William G. Eberhard and Flory Pereira.
The process of intromission in the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidiae).
Psyche 102:99-120, 1995.

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PSYCHE
Vol. 102 1995 NO. 3-4
THE PROCESS OF INTROMISSION IN THE
MEDITERRANEAN FRUIT FLY, CERATITIS CAPITATA (DIPTERA: TEPHRITIDIAE)
The distiphallus of the male of Ceratitis capitata is folded back 180å onto the basiphallus during the early stages of intromission, and is then unfolded within the female. Repeated folding and unfolding may occur within the female. Two membranous sacs on the distiphallus are capable of rhythmic cycles of inflation and deflation. Inflations of the sac near the base of the distiphallus probably help propel the aedeagus deeper into the female, along with periodic stiffening of the basiphallus; inflation of the larger, distal sac may drive the genital rod (which does not transfer sperm) into the ventral receptacle.
Millions of dollars are spent annually in attempts to control the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), but sur- prisingly little is known regarding many aspects of its basic repro- ductive biology. Male courtship behavior prior to mounting has been studied in detail (Feron, 1962; Prokopy and Hendrichs, 1979; Arita and Kaneshiro, 1985; Whittier, Kaneshiro, and Prescott, 1992; Bricefio, Ramos, and Eberhard, in press), but the process of 'smithsonian Tropical Research Institute, and Escuela de Biologia, Universidad de Costa Rica, Ciudad Universitaria, Costa Rica 2~niversidad de Costa Rica, Sede del Atlhntico, Tun-ialba, Costa Rica Manuscript received 2 November 1995.




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100 Psyche [vo~. 102
intromission has received little attention. Intromission would appear to be a relatively difficult task in these flies, because of the female's long, thin, folded vagina, one of the morphological char- acteristics that permit insertion of the ovipositor (sensu Norrbom and Kim, 1988) deep in to plants. The male intromittent genitalia, or aedeagus, consist of a long, thin, flexible, more or less tubular basiphallus (about 40% of the male's body length), with a smaller, morphologically complex distiphallus at its tip. The process that allows such a long flexible structure to be threaded through the female's cloaca and vagina to her bursa remains unknown. The pioneer work on genital structure and function in C. capi- tata is the detailed paper of Hanna (1938), and many statements in later publications (e.g., Feron, 1962) are apparently based on this account. Unfortunately, as noted by Griffiths (1972), it is often unclear which of Hanna's statements were based on direct observa- tions, and which were only speculations. Some of his statements are clearly wrong and, as will be shown below, he clearly did not observe portions of the processes he reported. It is thus difficult to interpret Hanna's descriptions without further work. There are several more recent studies of genitalic functional morphology. Monguira, Salom, and Muiioz (1983) and Valdez-Car- rasco and Prado-Beltran (1990) gave descriptions of the female reproductive anatomy of C. capitata, and Camacho (1991) described details of the morphology of the spermatheca and the spermathecal duct near the spermatheca. Solinas and Nuzzaci (1984) presented a detailed account in another tephritid, Bactro- cera (=Dams) oleae, of the functional anatomy of female repro- ductive structures and their mesh with male genitalia after intromission was complete. The genitalic surstyli of male C. capi- tata were found to remain outside the female's body, functioning as clamps with which the male seizes and holds the aculeus of the female in a strong grip (Eberhard and Pereira, 1993). The observa- tions reported here concern the portions of the male's genitalia that enter the female, and the roles they may play in the process of intromission. Sperm transfer will be described elsewhere. All flies were obtained from mass rearing facilities in San Jose, Costa Rica. The flies were quite tame, and pairs early in copulation



