DURING copulation the males of a number of insects enclose the semen

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1 Spermatophore Production in Rhodnius prolixus By K. G. DAVEY (From the Department of Zoology, University of Cambridge; present address: Department of Zoology, University of Toronto) With one plate (fig. 5) SUMMARY On the basis of experimental and histochemical evidence it has been shown that the main body of the spermatophore of Rhodnius is a mucoprotein or neutral mucopolysaccharide originating in the transparent accessory glands of the male. A study of the events during copulation demonstrated that the spermatophore is formed inside the spermatophore sac, the folded terminal portion of the intromittent organ. Normally folded inside the base of the penis, the spermatophore sac is everted by the influx of the transparent secretion and acts as a mould for the spermatophore. The solidification of the fluid, transparent secretion inside the sac is the result of a lowering in ph brought about by the secretion of the bulbus ejaculatorius. INTRODUCTION DURING copulation the males of a number of insects enclose the semen in a spermatophore, the product of the male accessory glands, before transferring the whole structure to the female. Some spermatophores have been described, and in a few cases the process of formation has been investigated, but a satisfactory explanation of the mechanism by which the fluid secretion of the accessory glands is converted to the solid material of the spermatophore has not emerged from these studies. This paper presents results which reveal the processes involved in the formation of the relatively simple spermatophore in the large blood-sucking bug, Rhodnius prolixus. RESULTS The spermatophore in Rhodnius The spermatophore of Rhodnius has been described in some detail by Khalifa (1950). Essentially it is a transparent, homogeneous, gelatinous, pear-shaped mass with a slit containing the semen in the dorsal surface of the anterior end (fig. 1). There is a slight opacity just posterior to the slit; this is the secretion of the opaque accessory glands of the male and its function is described elsewhere (Davey, 1958). In the female, the slit encloses a muscular pad on the dorsal surface of the bursa copulatrix, thereby holding the semen against the vestibulum of the oviduct. For details of the structure of the female tract the reader is referred to papers by Galliard (1935) and Khalifa (1950). The male genitalia In order to understand the events at copulation leading to the formation of the spermatophore, it is necessary to examine the structure of the external [Quarterly Journal of Microscopical Science, Vol. 100, part 2, pp , June 1959.]

2 222 Davey Spermatophore Production in Rhodnius prolixus genitalia of the male. The heteropteran intromittent organ is contained in the genital capsule (fig. 2), the floor of which is a heavily sclerotized cup variously interpreted as the remains of the ninth sternite (Snodgrass, 193^) and as the fused gonocoxopodites (Bonhag and Wick, 1953). On the dorso-lateral borders of the capsule is a pair of claspers. These are referred to as gonostyli by Bonhag and Wick (1953), but recent work by Snodgrass (1957) indicates that spermatozoa FIG. 1. A spermatophore of Rhodnius, dorsal view. they are more likely homologous with the parameres, or lobes of the ectodermal outgrowths from which the phallic organs develop. The roof of the genital capsule is formed by the anus and its associated sclerites. The terminal segments have been extended in fig. 2; normally the genital capsule lies telescoped within the horseshoe-shaped eighth sternite and the ring-shaped seventh sternite. The nomenclature of the various parts of the genitalia of insects is confused, but an attempt has been made in this study to adhere to the system of Snodgrass (1935). Potash preparations of the intromittent organ of Rhodnius are not particularly revealing and the external morphology of the genitalia can be seen in the photograph in fig. 5, A (opposite p. 225). The phallobase is a saddleshaped sclerite with two long basal apodemes as figured by Singh-Pruthi (1925) for other Reduviidae. The aedeagus appears to consist of a heavily sclerotized plate on the dorsal surface with rather more membranous ventral and lateral surfaces. A longitudinal section of the terminal segments of the male reveals a more complicated situation (fig. 3). The ejaculatory duct opens into a much-folded internal sac, the lumen of which has been stippled and which has been

3 Davey Spermatophore Production in Rhodnius prolixus 223 e/asper genital capsule terg/te W stern/te ~2K FIG. 2. The terminal segments of an adultmale Rhodnius: left lateral view with wings removed. stern/te X stern/te 21 spermatophore soc(.oedeogus+ endophallus') endophallus gen/to/ capsule ejaculatory duct s f ^tern/te M FIG. 3. Diagram of a median longitudinal section of the terminal segments of the male Rhodnius. designated as the spermatophore sac. Further, it is evident that the wall of the aedeagus is made up of two layers of cuticle bounding a blood-space and that the wall of the spermatophore sac is similarly double. It is suggested that only the outer wall of the aedeagus is in fact aedeagus and that the inner wall is the endophallus. Similarly, the wall of the spermatophore sac is composed of aedeagus on the outer side and of endophallus on the inner or lumen side.

