Estimating the differences in critical thermal maximum and metabolic rate of Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) across life stages

Introduction

Insects have a specific range of temperatures within which they can survive, grow and reproduce (Andrew, Hart & Terblanche, 2011; Huang & Li, 2015; DeVries, Kells & Appel, 2016), and have their distribution strongly defined by a thermal envelope (Gullan & Cranston, 2014; Li et al., 2019). Assessing the impact of extreme temperature exposures on insect distribution is vital to understanding insect ecology and the current and future effects of climate change (Diffenbaugh et al., 2008; Andrew, Hart & Terblanche, 2011; Andrew, 2013; Betini, Griswold & Norris, 2013; Andrew & Hill, 2017, Andrew & Terblanche, 2013). Within a rapidly changing climate, thermal limits are an essential part of assessing thermal physiology of insects, as they are the critical endpoints that an organism can survive or loses muscular control (Lighton & Turner, 2004; Andrew, Ghaedi & Groenewald, 2016), quantified in terms of critical thermal maximum (CT_max) and critical thermal minimum (CT_min) (Kingsolver & Umbanhowar, 2018).

Thermal limit measurements like CT_max and CT_min are commonly used to understand and predict species distributions among populations (Shik et al., 2019) and responses to environmental changes (Angilletta et al., 2003; Angilletta & Angilletta, 2009; Kingsolver & Umbanhowar, 2018; Angilletta, Michael & Dunham, 2003). CT_max is often aligned to the distribution of species, as it is a biologically significant and ecologically relevant thermal performance trait (Sunday et al., 2014; Shik et al., 2019). However, the CT_max of most insect groups is highly plastic, so can change depending on an organism’s pre-exposure conditions, making it difficult to forecast their response to a rapidly changing climate (Terblanche et al., 2005; Martínez, Cadena & Torres, 2016; Kellermann, van Heerwaarden & Sgrò, 2017).

CT_max has been shown to differ across life stages in some insects e.g. kelp fly (Paractora dreuxi), Sirex wasp (Sirex noctilio) and mealworm (Tenebrio molitor) (Marais, Terblanche & Chown, 2009; Vorhees & Bradley, 2012; Li et al., 2019), and can be influenced by body mass and surface area of the insect (Lighton & Turner, 2004). CT_max values depend substantially on how they are measured (Lighton & Turner, 2004; Shik et al., 2019); it is thus essential to determine a precise scale of temperature exposure for insects (Terblanche et al., 2007). Temperature ramping rates can influence the CT_max of an insect (Lighton & Turner, 2004; Terblanche et al., 2007). A slow temperature ramp rate of an organism leads to increased risk of heat shock, which is absent under the natural conditions (Lutterschmidt & Hutchison, 1997). The other process that can occur during a slow ramp is short-term physiological adjustments (e.g. induction of heat shock proteins) that protect cells from heat damage and might increase CT_max (Kingsolver, 2009; Shik et al., 2019). Fast temperature ramp leads to delay in thermal equilibrium between the air and the insect’s body. Because of this, under a fast temperature ramp, the inner temperature of the insect will be lower than the recorded air temperatures leading to an overestimation of their CT_max (Lighton & Turner, 2004; Andrew et al., 2013a; Agudelo-Cantero & Navas, 2019).

Intriguingly, only few studies have looked at thermal variation across different life stages (Le Lann et al., 2011). The few studies focused on a single unit of fitness in one environment (Le Lann et al., 2011). Thermal plasticity has been identified in many insect taxa e.g. ants (Iridomyrmex purpureus), Drosophila species and parasitic wasps (Aphidius rhopalosiphi) (Chown & Terblanche, 2006; Le Lann et al., 2011; Andrew et al., 2013a; Shik et al., 2019), but not all (Terblanche, Clusella-Trullas & Chown, 2010).