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were gently moved from a common cage into individual containers with little or no apparent disturbance. Copulating pairs were immobilized nearly instantaneously with a freezing spray (ethyl chloride) and then fixed in 70% ethyl alcohol. Whole mount prepa- rations of the genital tracts of coupled pairs were examined with the light microscope. Some specimens were soaked briefly in lac- tophenol before dissection. By sacrificing pairs after known peri- ods of genitalic contact, it was possible to deduce the order of some events during copulation. Specimens were prepared for scan- ning microscopy and sectioning using standard procedures. The genitalia of some males were pulled forcefully from the female during copulation and immediately fixed in 70% ethanol. Direct observations of the movements of male genitalia of living flies were made under a dissecting microscope by teasing the aedeagus from its resting place on the abdomen of the male after he had been decapitated to eliminate central control and mounted on a pin. Video recordings of behavior during copulation were made using a National Novicom camera (30 imageslsec) with +6 lenses.
A. Basic morphology and movements
The following descriptions emphasize only those parts of male and female morphology that are critical in the process of intromis- sion. Names for external structures are those of Norrbom and Kim (1988).
I. Female
The apical portion of the ovipositor, the aculeus, is a long, curved, apically pointed plate, that extends posteriorly slightly beyond a pair of slightly shorter, smaller ventral plates (8th ster- nites). The cloaca is located just anterior to the posterior edges of the sternites, and thus lies slightly farther anterior to the posterior tip of the aculeus. There are muscles joining the basal but not the apical portions of the 8th sternites to the aculeus (Valdez-Carrasco and Prado-Beltran 1990), so the female apparently cannot squeeze shut her cloaca1 opening.
Moving anteriorly, the tubular eversible membrane is joined at its posterior margin with the anterior end of the aculeus, and at its



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102 Psyche [vo~. 102
anterior margin with the posterior edge of the conical syntergoster- nite 7. When at rest, the ovipositor telescopes, and the aculeus and the eversible membrane lie largely or completely within the synter- gosternite 7. Just prior to intromission, the female extends the ovipositor so that the apical portion of the aculeus is exposed, and the male clamps the tip of the aculeus with his surstyli (Eberhard and Pereira, 1993).
Internally, the posterior portion of the vagina loops back on itself when the ovipositor is at rest, and straightens when the ovipositor is extended. A pair of more or less plate-like sclerotized structures (the "stigma" - Fig. 1) are apparently embedded in the wall of the vagina near the posterior end of a small expansion at the anterior end of the vagina (the bursa, or the "insemination pocket" of Solinas and Nuzzaci, 1984) (Figs. 1, 12). Projecting anteriorly from the upper end of the bursa is the ventral receptacle ("fertilization chamber" of Solinas and Nuzzaci, 1984), a smaller, blind-ended, cylindrical structure whose inner-most portion is expanded and covered with smooth, rounded projections (Figs. 1, 2). At the tip of the ventral receptacle is a small, heavily sclero- tized extension about the size of a single rounded projection (Fig. 2). -
SCLEROTIZED
POLYP
SPERM RECEPTACLE
LOBE CENTRAL
APICAL
SCLEROTIZED AREA LOBE.
Fig. 1. Diagrammatic cutaway view of positions of male genitalic structures in the female bursa when the male genital rod is inserted in the ventral receptacle of the female (see also Fig. 12).




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The wall of the bursa is more sclerotized in the area surrounding the entrance to the ventral receptacle, and has a truncated conical shape (the "receptacle cone" - Figs. 1, 2). The two spermathecal
ducts originate near the edge of the receptacle cone. They are long and thin-walled, except at their distal ends where there is a short Fig. 2. The ventral receptacle of a female and the sclerotized portion of the vagi- nal wall (receptacle cone) near its opening, into which projects the tip of the male's genilal rod. There are many finger-like projections of the distal portion of the recep- tacle, and a single sclerotized projection at the tip.



