Methods
Pea aphids are phloem feeding insects that generally reproduce through through parthenogenesis (MacKay et al. 1993), allowing them to rapidly increase in population size and sometimes reach economically damaging densities on crops. Multicolored Asian Lady beetles are aphidaphagous predators that were introduced to North America numerous times as biological control agents (Koch 2003). These lady beetles have become an important part of agroecosystems, and their predation helps generate a trophic cascade that can benefit crops (Cardinale et al. 2003). The lady beetles and pea aphids used in this study were obtained from laboratory colonies maintained at Mississippi State University. The lady beetle colony was established with wild-caught individuals from locations near Starkville, MS and was supplemented each year to maintain genetic variation and natural phenotypes. Because pea aphids are parthenogenic, all aphids in the study were genetically identical clones (clonal line a2a; pink/red color). Aphid colonies were maintained on fava bean plants (Windsor variety; Johnny’s Selected Seeds, Winslow, Maine, USA).
All experiments were conducted in plant growth chambers (Percival, Model: E41L2C8, Perry, Iowa, USA) at Mississippi State University in Spring 2018. The four temperature treatments were an “ambient” treatment with a 19℃ mean temperature (14/24℃, min/max) and three warming treatments that each had 23℃ means, but differed in their minimum and maximum temperatures in the following ways: constant (18/28℃), day (14/32℃), and night warming (22/24℃) (for additional details see Table 4.1). For our methods, ambient represents a reference temperature commonly encountered by these insects. All growth chambers were programmed to maintain 40% relative humidity and a 12/12h light/dark cycle.
To determine if temperature treatments had different effects on top-down control of aphid density and plant biomass, we conducted a 4 x 3 factorial experiment crossing temperature treatments (ambient, day, night, and constant warming) and trophic levels (plant (P), plant + herbivore (PH), and plant + herbivore + predator (PHP)). Four fava bean seeds were planted in 15 soil pots and placed into a growth chamber. Each pot was watered at least every other day throughout the duration of the experiment. After 14 days, we removed the weakest of the four plants, leaving three plants, and then covered the pot with a clear plastic cylinder (approximately 20 cm d x 30 cm ht) and insect mesh top. Additionally, one mesh-covered opening (5 cm x 7 cm) was installed into each cylinder to increase air flow.
Each pot was assigned to one of the three trophic-level treatments. Pots assigned to PH and PHP treatment pots received ten aphids per plant (30 aphids per pot). After six days of aphid population growth, we began acclimating lady beetles by placing them in petri dishes and randomly assigning them to growth chambers. On day seven, we counted the number of aphids in each pot to determine initial aphid abundance at the beginning of the experiment. Immediately afterwards, PHP treatments were stocked with one adult lady beetle per pot. One week later, lady beetles were removed and the number of aphids on each plant were counted. Plants were placed in paper bags and dried at 60° C for 48 hours. After 48 hours, dried plants were weighed on an electronic balance (Mettler Toledo PL602-S).
We performed another experiment to determine if consumption of aphids by lady beetles differed due to temperature treatments. We placed lady beetles (8 individuals per block x 3 blocks for each of 4 temperature treatments) in an individual deli container inside a growth chamber set at one of the four temperature treatments. Lady beetles were fed aphids ad libitum daily. After a five-day acclimation period, lady beetles were given 50 aphids in 12-hour intervals (06:00-18:00 and 18:00-06:00). After each twelve-hour interval, the number of aphids were counted. Any remaining aphids were removed, and 50 new aphids were given to each lady beetle. This process continued for three days. Lady beetles were returned to the lab colony after the experiment. This process was replicated three times for each temperature treatment. One lady beetle in the ambient treatment did not survive the acclimation period, leaving 23 total lady beetles in that treatment. All other treatments produced data from 24 lady beetles.
Data were analyzed in the R statistical programing environment (R Core Team 2016). We used a linear mixed-effects model to analyze aphid abundance and plant biomass. While count data are often Poisson distributed, this distribution can converge onto a normal distribution. Therefore, we compared a normal and Poisson model using the Akaike information criterion (AIC) estimator and selected the normally distributed model for further analyses. Warming type was a four-level (ambient, constant, day, or night) fixed effect and experimental block was used as a random effect. Post-hoc tests (least square means) for aphid abundance were separated and tested based on predator presence or absence. Post-hoc tests (least square means) for plant biomass were separated and tested based on trophic level (P, PH, and PHP).
We evaluated lady beetle predation rate using a general linear mixed-effects model with binomial error distribution. Time was a two-level (day or night) fixed effect, warming type was a four-level (ambient, constant, day, or night) fixed effect and experimental block was used as a random effect. A separate model was generated to compare lady beetle predation throughout the 24-hour day. For the 24-hour model warming type was a four-level (ambient, constant, day, or night) fixed effect and experimental block was used as a random effect. Inferences from the models are based on likelihood ratio tests comparing models with and without the specific predictor variables (e.g., temperature treatment and time of day). Post-hoc tests (least square means) for proportion of aphids consumed were tested to determine treatment level differences.