Insect herbivores that are found on the ground and plant surfaces in warmer and drier environments are exposed to substantial microclimate variability including seasonal and daily temperature variations at the soil and plant surface (Hodgson, 1991; Bale et al., 2002; Andrew, Ghaedi & Groenewald, 2016). How organisms respond to the variable microclimates depends on the thermal physiology e.g. CT_max (Somero, 2010; Shik et al., 2019). The physiology of insects can be modified by the microclimate they experience (Andrew et al., 2013a; Andrew, Ghaedi & Groenewald, 2016). Predicting the future in changing climates is made difficult by our limited understanding of the eco-physiological mechanisms and evolutionary process underlying thermal phenotypic plasticity (Shik et al., 2019; Arnold, Nicotra & Kruuk, 2019).

Helicoverpa punctigera is native to Australia and widely distributed across the continent (Gregg et al., 2016). It is a notable pest on crops like cotton, oilseeds, legumes and vegetables (Gregg et al., 2019). The adult can travel a long distance and can adapt locally to different environmental conditions including inland Australia (Gregg et al., 2016, 2019). Female adult H. punctigera lay eggs on leaves, flower buds and developing fruits of the host plant (Zalucki et al., 1986). The neonate (newly hatched) usually eat soft leaves; as they mature, they move about other feeding sites like flowers or flower buds, pods, fruits and seeds (Gregg et al., 2019). The last larval instar burrows into the soil and pupates. Inland habitats are characterised by extreme climatic conditions like very hot temperatures as high as 50 °C during summer (Bureau of Meteorology, 2018). The ability of this insect to survive the extreme summer temperatures in the inland depends on their thermal tolerances and physiological sensitivities e.g., CT_max and biochemical shifts that are temperature-dependent (Gaitán-Espitia et al., 2014). Female adults of H. punctigera are responsible for host selection and dispersal, whilst feeding activities of the larvae cause economic loss to the host plants, and a complete reorganisation of the body occurs within the pupal stage, understanding the metabolic rates among the different life stages are essential.

Energy is the crucial currency of life-history traits of animals including insects (Terblanche & Chown, 2007; Desforges et al., 2019). Metabolism combines organism’s energy supply and fitness-related activities (Shah et al., 2021). Measurement of metabolic rate is one way of measuring the energy cost related with different life-functions like growth, development, movement among others (Shah et al., 2021). In insects, metabolic rates can influence not only fitness in individuals but also geographic distributions and abundances (Shah et al., 2021). Therefore, estimating the metabolic rates of organisms will assist in identifying the cost of living and performance in a particular environment (Terblanche et al., 2005; Terblanche & Chown, 2007), and can help predict geographic variations in response to global warming (Shah et al., 2021).

Metabolic rate can respond to changes in ambient temperature, activity and body mass; however, age and ontogeny, sex, feeding status, season and time of day, among others, can also exert substantial influence (Terblanche & Chown, 2007; Harrison, 2009; Altermatt, 2010). For example, in the Australian field cricket (Teleogryllus oceanicus) and Thynnine wasp (Zaspilothynnus nigripes), males have a higher metabolic rate than females (Kolluru, Chappell & Zuk, 2004; Tomlinson & Phillips, 2015). Specific dynamic action is the amount of energy expenditure above the basal metabolic rate (RMR) due to the ingestion and digestion of food for use as energy or conversion to a storage form (Secor, 2009). One major metabolic variable is the temporary rise in metabolic rate following feeding status or swallowing food (Karasov & del Rio, 2007; McCue, 2012). This feeding status-associated rise in metabolic rate is a result of food breakdown and processing in the gut of the organism (Karasov & del Rio, 2007). High temperatures generally produce high metabolic rate, however the total energy assigned for specific dynamic actions is not fully dependent on temperature (McCue et al., 2016; Wang et al., 2002).

The temperature sensitivity (Q₁₀), defined as the rate ratio of a given process taking place at different temperatures_, is used to measure the degree of temperature dependence of a given biological process (Davidson, Janssens & Luo, 2006; Mundim et al., 2020). Q₁₀ is an important parameter in predicting the effects of temperature on carbon dioxide release (Mundim et al., 2020).

