30

On spermatophores in Simuliidae (Diptera)

By LEWIS DAVIES Deparimen! of Zoology, University of Durham

SYNOPSIS The form of the spermatophore of Simulium salopiense and its position in the

freshly-mated female are described. The inter-relationships of the male and female terminalia during copulation, and the probable course of events during the formation of the spermatophore are discussed.

THE purpose of the present paper is to provide details of the form and position of spermatophores in certain Simuliids, since this aspect of the biology of these insects has remained imperfectly known even to specialists. The existence of spermatophores in Simuliidae is briefly mentioned by Rubtzov (1956, 1959), but few facts and no illustra- tions were given. Indeed, Khalifa (1949) stated that spermatophores are unknown in the Diptera, an error repeated by Richards and Davies in their 1957 edition of Imms’ Textbook of Entomology, and by Davey (1960). The first certain demonstration of spermatophores in a Dipteron appears to date back to the detailed paper of Pomerant- zev (1932) on mating in Culicoides (Ceratopogonidae). Apart from this and the short account of an essentially similar sperm-transfer method in a member of the Chirono- midae given by Nielsen (1959), no other occurrences of spermatophores in Diptera seem to have been published.

A phenomenon of apparently different nature has been recorded for Anopheles gambiae Giles (Culicidae) by Gillies (1956), in which the male secretes during copula- tion what has been termed a “mating plug”, a solid elongate mass of albumen-like material resting within the common .oviduct of the newly-mated female and absorbed in the succeeding 36 hours or so. Similar plugs have since been recorded for four other species of Anopheles by Hamon (19631, but their precise nature and functions in the post-copulatory processes in female mosquitoes remain obscure and worthy of detailed study in view of the importance and amenability to laboratory maintenance of certain of these insects.

The Form of the spermatophores Lewis (1960) mentions briefly that in Simuliwn damnosum Theo. “an amorphous

object, which stained blue in Mallory’s triple stain and extended from inside the com- mon oviduct to the cerci, was seen in sections of one nulliparous fly”, and he surmised that it could be a mating-plug. My own observations show that a true spermatophore in the form of a translucent

hollow capsule is formed during copulation, as described below, in Simulium ( Wilheimia) salopiense Edw., freshly-emerged adults of which will mate within 2 x +“ glass tubes in the laboratory. Essentially similar spermatophores have been recovered from nulliparous females of a number of other species and these, together with the broadly uniform structure of the male and female terminalia throughout the Simuliidae, show that S. salopiense is not unusual in its sperm-transfer processes but is on the contrary typical of the family in this regard.

The spermatophore of S. sizlopiense (Plate I, fig 1) is a flattened hollow spheroid, 150-170 p at its greatest diameter, slightly narrower towards the anterior end and with a low anterodorsal swelling (fig. 1, c, e (S. venustum Say)). It contains a mass of sperm, which is partly divided in two by an internal posterior ridge of the spermato- Proc. R. ent. Soc. Lond. (A). 40 (1-3). Pp. 30-34, 2 figs., 1 Plate. 1965.

,

L. Davies on spermatophores in Simuliidae (Diptera) 31

phore wall. The sperms invariably show a considerable degree of parallel bunching, still largely retained after the spermatophore is burst by gentle pressure under a cover slip (Plate I, fig. 2), and are accompanied by a quantity of coarsely granular material, some of the granules being yellow in the fresh state. The wall of unfixed spermato- phores examined in 0.7 per cent. saline appeared to consist of two layers, an inner hyaline layer, which is thinner over part of the anterior dorsal swelling of the spheroid, and a thicker outer layer of more spongy appearance that varies in thickness from one part to another and often has irregular external projections.

a

e

100 microns - 0

FIG.' 1.-The form of the spmatophores of certain Simuliidae. Anterior end towards the top of the figure, except in c. The thickness of the outer and inner layers of the walls are as seen in optical sections. a, b, Prosimulium mixtum S. and D., showing variation in shape, with dorsal processes perforated and with the outer layer apparently absent in b ; c, e, S. venwtum Say, lateral and dorsal views respectively of separate spermatophores; d, S. jenningsi Mall.; f. S. salopiense.

