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Altitudinal and lowland gradients
At Saint Malo and Molloy, insects were collected along an altitudinal gradient up to 250 m above sea level (a.s.l.). At Saint Malo, three populations were collected from different elevations (0-5 m, 95-110 m and 190-210 m a.s.l.) and given the corresponding names: Saint Malo 0, Saint Malo 100 and Saint Malo 200. At Molloy, four populations were collected at three different elevations (0-5 m, 110-130 m and 245-250 m a.s.l.), with two distinct habitats being sampled at around 125 m a.s.l., one having less vegetation (Molloy 125) than the other (Molloy 125 Vegetation).
At Papous, a horizontal seashore – inland transect was completed, with sampling at different distances from the coast (approximately 2-15 m, 185-215 m, 385-415 m and 785-815 m): Papous 0, Papous 200, Papous 400 and Papous 800 respectively.
Measurement of the duration of recovery from cold and heat exposures
Empty Petri dishes lined with paper were placed into an incubator. After approximately 10 min, five individuals were added into each of 4-5 Petri dishes, and covered with lids; the cabinet was closed and a timer started. After 15 min exposure to a given temperature, the dishes were removed from the incubator, rapidly transferred into a walk-in chamber at 9 ± 1.5 °C, and a new timer was started. The number of insects in coma, and number of recovered insects (walking ability restored) were subsequently noted every minute, over 30 min (The recovery period was monitored for up to 120 min in a preliminary test, and no change in the number of recovered beetles was observed as compared with 30 min). Any individual that had not recovered after 30 min was considered dead. This procedure was repeated for each population and temperature treatment. All populations were exposed to -6, -7 or 37 °C for 15 min. The duration of exposure of 15 min was selected after having performed preliminary tests, with insects being exposed at these three temperatures for different durations (from 5 to 120 min; data not shown). Depending on the number of insects available for each population, the remaining adults were exposed to up to eight distinct temperatures (-5, -6, -7, -8, 35, 36, 37 and 38 °C) for 15 min; this additional experiment ensured that we investigated temperatures that were close to the thermal limits of the species, and that any difference in temperature tolerance would be identified.
To test for the effect of prolonged duration of exposure on the subsequent recovery, further assays conducted at -6 °C were completed with duration of exposure of the adult M. soledadinus of 1 or 2 h in addition to the 15 min (0.25 h) treatment for the populations of the altitudinal (Molloy, St Malo) and inland-shore (Papous) gradients. During the experiments, each insect was only used once (i.e. exposed to one temperature and duration combination on a single occasion).
Recovery of adult M. soledadinus sampled along an altitudinal gradient
The ability to recover from cold or heat exposure in insects sampled along an altitudinal gradient at Saint Malo and Molloy is presented in Figures 3 and 4, respectively. All populations collected from Saint Malo exhibited a similar ability to recover from cold or heat exposure (Figure 3A, B) (-7 °C: P=0.056; 37 °C: P=0.17). A trend was observed in the cold treatment recovery curves (Figure 3A) where, the higher altitude the population was collected from, the more individuals were able to recover.
As shown in Figure 4A, B, there were no significant differences in the ability to recover from extreme low or high temperature exposure in any of the Molloy populations (-7 °C: P=0.13 (4A; 37 °C: P=0.063). The recovery dynamics of the individuals from Saint Malo that were exposed to other temperature conditions (-5, -6, 36 or 38 °C) are presented in Supp Figures 2. The recovery dynamics of the individuals from Molloy that were exposed to other temperature conditions (-5, -6, -8, 35, 36 or 38 °C) are presented in Supp Figures 3.