Previous studies on Helicoverpa species (eg. H. armigera and H. punctigera) focused on the effect of temperature on development rate, survivorship and reproduction (Room, 1983; Qayyum & Zalucki, 1987; Mironidis & Savopoulou-Soultani, 2008; Zalucki & Furlong, 2005; Mironidis, 2014) but not on thermal physiology (CT_max and metabolic rate) of different life stages. For example, H. punctigera are distinctive in terms of their life stage variation and habitat (Gregg et al., 2016, 2019), just as other insects with stage-based niche changes. Across life stages, an individual will be exposed to a range of temperatures (Agudelo-Cantero & Navas, 2019) with different opportunities to escape extreme exposure. In the field, the larvae and adults of H. punctigera live on plant surfaces whiles the pupae lives in the soil. Based on this life stage and habitat variations, their tolerance to extreme temperatures may significantly vary among the different life stages. Our study is the first of H. punctigera thermal physiology across three life-stages–larvae, pupae and adult.

We assessed the thermal physiology (CT_max and metabolic rate) of three different life stages—late instar larvae, pupae, and adult—and sex of H. punctigera during temperature ramping using thermolimit respirometry, which employs a flow-through respirometry, with V́CO₂ as a key measure. The technique has an infrared activity detector to monitor moving activity (Lighton & Turner, 2004; Vorhees & Bradley, 2012). One important advantage of this technique is that CT_max can be identified directly from respiratory or activity data. In addition, it allows for CT_max to be determined without disturbing the organism (Vorhees & Bradley, 2012). The technique, in addition, allows for the measurement of V́CO₂ when exposed to biologically relevant temperature points.

The specific questions we asked are:

(i) what are the differences in V́CO₂ and CT_max across different life stages of H. punctigera considering the different field conditions each stage experiences? We predict that, larvae and adult of H. punctigera, which are exposed to the many microclimatic conditions on host plants surface, would have a higher metabolic rate and CT_max than pupae, because, the soil the pupae is burrowed in is more thermally stable than the leaves.

(ii) what are the metabolic rates of the life stages at five different temperatures at 5 °C bins: 25, 30, 35, 40 and 45 °C? Here, we predict that because exposure temperature has an overwhelming effect on the metabolic rate of insects, the metabolic rate among the life stages would be lower at 25, 30 and 35 °C compared with 40 and 45 °C.

(iii) does sex influence the metabolic rate and CT_max of adult and pupae of H. punctigera? We predict that because of the differences in activity (e.g., host selection and dispersal in the case of adults) between male and female, sex would influence metabolic rate and CT_max of H. punctigera pupae and adult.

Materials and Methods

Results

Metabolic rate (overall V́CO₂ (ml h⁻¹) across life stage of H. punctigera

There was a significant effect of life stages on overall V́CO₂ (ANOVA, DF = 2, F = 31.52, p < 0.0001). Pupae (n = 10) had an overall V́CO₂ of 41% lower than adults (n = 10) and 50% lower than larvae (n = 10) (p < 0.0001, and p < 0.0001, respectively; Fig. 1). The V́CO₂ of larvae was 9% higher than adults, but not statistically significant (p = 0.20; Fig. 1).

Upper thermal tolerance (CT_max) across life stages of H. punctigera

There was a significant life stage effect on CT_max (ANOVA, DF = 2, F = 43, p < 0.0001). Adult (n = 10) and pupae (n = 10) had a similar CT_max (p = 0.12; Fig. 2). However, larvae (n = 10) had a CT_max on average 2.2 °C higher than adults and 1.8 °C higher than pupae (p < 0.0001; p =< 0.0001, respectively; Fig. 2). Activity CT_max and CO₂ CT_max for adults were similar and not significant; unlike larvae, which had significantly higher CO₂ CT_max (Table 1).

Table 1:

Anova summary information for Activity CT_max, CO₂ CT_max and mean CT_max between two life stages (Larvae and Adults) of H. punctigera following thermolimit respirometry.

