Experimental demonstration of possible cryptic female choice on male tsetse fly genitalia

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Abstract

A possible explanation for one of the most general trends in animal evolution – rapid divergent evolution of animal genitalia – is that male genitalia are used as courtship devices that influence cryptic female choice. But experimental demonstrations of stimulatory effects of male genitalia on female reproductive processes have generally been lacking. Previous studies of female reproductive physiology in the tsetse fly Glossina morsitans suggested that stimulation during copulation triggers ovulation and resistance to remating. In this study we altered the form of two male genital structures that squeeze the female's abdomen rhythmically in G. morsitans centralis and induced, as predicted, cryptic female choice against the male: sperm storage decreased, while female remating increased. Further experiments in which we altered the female sensory abilities at the site contacted by these male structures during copulation, and severely altered or eliminated the stimuli the male received from this portion of his genitalia, suggested that the effects of genital alteration on sperm storage were due to changes in tactile stimuli received by the female, rather than altered male behavior. These data support the hypothesis that sexual selection by cryptic female choice has been responsible for the rapid divergent evolution of male genitalia in Glossina; limitations of this support are discussed. It appears that a complex combination of stimuli trigger female ovulation, sperm storage, and remating, and different stimuli affect different processes in G. morsitans, and that the same processes are controlled differently in G. pallidipes. This puzzling diversity in female triggering mechanisms may be due to the action of sexual selection.

Introduction

One of the most sweeping of all evolutionary patterns in animal morphology is for male genitalia to diverge especially rapidly compared with other body parts (Eberhard, 1985, Eberhard, in press, Hosken and Stockley, 2003). One hypothesis to explain this pattern is that male genitalia function as courtship devices, and diverge rapidly because they are under sexual selection by cryptic female choice (Eberhard, 1985). Sexual selection by cryptic female choice (CFC) can occur when the females of a species modulate reproductive processes under their control that occur after copulation has begun, and thus favor the paternity of males that have certain traits (such as a particular genital morphology) over that of others (Thornhill, 1983, Eberhard, 1996). The female could gain from biasing paternity by producing sons whose genitalia are better able to induce such female responses. An alternative hypothesis to explain this divergence is that male–male competition to manipulate female reproductive processes results in male-imposed damage to the female's reproduction, and that selection on females to avoid this damage results in females and males being engaged in sexually antagonistic coevolution (SAC) (Arnqvist and Rowe, 2005).

Experimental modification of the male's genitalia in the tsetse fly Glossina pallidipes, and of the receptors in the portions of the female that they contact during copulation showed that stimuli from two male genital structures trigger three different female reproductive processes that could result in cryptic female choice: ovulation; sperm storage; and female avoidance of remating (Briceño and Eberhard, 2009). The present study describes the results of a complementary set of experimental alterations of male genital form and of corresponding female receptors in a second species of tsetse fly in the same subgenus, G. morsitans centralis. These modifications included removal of a derived male genital structure, the median cercal hook, which is present only in G. morsitans and its sister species G. submorsitans.

Glossina morsitans is widely though somewhat patchily distributed in Africa, where it is an important vector of trypanosomiasis in humans and domestic animals. It shows clinal variation, differences among different geographic populations, and gene flow between such populations; different forms have been variously recognized as species, subspecies, and races of subspecies (Buxton, 1955, Gooding and Krafsur, 2005). Copulation in G. morsitans lasts about 45–120 min (Saunders and Dodd, 1972, Wall and Langley, 1993). A spermatophore is transferred toward the end of copulation (almost never before 45 min) (Saunders and Dodd, 1972). The mouth of the spermatophore is placed in the mouth of the spermathecal duct in Glossina, which is distant from the spermathecae (Buxton, 1955, Pollock, 1974). Transfer of a spermatophore may not always associated with transfer of sperm to the spermatheace, as some discarded spermatophores of G. austeni contained “considerable quantities” of sperm (Pollock, 1970). Only a single egg is ovulated in each reproductive cycle, and ovulation of the female's first egg is triggered by her first copulation. The egg is fertilized in the female's uterus, where the larva hatches and feeds and develops, leaving only when it is mature and ready to pupate (Newstead et al., 1924).

Previous experiments concerning induction of ovulation employed interrupted copulations, copulations with and without spermatophore transfer, insertion of glass beads into the uterus, haemolymph transfusions from mated females, copulations with males rendered aspermic by either repeated previous copulations and or severed ejaculatory ducts, males with modified genitalia, implants of male fat body, testes, ejaculatory ducts, and accessory glands, and implantations of full and empty spermathecae from other females (Saunders and Dodd, 1972, Dodd, 1973, Chaudhury and Dhadialla, 1976, Gillott and Langley, 1981). They showed that the stimuli which induce ovulation in G. morsitans are not chemical. Ovulation was not triggered by transfer of sperm, deposition of the spermatophore in the female, male fat body, secretions of the male's testes, accessory glands or ejaculatory ducts, or from humeral factors from spermathecae of inseminated females (Saunders and Dodd, 1972, Gillott and Langley, 1981). Instead, mechanical stimulation received during copulation seemed to induce ovulation, with the effects accumulating gradually during copulation (Saunders and Dodd, 1972). The nature of these mechanical stimuli was not determined. Artificial stimulation of the uterus with a glass bead increased ovulation, but not as much as natural copulation (Chaudhury and Dhadialla, 1976).

