DOI: 10.5061/dryad.1c59zw413
title: Increased pupal temperature has reversible effects on thermal performance and irreversible effects on immune system and fecundity in adult ladybirds

description: 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.
description: Ladybird 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
 (CC<sub>Rt</sub>) and heat knockdown time
 (HK<sub>Dt</sub>). 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 HK<sub>Dt</sub>) 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 CC<sub>Rt</sub> and 270
 for HK<sub>Dt</sub>. As a measure of body size, we recorded
 the live body mass after eclosion of beetles assayed
 for CC<sub>Rt</sub> using a Sartorius balance with a precision
 of 10<sup>−4</sup>g. All beetles measured for
 HK<sub>Dt</sub> were dried for 48 hours at 42°C after the
 HK<sub>Dt</sub> 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 Na<sub>2</sub>HPO<sub>4</sub>,
 and 1.8 mM KH<sub>2</sub>PO<sub>4</sub>), 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<sup>−4</sup> 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.