The form of spermatophores from various Simuliidae is shown in figure 1. They differ from that of S. salopiense in minor features only, particularly in that (in some species) the anterodorsal swelling is more conspicuous. This swelling, sometimes perforated in empty spermatophores (fig. 1, a, b), appears to lie against the membrane joining the two arms of the so-called genital fork (termed the vaginal apodeme in other Nematocera by Abul-Nasr (1950) and other anatomists). This means that when the

32 L. Davies on spermatophores in Simuliidae (Diptera)

spermatophore is in situ the thinner walled parts are adjacent to the opening of the spermathecal duct.

Position of the Spermatophore in the Freshly-mated Female Figure 2 (a, b) shows the position of the terminalia in a freshly-mated female of

S. salopiense. In ventral view the relatively large spermatophore lies with its posterior edge dorsal to the so-called anal lobes (part of the tenth segment),-and its anterior end lies dorsal to the eighth sternite. It thus lies in a deep cavity and is embraced anterior- ly by the two arms of the genital fork, so that the membrane connecting these two arms is stretched dorsally. The large anteropostero extent of the cavity so formed is striking and ieads, as figure 2 (a, b) shows, to the considerable backwards displacement of the anal lobes from their normal approximation to the posterior edge of the eighth sternite seen in the unmated female. The considerable depth of the cavity is emphasised by the fact that in lateral view the spermatophore itself is not visible.

FIG. Z-S. sufopiense. u, Female terminalia, ventral view, showing position of spermatophore; b, the same, lateral view, broken line indicating the spermatophore in siru; c, terminalia of male and female in copulo. A, anal lobe; CF, arm of genital fork; 82'. eighth tergite of female; 9TF, ninth tergite of female; lOT, tenth tergite of female; SS, eighth sternite of female; CX, coxite of male; AE, aedeagus; P, paramere; PH, paramera1 hooks; CE, wrci of male; 9TM, ninth tergite of male.

The precise homologies of the region of the cavity mentioned above are difficult to determine. The pyramid-shaped chamber housing the anterior end of the spermato- phore may be equivalent to the atrium of mosquitoes as defined by Christophers (1960). since it receives the spermathecal duct opening on its dorsal surface. Its posterior boundaries do not seem to be marked by any discontinuity of its membranous wall. Certainly that part of the cavity housing the posterior part of the spermatophore, and bounded posteriorly by the anal lobes and IateralIy by the terminal expansion of the arms of the genital fork, must be considered anatomically external and corresponding to the cloaca-like hollow behind the eighth sternite described by Christophers for Aedes aegypti (L.). The difficulty of determining precise homologies seems to result from the modification of the region of the gonopore in the female Simuliid by expan- sion of membranous parts, specifically to house the relatively large spermatophore.

L. Davies on spermatophores in Simuliidae (Diptera) 33

The Position of the Terminalia of the Two Sexes During Copulation In order to understand how the spermatophore comes to lie in the position described

above in the mated female, it is necessary to consider the inter-relations of the male and female terminalia during copulation. This question merits attention since, although taxonomists have long described genitalic form in Simuliids, there is as far as I am aware no account of what happens to the various structures during the mating process.

A number of pairs of S. salopiense fixed in copula have been examined, and the facts disclosed are illustrated in figure 2, c. The terminology for the male genitalia pro- posed by Freeman (1950) and, for the female parts, names widely used by black-fly specialists (e.g. Stone and Jamnback, 1955), will be used.

The outstanding feature shown by figure 2, c, is that the male coxites embrace the female tenth segment, so that the lateroventral parts of the tenth tergite, the anal lobes (A in fig. 2, a and b), are pulled back. The styles, which in this species are very small relative to the coxites, are not visible in figure 2c, and seemed to be bent between the coxites and the. female tenth tergite. It would be valuable to find out what is the position during mating of the large styles found in many Simuliidae. The aedeagus iseverted by distension of its membranouswalls, to anextent comparable with that shown in Freeman (1950: fig. 1) for the same species, and the parameral hooks are erected, being engaged with and pushing ventrally the posterior edge, or ovipositor lobes, of the female eighth sternite. Allowing for the probability that the terminalia of the two sexes became slightly separated from the natural copulatory position as a result of fixing the mating pair, figure 2c shows that a virtually closed inter-genitalic cavity is formed, into which protrudes the blunt and largely membranous aedeagus.

