Global changes and human activities have increased the likelihood of transport of non-native insect species all around the globe. When established, the spread of organisms leads to the spatial sorting of the populations, progressively contributing to the selection of individuals with enhanced dispersal performance at the edges of the invaded range. During dispersal, propagules are also subjected to contrasting environmental conditions that can be stressful. These include temperature extremes and variations, desiccation and the quantity and quality of food, which can impose physiological constraints. As a consequence, dispersal syndrome may be promoted along invasion gradients with individuals being characterized by higher stress tolerance in addition to higher dispersal capacities. However, only few studies have examined the differences in stress resistance of non-native insect populations along their invasion gradient. Merizodus soledadinus (Guérin-Méneville, 1830) is a non-native insect species invading the subantarctic Kerguelen Islands, where its quick spread highly impacts the native fauna. On the Kerguelen Islands, its invasion history is precisely known. This offers a unique opportunity to study its stress resistance in relation to the residence time. In this study, we investigated the effects of the residence time of populations of M. soledadinus on its resistance to heat, desiccation, food deprivation and the combination of these three stresses in the laboratory. We found that desiccation and multiple stress treatments caused the highest mortality rates. Range edge populations showed a longer survival compared to core populations. However, the dynamics of survival were different: core populations experienced a steady decline in survival, while range edge populations initially experienced a slow mortality decline followed by a rapid mortality. This suggests greater stress resistance for individuals at the invasion front, potentially explaining the intense expansion of M. soledadinus on Kerguelen Islands. Further research could explore the bioenergetic consequences of these differences in stress resistance.

Insect collection

Adults of M. soledadinus were hand-collected from six distinct localities of the Kerguelen Islands (48°25’ – 50°S; 68°27’ – 70°35’E) during the austral summer 2015 - 2016: Port Couvreux (the unique site of introduction of the beetle), Cataractes, Anse du Pacha, Pointe Suzanne, Isthme Bas and Val Studer (all localities are at least 8 km apart, with the furthest being more than 50 km apart, Figure 1). The austral summer (from September to April) corresponds to the maximum period of emergence for M. soledadinus on the Kerguelen Islands (Ouisse et al. 2017). We collected adults in December 2015 - January 2016, to maximize the collection of newly emerged individuals. All of the localities where specimens of M. soledadinus were collected are coastal, except Val Studer which lies around 7.2 km from the coastline. All sampling sites host large densities of M. soledadinus, for which the year of establishment is known, and therefore the time elapsed since colonization (hereafter “residence time”, Lebouvier et al. 2020). At the time of collection, populations of M. soledadinus were established for 103 years at Port Couvreux, 36 years at Cataractes, 21 years at Anse du Pacha and Port-aux-Français, 10 years at Pointe Suzanne, five years at Isthme Bas, and four years at Val Studer (Figure 1) (Source: project 136 - SUBANTECO, French polar institute, also see Lebouvier et al. 2020). Adult longevity of M. soledadinus is eight months on average (Ouisse et al. 2017). This beetle species can produce up to two generations per year. The number of generations since the establishment of the insect in each locality is thus expected to vary from a minimum of six (for the population with the shortest residence time: Val Studer) to 154 (for the population with the longest residence time: Port Couvreux).

Maintenance of the insect populations

More than 500 adults were collected from each locality and directly placed into sealed plastic boxes before being brought back to the laboratory of the research station (Port-aux-Français, Kerguelen Islands) within 24 - 48 hours after sampling. At the laboratory, insects were transferred to plastic boxes (18 x 12 x 7 cm) at standardized densities of approximately 100 individuals per box for each population. The bottom of all plastic boxes was covered with filter paper moistened with tap water. Insects were fed ad libitum with larvae of the invasive non-native fly Fucellia maritima (Haliday 1838) (Diptera: Anthomyiidae), and they were provided drinkable water by the addition of 2 mL microtubes filled with tap water. The microtube caps were kept open, and the opening of the tubes was plugged with cotton for a slow diffusion of the water. All rearing boxes were maintained during seven days in a walk-in climatic chamber at 8 °C, photoperiod 12:12h (Light:Dark).

Range expansion and evolution of stress tolerance

Transparent Petri dishes (ø 9 x 1.5 cm) were used for all survival assays described below.

Assessment of rearing conditions

We used insects from the population of Port-aux-Français to monitor the survival of the insects under controlled – supposedly non-stressful – conditions. We decided to use insects from Port-aux-Français as the longevity of this population was formerly assessed by Ouisse et al. (2017) under laboratory conditions, thus allowing to determine if lifespan remained in a similar time range. Moreover, this population is not used in the stress treatments. In total, ten Petri dishes were prepared, each containing 10 individuals of M. soledadinus (n = 100 M. soledadinus individuals).

The Petri dishes were maintained at 8 °C in the walk-in climatic chamber (photoperiod 12:12h (Light:Dark)) corresponding to the mean monthly temperature of the austral summer on the Kerguelen Islands (Supplementary information 1, Table S1-1; Lebouvier et al. 2011). The bottom of each Petri dish was covered with moistened filter paper and a microtube filled with tap water was added to provide drinkable water and suitable humidity conditions for M. soledadinus individuals. Every 10 days, the filter paper and Eppendorf of the Petri dishes were checked to ensure the presence of drinkable water and suitable humidity. The filter paper was changed every two months. Once a month, adults of M. soledadinus were fed with larvae of F. maritima. The survival of adult M. soledadinus under the rearing conditions was monitored fortnightly.