Duration of recovery of adult M. soledadinus exposed for different time periods
The recovery times of 25 individuals from the Molloy populations after they were exposed to -6 °C for 0.25 h, 1 h or 2 h are shown in Figure 5. For populations sampled at Molloy 0 (Figure 5A), Molloy 125 (Figure 5B) and Molloy 125 Vegetation (Figure 5C), the recovery dynamics of the insects exposed to cold for the three durations did not differ significantly (0 m: P=0.17; 125 m rocky habitat: P=0.076; 125 m vegetation: P=0.19). However, there was a significant difference between recovery curves from the Molloy 250 population after 1 h exposure as compared with those exposed for 0.25 h at -6 °C (P=0.002). This latter population was not exposed to -6 °C for 2 h, due to lack of available individuals. No significant difference was observed among the recovery curves when the insects from the altitudinal transect at St Malo and the seashore-inland transect at Papous were exposed to – 6 °C for different durations (Supp Figures 4 and 5) (0 m [Supp Fig. 4A]: P=0.29; 100 m [Supp Fig. 4B]: P=0.08; 200 m [Supp Fig. 4C]: P=0.56; 0 m [Supp Fig. 5A]: P=0.2; 200 m [Supp Fig. 5B]: P=0.48; 400 m [Supp Fig. 5C]: P=0.24; 800 m [Supp Fig. 5D]: P=0.44).
Adult M. soledadinus sampled along an altitudinal gradient
The colonization of higher altitude habitats may have been favoured by recent climate change. However, the climatic conditions encountered at moderate elevations at the Kerguelen Islands may remain sub-optimal for adult M. soledadinus. We thus hypothesized that populations from higher altitudes would be more cold tolerant than those collected closer to sea level. Although no statistically significant difference was detected among the recovery curves of the different populations sampled along the altitudinal gradient at Saint Malo, the curves fall in sequence from the highest location (Saint Malo 200) to the location closest to sea level (Saint Malo 0) in terms of recovery ability from cold exposure. In bumble bees, the critical thermal minimum was lower and the insects recovered at lower ambient temperatures when they were sampled at high altitudes (Oyen et al. 2016), as also reported in the copper butterfly Lycaena tityrus (Karl et al. 2008); however, these two studies compared the thermal tolerance of specimens collected from low to high altitudes on a larger altitudinal gradient than in the present work.
A different recovery pattern was observed for the individuals collected from the second altitudinal transect (Molloy), with fewer individuals from Molloy 250 and Molloy 125 recovering from exposure to either high or low temperature compared with those collected at Molloy 0 and Molloy 125 vegetation. These findings suggest that habitat quality influenced temperature tolerance in these populations as distinct from elevation per se. The locations where Molloy 0 and Molloy 125 Vegetation were collected had a larger proportion of vegetation, and can therefore also be expected to give access to more nutrient-rich and varied habitats. Thermal tolerance physiology can be influenced by the quality of trophic resources as reported by Colinet et al. (2013), who found that sugar-enriched diets increased chill coma recovery ability in the fruit fly Drosophila melanogaster. In other studies, low diet quality had no effect on the critical thermal maximum of the chrysomelid Cephaloleia belti (Garcia-Robledo et al., 2018), while the feeding status (fed versus starved insects) of the fly Ceratitis capitata (Nyamukondiwa and Terblanche, 2009) affected their thermal tolerance, and starvation lowered the supercooling point – and increased cold tolerance – in the beetle Alphitobius diaperinus (Salin et al., 2000). The overall quality of the environment can contribute to temperature tolerance in insects, either directly, or by eliminating some level of multi-stress exposure occurring when factors such as starvation or dehydration coincide. Finally, vegetation cover makes ground temperature more similar to air temperature than in rocky habitats where insects may be more exposed to heat stress because of high level of radiation (Buckley et al. 2013).
Adult M. soledadinus sampled along a seashore-inland transect and exposed for different time periods
The seashore-inland transect was established in order to identify any effects of the distance to the sea on the thermal tolerance of adult M. soledadinus. The distance to the coast did not systematically affect temperature tolerance: the four populations sampled at Papous had very similar recovery curves after exposure to high temperature, but the pattern of the graphs could suggest a trade-off with cold tolerance. As the different populations sampled at Papous were physically closer to each other compared to the other studied populations, and the ground between them apparently without obstruction, it is possible that movement of individuals between populations at Papous 0, Papous 200 and Papous 400 was sufficient to obscure any differences between samples collected from the different locations. Moreover, while landscape composition has been reported to affect the thermal tolerance of parasitic wasps (Tougeron et al. 2016), micro-scale characteristics of the sampled habitats may have remained under oceanic influence, and thus under salinity gradients (Herbst 2001) that constrain the thermal tolerance of adult M. soledadinus. Similarly, Warren et al. (2018) reported that the thermal tolerance of ants from coastal areas was lower as compared with the specimens collected further inland. It is thus possible that insects from Papous 800 were sufficiently well separated from the other populations and far away for the coast to lead to different recovery patterns after extreme cold exposure. As differences in thermal tolerance sometimes become apparent when animals are exposed to high stress levels, it is interesting to note that increasing the duration of exposure to -6 °C did not have a significant effect on the recovery dynamics of all but one of the tested populations. In insects, chill coma results from the progressive depolarization muscle resting membrane potential (MacMillan et al., 2012; Andersen et al. 2015). Our results suggest that the 15 min exposure at cold temperature was long enough for altering the membrane potential over the critical threshold associated with the onset of chill coma, as the recovery dynamics and number of recovered beetles did not change significantly with increasing exposure duration. The exception to this generalisation was provided by the population from the highest elevation at Molloy (Molloy 250), which showed a significant reduction in tolerance to exposure to -6 °C when the duration was increased from 15 min to 1 h.