Stage	DF	Sum Sq.	Mean Sq.	F	p	Mean CTmax
						Life stage
						Larvae	Adults
Activity CTmax	1	8.3	8.28	0.21	0.65	44.1 ± 1.3	43.1 ± 2.5
CO2 CTmax	1	133.1	133.08	4.27	0.05	49.0 ± 0.3	43.5 ± 2.4

DOI: 10.7717/peerj.12479/table-1

V́CO₂ of H. punctigera life stages at different temperature points

Temperature effect on mean V́CO₂ was high in adults and larvae compared to pupae (Table 2) at the different temperature points. The mean V́CO₂ of pupae was significantly different from that of adult and larvae at all temperature points except at 25 °C (Table 2). The mean V́CO₂ of each of the life stages doubled at both 40 °C and 45 °C (Table 2) before CT_max. Although the mean V́CO₂ between adults and larvae was not significantly different at 40 °C, adults had a 0.01 µl h⁻¹ higher mean V́CO₂ than larvae (Table 2). At equilibrium (25 °C) and 30–35 °C larvae had 0.19 µl h⁻¹, 0.13 µl h⁻¹ and 0.16 µl h⁻¹ higher than adult (Table 2) respectively. The Q₁₀ 25–35 °C values for larvae and adults were lower compared to Q₁₀ 35–45 °C–28% lower for larvae (Q₁₀: 25–35 °C 1.40 ± 0.06 to Q₁₀ 35–45 °C 1.95 ± 0.14), 41% lower for adults (2.01 ± 0.22 to 3.42 ± 0.24) but 32% higher for pupae (1.74 ± 0.21 to 1.42 ± 0.98) (Fig. 3). Among life stages the Q₁₀: 25–35 °C for adults was significantly higher (p = 0.04) compared with larvae, but not pupae (p = 0.53; Fig. 3). The Q₁₀: 25–35 °C was not significant between larvae and pupae (p = 0.35; Fig. 3). The Q₁₀: 35–45 °C values were not statistically different among larvae and pupae (p = 0.07; Fig. 3) but significant among larvae and adults as well as pupae and adults (p = 0.00, p = 0.00; Fig. 3) respectively.

Table 2:

Mean ± s.e. V́CO₂ (µl CO₂ h⁻¹) over different temperature points among life stages (larvae, pupae and adult).

Life Stage	Mean V́CO2 (µl CO2 h−1)
	25 °C	95% CL	30 °C	95% CL	35 °C	95% CL	40 °C	95% CL	45 °C	95% CL	CTmax	95% CL
Larvae	0.40 ± 0.04a	[0.31–0.50]	0.44 ± 0.04a	[0.43–0.62]	0.53 ± 0.04a	[0.57–0.76]	0.70 ± 0.06a	[0.54–0.81]	1.03 ± 0.22a	[0.83–1.78]	1.03 ± 0.14a	[0.90–1.50]
Pupae	0.08±0.03b	[−0.03 to 0.11]	0.11 ± 0.03b	[0.02–0.15]	0.14 ± 0.03b	[0.08–0.22]	0.19 ± 0.04b	[−0.01 to 0.16]	0.24 ± 0.16b	[−0.09 to 0.60]	0.23 ± 0.10b	[−0.05 to 0.37]
Adult	0.21±0.04b	[0.16–0.36]	0.31 ± 0.04c	[0.21–0.41]	0.37 ± 0.04c	[0.22–0.43]	0.71 ± 0.06a	[0.71–1.01]	1.47 ± 0.24c	[0.91–1.92]	1.23 ± 0.15a	[1.01–1.65]

DOI: 10.7717/peerj.12479/table-2

Note:

Values in bold indicate significant differences. Letters indicate significant differences among life stages. The 95% CL is 95% confidence limit.

