Non-choice feeding bioassays
To study the effect of short and long term attack, MCB larvae were fed
with stems of plants infested 48 hours (short-term attack) or nine days
(long-term attack) before bioassay establishment or non-infested plants.
1st instar MCB larvae were initially weighted and
individually placed in plastic tubes on 2 cm sections of the stem below
the main ear. Larvae were previously fed on maize-based artificial diet
and maintained at starvation for 24 h, presenting weights of 1-3 mg at
the beginning of the bioassay. Sixty larvae per treatment and genotype
were set and maintained in a growth chamber under controlled conditions
of temperature and humidity (22 °C, 80% RH) and a photoperiod of
16L:8D. When necessary, new fresh stem portions of plants infested 48
hours and 9 days before and control plants were provided to the bioassay
larvae. Larval weights and data related to dead larvae were recorded at
7, 11, 14, 18, 22, and 26 after bioassay establishment.
A repeated-measure analysis was performed to test differences for larval
weights using the PROC GLIMMIX procedure of SAS software (SAS, 2008,
Stroup, 2013). Initial larval weight was included as covariate, genotype
and treatment were set as fixed factors, and a first-order
autoregressive covariance structure (AR-1) was chosen in the
within-subject correlation. Within each genotype, differences for larval
weight of treatments were tested at each time using least square (LS)
means adjusted by the initial larval weight. Additionally, linear and
quadratic coefficients of regression of larval weight on time were
obtained for each treatment-genotype combination. Within each genotype,
the comparison of the larval growth curves of treatments was performed
by making orthogonal contrasts among treatment regression parameters
(intercept, linear and quadratic components, respectively) (p ≤
0.05)(Littell, Milliken, Stroup, Wolfinger & O, 2006). The PROC
LIFETEST procedure of SAS software was used to test differences of
larval survival among treatments applied to the same genotype using the
Kaplan-Meier method (SAS, 2008). The death of larvae was the event of
interest and the missing and alive larvae at the end of the bioassay
were treated as censored data. The homogeneity of the survival
distributions was tested using the Šidák multiple-comparison adjustment
for log-rank test (p ≤ 0.05).