4 224 Davey Spermatophore Production in Rhodnius prolixus A consideration of fig. 4 makes the reasons for this suggestion more clear. Fig. 4, A represents the condition found in many insects a basiphallus and tubular aedeagus with a membranous endophallus. In fig. 4, B the terminal part of the entire phallic structure has invaginated to form an internal sac, and it is suggested that this is what has happened in Rhodnius.! pholjotreme endophollus endophollus spermatophore phallotreme ej'oculoiory bosiphallus duct basiphallus ejaculotory duct FIG. 4. Diagram illustrating the development of the spermatophore sac from the generalized condition. The primitive arrangement is represented in A (after Snodgrass, 1935). B, achieved by the folding in of the terminal portion of the penis in A, is a diagram of the condition found in Rhodnius. The events at copulation In order to study the processes occurring at copulation, it was necessary to kill the mating pairs at various times after the initiation of copulation. To this end, pairs of recently fed Rhodnius were confined in glass vials in an incubator at 28 C, and the progress of mating observed through the glass front of the incubator at intervals of 3 min. When the desired stage had been reached, the vial was removed from the incubator and boiling aqueous Bouin's fluid was poured over the insects. The pair was then dissected to free the male genitalia from the female. The following description is based on material obtained in this way. During copulation the male takes up a position beside and slightly ventral to the female. Since the penis is normally directed towards the posterior, this position requires that the genitalia, including the genital capsule, be rotated through almost 180 0, as has been described in some detail for Oncopeltus (Bonhag and Wick, 1953). Within 3 min of the beginning of copulation, the aedeagus has been extended into the bursa copulatrix of the female (fig. 5, A). Later, at 6 min, the spermatophore sac becomes visible (fig. 5, B), having been everted out of the aedeagus. Eversion of the spermatophore sac continues

5 FIG. s K. G. DAVEY

6 Davey Spermatophore Production in Rhodnius prolixus 225 until, at 10 min after the beginning of copulation, it reaches the fully everted condition shown in fig. 5, c. Dissection of the spermatophore sac at this stage reveals that it contains a fully formed spermatophore. Within about 12 min the spermatophore sac has collapsed after ejecting the spermatophore (fig. 5, D). Copulation normally occupies about 30 min; the remainder of the time is spermatophore sac basiphallus FIG. 6. Drawing of a partially erect penis of Rhodnius, left lateral view. taken up with the return of the spermatophore sac to its normal position inside the aedeagus. This process has not been studied. The intromittent organ is difficult to dislodge from the female when the spermatophore sac is fully everted. This is presumably brought about by two lateral extensions of the spermatophore sac at its base, referred to here as wings (fig. 6). Since the distance between the tips of the wings is greater than the diameter of the female gonopore, the male genitalia cannot be withdrawn until the spermatophore sac has collapsed. The spermatophore sac also bears FIG. 5 (plate). Photographs of the male genitalia of Rhodnius at various times after the initiation of mating. A, 3 min after; only the aedeagus is visible. B, 6 min after; the spermatophore sac is partially everted. C, 10 min after; the sac is completely everted. D, 12 min after; the sac has collapsed and the spermatophore has been ejected. aed, aedeagus; bph, basiphallus; cl, clasper; gen, genital capsule; sp, spermatophore sac; sph, spermatophore; v, virga; VIII, eighth sternite.

226 Davey Spermatophore Production in Rhodnius prolixus three sclerotized plates, or virgae, consisting of a spade-like terminal plate and a pair of longer lateral plates (figs. 5, c; 6).

7 226 Davey Spermatophore Production in Rhodnius prolixus three sclerotized plates, or virgae, consisting of a spade-like terminal plate and a pair of longer lateral plates (figs. 5, c; 6). A median longitudinal section of the everted genitalia reveals certain details (fig. 7). The fully formed spermatophore may be seen within the spermatophore sac and this diagram shows the phallotreme, or terminal opening of the spermatophore sac through which the spermatophore is.spermotozoo spermatophore sac ^transparent accessory secretion \dophallus -.ulatory duct -blood space aedeagus 'basiphallus, bulbus 'e/acu/ator/us \ital ipsule FIG. 7. Diagram of a median longitudinal section of the terminal segments of a male Rhodnius with a fully everted spermatophore sac. finally released. The folds of the sperrriatophore sac projecting into the lumen appear to be responsible for the formation of the slit which contains the semen. Among the stains used for these sections was Mann's methyl blue / eosin. According to Pantin (1948), methyl blue takes the properties of a basic stain in the presence of eosin. In the spermatophore sac the material forming the main body of the spermatophore is strongly eosinophil (cross-hatched portion in fig. 7). In the common duct within the bulbus ejaculatorius and in the accessory gland and its duct, however, the material which is continuous with the spermatophore stains strongly with methyl blue. It is apparent that some change occurs in this material at the level of the intromittent organ. The transparent accessory glands The internal organs of reproduction of the male of Rhodnius can be seen in fig. 8. They consist on each side of a testis, a vas deferens which swells in its

