Research Project

Does Mating Cause Eggs to Mature?

(see also Oberhauser and Hampton 1995)

Abstract

We studied the relationship between the timing of mating and oogenesis in monarch butterflies to determine 1) the potential for male nutrient input into eggs and 2) whether mating stimulates egg development. We measured the effects of mating on both the presence or absence of eggs, and on egg numbers. Most females mated soon after they started maturing eggs. One and two days after mating, females contained the same number of mature oocytes as virgin females of the same age, while three days after mating they contained more mature oocytes than virgins. These results confirm the potential for male-derived nutrients to augment oocyte production, but indicate that mating is not required for oocyte maturation to occur.

Introduction

In many Lepidoptera, fecundity appears to be limited by protein obtained as larvae; females deplete both nutrients and oocytes (eggs) throughout their adult lives, and die with few oocytes left (Norris 1932, Labine 1968, Dunlap-Pianka et al. 1977). However, some species obtain protein as adults, and their fecundity need not be restrained by larval reserves (Gilbert 1972, Dunlap-Pianka et al. 1977, Boggs 1990). These species might be expected to continue to manufacture eggs throughout their lives. One source of protein is the adult food supply. For example, Heliconius butterflies extract protein from the pollen on which they feed (Gilbert 1972, Boggs et al. 1981, Boggs 1987). In addition, many female Lepidoptera obtain protein from the spermatophores transferred by males during mating (Boggs and Gilbert 1979, Boggs 1981, Boggs and Watt 1981, Greenfield 1982, Boggs 1990, Wells et al. 1993, What factors affect the size and composition of monarch spermatophores?), and in many species there is a positive relationship between the amount of spermatophore material received and fecundity (Rutowski et al. 1987, Watanabe 1988, Oberhauser 1989, Tamhankar et al. 1993, Wiklund et al. 1993).

Whether females use male-derived nutrients in egg production will depend in part on the state of oocyte development when they are received (Boggs 1990). If females mate after most of their eggs have been yolked, there is less potential for male nutrient input than when females mate when their oocytes are less mature. The timing of vitellogenesis (deposition of yolk in the oocyte, see Egg production) and mating vary widely in the Lepidoptera. In some species, females eclose with a full complement of oocytes, many of which are already yolked, while in others they eclose without developed oocytes (e.g. Norris 1932, Labine 1968, Dunlap-Pianka et al. 1977). Some females mate while still in the pupal stage or as newly-eclosed adults, while others remain unmated for several days after eclosion (Gilbert 1976, Ehrlich and Ehrlich 1978).

In this study we examined the relationship between the timing of mating and oocyte development in the monarch butterfly. This relationship is relevant to questions about the role of mating and male-derived nutrients in oocyte maturation. First, it will determine the potential for male-derived nutrient input into eggs. Several lines of evidence indicate that male-derived nutrients are important to female monarchs. Monarch spermatophores contain large quantities of nitrogen (Oberhauser 1992, What factors affect the size and composition of monarch spermatophores?) that can be traced to both reproductive and somatic tissue in the female after mating (Boggs and Gilbert 1979, Wells et al. 1993), and these nutrients probably contribute to female reproductive output (Oberhauser 1989 and What factors affect the number of eggs that female monarchs lay?). Captive monarchs show a fecundity pattern similar to that found in Heliconius butterflies that obtain protein as adults from pollen, despite the fact that their adult food source (nectar) contains little protein (Baker and Baker 1973). They have high lifetime fecundities, show a relatively steady output of eggs over a three to four week period, and die with oocytes remaining in their ovaries (Oberhauser 1989, 1997 and What factors affect the number of eggs that female monarchs lay?). A second question is whether there is a causal relationship between mating and oogenesis. Unmated female monarchs captured in the wild usually have no visible oocytes (Ehrlich and Ehrlich 1978, personal observations), and some authors have suggested that mating is required to stimulate egg production in this species (Ehrlich and Ehrlich 1978, Drummond 1984). Our study tests this hypothesis.

Methods

Experimental butterflies were F2 offspring of wild females caught in Tennessee in April 1993. Larvae were reared on cuttings of common milkweed (Asclepias syriaca) in screen cages kept outdoors in St. Paul, Minnesota. All adults were weighed to the nearest tenth of a milligram the morning after they eclosed, before being fed for the first time. At this point, the meconium has been discharged and masses remain relatively constant (Oberhauser unpublished). Females that eclosed over a four day period (18-21 July 1993) were assigned to mating and non-mating treatments, with sample sizes of 60 and 49, respectively. All females were kept in walk-in screen cages (2m x 2m x 2m) from the day of eclosion until they were dissected at various ages. They were removed for one to two hours every morning to be fed a 1:4 honey:water solution ad libitum. No males were ever put into the cage with nonmating females. Mating cages always contained equal numbers of four- to six-day-old virgin males and experimental females. We controlled male age and mating history to eliminate the possibility that female mating age was affected by the type of male in the cage and to control spermatophore size. Male age, mating history and size all affect spermatophore size, with most variation being explained by age in virgin males (see Oberhauser 1988, What factors affect the size and composition of monarch spermatophores?). Densities in mating cages were 25 or fewer of each sex.

No host plants were available to females in either treatment to control for any effects of oviposition (virgins rarely lay eggs and oviposition rarely occurs in the absence of host plants, personal observations). Previous work on monarchs had shown that host plant availability is not required for oogenesis to occur; females held in glassine envelopes from the day of eclosion produce mature oocytes (Herman and Barker 1977, personal observations).

We checked for mating pairs at two hours intervals throughout the day. This frequency ensured observation of all matings because monarchs begin mating in mid- to late afternoon and remain in copula for several hours, rarely separating before 0200h (Svärd and Wiklund 1988a, Oberhauser 1989b). After mating, females were moved to the cage with nonmating females until they were dissected. Due to environmental factors we stopped measuring the timing of mating after females had been in mating cages for eight days; cool, wet weather for the following two days made mating unlikely, and the timing of later matings could have been due to weather conditions, and not female receptivity.

From ages one to ten days, we dissected six nonmating females every day under 6 and 12x magnification on a Wild dissecting microscope and counted mature oocytes (oocytes that are both yolked and have a ridged chorion, see Egg production) in the ovaries and common oviduct. We did not make total egg counts because immature oocytes, especially at the tops of ovarioles, are tiny and hard to count accurately. We noted the presence of visible immature oocytes, distinguishing between those that were not yolked and those that were yolked.

Females in the mating treatment were dissected after mating. We determined that matings had been successful by checking the bursa copulatrix for the presence of a spermatophore. Oocytes were counted as described above. We initially planned to limit these dissections to one and two days after mating, but it was clear after five days that these females did not differ in oocyte development from virgin females of the same age. We then dissected additional females three days after mating. Times of dissection refer to the number of days after the day mating began.

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