Background
We say that animals are sexually mature when they can produce offspring; most animals require time to develop before they reach this stage. In humans, the onset of sexual maturity is a gradual process caused by hormones released by the hypothalamus and the pituitary (parts of the brain). A similar process occurs in monarchs. A pair of glands called the corpora allata release a hormone called juvenile hormone. High levels of this hormone circulating in adult butterflies cause eggs to mature in females and cause the male reproductive tract to develop. The reproductive organs in most animals begin to develop a long time before sexual maturity. Monarch reproductive organs actually begin to develop during the larval stage, but it isn’t until they receive hormonal messages that they complete development. Research by Dr. Bill Herman (1975) at the University of Minnesota has shown that diapause monarchs that reach sexual maturity after the overwintering period have low levels of juvenile hormone in their blood.
In most insects, sexual maturity coincides with the final molt to the adult stage. However, it may be delayed for several days after the beginning of the adult stage, and in species with an adult diapause, maturity may even be delayed for several months. In monarchs, breeding season individuals are sexually mature four to five days after they emerge as adults, and the generation that migrates is not sexually mature until after the overwintering period. When monarchs mate, the male uses the claspers on the end of his abdomen to attach to the vaginal groove (ostium bursa) of the female. Once attached, the female cannot get away, and the male transfers spermatophore components to the female in a process that can take up to 16 hours.
Male Anatomy
Testes: Two testes in monarchs (and other animals) produce sperm. Monarch testes are bright red and are held together in a single sac.
Vas deferens: The vas deferens is a tube with a fairly thick covering that connects the testes to the ejaculatory duct.
Accessory glands: Lepidopteran accessory glands produce secretions that may facilitate sperm transfer, produce cues to prevent remating by the female, and provide nutrients for the female.
Ejaculatory duct: The ejaculatory duct is a long tube that carries sperm from the vas deferens and secretions from the accessory glands to the aedeagus (penis). The ejaculatory duct is quite complex in insects in which a complex spermatophore is formed, like monarchs.
Aedeagus: The insect intromittent organ (penis) is called the aedeagus. In Lepidoptera, this is held within the last abdominal segments except during mating. It is a continuation of the ejaculatory duct and is inserted into the female during mating. Sperm and accessory gland materials move through the aedeagus into the bursa copulatrix of the female.
Sperm and Sperm Production
Sperm begin to mature during the third and fourth larval instars; the bright red testes are easily visible in dissected male larvae. Sperm production (spermatogenesis) occurs through a series of mitotic and meiotic divisions. A single original cell, called a spermatogonium, is enclosed in a cyst that is formed from other cells in the testes. This original cell undergoes six mitotic divisions to produce 64 spermatocytes. The spermatocytes undergo two meiotic divisions, resulting in a total of 256 sperm cells. These divisions take place during the larval and pupal stages. The last part of sperm development involves transformation into a cell with a head, which contains the nucleus and DNA, and a filamentous tail, which propels the sperm forward. The sperm stay in bundles of 256 until after they have moved out of the testes.
All butterflies and moths produce two kinds of sperm; eupyrene sperm have a nucleus and can fertilize eggs, while apyrene sperm do not have a nucleus. The function of the apyrene sperm is unknown, but many researchers think that they may increase the chances that the eupyrene sperm from a particular male are actually used to fertilize the female’s eggs. The bundles of apyrene sperm separate before being transferred to the female, but the eupyrene bundles stay together and can be observed under a microscope shortly after copulation ends.
Lepidopteran sperm are transferred within a protein-rich ejaculate called a spermatophore. This spermatophore can represent a significant investment by the male; some male monarchs transfer spermatophores that weigh up to 10% of their own mass! But this isn’t the lepidopteran record; males in another species (Pieris napi) can transfer up to 23% of their mass during mating (Forsberg and Wiklund 1989). The spermatophore is not transferred intact to the female; most of it forms during mating within an organ in the female called the bursa copulatrix. The roundish body of the spermatophore is covered with a tough, white sac, and contains a granular substance. The stem-like structure is called the collum. It forms within the male’s aedeagus and is transferred with the sperm at the very end of copulation. The collum has an opening that is positioned next to the opening of a duct in the female that leads to the sperm storage organ. The sperm are contained in a discrete sac in the pointed end of the spermatophore. It takes a long time to transfer all of this material to the female; mating monarchs often remain paired for 16 hours or longer.
