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 themain body of the spermatophore of Rhodnius is a mucoprotein or neutral mucopoly-saccharide originating in the transparent accessory glands of the male. A study of theevents during copulation demonstrated that the spermatophore is formed inside thespermatophore sac, the folded terminal portion of the intromittent organ. Normallyfolded inside the base of the penis, the spermatophore sac is everted by the influx ofthe transparent secretion and acts as a mould for the spermatophore. The solidificationof the fluid, transparent secretion inside the sac is the result of a lowering in pH broughtabout by the secretion of the bulbus ejaculatorius.

INTRODUCTION

DURING copulation the males of a number of insects enclose the semenin a spermatophore, the product of the male accessory glands, before

transferring the whole structure to the female. Some spermatophores havebeen described, and in a few cases the process of formation has been investi-gated, but a satisfactory explanation of the mechanism by which the fluidsecretion of the accessory glands is converted to the solid material of thespermatophore has not emerged from these studies. This paper presentsresults which reveal the processes involved in the formation of the relativelysimple spermatophore in the large blood-sucking bug, Rhodnius prolixus.

RESULTSThe spermatophore in Rhodnius

The spermatophore of Rhodnius has been described in some detail byKhalifa (1950). Essentially it is a transparent, homogeneous, gelatinous,pear-shaped mass with a slit containing the semen in the dorsal surface of theanterior end (fig. 1). There is a slight opacity just posterior to the slit; this isthe secretion of the opaque accessory glands of the male and its function isdescribed elsewhere (Davey, 1958). In the female, the slit encloses a muscularpad on the dorsal surface of the bursa copulatrix, thereby holding the semenagainst the vestibulum of the oviduct. For details of the structure of the femaletract 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 ofthe spermatophore, it is necessary to examine the structure of the external[Quarterly Journal of Microscopical Science, Vol. 100, part 2, pp. 221-230, June 1959.]

222 Davey—Spermatophore Production in Rhodnius prolixus

genitalia of the male. The heteropteran intromittent organ is contained in thegenital capsule (fig. 2), the floor of which is a heavily sclerotized cup variouslyinterpreted as the remains of the ninth sternite (Snodgrass, 193^) and as thefused gonocoxopodites (Bonhag and Wick, 1953). On the dorso-lateral bordersof the capsule is a pair of claspers. These are referred to as gonostyli byBonhag 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 ecto-dermal outgrowths from which the phallic organs develop. The roof of thegenital capsule is formed by the anus and its associated sclerites. The terminalsegments have been extended in fig. 2; normally the genital capsule liestelescoped within the horseshoe-shaped eighth sternite and the ring-shapedseventh 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 Snod-grass (1935). Potash preparations of the intromittent organ of Rhodnius are notparticularly revealing and the external morphology of the genitalia can beseen in the photograph in fig. 5, A (opposite p. 225). The phallobase is a saddle-shaped sclerite with two long basal apodemes as figured by Singh-Pruthi(1925) for other Reduviidae. The aedeagus appears to consist of a heavilysclerotized plate on the dorsal surface with rather more membranous ventraland lateral surfaces.

A longitudinal section of the terminal segments of the male reveals a morecomplicated situation (fig. 3). The ejaculatory duct opens into a much-foldedinternal sac, the lumen of which has been stippled and which has been

Davey—Spermatophore Production in Rhodnius prolixus 223

e/asper

genital capsule

FIG. 2. The terminal segments of an adultmale Rhodnius:left lateral view with wings removed.

terg/te W stern/te ~2K

spermatophore sOc(.oedeogus+endophallus')

endophallusstern/te X

stern/te 21

gen/to/capsule

ejaculatoryduct s ™ f

^tern/te M

FIG. 3. Diagram of a median longitudinal section of the terminal segments of the maleRhodnius.

designated as the spermatophore sac. Further, it is evident that the wall of theaedeagus is made up of two layers of cuticle bounding a blood-space and thatthe wall of the spermatophore sac is similarly double. It is suggested that onlythe outer wall of the aedeagus is in fact aedeagus and that the inner wall is theendophallus. Similarly, the wall of the spermatophore sac is composed ofaedeagus on the outer side and of endophallus on the inner or lumen side.

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 andtubular aedeagus with a membranous endophallus. In fig. 4, B the terminalpart 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

ej'oculoiory duct

•bosiphallus

endophollus

spermatophore

•phallotreme

•basiphallus

ejaculotory duct

FIG. 4. Diagram illustrating the development of the spermatophore sac from the generalizedcondition. The primitive arrangement is represented in A (after Snodgrass, 1935). B, achievedby 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 tokill the mating pairs at various times after the initiation of copulation. To thisend, pairs of recently fed Rhodnius were confined in glass vials in an incubatorat 28° C, and the progress of mating observed through the glass front of theincubator at intervals of 3 min. When the desired stage had been reached, thevial was removed from the incubator and boiling aqueous Bouin's fluid waspoured over the insects. The pair was then dissected to free the male genitaliafrom the female. The following description is based on material obtained inthis way.