8 Davey Spermatophore Production in Rhodnius prolixus 227 middle third to form the seminal vesicle, and the accessory glands with their duct, which joins the vas deferens of the corresponding side in the bulbus ejaculatorius. The two bulbi unite in the mid-line to form the ejaculatory duct opening at the base of the penis. The accessory glands are of two types: three of the four lobes on each side are filled with a transparent mucilaginous fluid; the fourth contains the opaque secretion referred to earlier. Khalifa (1950) noted that the transparent glands contain little secretion in duel or the accessory gland bulbus e/aculotorius ejaculatory duct- FIG. 8. Drawing of the left side of the male reproductive system of Rhodnius, dorsal view. starved males, which are unable to produce spermatophores, and he inferred from this that the transparent secretion made up the main body of the spermatophore. A more direct demonstration of this was obtained in the present study by mating normal females to males which had one or more lobes of the transparent accessory glands removed. Such matings resulted in the production of smaller, but otherwise normal spermatophores. Removal of the opaque glands, on the other hand, had no effect on the main body of the spermatophore. The transparent secretion resembles the material making up the main body of the spermatophore in every apparent way except fluidity. Simple histochemical tests, performed as outlined in Pearse (1953), on whole spermatophores and on whole and sectioned transparent accessory glands fixed in neutral formaldehyde solution revealed a further correspondence in properties. Both were strongly positive to Millon's test, demonstrating the presence of protein. Fat was absent from both spermatophores and accessory glands, as shown by their failure to stain with Sudan black B or Sudan red colouring agents. The presence of polysaccharide in both materials was demonstrated

9 228 Davey Spermatophore Production in Rhodnius prolixus by a very strong periodic acid / Schiff reaction. These results pointed to a protein-polysaccharide complex, and additional tests were performed with a view to characterizing the substance. Neither exhibited metachromatic staining with toluidine blue, and a methylene blue extinction test performed on spermatophores only gave a methylene blue extinction value which was indefinite, but certainly greater than 5. According to Pearse (1953), materials exhibiting these properties can be designated as mucoproteins or neutral mucopolysaccharides. At any rate, the transparent accessory secretion appears to differ from the material in the spermatophore only in consistency; the spermatophore is simply a more solid transparent secretion. The importance of ph A clue to the processes involved in the solidification of the transparent secretion to form the spermatophore was gained from a consideration of the behaviour of the freshly formed spermatophore in various environments. A spermatophore removed from a recently mated female swelled slightly in distilled water, and slowly liquefied in more basic solutions, such as tap water. In slightly acid solutions, the spermatophore remains unchanged. The effect of ph on the consistency of the transparent secretion was studied by placing a single lobe of the gland in insect Ringer which had been buffered to the desired ph with isotonic sodium hydrogen phosphate / potassium dihydrogen phosphate buffer. After 10 min the gland was dissected and the condition of the contents noted. The ph values employed lay between 5-0 and 6*8 and were separated by 0-2. Above ph 5-6 the material was quite fluid, but at 5-4 and 5-6 the secretion had gelled to form a material closely resembling that in the spermatophore. At ph 5*2, the material was again fluid. This experiment was repeated twice and a fourth trial was performed with Mcllvaine's standard buffer; identical results were obtained in each case. It is clear that the mucoprotein of the accessory gland will solidify at a ph of about 5-5 to form a material like that in the spermatophore. The ph of the reproductive ducts It is in the male system that the spermatophore material exists as both a fluid and a solid, so that the ph of the various ducts is of interest. Since the amount of fluid in these ducts is very small, the organ in question was crushed on to a tiny piece of B.D.H. indicator paper. In this way it was found that the accessory glands, testis, and ducts down to the level of the bulbus ejaculatorius were at or near neutrality, while the ph of the fluid in the lumen of the bulbus was about 5"5. Only the bursa copulatrix of the female was examined; it also was at ph 5-5. In order to confirm these observations, certain properties of methyl red were employed. In addition to its well-known action as a ph indicator with an end point near 5-5, this dye can also be used as a vital stain. A 1% solution in 0*9% NaCl was injected into the haemocoele of 10 males, which were dissected in 0^9% NaCl 5 h after injection. The ducts and organs of the

