(Continued)

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Results

Effect of mating on the presence of mature oocytes

Figure 1 summarizes the state of oocyte maturation in both virgin and mated females. Egg maturation takes from two to three days; at age one day no oocytes were visible in any females, by age three days half of the females had yolked oocytes, and by age four some females had mature oocytes. We used a log-linear model to test for independence of oocyte development state and mating treatment. For this test, we combined ages four and five; six and seven; and eight, nine and ten to make three age categories. Females dissected at ages one, two and three were not included in the test, because of the small number of mated females dissected at these ages. Given age, oocyte development state is independent of mating treatment (Pearson X² coefficient = 5.28, df = 9, p = 0.809).

Figure 1: State of oocyte development in female monarchs dissected at different ages. Females were categorized according to their most mature oocytes. For example, those with mature oocytes also contained yolked and unyolked oocytes, but they are only counted as having mature oocytes.

Effect of mating on the number of mature oocytes

Figure 2 shows the number of mature oocytes as a function of virgin female age. Females continue to produce mature oocytes over their first ten days, even when they do not mate. A linear regression of number of mature oocytes on female age and mass (Table 1a) indicates that oocyte number increases with age. The effect of virgin female mass on oocyte number is not significant at the 0.05 level of confidence. We tested for effects of female age, mass, and the number of days that had elapsed since mating on mature oocyte number in mated females. Time since mating was included in the model in two different ways; once with one, two, and three days separated (Table 1b), and once with one and two days combined (Table 1c). This was done because visual (see Figure 3) and statistical (see below) analyses of the data suggested that, for a given age, there is no difference in the number of mature oocytes in mated females dissected one or two days after mating. Again, there is a strong effect of female age on mature oocyte number. Initial female mass had a small, but statistically significant, negative effect. Time since mating had a positive effect in both models, but the adjusted R² value for the model that combined one and two days is higher.

Figure 2:  Number of mature oocytes as a function of virgin female age. The purple stars indicate means, which were calculated using both zero and positive values. Symbols at zero include females with immature oocytes, and some represent more than one female.

TABLE 1. EFFECTS OF AGE^a, FEMALE MASS, AND TIME SINCE MATING ON NUMBER OF MATURE OOCYTES

a. Virgin females

b. Mated females (1, 2, and 3 days after mating separate)

c. Mated females (1 and 2 days after mating combined)^d

^aOnly females aged four days and older were included in regressions, since no eggs are mature before this age.
^bAdjusted R² from stepwise analysis of variance
^cTime Since Mating; number of days after mating that female was dissected
^dTsm included as dummy variable, with 1 and 2 days combined

There is no difference between the number of mature oocytes in mated females dissected one or two days after mating and in virgin females dissected at the same age (Figure 3; p > 0.15 for ages four to ten days, t-tests for unequal sample sizes and variances). However, females dissected three days after mating (at ages eight, nine, and ten days) had more mature oocytes than both virgin females and mated females of the same age dissected one or two days after mating (p < 0.02 for all ages; t-tests for ages eight and nine, sign test age ten).

We observed no oviposition behavior on the part of mated females before they were dissected, nor did we detect any eggs on either the sides of the cage or non-hostplants (mostly grass) in the cage. While it is possible that some oviposition on inappropriate substrates did occur, we have no evidence of this.

Figure 3: Number of mature oocytes as a function of mated female age. Purple stars indicate means for virgin females of the same age for comparison. Other symbols refer to the number of days after mating that females were dissected.

Timing of mating

Forty-two of the mating-treatment females (86%) had mated by age eight days, when we stopped putting males in the mating cage. The percentage of females that mated at each age is shown in Figure 4. Before age four, when no females had mature oocytes, only 14% of the females had mated. By age six days, when all virgin females had either yolked or mature oocytes, 70% of them had mated.

Figure 4: Absolute and cumulative percentages of females that mated as a function of age

Discussion

Effects of mating on oocyte development

Experimental virgin females contained mature eggs (Figures 1 and 2), clearly indicating that mating is not required to stimulate oogenesis in monarchs. This is contrary to Ehrlich and Ehrlich (1978) and Drummond (1984) who stated that mating is required to induce oogenesis in monarchs. Our results do not tell us what does stimulate oogenesis in monarch. The induction of oogenesis in Lepidoptera is often caused by increased levels of   juvenile hormone (e.g. Sroka and Gilbert 1971; Pan and Wyatt 1971, 1976; Herman and Bennett 1975; Herman and Barker 1977; Hagedorn and Kunkel 1979; Lessman et al. 1981, Egg production), but how and when the corpora allata are activated to release juvenile hormone are not clearly understood. There are often complex relationships between oocyte development and several factors, including female nutrition, the presence of a spermatophore and viable sperm, and oviposition (e.g. Benz 1969, Sasaki and Riddiford 1984).