During copulation the male takes up a position beside and slightly ventralto the female. Since the penis is normally directed towards the posterior, thisposition requires that the genitalia, including the genital capsule, be rotatedthrough almost 1800, as has been described in some detail for Oncopeltus(Bonhag and Wick, 1953). Within 3 min of the beginning of copulation, theaedeagus 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 beeneverted out of the aedeagus. Eversion of the spermatophore sac continues

FIG. s

K. G. DAVEY

Davey—Spermatophore Production in Rhodnius prolixus 225

until, at 10 min after the beginning of copulation, it reaches the fully evertedcondition shown in fig. 5, c. Dissection of the spermatophore sac at this stagereveals that it contains a fully formed spermatophore. Within about 12 minthe 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 positioninside the aedeagus. This process has not been studied.

The intromittent organ is difficult to dislodge from the female when thespermatophore sac is fully everted. This is presumably brought about by twolateral extensions of the spermatophore sac at its base, referred to here aswings (fig. 6). Since the distance between the tips of the wings is greater thanthe diameter of the female gonopore, the male genitalia cannot be withdrawnuntil the spermatophore sac has collapsed. The spermatophore sac also bears

FIG. 5 (plate). Photographs of the male genitalia of Rhodnius at various times after theinitiation 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 anda pair of longer lateral plates (figs. 5, c; 6).

A median longitudinal section of the everted genitalia reveals certaindetails (fig. 7). The fully formed spermatophore may be seen within thespermatophore sac and this diagram shows the phallotreme, or terminalopening of the spermatophore sac through which the spermatophore is

.spermotozoospermatophore sac

^transparent accessorysecretion

\dophallus-.ulatory duct-blood space

aedeagus'basiphallus

, bulbus'e/acu/ator/us

\italipsule

FIG. 7. Diagram of a median longitudinal section of the terminal segments of a male Rhodniuswith a fully everted spermatophore sac.

finally released. The folds of the sperrriatophore sac projecting into the lumenappear 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 stainin the presence of eosin. In the spermatophore sac the material forming themain body of the spermatophore is strongly eosinophil (cross-hatched portionin fig. 7). In the common duct within the bulbus ejaculatorius and in theaccessory gland and its duct, however, the material which is continuous withthe spermatophore stains strongly with methyl blue. It is apparent that somechange 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 infig. 8. They consist on each side of a testis, a vas deferens which swells in its

Davey—Spermatophore Production in Rhodnius prolixus 227

middle third to form the seminal vesicle, and the accessory glands with theirduct, which joins the vas deferens of the corresponding side in the bulbusejaculatorius. The two bulbi unite in the mid-line to form the ejaculatory ductopening at the base of the penis. The accessory glands are of two types: threeof 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 theaccessory gland

bulbus e/aculotorius

ejaculatory duct-

FIG. 8. Drawing of the left side of the male reproductivesystem of Rhodnius, dorsal view.

starved males, which are unable to produce spermatophores, and he inferredfrom this that the transparent secretion made up the main body of thespermatophore. A more direct demonstration of this was obtained in thepresent study by mating normal females to males which had one or morelobes of the transparent accessory glands removed. Such matings resulted inthe production of smaller, but otherwise normal spermatophores. Removal ofthe opaque glands, on the other hand, had no effect on the main body of thespermatophore.

The transparent secretion resembles the material making up the main bodyof the spermatophore in every apparent way except fluidity. Simple histo-chemical tests, performed as outlined in Pearse (1953), on whole spermato-phores and on whole and sectioned transparent accessory glands fixed inneutral formaldehyde solution revealed a further correspondence in proper-ties. Both were strongly positive to Millon's test, demonstrating the presenceof protein. Fat was absent from both spermatophores and accessory glands,as shown by their failure to stain with Sudan black B or Sudan red colouringagents. The presence of polysaccharide in both materials was demonstrated

228 Davey—Spermatophore Production in Rhodnius prolixus

by a very strong periodic acid / Schiff reaction. These results pointed to aprotein-polysaccharide complex, and additional tests were performed with aview to characterizing the substance. Neither exhibited metachromatic stain-ing with toluidine blue, and a methylene blue extinction test performed onspermatophores only gave a methylene blue extinction value which wasindefinite, but certainly greater than 5. According to Pearse (1953), materialsexhibiting these properties can be designated as mucoproteins or neutralmucopolysaccharides. At any rate, the transparent accessory secretion appearsto differ from the material in the spermatophore only in consistency; thespermatophore is simply a more solid transparent secretion.