Effect of daily heat spikes and cyfluthrin exposure
Here, we combined two abiotic stresses by jointly exposing adult A. diaperinus to both moderately stressful heat spikes and insecticide for eight days, before assessing any visible impairment or death of exposed individuals. One temperature cabinet was set to constant 26 °C (control temperature), and another cabinet varied the temperature by holding a 6h period at 38 °C, followed by 18h at 26 °C (Supplementary File 2); the two cabinets were MIR 154 Panasonic programmable incubators. The experiment was run in dark conditions.
Insects were placed in glass Petri dishes (10 cm diameter) with a fitting filter paper covering the bottom. For insecticide application, cyfluthrin (pure molecule standard, CAS-number 68359-37-5, Sigma-Aldrich) was dissolved in acetone, and a volume of 2 mL of appropriately diluted solution was transferred to the filter paper. Pre-testing the insects’ susceptibility to cyfluthrin led to preparation of two distinct insecticide concentrations, so that the applied dose was 20 mg cyfluthrin/m2 for populations FARM1, 2 and 3, and 0.5 mg cyfluthrin/m2 for LAB. We had to reduce the cyfluthrin dose significantly for the LAB population, as the 20 mg cyfluthrin / m² concentration killed all adults within a day at our experimental temperatures. These cyfluthrin concentrations induced negligible knockdown (<5%) over prolonged exposures in the works of Desvignes-Labarthe (2018) and Renault and Colinet (Submitted). These concentrations also range in the values that may be encountered by the insects in poultry houses (Salin, Delettre, and Vernon 2003). In the other Petri dishes, 2 mL of pure acetone was added to control for any effect of the solvent. All Petri dishes were left to dry under a fume hood for at least 10 min before the tests, which effectively evaporated all acetone. The exiting literature reveals that cyfluthrin remains efficient over seven days (Guillebeau, All, and Javid 1989), with knockdown effects remaining unchanged when the beetle Tribolium castaneaum were assessed eight weeks after the chemical was applied (Arthur 1999) Finally, starting from an initial concentration of 17.3 mg / m², (Nakagawa et al. 2017) reported that cyfluthrin concentration decreased by ca. 17% after 56 days. Thus, even if changes in cyfluthrin concentration were not monitored here, it is assumed that it remained close to the nominal concentration.
For all treatments, the insects were supplied with a water source (0.5 mL microtube containing water and cotton) and a food source (approx. 0.8 g dog food pellet). For each population, a set of Petri dishes treated with the insecticide and a set of dishes without pesticide treatment were placed in the temperature cabinets (8 to 30 dishes of each, as specified in Table 1) along with an iButton® (iButtonLink, LLC., Whitewater, USA) measuring both temperature and humidity. Each Petri dish in experiments with poultry house collected populations contained 30 individuals, and for the LAB population, each Petri dish contained 20 individuals, due to limited number of individuals (Table 1). Previous experiments that examined the sensitivity of A. diaperinus to insecticides (including cyfluthrin) revealed that densities from 10 to 30 adults per Petri dish had no effects on the results (Desvignes-Labarthe 2018; Renault and Colinet Submitted).
Table of contents :
Chapter 1 Background
Thermal performance curve
What is stress?