While there was no detectable increase in the rate of oocyte maturation for two days after mating, three days after mating females contained more mature oocytes than virgin females of the same age. This suggests two (non-exclusive) explanations: 1) mating provides a hormonal or mechanical stimulus to mature additional oocytes, and 2) females utilize nutrients from spermatophores to produce more eggs. We think that the second explanation is likely to be important in monarchs for several reasons. Monarch spermatophores contain protein (Oberhauser 1992) which limit reproduction in many Lepidoptera (Norris 1932, Labine 1968, Dunlap-Pianka et al. 1977). Labeled amino acids from spermatophores are incorporated into eggs (Boggs and Gilbert 1979) and other female tissues (Wells et al. 1993) after mating, and females that receive more spermatophore material show higher fecundity over the time that they are degrading this male-derived material (Oberhauser 1989a, 1992). It is likely that the effect of these nutrients could take three days, given the rate at which oocytes are matured (Table I). There is some correspondence between this period and the results of Boggs and Gilbert (1979), who found peak incorporation of male-derived material into monarch eggs two to four days after mating in one of the two matings they analyzed. Wiklund et al. (1993) also found that peak incorporation of male-derived materials occurred three to four days after mating in Pieris napi.

The lack of a positive effect of initial female mass on number of mature oocytes in both treatments is consistent with previous work on monarchs (Oberhauser 1989a) that showed no effect of female mass on lifetime fecundity. Fecundity in insects is often correlated with mass (Suzuki 1978, Lederhouse 1981, Cushman et al. 1994 include data on Lepidoptera), but this effect should be less important with increasing dependence on adult, rather than larval, nutrient sources for egg production (Boggs in review). This provides further support for the importance of male-derived nutrients to female monarchs.

Timing of mating and oocyte development

Our results support three conclusions about the relationship between the timing of mating and oocyte maturation in monarchs. 1) Females mate for the first time early in the process of vitellogenesis, when they have a few mature oocytes ready to be fertilized. This suggests that females reject courting males before they have begun to mature eggs, or that males avoid mating with these females (see How is monarch mating different from that in other butterflies?). 2) Because females delay mating until vitellogenesis has begun, male-derived nutrients are likely to be more important for vitellogenesis than for other activities, such as somatic maintenance and foraging activity. This makes sense from a resource allocation viewpoint. Yolk is protein-rich, making male-derived nutrients likely to be important in vitellogenesis, while sugars in the adult diet may suffice for activities such as somatic maintenance and foraging (Boggs and Ross 1993). 3) The number of mature oocytes at the time of a female's first mating is small relative to average daily fecundities (Oberhauser 1989a, 1989b, and unpublished), so while the first eggs laid after mating utilize nutrients obtained solely from larval reserves, male-derived nutrients can supplement these reserves early in egg-laying. The high degree of female multiple mating in monarchs (Pliske 1973, Suzuki and Zalucki 1986, Oberhauser 1989, How often do males and females mate?) results in constant replenishment of male-derived nutrients during egg laying.

The timing of egg maturation could have important fitness implications for females. An obvious strategy is to link egg maturation to host plant presence; it may be costly to divert resources to egg production before oviposition is possible. In some Lepidoptera egg maturation has been shown to be dependent on host plant access (McNeil and Delisle 1989, McNeil 1991, Tamhankar et al. 1993). Monarchs, however, lack a linkage of egg maturation and mating to host plant presence. Females probably find oviposition sites easily due to the abundance of hostplants throughout monarch summer ranges and their ability to fly long distances. Overwintering in large congregations in sites without host plants could also uncouple mating and egg production from hostplant presence. Females usually mate before they leave these congregations (Hill et al. 1976, Brower 1985, Van Hook 1993, Wells et al. 1993). This allows them to take advantage of the presence of many males; population densities of the first butterflies to recolonize summer habitat might be low enough to make it risky to migrate before mating.

Acknowledgements

We thank Don Alstad, Sarah Stai and Liz Goehring for help making cages, rearing, dissecting, and observing butterflies; and Don Alstad, Carol Boggs, Dennis Frey, Sonia Altizer, Liz Goehring, Dann Siems, Christer Wiklund and an anonymous reviewer for comments on earlier versions of the manuscript. Research was supported by NSF DEB-9220829 to KSO.

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