10 Davey Spermatophore Production in Rhodnius prolixus 229 reproductive system of such males were stained yellow, indicating a ph considerably above the end point, whereas the fluid in the bulbus was stained orange, indicating the end point ph of 5-5. To confirm the ph of the bursa of the female, a small crystal of methyl red was inserted into the bursa through the genital opening. After 5 h the bursa was stained with the end-point colour. The bulbus ejaculatorius It appears from the foregoing that the lowering of ph at the level of the bulbus may be responsible for the solidification of the transparent accessory outer epithelium vas deferens muscle ejaculatory duct FIG. 9. Diagram of a median longitudinal section of the bulbus ejaculatorius of the male Rhodnius. secretion to form the spermatophore. In order to test the ability of the bulbus to clot the transparent secretion, the bulbi from 20 males were macerated with forceps in 1 ml of insect Ringer previously adjusted to ph 7 with o- 2 N NaOH. Bits of the vas deferens and accessory duct from the same animals were macerated in another sample of the same Ringer. A lobe of the transparent accessory gland was placed in each suspension. After 10 min the secretion in the Ringer containing the bulbi had solidified, while the other remained fluid. Moreover, the ph of the Ringer containing the bulbi had fallen to 5*5, while that of the other suspension remained near neutrality. Because of the large numbers of insects required, this experiment could be repeated only once; on that occasion similar results were obtained. Other experiments using substantially fewer insects were unsuccessful. The anatomy of the bulbus (fig. 9) is so arranged that the products of the accessory glands are kept separate from the secretion of the bulbus up to the level of the penis. The outer wall of the bulbus is a single layer of epithelium

230 Davey Spermatophore Production in Rhodnius prolixus lined with an intima, except on the medial side, where the wall is formed b) muscle within which the combined accessory and sperm ducts run.

11 230 Davey Spermatophore Production in Rhodnius prolixus lined with an intima, except on the medial side, where the wall is formed b) muscle within which the combined accessory and sperm ducts run. Projecting from this common duct into the lumen is the inner epithelium, which presumably secretes the greenish, watery fluid that fills the lumen of the bulbus. This fluid was not fixed by any of the fixatives used, and, as we have already seen, has a ph of 5-5. The structure is, of course, repeated on the other side of the animal and the outer epithelial layers from each bulbus unite to form the short ejaculatory duct opening into the base of the penis. The common ducts from each side empty separately into the ejaculatory duct. Thus the fluid secretion from the transparent glands is kept separate from the acid secretion of the bulbus until they are allowed to mix in the penis. It is worth remembering that this is the region in which the transparent secretion becomes eosinophil in Mann's stain. CONCLUSION On the basis of these observations it is concluded that the spermatophore of Rhodnius is composed of a mucoprotein or neutral mucopolysaccharide originating in the transparent accessory glands of the male and that this secretion solidifies in the spermatophore sac of the male intromittent organ as a result of a change in ph from near neutrality to 5*5. This change in ph is brought about by the secretion of the bulbus ejaculatorius of the male. The entry of the transparent secretion into the spermatophore sac causes the eversion of that organ, which acts as a mould for the spermatophore. I am indebted to Professor Wigglesworth of the Department of Zoology at Cambridge for suggesting and supervising the problem, and for reading the manuscript. Generous financial assistance in the form of a fellowship came from the Ontario Research Foundation. REFERENCES BONHAG, P. F., and J. R. WICK, 'The functional anatomy of the male and female reproductive systems of the milkweed bug.' J. Morph., 93, 171. DAVEY, K. G., 'The migration of spermatozoa in the female of Rhodnius prolixus.' J. exp. Biol., in the press. GALLIARD, H., Recherches biologiques et morphologiques sur la reproduction des rdduviides hematophages {Rhodnius et Triatoma). These Fac. Sci. Univ. Paris. KHALIFA, A., 'Spermatophore production and egg-laying behaviour in Rhodnius prolixus.' Parasitology, 40, 283. PANTIN, C. F. A., Notes on microscopical technique for zoologists. Cambridge (University Press). PEARSE, A. G. E., Histochemistry, theoretical and applied. London (Churchill). SINGH-PRUTHI, H., 'Morphology of the male genitalia in Rhynchota.' Trans, ent. Soc. Lond., 1925, 127. SNODGRASS, R. E., Principles of insect morphology. New York (McGraw-Hill). I 957' 'A revised interpretation of the external reproductive organs of male insects.' Smithsonian misc. Coll., 135, 66.

THE MIGRATION OF SPERMATOZOA IN THE FEMALE OF RHODNIUS PROLIXUS STAL.

694 THE MIGRATION OF SPERMATOZOA IN THE FEMALE OF RHODNIUS PROLIXUS STAL. BY K. G. DAVEY Department of Zoology, University of Cambridge (Received 29 March 1958) INTRODUCTION In Rhodnius, as in many insects,