Research Project

What Factors Affect Number of Eggs that Female Monarchs Lay?

(see also Oberhauser 1988, 1997)

Abstract

I conducted three experiments to measure how several factors affect female fecundity and lifespan. These factors included female mass at eclosion (which is assumed to be an indication of the quality and quantity of the larval diet), the adult diet, and nutrients transferred by males during mating (see Spermatophores). Lifetime fecundity was higher when females received a large spermatophore early in egg-laying, or when females mated several times. When lifespan was controlled, there was no effect of female size on fecundity, nor did the concentration of sugar in the adult diet affect fecundity. Egg laying lifespan (the time from eclosion to the last day of egg laying) was positively correlated with female size in one experiment, but was not affected by the amount of male-derived nutrients received nor the quality of the adult diet. These results suggests that larval reserves are more important than nutrients received during the adult stage in determining how long a female lives, but that male-derived nutrients are used by females to increase their output of eggs. At the end of their egg-laying lifespans, females contained unlaid eggs, suggesting that females run out of the reserves required for somatic maintenance before they stop producing eggs, and that lifetime reproductive success is limited by the ability to lay, rather than manufacture, eggs. Thus, even though larval reserves may not be used to increase egg output, they probably contribute to reproductive success indirectly, by increasing lifespan.

Background

In many insects, nutrients for egg production are available from three sources: larval feeding, the adult diet, and materials transferred by males during mating. This is represented by the following equation (Boggs 1990):

(a) mass into eggs = mass from larval reserves + mass from adult diet + mass from male-derived nutrients, or

(b) mass into eggs = mass from larval reserves + mass from adult income.

It is useful to think of the nutrients ingested as adults or received from males as "income", since they can be replaced, while those received during the larval stage cannot. Boggs (1986) predicted that the relative importance of these materials for egg production will vary with 1) the timing of egg production, 2) the quality of the adult diet, and 3) the quality and quantity of male donations (see spermatophores). Larval nutrients are expected to be most important when females eclose with most of their eggs mature. When females eclose without mature eggs, adult income can contribute significantly to egg production; in these species, male-derived nutrients should be most important when females mate multiply and the adult diet lacks protein, and least important when females mate only once and the adult diet contains protein-rich foods like pollen.

Larval reserves and income must also be used for things other than reproduction. Females need to fly to get nectar and lay eggs, and these activities use up nutrients. The things that an organism needs to do simply to stay alive are often called "somatic maintenance," which is different from reproduction. Since lifespan will also affect an organism’s fitness, there might be trade-offs in the use of nutrients for reproduction or somatic maintenance.

In order to test the importance of different sources of materials for egg production in monarch butterflies, I measured how fecundity varies with differences in the amounts of material available from the three potential sources. If larval resources are important to egg production, large females should lay more eggs. On the other hand, when adult income contributes substantially to fecundity, the association between female size and fecundity should be weak or nonexistent. Instead, fecundity should vary with the amount of adult income (food or male-derived nutrients) received.

There is a great deal of variation within the Lepidoptera in the timing of oogenesis and the quality and quantity of adult income, which makes this an ideal group for studying the sources of materials for egg production. Females of some species eclose with most of their eggs mature, while others eclose with no mature eggs. Some adults ingest protein-rich pollen, while others do not eat at all. Males transfer protein-rich spermatophores to females during mating, and both the number and size of spermatophores vary among species. I hypothesized that adult income, particularly nutrients received from males, should be important to monarch fecundity. Females mate multiply and receive relatively large spermatophores from males, and the adult diet of nectar is relatively poor in quality. Furthermore, females eclose with no mature eggs (Barker & Herman 1973, Oberhauser & Hampton 1995), and continue to produce eggs over a relatively long lifespan

Methods

General Rearing Methods

All experimental monarchs were the offspring of adults collected in the wild. I reared larvae on fresh cuttings of Asclepias syriaca under ambient photoperiod and temperature conditions. The day after eclosion they were individually marked and weighed to the nearest 0.01 mg, and their forewings measured to the nearest 0.1 mm. I kept adults in glassine envelopes and fed them a 20% honey solution every other day until they were ready to mate. The first matings for all females took place in 2m x 2m x 2m screen cages outside, and females were kept in 65 x 65 x 65 cm cages while they were ovipositing and for subsequent matings. Females oviposited on either cuttings of common milkweed or potted tropical milkweed plants which were replaced daily. I counted the number of eggs that they laid every day. Except as noted, all butterflies were fed a 20% honey solution daily while they were kept in cages.