Ecological consequences of climate stress
Increased temperature and thermal fluctuations
Invasive species
Mild winters
Climate stress tolerance strategies
Heat shock proteins and chaperones
Oxidative stress
Desiccation
Other extreme temperature tolerance strategies
Metabolomics – finding the players of physiological responses to stress exposure
Pesticides
Measuring toxicity
Types of insecticides
Pathways of uptake and excretion
Ecological consequences of pesticides in the environment
Pesticide and temperature interactions in insects
Effect of temperature on pesticide sensitivity in insects
Pesticide effect on temperature sensitivity in insects
This thesis
Aims and hypotheses
Biological models
Merizodus soledadinus
Alphitobius diaperinus
References
Chapter 2 Thermal tolerance patterns of a carabid beetle sampled along invasion and altitudinal gradients at a sub-Antarctic island
1 Introduction
2 Methods
2. 1 Insect collection
2. 1. 1 Invasion gradient
2. 1. 2 Altitudinal and lowland gradients
2. 2 Measurement of the duration of recovery from cold and heat exposures
2. 3 Statistical analysis
3 Results
3. 1 Recovery of adult M. soledadinus sampled along an invasion gradient transect
3. 2 Recovery of adult M. soledadinus sampled along an altitudinal gradient
3. 3 Duration of recovery of adult M. soledadinus exposed for different time periods
3. 4 Duration of recovery of adult M. soledadinus sampled along a seashore-inland transect 64
4 Discussion
4. 1 Thermal sensitivity of M. soledadinus adults sampled along an invasion gradient
4. 2 Adult M. soledadinus sampled along an altitudinal gradient
4. 3 Adult M. soledadinus sampled along a seashore-inland transect and exposed for different time periods
5 Conclusions
Supplementary material
References
Chapter 3 Thermal plasticity and sensitivity to insecticides in populations of an invasive beetle: Cyfluthrin increases vulnerability to extreme temperature
1. Introduction
2. Materials and methods
2.1. Rearing of the insects
2.2 Experimental design
2.2.1. Characterization of the thermal tolerance of the insects from different populations
2.2.2. Effect of daily heat spikes and cyfluthrin exposure
2.2.3. Effect of daily heat spikes and cyfluthrin exposure on recovery from exposure to extreme temperatures
2.3. Data treatment
3. Results
3.1. Characterization of the thermal tolerance of the insects from different populations
3.2. Effect of daily heat spikes and cyfluthrin exposure
3.3. Effect of daily heat spikes and cyfluthrin exposure on recovery from exposure to extreme temperatures
4. Discussion
4.1 Characterization of the thermal tolerance of the insects from different populations
4.2. Effect of daily heat spikes and cyfluthrin exposure
4.3. Effect of daily heat spikes and cyfluthrin exposure on recovery from exposure to extreme temperatures
5. Conclusion
Supplementary File 1
Supplementary File 2
Supplementary File 3
References
Effect of heat and pesticide exposure on Alphitobius diaperinus
1 Introduction
2 Methods
2.1. Rearing of the insects
2.2 Experimental chamber preparation
2.3 Exposure treatment
2.4 Physiological Analyses
2.5 Reproduction
3 Results
4 Discussion
References
Chapter 4 Desiccation in the lesser mealworm Alphitobius diaperinus
1 Introduction
2 Methods
2.1 Collection and rearing of insects
2.2 Effects of desiccating conditions on the survival and physiology of the insects
2.2.1 Survival of the insects exposed to desiccating conditions
2.2.2 Measurements of body water and sugar content
2.2.3 Metabolomics
2.3 Effects of desiccating conditions on activity and reproductive capacities of the insects
2.3.1 Measurement of locomotor activity
2.3.2 Measurement of the reproduction capacities of the beetles
2.4 Statistics
3 Results
3.1 Survival of the insects exposed to desiccating conditions
3.2 Effects of desiccating conditions on sugar content and metabolic profiles
3.3 Locomotor activity of the beetles
3.4 Reproductive capacities
4 Discussion
Conclusion
Supplementary Material
References
Chapter 5 General discussion on the scientific contributions of the thesis’ results and future perspectives Warming and geographic expansion of alien species
Thermal tolerance breadth
Effects of climate change and pesticides on A. diaperinus
References
