperature in response to low or high ambient temperature and must conform to the surrounding thermal environment. This result poses the question of whether larvae are behaviourally and/or physiologically constrained from thermoregulating during the moulting process.

Given the inability of the larvae to move during moulting, a behavioural constraint seems obvious; however, questions still remain about behavioural aspects of selecting moulting sites. From what is known about the benefits of larval thermoregulation, such constraints would seem to be a disadvantage, with a negative impact on growth and development. For example, a study of tobacco hornworms (Stamp 1990) demonstrated that lower temperature significantly increases the overall proportion of time larvae spend moulting; this disproportionate increase in time spent moulting decreases the time spent feeding, therefore contributing to a reduction in growth rate. How larvae cope with such limitations has not been examined to date.

As a rare study of thermoregulation during moulting, the results of this study open the door for additional research. More extensive experimental studies are needed to test for both behavioural and physiological constraints on thermoregulation during moulting, especially microhabitat choice. In this study, there was nearly complete overlap of environmental temperatures experienced by moulters and feeders; however, the range of choices available at the start of feeding and moulting was not measured. Future studies should investigate this aspect of microhabitat choice. Other aspects of microhabitat choice, including the modulating effects of CO2 (O'Neill et al. 2011) and relative humidity (Yan et al. 2010) on air and leaf temperature, and the influence of insect body size on the actual thermal environment experienced (Woods 2012), are also worth investigating. The results presented here also raise questions about the effects of limited/no thermoregulation during moulting and its impact on the growth of each instar and the length of the larval period. Additional studies on these and other questions will help to elucidate the impact of thermoregulation of moulting larvae on the ecology and evolution of insects.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Wes Leid and Tony Ives for fruitful discussions and helpful comments. This research was supported by a grant from the National Science Foundation (EF-0328594) to PAC, and partially fulfilled the requirements for the BS in Zoology with Honours at Washington State University for VRS.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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