The longevity of the individuals of M. soledadinus of Port-aux-Français, calculated for the 10 replicates, averaged 705 days. Longevity ranged from a minimum of 190 days and a maximum of 884 days (Supplementary information 2, Figure S2). A similar lifespan was reported for this species by Ouisse et al. (2017), and is also consistent with former observations in other ground beetle species (Lövei and Sunderland 1996), attesting that our laboratory conditions did not lead to a reduced survival.

Abiotic stress

The tolerance of M. soledadinus individuals from the six populations (Port Couvreux, Cataractes, Anse du Pacha, Pointe Suzanne, Isthme Bas and Val Studer) to four experimental stress was examined: heat (I), desiccation (II), starvation (III) and exposure to multiple stress which combined heat, desiccation and starvation (IV). For each experimental stress, 10 Petri dishes, each containing 10 insects of one of the six populations, were prepared (with few exceptions, 9 - 12 Petri dishes replicates, n = 248 Petri dishes in total, see Supplementary information 3, Table S3-1 and Figure S3-2). The experimental stress started at the end of a 7-day maintenance period following field capture.

(I) Heat stress treatment: Petri dishes were transferred to an incubator (Panasonic, MIR154) that allowed an accurate control of the exposure duration at each desired experimental temperature: temperature remained at 8 °C for six hours before gradually increasing with one-hour increments to 10, 12, 16 °C, and with two-hour increment to 20 °C, and finally peaking at 28 °C for two hours. Then, the temperature gradually decreased on an hourly basis to 20, 16, 12, 10 and finally 8 °C which was kept constant for 6h (Supplementary information 3, Figure S3-1). This daily variation of the temperature, and the resulting heat stress, represented an intense warm event for the insects. So far, the maximum mean air temperature measured at 2 m above the ground level on the Kerguelen Islands is 25.8 °C (Lebouvier et al. 2011, project 136 - SUBANTECO, French polar institute). A 12:12h (Light/Dark) photoperiod was applied. To reproduce a diurnal increase in temperature, the periods during which the temperature started to increase above 8 °C coincided with the sunrise. Humidity conditions and food supply of the insects were similar to those used for the assessment of the rearing conditions (Supplementary information 1, Table S1-1).

(II) Desiccation stress treatment: no source of humidity and no drinkable water were provided to M. soledadinus individuals. The feeding regime, temperature and photoperiod were the same as those used for the assessment of the rearing conditions (Supplementary information 1, Table S1-1).

(III) Starvation stress treatment: for this treatment, M. soledadinus individuals were deprived from food for three weeks before the start of the experiment to ensure that the beetles had empty guts (Laparie et al. 2012) and that the food stress started at the beginning of the assay. The insects were then starved until death but given drinking water as previously explained (moistened filter paper and microtube filled with tap water). Humidity level was weekly checked. Temperature and photoperiod were the same to those used for the assessment of the rearing conditions (Supplementary information 1, Table S1-1).

(IV) Multiple stress treatment: this treatment corresponded to the combination of heat, desiccation and starvation stress treatments. The temperature and photoperiod conditions corresponded to those applied to the heat treatment (Supplementary information 3, Figure S3-1). Humidity conditions were the same as used in the desiccation treatment. The feeding regime was the same as the one applied to the starving insects, including the three-week deprivation period before the experiment started. During these three weeks, water was provided to the individuals with the same set-up as previously explained (moistened filter paper and microtube filled with tap water), and humidity level was weekly checked (Supplementary information 1, Table S1-1).

Assessment of survival

The survival of adult M. soledadinus was monitored:

(i) every 12 hours for Petri dishes exposed to the desiccation and multiple stress treatments, as they were expected to be the most stressful conditions for the beetles (Ouisse et al. 2016),

(ii) once a day for the heat and starvation stress treatments.

During these checks, hereafter referred to as “experiment time points”, the status of all M. soledadinus individuals was determined with a binocular magnifier installed in the walk-in climatic chamber (8 °C): individuals were classified as alive, comatose, or dead. Alive individuals were moving and were standing on their legs. Individuals in a coma were no longer on their legs but displayed movements of their appendages (antennas, mandibles, legs); individuals in a coma were categorized as alive individuals. Dead individuals were on their backs with their legs folded, and exhibited no movement of appendages even if stimulated with a paintbrush. At each experiment time point, the individuals identified as dead, and if necessary, larvae of M. soledadinus were removed from the Petri dishes. For each Petri dish, the observations ended when all adults had died. Therefore, the duration of the experiment was different between each Petri dish. Predation events among M. soledadinus conspecifics were observed by individuals found dead and which had been partially attacked (e.g., missing or damaged body parts). Predation events were observed in low numbers (mostly once per Petri Dish), only for the longest stress treatments: starvation and heat stress treatments, and for all the studied populations (Supplementary information 4, Table S4).  

Proportion of dead M. soledadinus

At each experiment time point, the number of dead and alive individuals was counted for each Petri dish. We tested the effects of the duration of the experiment (continuous covariable), the residence time (continuous) and the stress treatment (categorical) on the proportion of dead individuals. These effects were first tested across the four stress treatments, then on each stress treatment independently. We used a binomial denominator written as a vector: number of successes, number of failures (Crawley 2013), corresponding respectively to the number of dead individuals and the number of living individuals.