Increased pupal temperature has reversible effects on thermal performance and irreversible effects on immune system and fecundity in adult ladybirds

The environmental conditions an organism encounters during development vary in their lasting impact on adult phenotypes. In the context of ongoing climate change, it is particularly relevant to understand how high developmental temperatures can impact adult traits, and whether these effects persist or diminish during adulthood. Here, we assessed the effects of pupal temperature (17°C – normal temperature, 26°C – increased temperature, or 35°C – heat wave) on adult Harmonia axyridis thermal stress tolerance, immune function, starvation resistance, and fecundity. Temperature during pupation significantly affected all investigated traits in fresh adults. Heat acclimation decreased adult haemocyte concentration, cold tolerance, and total egg production, and had a positive effect on heat tolerance and starvation resistance. The negative effects of heat acclimation on cold tolerance diminished after seven days. In contrast, heat acclimation had a lasting positive effect on adult heat tolerance. Our results provide a broad assessment of the effects of developmental thermal acclimation on H. axyridis adult phenotypes. The relative plasticity of several adult traits after thermal acclimation may be consequential for the future geographic distribution and local performance of various insect species.Funding provided by: Ministerstvo Školství, Mládeže a TělovýchovyCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001823Award Number: CZ.02.2.69/0.0/0.0/18_053/0016979Ladybird rearing and experimental setup Individuals assayed in our experiment were the offspring of field-collected adults from České Budějovice, Czech Republic (48.9794344 N, 14.4451989 E). Seven parental pairs were placed into separate Petri dishes (9 cm in diameter) containing folded paper strips as substrate for egg laying. Parental pairs were kept in a climatic chamber (Sanyo® MIR-155) at 26 °C, relative humidity of 70%, and long day photoperiod (16L:8D). Ladybirds were provided with fresh pea aphids and water ad libitum daily. Eggs from each parental pair were collected, each clutch placed into separate Petri dishes, and kept under the same conditions as parental pairs. After hatching, larvae were separated into smaller groups of 4–5 individuals, placed into new Petri dishes, and provided with fresh pea aphids and water ad libitum. The last instar larvae were placed individually into new Petri dishes, fed until the prepupal stage, and then checked twice a day to collect fresh pupae as soon as possible. Petri dishes with fresh pupae were immediately distributed among three climatic chambers set to one of three thermal regimes: normal temperature (17°C), increased temperature (26°C), or heat wave (35°C). Pupae were checked twice a day (morning and evening) to collect freshly eclosed adults. The pupal stage was selected because of its lack of mobility, and therefore its inability to escape such high stressful temperatures under natural conditions. New adults were moved to the 26°C climatic chamber and provided with pea aphids and water ad libitum. After 24 hours, the first subset of adults was used to measure haemocyte concentration and thermal performance. Seven days after eclosion, the same variables were measured for the second subset of adults. The third subset of ladybirds was used to assay ladybird longevity without food. Finally, the fourth subset was used to assay fecundity (e.g., time until the first clutch, number of clutches, etc.). Measurement of response variables Thermal performance To investigate high- and low-temperature performance of ladybirds subjected to various pupal temperatures, we measured chill coma recovery time (CCRt) and heat knockdown time (HKDt). To measure CCRt, sets of six ladybirds in separate Petri dishes were exposed to -4°C in a climatic chamber for 120 minutes and then transferred to a climatic chamber set to 26°C for recovering. Beetles were turned onto their dorsum and time until the first coordinated movement was measured (e.g., flipping to their ventral side). Experimental ladybirds were stimulated to move by gentle teasing with an entomological pin every fifteen seconds to identify beetles that remained motionless despite being already able to move. To measure HKDt, sets of maximally 12 individuals were placed in clean Petri dishes (without food and water) in a climatic chamber set to 42°C. Ladybirds were checked every 15 minutes using a webcam placed inside the chamber. Individuals that had become immobile or dead (reached their HKDt) were easily identified as motionless, typically with spread wings. We aimed to analyse up to six individuals for each combination of variables (sex, parental pair, pupal temperature, and sampling time; n = 336 ladybirds). However, some parental pairs produced an insufficient number of offspring, resulting in 289 beetles measured for CCRt and 270 for HKDt. As a measure of body size, we recorded the live body mass after eclosion of beetles assayed for CCRt using a Sartorius balance with a precision of 10−4g. All beetles measured for HKDt were dried for 48 hours at 42°C after the HKDt assay and their dry mass recorded. Haemocyte concentration Haemolymph was collected via puncture sampling. Haemolymph was collected using a glass microcapillary (Hirschmann, Germany) and measured using a digital calliper with a precision of 0.01 mm. Collected haemolymph (~1μl) was immediately diluted (100× dilution) in anticoagulant buffer (Phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4), and the total haemocyte concentration was recorded immediately using a Bürker chamber under a Carl Zeiss Primo Star microscope (set to 100× magnification). We aimed to analyse four individuals for each combination of sex, parental pair, pupal temperature, and sampling time (1 day or 7 days; n =336 ladybirds). However, problems sampling several individuals (e.g., haemolymph coagulation) resulted in a smaller dataset of 308 beetles. Starvation resistance We indirectly tracked the whole-body effects of developmental thermal acclimation on ladybird physiology by assessing longevity under a physiologically stressful condition, food deprivation. Ladybirds used to investigate the effects of pupal temperature on starvation resistance (longevity without food) were placed in Petri dishes with watered pieces of cotton wool and moved to a climatic chamber set to 26°C. Survival of these ladybirds was checked daily, and new water was added ad libitum. In total, starvation resistance was recorded for 126 individuals, i.e., three individuals per combination of sex (2), parental pair (7), and pupal temperature (3). Their live body mass after eclosion was measured using a Sartorius balance with a precision of 10−4 g since body mass is known to influence starvation resistance in ladybirds. Fecundity Finally, we evaluated the fitness consequences of thermal acclimation during pupation by tracking the cumulative egg production and days until the first clutch of females after eclosion. We recorded egg production each of 28 days after eclosion. Experimental pairs (n = 63 males and females) were established from a combination of individuals that eclosed on the same day and originated from different parental pairs. Each experimental pair was placed in one Petri dish for mating, placed in a climatic chamber set to 26°C, relative humidity of 70%, and long day photoperiod (16L:8D; same as the parental generation), and provided with pea aphids and water ad libitum. Every day folded paper strips in Petri dishes were changed and the total number of eggs was counted. Note that ladybirds typically lay eggs on the underside of leaves, which is mimicked by folded paper strips. Live body mass was measured for each experimental female after eclosion