The influence of sex on upper thermal limit (CT_max) and metabolic rates V́CO₂ (ml h^-1) of adult moth and pupae

There was no sex effect (ANOVA, DF = 3, F = 0.15, p = 0.92; Fig. 4) on the CT_max of adults (males: 46.92 °C ± 0.29 °C and females: 46.72 °C ± 0.29 °C) and pupae (males: 47.47 °C ± 0.29 °C and females: 47.32 °C ± 0.29 °C). For metabolic rate assessments, there was no significant difference between female and male adults as well as between female and male pupae (t = −0.57, p = 0.93; Fig. 5) and (t = 1.38, p = 0.52; Fig. 5) respectively.

Discussion

Here, we measured the upper thermal tolerance limits (CT_max) and metabolic rates at different temperature exposures across three life stages larvae, pupae, and adults and between sexes of H. punctigera using thermolimit respirometry. These life stages experience different temperatures because of the different habitats they naturally occupy, enabling an ecologically relevant investigation of their metabolic rates at different temperatures. We found that CT_max differed among the life stages of the insect with larvae exhibiting significantly higher CT_max compared with pupae and adults (Fig. 2). Similar to the findings presented here, studies by other authors have shown that CT_max differs among life stages of insects including Paractora dreuxi, Sirex noctilio and Tenebrio molitor (Marais, Terblanche & Chown, 2009; Vorhees & Bradley, 2012; Li et al., 2019). The higher CT_max of the larvae compared with adults however, contradicts the findings of Vorhees & Bradley (2012) and Li et al. (2019) in T. molitor and S. noctilio respectively, where adults had a higher CT_max than larvae, although, the insects are of different taxa. The differences in the CT_max between the life stages suggest differences in upper thermal tolerance in this species with larvae having the highest thermal tolerance to high temperature (Marais, Terblanche & Chown, 2009).

The CT_max differences among the life stages could be caused by several different factors such as interactions of the microclimatic conditions experienced by the life stages and the level to which they compensate their physiology to avoid harsh or extreme temperatures (Vorhees & Bradley, 2012). However, this would not be the case here, as all insects were kept at a constant 25 °C, so a more evolutionarily relevant mechanism must be in train here. The results imply that, larvae, pupae and adults have the ability in the wild to adapt and cope differently.

The significantly higher CT_max exhibited by larvae is not surprising because larvae in their natural environment are generally more exposed to extreme/hot temperatures, just above the soil surface and while feeding on plants, and, less able to behaviourally adapt to extreme periods. Hence the need to modify their physiological response to such extreme periods of heat conditions for survival (Andrew et al., 2013a, 2013b; Andrew, Ghaedi & Groenewald, 2016). Even though the insects used in the study were laboratory-reared, they were most likely to exhibit similar characteristics like those in the field, because there is little genetic variability among H. punctigera population (Daly & Gregg, 1985). The limitation of our study is that; the low genetic diversity in the wild in the insect does not imply that laboratory populations will not be adapted after many generations. We acknowledge that the climatic variability experienced in the wild could further influence in these thermal tolerance traits, but is unlikely to affect the clear life-stage differences.

The decline in the upper thermal limit between larvae, pupae and adults in that order (Fig. 2) suggests that the insects upper thermal limit could be influenced by ontogeny as observed in Drosophila melanogaster (Bowler & Terblanche, 2008), although we did not test for age effects in a single life stage. This upper thermal limit decline across the life stages however, does not always follow a logical order as in some insect’s e.g. Drosophila buzzatii; heat tolerance was high in pupae followed by eggs and larvae (as cited in Vorhees & Bradley, 2012). Therefore, caution must be made against the generalisation concerning the effects of ontogeny on heat resistance (Vorhees & Bradley, 2012), although we should expect ontogenetic variation (Bowler & Terblanche, 2008). One would have expected in part, no significant difference in the CT_max between larvae and adults because of the several factors like the microclimate conditions they experienced and the extent to which they can use their physiology to avoid the extreme temperatures. The lower CT_max of the adults compared with the larvae may be because the adults have a higher ability to avoid extreme temperatures by flying away from areas of extreme temperatures compared to larvae. The results suggest that there are ecological reasons for different thermal preferences: larvae are more sedentary so it is harder for them to move away from hotter temperatures they may be exposed to; adults are more mobile, so can move to preferred temperatures refuges in the evening so have a lower thermal threshold. Because of the ectothermic and regional heterothermic nature of insects, high temperatures could speed up growth and development of insects (Andrew, Hart & Terblanche, 2011; DeVries, Kells & Appel, 2016). The higher CT_max of the larvae could assist them to grow faster in warmer conditions like summer if food, and most importantly water, are not limiting factors.