A second response of female G. morsitans to copulation is a diminished receptivity to additional mating attempts by males. Undetermined mechanical stimuli during copulation (as well as male accessory gland substances and distension of the uterus) also trigger this female response (Gillott and Langley, 1981). Still another possible female response to copulation is transfer of sperm to the spermathecae, as suggested by evidence from G. pallidipes; modification of female ability to sense male genital structures resulted in reduced sperm storage in the spermathecae (Briceño and Eberhard, 2009). Both ovulation and sperm transfer to the spermathecae sometimes fail to occur in otherwise apparently normal copulations of G. morsitans (Buxton, 1955, Saunders and Dodd, 1972). There are also intimations that female G. morsitans affect sperm transfer to the spermathecae; when Saunders and Dodd (1972) interrupted copulations after 2 h, 19 of 26 females entirely lacked sperm in their spermathecae, while only 1 of 19 pairings that separated spontaneously in the same period (1–2 h after initiation) failed to result in insemination (Chi2 = 20.4, p < 0.001).

Numerous stimuli associated with copulation could induce these female responses. Males of G. morsitans perform energetic and sustained courtship behavior during copulation (Wall and Langley, 1993), and males also squeeze the female with vigorous, rhythmic, sustained movements of their genitalia; the temporal pattern of genital squeezing differs from that in G. pallidipes (Briceño and Eberhard, unpub.), as would be expected if genital squeezing is under sexual selection. Several portions of the male's genitalia that contact the female have morphological modifications that appear designed to stimulate the female, including the cerci, the surstyli, the inferior claspers, and the abdominal sternite 5 (Briceño et al., 2007; Briceño and Eberhard, unpub.).

In nature Glossina copulate near feeding sites (large mammals) (Wall and Langley, 1993). Field data are not sufficient to determine whether female G. morsitans mate more than once during a normal lifetime in the field, but they do remate in captivity (Gillott and Langley, 1981; below). Flash-freezing of copulating pairs as well as direct behavioral observations show that the male genitalia of G. morsitans perform the same two basic mechanical functions (in addition to possible stimulation) that have been documented in G. pallidipes: one set of structures squeezes the external surface of the tip of the female's abdomen in a powerful grip; a second set is introduced deep into the female's vagina (VanderPlank, 1948; Briceño and Eberhard, unpub.). The present study concerns the structures that squeeze the male's cerci (Fig. 1, Fig. 2), whose distal margins press powerfully against the featureless membrane on the ventral surface of the female's abdomen; and his highly modified, sexually dimorphic sternite 5 (Fig. 1, Fig. 3), whose dense covering of stout setae (the “hectors” of older publications—Buxton, 1955) is pressed against the posterior dorsal surface of her tergite 6 by the squeezing action of his cerci. The male's cerci rhythmically squeeze the female during much of the copulation (Briceño and Eberhard, unpub.). The substantial force exerted by cercal squeezing causes the ventral wall of the female's abdomen to bend inward so sharply and deeply that the entire cercus is generally hidden from view (VanderPlank, 1948, Briceño et al., 2007).

The male cerci of G. morsitans are plate-like structures joined medially by a membrane, with strong setae along their distal margins (Fig. 1a). Each cercus has a sharp hook-like, laterally directed projection near its distal median corner (Fig. 1, Fig. 2). This structure (the “median cercal hook” hereafter) has small setae on its base, but lacks setae distally. This hook is an apparently derived structure within the genus Glossina, and is present only in the sister species G. morsitans and G. submorsitans (Fig. 2). The cerci of G. morsitans apparently articulate against each other near their tips, and are moved by muscles connecting their bases (Eberhard and Briceño, unpub.).

Section snippets

Flies

All flies were 10–12-day-old virgins of a mass reared stock at the FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, Austria, which was founded at least 10 years previously with specimens collected in Tanzania. All experimental flies were kept at 23.5 ± 24 °C and 75 ± 78% relative humidity, with lights on at 08:00 and off at 16:00, and were offered a blood meal of frozen and thawed bovine blood through a silicone membrane three times per week throughout the experiments. Copulations

Results

Most results are summarized in Table 1. They will be discussed separately for each experiment.

Effects of median cercal hooks

Removal of the median cercal hooks in male G. morsitans centralis resulted in an increase in female receptivity to subsequent mating, and a reduction in two variables associated with female sperm storage: a decrease in the frequency with which sperm were present in the spermathecae decreased; and a decrease in the degree of filling of the spermathecae in those females that had sperm. The frequency with which sperm were stored was also reduced in a sensory blinding experiment in which the area

Acknowledgements

We thank the International Atomic Energy Agency for the use of flies and facilities, Andrew Parker and Marc Vreysen for logistic support, Rudolf Boigner and Carmen Marin for help rearing flies, Mary Jane West-Eberhard for comments on the manuscript, and the International Atomic Energy Agency, the Smithsonian Tropical Research Institute, and the Universidad de Costa Rica for financial support.

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