Effects of benign and heat-stress conditions on parent clones and their selfed offspring of the pea aphid

In the face of global climate change, understanding the interaction between heat stress and inbreeding depression is a critical issue. We evaluated the magnitude of inbreeding depression under benign and heat-stress conditions using parent clones and their selfed offspring of Acyrthosiphon pisum. 

The number of nymphs produced by an adult during the first seven days was counted, and the total area of all nymphs was measured as fitness components of a clone. Inbreeding depression was observed during the development of the fundatrix and was exemplified by the occurrence of sterile fundatrices, which accounted for 28.6% of the adults. 

Once clonal lines were established, no significant overall differences were detected in these fitness components between a parent clone and its 30 selfed-offspring clones at a constant 20 °C. However, pairwise comparisons revealed a significant reduction in the fitness components in a few selfed clones, whereas one selfed clone significantly outperformed the parent in terms of the total area of the nymphs. These results demonstrate substantial variation in the fitness components among sibling clones derived from selfing. 

However, when clones were exposed to either a single heat stress (35 °C for 6 h) or repetitive heat stresses (35 °C for 6 h over three consecutive days), inbreeding depression was exacerbated under the more intense stress conditions. These results suggest that inbred clones could spread in the population in regions with mild climates, but are likely to be largely eliminated when exposed to high summer temperatures.
Methods
In the first experiment, a single clone of the pea aphid (Acyrthosiphon pisum, clone 05E) and its selfed offspring clones were prepared. We evaluated the fecundity and growth rate of each clone at 20 °C under a 16L:8D photoperiod. From each clone, a single first-instar nymph was selected as the test aphid and transferred to a new seedling, where it was reared to adulthood. The first day of larviposition was recorded as day 1, and on day 7, all offspring produced were collected and preserved in 80% ethanol. For each of the 30 selfed clones, we prepared 10 to 12 replicates (mean = 11.4), and for the parental clone 05E, we prepared 13 replicates. The number of nymphs produced over the first seven days (7-day fecundity) was counted. All nymphs were placed in a petri dish containing a small amount of 80% ethanol, and their image was captured using a digital microscope (Dino-Lite Edge, AnMo Electronics) under standardized lighting conditions. Images were binarized using a consistent threshold in ImageJ, and the total area of all nymphs was calculated based on the processed images (https://imagej.net/ij/).

The second experiment involved two families: the parental clone 05E and its selfed offspring (05E-self), and the parental clone 07C and its selfed offspring (07C-self). In each of the four clones, five first-instar nymphs were randomly selected within 6 hours of birth and transferred onto a new seedling inside a plastic cage. Seedlings with nymphs were randomly assigned to one of two heat stress treatments or a control. In the single heat stress treatment (SH), cages containing first instars were transferred from 20 °C to 35 °C and maintained at this temperature for 6 hours. After treatment, they were returned to 20 °C under a 16L:8D photoperiod. In the repetitive heat stress treatment (RH), the SH treatment was repeated for three consecutive days, with intervals at 20 °C between heat exposures. In the control (CT), first instars were reared on seedlings continuously at 20 °C. For the SH, RH, and CT treatments, 20–51 aphids (mean = 35.5), 35–40 aphids (mean = 37.8), and 25–35 aphids (mean = 29.3) were used, respectively. After completion of the SH and RH treatments, surviving nymphs were individually transferred to new seedlings and maintained at 20 °C. This transfer was performed because heat stress may have adversely affected the broad bean seedlings. When the nymphs reached the fourth-instar stage, all test aphids—including those in the control group—were transferred individually to new seedlings and allowed to reproduce. Larviposition and mortality were monitored twice daily. On day 7, all nymphs produced by each test aphid were collected and preserved in 80% ethanol. The 7-day fecundity was thus recorded for each aphid. To evaluate the effects of heat stress on each clone, the 7-day fecundity of the selfed offspring was compared with that of the parental clone. The coefficient of inbreeding depression (δ) was calculated for each treatment and family as follows (Lynch & Walsh, 1998):
δ = 1- XI/XO
where XO is the mean 7-day fecundity of the parental clone and XI is that of its selfed offspring. Parental clones are assumed to be derived from outbreeding.