I conducted three experiments, varying the amount of times females were allowed to mate, and in one case, the concentration of honey water fed to adults. The mating treatments in these three experiments are summarized in figure 1.

Experiment 1: Large vs. Small Spermatophore

In the summer of 1986, twenty-five females received either a large or small spermatophore, mating to eight day old unmated males, or males that had mated two days previously. These two male groups transfer spermatophores of approximately 35 and 17 mg respectively (see Material investment in mating by male monarch butterflies, Oberhauser 1988). All females mated on the same day. I kept the milkweed cuttings on which females laid the eggs until larvae hatched so that I could determine whether the eggs were fertile.

Experiment 2: Single vs. Multiple Mating, and Food Quality

In the summer of 1988, I conducted another experiment in which I increased the difference between spermatophore treatments, and determined if the quality of the adult food source affects female fecundity. I used two mating treatments, single and multiple mating, and two feeding treatments, low and high food concentration. I used all possible combinations of these treatments in a 2x2 factorial experiment with four treatments: 1) single mating, low food concentration; 2) single mating, high food concentration; 3) multiple mating, low food concentration; 4) multiple mating, high food concentration. All females mated for the first time at age five to seven days to previously unmated males. Single mating females were not exposed to males after their first mating. After three days, males were put into the cages with the multiple mating females. If a female mated, no male was put into her cage for three more days, and if she didn’t mate, a different male was added the following day, until she mated. Three days after each mating, another male was added, and the process repeated. Females in the low and high food concentration treatments were fed either a 15% or 30% honey solution daily. I weighed females every third day until they died, and measured the fertility of a subsample of 20 eggs per female each day.

Initial sample sizes in each treatment were nine females.

Experiment 3: One and Two Matings

In the summer of 1994, I conducted a final experiment with more controlled differences in the amount of male-derived nutrients females received. 60 four to five day old females first mated with males that ranged in age from five to 11 days, and were either virgins, or had mated the day before the experimental mating. These two male types transfer spermatophores of over 25 mg and approximately 7 mg, respectively (Oberhauser 1988), and are designated large and small spermatophore donors.

The morning after their first mating, I put females into separate cages with potted Asclepias curassavica plants and assigned each to one of five mating treatments: a) two unmated males (large, large), b) an unmated male followed by a mated male (large, small), c) a mated male followed by an unmated male (small, large), d) two mated males (small, small), or e) one mated male (small) (the words in parentheses after each treatment refer to the order and sizes of spermatophores received, see figure 1). Beginning on the third day of egg-laying, one or two males of the assigned type for the second mating were put into each female's oviposition cage in the afternoon. This was repeated until females remated. Any that had not remated within seven days were removed from the experiment.

Every other day I weighed females just before being fed. They were kept until they had laid no eggs for seven days, could no longer fly, or died in the cage. Ten eggs from each female, in batches of five, were weighed to the nearest 0.01 mg every day of laying.

After they died or were removed from the experiment, females were frozen for later dissection to determine whether they contained oocytes and fat bodies. I counted the number of mature oocytes in the most intact ovariole, and multiplied this number by eight (the total number of ovarioles) to estimate the total number of oocytes. The state of fat bodies was categorized as none (no fat bodies visible) or some (a few fat bodies visible).

Initial sample sizes were 12 females in each of the double mating treatments (treatments a-d), and six in treatment 'e' (small). Nine females were removed from the experiment because they did not remate, laid no eggs, or laid eggs for fewer than six days. The final total sample size was 47 females, with six to 12 females per treatment.

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