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section of strong muscles near the entrance of each teardrop- shaped, highly sclerotized spermatheca (Monguira et al., 1983; Camacho, 199 1).
11. Male
Tergite 9 (epiandrium) of the male abdomen is U-shaped, and ventrally forms two pairs of closely associated surstyli that clasp the aculeus when copulation begins. The long aedeagus (about 40% as long as the male's total length) arises from the ventral sur- face of the abdomen just anterior to the surstyli. The complex dis- tiphallus comprises the tip of the aedeagus; the rest of the aedeagus is the more or less cylindrical basiphallus. Nearly all of the dorsal side of the basiphallus (posterior side of Hanna, 1938) is strengthened by a pair of chitinous strips, while the ventral side is a stiff membrane, with a series of transverse folds (e.g., Figs. 1, 10). At rest the basiphallus curls spontaneously toward its membra- nous side, and is kept between folds of the intersegmental mem- brane between abdominal tergite 5 and the postabdomen (segments 6-8). When the aedeagus of a live decapitated male was teased free, the basiphallus repeatedly straightened partially in brief rhythmic movements about twicelsec.; extensions occurred in syn- chrony with inflations of the basal sac of the distiphallus (below). The ejaculatory apodeme and sperm sac ("erecting and pumping organ" of Hanna, 1938) may produce increases in fluid pressure within the aedeagus that result in this and other movements of the aedeagus.
The distiphallus at the tip of the aedeagus is complex. At its basal end is a dorsal, basal lobe or eversible sac, with small, coni- cal, scale-like teeth covering part of its surface (Figs. 3, 8) (the "oval bladder" of Hanna, 1938). When the distiphallus was touched persistently with the tip of a pin, the basal sac repeatedly expanded and collapsed. Typical frequencies averaged about two expansionslsec, but there was much variation. As the sac was everted, the direction it projected changed, causing it to move rear- ward, from a distal to a basal orientation (Fig. 5). Some eversions were "double," with only a partial collapse followed immediately by a second eversion. Eversion of the basal sac was always accom- panied by a ventral flexion of the rest of the distiphallus in the region near the base of the sac (upper arrow in Fig. 5).



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Fig. 3. The distiphailus of a male Ceratitis capitata. Distal to the basal sac is the exceedingly complex central por- don of the distiphallus (Fig. 3), covered by a relatively smooth, featureless membrane (Fig. 4). A single rod-like sclerotized struc- ture (the "genital rod" of Hanna, 1938) extends dorsally from the central sclerotized area (Figs. 1, 3, 4). The tip of the rod flares, somewhat like the bell of a trumpet, and is more flexible than the rest of the rod (compare Figs. 1 4 and 13). The rod is apparently hollow (Fig. 3, and its tip is covered with a featureless membrane (Fig. 6).
Near the tip of the distiphallus is a large distal lobe or sac (the "toothed bladder" of Hanna, 1938) (Figs. 3, 4). The conical teeth that are especially prominent on its ventral and lateral surfaces are similar to but generally larger than those on the basal sac. The dis- tal sac was always partially inflated both in slide preparations of copulating pairs and in living males (contrary to the description of Hanna, 1938). In living flies the sac repeatedly inflated and



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Fig. 4. Scanning electron micrograph of the distiphallus of a male Ceratitis cap- titata.
deflated. Maximum inflation increased the diameter an estimated 50-100% over that when the sac was minimally inflated. Inflation of the distal sac caused it to expand mostly in the ven- tral direction (away from the central portion of the distiphallus) rather than basally or distally. Inflation of the distal sac was not synchronous or coordinated in any obvious way with that of the basal sac. The two sacs are apparently hydraulically independent. When pricked with a pin, the distal sac of a living male deflated



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Fig. 5. Diagrammatic representation of the movements of the basal sac of the dis- tiphallus when it inflates (dotted lines). As the sac inflates the distiphallus moves in the opposite direction (arrow).
immediately and permanently (and much more completely than in any other preparation), and released a drop of fluid. The basal sac continued to inflate and deflate as before. No obvious openings through which sperm could emerge from the distiphallus were seen in either light microscope or SEM preparations. There were sperm on the surface of the distiphallus in two SEM preparations of male genitalia that had been pulled from females after about 30 min of copulation. In both cases the sperm were in the same area, near the base of the genital rod, where multiple fine folds (Figs. 6, 7) may have concealed open- ing(~) from which sperm could emerge.
No structures were observed that even remotely resembled sev- eral of those described by Hanna (1938), including the heavily sclerotized ductus ejaculatorius, the valve, the large triangular sclerite to keep the genital rod in place, and the relieving bladder.



