Individuals used for the experiments were derived from approximately 100 adult B. dorsalis emerged from G. japonica seeds collected in June, 1997, at the bank of the Hozu River, Kameoka City, Kyoto Pref., Japan. Before experiments, the insects were reared for 2 generations within a growth chamber regulated at 25°C and 16L–8D.
Female preference for greater investing or larger males. The adults used for this experiment were prevented from mating for 3 days after emergence until examination, provided with water, sugar and dry yeast. The body weight of males was measured with an electric balance to the nearest 10m g, and then 36 pairs of males with body mass in each pair differing approximately by 1 mg were carefully selected. Subsequently, each of the male pairs and a single female were introduced into a Petri dish (f 4 cm) and the male to which the female courted was observed for 30 min. When the female started to mate with either of the males, the other male was removed from the dish and introduced into another dish with another female. After copulation, body weight was measured for both males again. The weight of the seminal fluid transferred was estimated in terms of the decrease in the male’s body weight after copulation. The weight loss due to evaporation was disregarded because of the short interval between the two measurements. I tested first whether the females chose larger males by the binomial test and, subsequently, compared the weight of the seminal fluid between the males chosen and not chosen by the first females by Student’s t-test.
Relationship between the amount of seminal fluid obtained by a female and the female’s refractory period. This experiment used only virgin adults 3 days after emergence from the host seeds. A male and a female were introduced into a Petri dish (f 4 cm) to mate after the male’s body weight was measured. The weight of the seminal fluid transferred was measured in the same way as in the female preference experiment. Each of those females that had experienced mating was introduced into another dish with another male to observe mating behavior. The behavior of each pair was recorded by a time lapse video recorder (SONY EVT-820) to measure the refractory period, estimated in terms of the period from the end of the first mating to the beginning of the courtship behavior at the second mating. Copulation in B. dorsalis is always accompanied by female courtship behavior; the female has the initiative to start the mating process (Takakura, 1999). Therefore, the refractory period defined above corresponds acculately with the period that females need to remate.
To manipulate male investment, males were reared under two dietary regimes from emergence to/in this experiment: half of the males used for the first mating were reared on water and sugar (poorly-fed males), while the remainders were fed on water, sugar and dry yeast (well-fed males). Data analysis was conducted with ANCOVA (analysis of covariance) where the dependent variable was the refractory period, and the independent variables were the weight of seminal fluid (as a covariant) and the feeding regime (as a factor). In addition, the relationship between the weight of the male body and the seminal fluid was examined by correlation analysis.
Sperm precedence. The sperm precedence of the last mated male (P2 value) was measured. The host seeds containing a pupa were exposed to gamma rays of 75 Gr radiated from 60Co. Among the adults that emerged from these seeds, only 7- day-old males were used for the measurement. A virgin female was allowed to mate first with the irradiated male (R male), second with the non-irradiated male (NR male) 24 h later, or vice versa. The females after experiencing the double mating were individually introduced into Petri dishes (f 11 cm) with water, sugar and dry yeast, and kept in a growth chamber regulated at 25°C and 16L–8D. Up to 10th day after the second mating, the eggs laid by those females were examined for development, which was distinctive and easily identified by the appearance of the dark-colored head capsule with development of the eggs. As a control, the development rate, defined as the proportion of developing eggs, was checked for eggs laid by females which mated only with either an R male or NR male. Based on Boorman and Parker (1976), the P2 value was calculated from the egg development rate.