Insect rearing, body weighing and sperm count
Insect rearing and weighing:
Insects were maintained in plastic cylinders (8 cm diameter × 10 cm height),
each filled with 50 g of a standard diet (43.5% wholemeal wheat flour, 43.5% maize
meal, 3.0% yeast, and 10% glycerine). Cylinders were covered with two layers of
nylon mesh. To start the colonies, 116 newly laid eggs (< 24 h old) [1] were introduced
into each cylinder with the standard diet. Two crumpled paper towels (25 × 25 cm)
were placed in each cylinder for pupation. Adults were not given food or water as they
do not feed [2]. The insect colony was kept and all experiments were carried out at 25 ±
1°C and 70 ± 10% RH, with a photoperiod of 14:10 h light:dark cycle.
Mature pupae were collected from the crumpled paper towels and weighed using
an electronic dual range balance (Mettler AE100, Switzerland) with a readability of
0.0001 g. The weighed pupae were kept individually in glass tubes (2 cm diameter × 7.5
cm height) until adult emergence to ensure virginity and age. To minimize weight
changes over time, only those insects that emerged within 12 h after pupal weighing
were used for experiments. The emerged moths were sexed and kept in the same glass
vials before being used for experiments.
In Ephestia kuehniella, the pupal and adult weight had a strong linear
relationship in both sexes (analysis of regression: DF = 1,59, F = 192.0, P < 0.0001 for
males and DF = 1,59, F = 634.0, P < 0.0001 for females) (Fig. s1) [3], indicating that
pupal weight is an honest reflection of adult weight in this species. Consequently, we
used pupal weight as the index of adult weight in this study.
Mean pupal weight (mean ± SD) was 22.8 ± 1.9 mg and 25.1 ± 2.3 mg for male
and female, respectively. Body weight was categorized as average, light (less than 1
1
standard deviation from the mean), or heavy (greater than 1 standard deviation from the
mean).
Sperm count protocol:
Lepidopteran males produce two types of sperm, eupyrene (nucleate) and apyrene
(anucleate) spermatozoa. Although both eupyrene and apyrene sperm reach the
spermathecae of the inseminated females, only eupyrene sperm can fertilise eggs.
We dissected the copulated female under a stereo microscope (Olympus SZ III,
Japan) immediately after the copulation ended, and counted sperm she received using
the methods outlined in Cook and Wedell [4]. Briefly, the spermatophore was placed in
a drop of saline on a slide and ruptured with a needle to release the sperm. In E.
kuehniella, eupyrene sperm are transferred to the female in bundles (there are 256
eupyrene sperm in each bundle) which later break apart [5]. Therefore, the number of
bundles was multiplied by 256 to give the total number of eupyrene sperm. To count the
apyrene sperm, the sample was washed from the slide with saline into a 30 ml specimen
tube and diluted with distilled water. A total of four 10 μ1 subsamples were removed
from each sample and allowed to dry on slides. The dried smears were then examined
using dark field phase contrast microscopy at ×100 magnification and all apyrene
sperm in each smear were counted. The total number of apyrene sperm per
spermatophore was calculated by multiplying the average 10 μ1 sperm count by its
dilution factor.
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References:
1. Xu J., Wang Q. 2009 Male moths undertake both pre- and in-copulation mate
choice based on female age and weight. Behav Ecol Sociobiol 63, 801-808.
2. Norris M.J. 1934 Contributions towards the study of insect fertility - III. Adult
nutrition, fecundity, and longevity in the genus Ephestia (Lepidoptera, Phycitidae).
Proc Zool Soc Lond 104, 333-360.
3. Xu J., Wang Q. 2013 Trade-off between adult body size and juvenile survival: an
experimental test of parental effects in the Mediterranean flour moth. Aust J Entomol
52, 403-406.
4. Cook P.A., Wedell N. 1996 Ejaculate dynamics in butterflies: A strategy for
maximizing fertilization success? Proc Roy Soc Lond B Bio 263(1373), 1047-1051.
5. Koudelova J., Cook P.A. 2001 Effect of gamma radiation and sex-linked recessive
lethal mutations on sperm transfer in Ephestia kuehniella (Lepidoptera: Pyralidae). Fla
Entomol 84, 172-182.
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