The CT_max exhibited by the life stages is significantly greater than the highest recorded maximum daily mean ambient temperature in Tamworth region where the population originated from (40.9 °C) (NicheMapR). However, the microclimatic conditions that H. punctigera could be exposed to may be much higher than this, particularly during the middle of the day (Andrew et al., 2013a), and can put the insects under physiological stress that can change their interactions under changing climate (Dawson et al., 2011). The higher CT_max exhibited by the insect could be the reason for which it is able to occupy a wide range of climatic conditions including inland of Australia, and spread in all states of Australia (Gregg et al., 2016, 2019).

Our results show that upper thermal limits were not significantly different between males and females. The similar thermal limits demonstrated by males and females indicates that sex has minimal influence on CT_max (Fig. 4), similar to the findings in bumble bees (Bombus terrestris) (Oyen, Giri & Dillon, 2016); suggesting that H. punctigera sex ratios will not change with adult exposure to climatic extremes.

Insects as other animals require energy to forage, survive, grow and reproduce (Terblanche, Klok & Chown, 2004; White et al., 2019). The processes that cause variation in metabolic rate apart from ambient temperature, body mass is also an important factor that contributes to metabolic rate of insects (White et al., 2019). Insects show a range of thermoregulatory capacities: ectothermic, regional heterothermic, poikilothermic and endothermic (Heinrich, 1993; Gullan & Cranston, 2014; O’Neill, Kemp & Johnson, 1990) their activity and physiological processes are primarily influenced by ambient temperature (Nguyen et al., 2014). Our results revealed V́CO₂ varied among the life stages at the different temperature points. The differences in the mean V́CO₂ among the life stages suggests that the metabolic demands of the life stages differ. Several factors may account for the differences in the metabolic rates among the life stages; including ontogeny, upper thermal tolerance, activity, body mass and feeding (Terblanche, Klok & Chown, 2005).

Fat body tissues play crucial roles in the life of insects, as they are involved in multiple metabolic functions like energy storage and utilisation in reply to the energy call or request of the insect (Arrese & Soulages, 2010). Muscle tissue usually has a higher metabolic rate than fat body tissue. Muscle tissues are active and burns calories even at rest unlike fat body for storing excess energy.

The significantly higher V́CO₂ of larvae and adult compared with pupae could be due to low amount of fat body tissues in the larvae and high muscle tissues in the case of adult. Another reason for the high metabolic rate may be due to the high activities of larvae and adult compared with pupae, similar to that reported by DeVries, Kells & Appel (2013) for the bed bug, Cimex lectularius. Size also affects the metabolic rate of an organism (Terblanche, Klok & Chown, 2004; Quinlan & Gibbs, 2006; DeVries, Kells & Appel, 2013), and was similar in our case, as size was significant (Figure 3 in Supplemental Material). Pupae, which has a higher CT_max than adults, had a comparative lower V́CO₂. The lower V́CO₂ may be an indication of lower stress at the higher temperatures and or maybe due to more fat tissues in pupae. It is plausible that it may be an adaption strategy for survival to dry conditions (Terblanche, Klok & Chown, 2004; Quinlan & Gibbs, 2006). The high amount of fat tissues in the pupae because of lipid reserves needed to provide energy during the extended non-feeding periods could be the reason for the low metabolism (Arrese & Soulages, 2010). Because pupae do not have a source of free water underground, they must conserve water by reducing respiration (Table 1 in Supplemental Material) (Harrison, 2009).

