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.