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Fig. 6. Scanning ctcctmn micrograph of the lip nf the genilai rod, showing the membrane covering its tip. The arrow indicates the area where sperm may emerge (see Fig. 7).
Fig. 7. Scanning electron micrograph of the distiphallus near the base of the gen- ita! rod, showing possible apertures through which sperm may emerge.



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B. Behavior of genitalia during intromission Following elaborate courtship behavior (e.g., Feron, 1962; Bricefio et al., in press), the male mounted the female, and in suc- cessful pairings seized the female aculeus with his surstyli when she extended it (Eberhard and Pereira, 1993; Headrick and Goeden, 1994). In two pairs observed dorsally under a dissecting microscope at the moment the exposed aculeus was clamped, the male's dis- tiphallus was in clear view as the female's ovipositor was extended. The distiphallus quickly flipped ventrally, where it was lost from sight (Eberhard and Pereira, 1993). Presumably it was positioned near the tip of the aculeus, as it was in this position in three of the four males frozen 0.5-2.0 min after pairing had begun and in which the male separated from the female after being placed in alcohol, indicating that intromission had not yet occurred (Fig. 8). Typically the male then appeared to pull posteriorly repeatedly, so that the female's ovipositor was further extended. Soon after achieving intromission the female's ovipositor was extended until the eversible membrane had entirely emerged from syntergosternite 7 and the aculeus had nearly completely emerged from the eversi- ble membrane. Probably this extension caused the female's nor- mally S-shaped vagina to straighten, and may have facilitated the movement of the male's aedeagus to her bursa. The male also made repeated pushing or thrusting movements with his abdomen that caused the female ovipositor to fold or buckle briefly. The tibiae of the male's middle and hind legs were often crossed and pressed against the ventral surface of the syntergosternite during these movements.
An alternative interpretation, that the female actively extends her ovipositor rather than having it pulled by the male, cannot be discarded with complete confidence, but pulling seems more likely for two reasons. Observations under a dissecting microscope showed that in some cases the anterior portion of the female's abdomen was also extended to about twice its resting length, implying that the male had pulled forcefully. In addition, when a male mounted a female but failed to clamp her aculeus, he often performed repeated "pulling" movements by raising the tip of his abdomen and moving it rearward (see also Eberhard and Pereira, 1993).
Eventually (after about 1 - 10 min) the aculeus moved anteriorly and was partially engulfed by the tubular eversible membrane; the



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Fig. 8. Posterior portion of the abdomen of a male which was frozen 30 sec after he established genital contact with a female. The male fell free of the female, indi- cating that he had not introduced his aedeagus in the female's vagina or had achieved only very shallow intromission. His basiphallus was sharply bent, and the distiphallus (not folded back on the basiphallus-compare with Fig. 10) was posi- tioned between the surstyli, near the site of the genital opening of the female when her aculeus was held by the surstyli.
eversible membrane remained completely outside syntergosternite 7. This was the position during most of copulation. The male often made long series of "thrusting" movements of various rhythms and magnitudes (Fig. 9). With each "thrust" the female aculeus moved anteriorly, deeper into the tubular eversible membrane. In at least some pairs it appeared (from a slight increase in the degree of extension of the male's abdomen as the aculeus returned to its