As the effect of temperature on metabolic rates of insects is widely reported (Terblanche, Klok & Chown, 2004; Terblanche & Chown, 2007; Hill et al., 2016), only a few studies looked at that at different temperature points for different life stages for H. punctigera. Our results on metabolic rate between life stages considering the different temperatures (Table 2) and Q₁₀ (Fig. 3) indicates that there was a correlation between temperature rise and V́CO₂ release on the life stages during the thermolimit respiratory (Table 2). The Q₁₀ values for all the life stages except pupae almost doubled from Q₁₀: 25–35 °C to Q₁₀: 35–45 °C, with adults having the highest thermal sensitivity. This increase in metabolic rate as temperature increases is consistent with other studies (Terblanche et al., 2005; Terblanche & Chown, 2007; Andrew, Ghaedi & Groenewald, 2016). However, the negative effects associated with increasing temperatures on metabolic efficiency could be reduced on adults, as they are able to compensate for short-term extreme microclimate temperatures by flying away. Although, increase in temperatures result in high metabolic rate, the total energy assigned for certain actions, as specific dynamic actions are not dependent on temperature (McCue et al., 2016; Wang et al., 2002). It is possible that as insects enlarge their geographic radius or scale, they will face abiotic and biotic stress.

For better comparison among life stages, we measured V́CO₂ at CT_max, and in all cases, V́CO₂ among life stages was higher. Even though the V́CO₂ at CT_max increased in all the life stages, adults V́CO₂ were higher than larvae and pupae. Harrison (2009) reported that tracheated insects have the ability to carry or move gases by opening their spiracles. Short periods of spiracles closure follow a continuous carbon dioxide exchange (Harrison, 2009). The higher metabolic rates due to higher stress levels may be due to a decrease between spiracle opening and closing. Therefore, the higher V́CO₂ of adults at CT_max may be due to their high activity rate and a decrease between its spiracle opening and closing compared to larvae and pupae (Harrison, 2009; Boardman & Terblanche, 2015). The lower metabolic rate of pupae at CT_max compared to larvae and adults could not be explained beyond a physiological adaption strategy to reduce physiological cost by reducing respiration and conserve water at this stage (Harrison, 2009), similar to what Merkey et al. (2011) reported in Drosophila melanogaster pupae.

The lower V́CO₂ of adults compared to larvae at 25 °C could suggest that adults have a lower resting metabolic rate than larvae at that point. The higher resting metabolic rate of larvae could be the result of modification of the energy allocation between competing functions such as maintenance for continuous development. Additionally, the lower V́CO₂ of adults may be as result of the occurrence of discontinuous gas exchange in the adults at 25 °C. Discontinuous gas exchange is associated with low metabolic rates. In contrast, age, as proven to affect metabolic rates in some insects like Glossina pallidipes (Terblanche, Klok & Chown, 2004) did not apply in our case, as larvae had a lower V́CO₂. The high V́CO₂ produced by the larvae could imply a high-energy utilisation (Terblanche, Klok & Chown, 2004) for development. This high-energy utilisation can provide a deep understanding into the life histories of the life stage across a variety of environments. Therefore, this high-energy utilisation may translate to an increase in food consumption and more considerable plant damage (Deutsch et al., 2018). The metabolic rate of pupae and adults was not influenced by sex, suggesting that the cost of living of the dispersing adults and pupae of both sexes are similar. Similar work by Terblanche, Klok & Chown (2004) found out that metabolic rate did not differ between males and females of Glossina pallidipes and G. morsitans.

Conclusion

The present study provides vital information on how the different life stages of H. punctigera will physiologically perform in a changing thermal environment. It also provides data for predictions about metabolic rates of H. punctigera among life stages. The study revealed that CT_max of larvae was significantly higher than those of pupae and adults, and the metabolic rate of pupae was significantly different from those of larvae and adults. Also, sex did not influence CT_max and metabolic rate. The results suggest that the performance and survival of the different life stages of H. punctigera could differ. This indicates that life-stage needs to be taken into account when assessing responses to a rapidly changing change, as ontogeny influences vulnerability and exposure. This finding can be used to predict the potential spread of the insect to other new environments and can indirectly help in management planning.

Supplemental Information