Female Anatomy
Bursa copulatrix: The bursa copulatrix is a sac-like organ in female Lepidoptera in which the spermatophore is stored immediately after mating. It secretes enzymes that break the spermatophore down into nutrients used by the female.
Sperm duct: The ductus seminalis, or sperm duct, is a tube that connects the bursa copulatrix to the common oviduct. Sperm move through it to get to the spermatheca.
Spermatheca: Sperm are stored in the spermatheca, a storage pouch at the end of a long, tubular gland called the spermathecal gland. This gland produces secretions that probably provide nutrients for the sperm.
Ovaries and Ovarioles: Lepidoptera have two ovaries, each of which consists of four ovarioles. Oocytes (eggs) are produced here.
Common oviduct: The two ovaries connect to a tube called the common oviduct.
Vulva: The opening through which eggs are laid is the vulva.
Egg Production (Oogenesis)
The end of the ovariole is called the germarium, where oocytes are produced from the original germ cells. This process begins during the larval stage and continues in the adult. After the female monarch ecloses, the oocytes begin to move down the ovariole, enlarging as they go through the vitellarium, where the yolk is deposited on them. This process is called vitellogenesis. The yolk contains both protein and lipids. In some Lepidoptera, vitellogenesis occurs before the adult stage; in these species, females eclose with their eggs fully developed and can mate and lay eggs soon afterward. Vitellogenesis doesn’t begin in monarchs until the adult stage.
The last stage of oogenesis is the formation of the egg-shell or chorion. The chorion is a protective layer produced by cells in the ovarioles. It contains small, water-repellent pores through which air is exchanged and small openings called micropyles through which the sperm will enter the egg to fertilize it. The monarch chorion is covered with vertical ridges. Oogenesis continues throughout female monarchs’ lives, so their ovarioles usually contain a series of oocytes in successive stages of development.
Fertilization
Mating monarchs can remain together for 16 hours or longer, and it is only at the very end of this period that sperm are transferred. We have studied sperm movement in female monarchs by dissecting females at different time intervals following the end of mating. Immediately after the pair separates, sperm are in the end of the spermatophore. They move out of the collum and into the sperm duct about 10 minutes later. The bundles of eupyrene sperm break apart after the sperm leave the spermatophore, an amazing process that can be observed under a microscope.
Sperm are probably propelled through the sperm duct and into the spermatheca by a combination of their own movement and muscular contractions by the female. They are stored in the spermatheca until they are released to fertilize eggs, which may occur weeks or even months after mating. Both apyrene and eupyrene sperm move into the spermatheca, even though only the eupyrene sperm can fertilize eggs.
Fertilization occurs just before an egg is about to be laid. As the egg passes down the common oviduct, a few sperm are released from the spermatheca. It’s not clear exactly how this happens, but it is likely that the spermatheca has muscles that contract and force out the sperm. The egg is oriented so that the micropyle is opposite the opening of the spermatheca. When the sperm reach the egg, they move through the micropyle and fertilization occurs. We don’t know if more than a single sperm actually go into the egg, but even if they do, only one would fertilize it and the others would break down.
Once the egg is fertilized, the new monarch starts to form and the female deposits the egg (oviposits) on an appropriate hostplant. Work by Haribal and Renwick (1995) at Cornell University has shown that flavonol glycosides in the milkweed stimulate oviposition by female monarchs. They probably detect these chemicals with taste receptors on their feet and possibly the ovipositor at the end of their abdomen. Females are probably quite choosy about the characteristics of the plants on which they lay eggs, since hostplant quality can have a big effect on larval development and survival.