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-2. -. I 1 L - - L .
LARGE EXTENSION OF FEMALFS OVIPOSTOR
10 20 30 4.0 ' 50
TIME WtN>
Fig. 9. Male behavior duriug a copulation lasting 54 min. The widths of the bars indicate the duration of different activities. original position) that the "thrusts" were actually brief, rhythmic pulls on the aculeus, and that after each pull the aculeus was retracted by the female(?) back into her body. Occasionally the male pulled forcefully and extended her ovipositor briefly (Fig. 9). The internal events associated with intromission were deduced from 33 different pairs frozen at different times after original geni- talic contact in which the aedeagus was at least partially inserted in the female's vagina: 2 after 30 sec; 1 after 1 min; 8 after 2 min; 8 after 3 min; 6 after 5 min; and 8 after 10 min. Penetration did not follow a strict timetable. The genitalia of three males frozen after 2 min had reached the inner end of the bursa, while those of another male were still short of the stigma after ten min. In eight cases the distiphallus was folded back on the basiphallus (Fig. 10). In three of the pairs with the distiphallus folded back the male's genitalia were still substantially short of the stigma? while in three others they had passed the stigma, and were within the bursa. In one case the basiphallus was short of the stigma and apparently in the process of unfolding (Fig. 11). In all eight cases in which the male genitalia was not folded back and had not entered the bursa, the distal sac of the distiphal- lus was flexed anteriorly, so that it was the leading portion of the male's genitalia (Fig. 12, Left).




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In all 10 cases in which the male's distiphallus was not folded back and had not passed the stigma, the genital rod was pressed against the side of the central portion of the distiphallus (as in Fig. 12, Left). In contrast, in 12 of the 16 cases in which the male's genitalia had entered the bursa, the genital rod was flexed at least 30å from the axis of the central portion (Fig. 12, Right); in 3 of these pairs its tip was within the ventral receptacle (Fig. 1). The soft tip of the genital rod apparently folded back on itself as the rod entered the receptacle (Fig. 13).




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Fig. 10, Wholemount of the aedeagus of a male in the vagina of a female posfe- rior 10 the stigma after 2 min of genital contact. The male's distiphallus ks folded back 180' to lie dong the terminai portion of the basiphahs.



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Fig. 11. Wholemount of male genitalia in the vagina posterior to the stigma after 2 mh of copulation. The distiphallus appears to be in the process of unfolding. Fig. 12. Positions of he male aedeagus in the female's vagina. Left. The dis- tiphailus is just passing the stigma, with the genital rod pressed against the central portion of the distiphallas, and the distal sac positioned so that it penetrates the female most deeply. Right. The distiphallus is deeper in the female, at the anterior end of the bursa. The tip of the genital rod is within the receptacle cone, and the distal sac presses against the opposite wall.



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[Vol. 102
SPERM
ROD
RECEPTACLE CONE
Fig. 13. Diagrammatic view of the male genital rod inserted into the female's ventral receptacle. Note the distortion of the tip of the rod (compare with Figs. 1-4).
A tentative sequence of the events in a successful intromission can be deduced from these observations and previous publications. The male first clamps the female aculeus with his surstyli. The male's median prensisetae are inserted between the lateral edges of the female's aculeus and her 8th sternites (Eberhard and Pereira, 1993), probably thereby lifting the distal portions of the sternites from the aculeus and exposing the cloacal opening for insertion of the aedeagus. The male also apparently extends the female's ovipositor by pulling the aculeus posteriorly. While the female's ovipositor is extended, the male flips his aedeagus ventrally. The basiphallus is folded sharply (Fig. 8), perhaps by catching the dis- tiphallus between his surstyli or on the aculeus. Probably the next step is the insertion of the folded distiphallus and the tip of the basiphallus into the female's cloacal opening, but the details of this process are not clear.
The distiphallus is susequently unfolded after entering the female. Unfolding occurs when the male genitalia have still not reached the bursa, and it is possible that there are several cycles of folding and unfolding as the male's genitalia move deeper into the female. Unfolding may be accompanied by the male pulling his



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basiphallus posteriorly, and refolding may occur as he thrusts it anteriorly. The genital rod is pressed against the central portion of the distiphallus, and the distal sac is oriented directly anteriorly in the female. The movement of the male's genitalia deeper into the female is probably brought about by a combination of periodic