The importance of pH

A clue to the processes involved in the solidification of the transparentsecretion to form the spermatophore was gained from a consideration of thebehaviour of the freshly formed spermatophore in various environments. Aspermatophore removed from a recently mated female swelled slightly indistilled 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 studiedby placing a single lobe of the gland in insect Ringer which had been bufferedto the desired pH with isotonic sodium hydrogen phosphate / potassiumdihydrogen phosphate buffer. After 10 min the gland was dissected and thecondition of the contents noted. The pH values employed lay between 5-0 and6*8 and were separated by 0-2. Above pH 5-6 the material was quite fluid, butat 5-4 and 5-6 the secretion had gelled to form a material closely resemblingthat in the spermatophore. At pH 5*2, the material was again fluid. Thisexperiment was repeated twice and a fourth trial was performed with Mcll-vaine's standard buffer; identical results were obtained in each case. It isclear that the mucoprotein of the accessory gland will solidify at a pH of about5-5 to form a material like that in the spermatophore.

The pH of the reproductive ductsIt 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 theamount of fluid in these ducts is very small, the organ in question was crushedon to a tiny piece of B.D.H. indicator paper. In this way it was found that theaccessory glands, testis, and ducts down to the level of the bulbus ejaculatoriuswere at or near neutrality, while the pH of the fluid in the lumen of the bulbuswas about 5"5. Only the bursa copulatrix of the female was examined; it alsowas at pH 5-5.

In order to confirm these observations, certain properties of methyl redwere employed. In addition to its well-known action as a pH indicator with anend point near 5-5, this dye can also be used as a vital stain. A 1% solutionin 0*9% NaCl was injected into the haemocoele of 10 males, which weredissected in 0^9% NaCl 5 h after injection. The ducts and organs of the

Davey—Spermatophore Production in Rhodnius prolixus 229

reproductive system of such males were stained yellow, indicating a pHconsiderably above the end point, whereas the fluid in the bulbus was stainedorange, indicating the end point pH of 5-5. To confirm the pH of the bursaof the female, a small crystal of methyl red was inserted into the bursa throughthe 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 thebulbus may be responsible for the solidification of the transparent accessory

outerepithelium

vas deferens

muscle

ejaculatory ductFIG. 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 bulbusto clot the transparent secretion, the bulbi from 20 males were macerated withforceps 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 weremacerated in another sample of the same Ringer. A lobe of the transparentaccessory gland was placed in each suspension. After 10 min the secretion inthe 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, whilethat of the other suspension remained near neutrality. Because of the largenumbers of insects required, this experiment could be repeated only once; onthat occasion similar results were obtained. Other experiments using sub-stantially fewer insects were unsuccessful.

The anatomy of the bulbus (fig. 9) is so arranged that the products of theaccessory glands are kept separate from the secretion of the bulbus up to thelevel 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. Projectingfrom this common duct into the lumen is the inner epithelium, which presum-ably secretes the greenish, watery fluid that fills the lumen of the bulbus. Thisfluid 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 theanimal and the outer epithelial layers from each bulbus unite to form the shortejaculatory duct opening into the base of the penis. The common ducts fromeach side empty separately into the ejaculatory duct. Thus the fluid secretionfrom the transparent glands is kept separate from the acid secretion of thebulbus until they are allowed to mix in the penis. It is worth rememberingthat this is the region in which the transparent secretion becomes eosinophilin Mann's stain.

CONCLUSION

On the basis of these observations it is concluded that the spermatophoreof Rhodnius is composed of a mucoprotein or neutral mucopolysaccharideoriginating in the transparent accessory glands of the male and that this secre-tion solidifies in the spermatophore sac of the male intromittent organ as aresult of a change in pH from near neutrality to 5*5. This change in pH isbrought about by the secretion of the bulbus ejaculatorius of the male. Theentry of the transparent secretion into the spermatophore sac causes theeversion of that organ, which acts as a mould for the spermatophore.

I am indebted to Professor Wigglesworth of the Department of Zoology atCambridge for suggesting and supervising the problem, and for reading themanuscript. Generous financial assistance in the form of a fellowship camefrom the Ontario Research Foundation.

REFERENCESBONHAG, P. F., and J. R. WICK, 1953. 'The functional anatomy of the male and female

reproductive systems of the milkweed bug.' J. Morph., 93, 171.DAVEY, K. G., 1958. 'The migration of spermatozoa in the female of Rhodnius prolixus.' J.

exp. Biol., in the press.GALLIARD, H., 1935. Recherches biologiques et morphologiques sur la reproduction des rdduviides

hematophages {Rhodnius et Triatoma). These Fac. Sci. Univ. Paris.KHALIFA, A., 1950. 'Spermatophore production and egg-laying behaviour in Rhodnius

prolixus.' Parasitology, 40, 283.PANTIN, C. F. A., 1948. Notes on microscopical technique for zoologists. Cambridge (University

Press).PEARSE, A. G. E., 1953. Histochemistry, theoretical and applied. London (Churchill).SINGH-PRUTHI, H., 1925. 'Morphology of the male genitalia in Rhynchota.' Trans, ent. Soc.

Lond., 1925, 127.SNODGRASS, R. E., 1935. Principles of insect morphology. New York (McGraw-Hill).

I957' 'A revised interpretation of the external reproductive organs of male insects.'Smithsonian misc. Coll., 135, 66.