Probable Events During Formation of the Spermatophore Sufficient material has not been available to fix pairs at intervals during copulation

so that different stages in spermatophore formation can be seen. The relations of the parts described above and the facts given below leave little doubt that the male ejects and partly fills the inter-genitalic cavity of the pair with a viscous mass, into which is ejaculated the sperm-mass plus the granular material mentioned earlier. The viscous mass is thus presumably hollowed out and then hardens to form a continuous invest- ment around the sperm. The apparent inner hyaline layer of the spermatophore wall (page 31) is probably formed by compaction as a result of the injection of the more liquid sperm-mass under pressure into the viscous material. The more spongy outer layer may result from reduced compaction, since the largely membranous walls of those parts of the inter-genitalic cavity bounded by the female terminalia would expand or unfold with the increasing size of the spermatophore as it is formed.

Copulation in S. sulopiense lasted two to three minutes, long enough for the above events to take place. There was no evidence that the male formed a spermatophore within his genital tube or outside the aedeagus either before copulation or during its initial stages and then inserted the complete structure into the female. Examination of fixed and unfixed males of various species immediately after capture from swarms in the field disclosed no spermatophores in process of formation. They have been found in the position described only after at least two minutes coupling.

The internal anatomy of the male genital system in specimens taken from mating swarms supports the above interpretation that the spermatophore is formed in situ in the cavity opened up within the female terminalia during mating. The paired so-called seminal vesicles (see Rubtzov, 1956, fig. lo), divided into a lower thick-walled tube and an upper chamber nearer the vas deferens, was in all instances filled with a slightly cloudy, white and viscous material that was quite sperm-free and hardened within five minutes in air on a slide. This material is probably that which forms the spermato- phore wall. Sperm was always confined to, and tightly filled, the vasa deferentia and the testes themselves. There is thus the correct order of components: first the viscous material that would be expected to be ejected first, followed by the sperm. By analogy with other insects (Khalifa, 1949), it is to be expected that ejection of viscous material,

34 L. Davies on spermatophores in Simuliidae (Diptera)

followed by ejaculation of sperm into it, would lead to the formation of a vesicular spermatophore.

DISCUSSION The simuliid spermatophore immediately after it is formed is a closed capsule. It

should be noted that part of the anterodorsal wall of the spermatophore is thinner than elsewhere, and forms a lobe or swelling that projects against the membrane bear- ing the spermathecal duct opening, between the arms of the genital fork. In empty spermatophores taken from nulliparous females that had been attracted to man in the field and had copulated at an unknown time before capture, the wall was sometimes perforated in this region. Presumably the sperm had escaped by this route, but the agency responsible for the formation of the hole is unknown.

Empty spermatophores were found in 5-15 per cent. of nulliparous females of S. venustum Say attracted to man in the field, but although over 4000 parous females of the same species were examined not a single one bore a spermatophore. This suggests that in this species parous females very rarely copulate. The position of the spermatophore in the species mentioned in this paper shows that if it were still present in the gravid female it would be pushed out by the first egg to be laid. The fact that there was no empty spermatophore in the majority of nulliparous females (although sperm was usually present in the spermatheca), shows that the spermatophore must be either actively ejected or passively dropped out some time after mating. There was no evidence that it is absorbed by the female. In S. venustum the presence of a spermato- phore is a useful indication that the female is nulliparous, although clearly its absence gives no indication of the history of the fly. It may well be that the same is trueof other species, and that the presence of a spermatophore can be made use of without dissection in separating a part of the nulliparous component of field catches from the remainder.

Spermatophores are now known to occur in three families of Diptera, all in the Nematocera. If the “mating plugs” of Anopheles spp. prove to be spermatophores, then members of four families of Nematocera employ them. The doubt in this last case remains, since the plug does not appear to invest the sperm mass and is sometimes placed in the common oviduct, and thus in a deeper part of the female genital tract than the part bearing the spermathecal opening, namely the atrium.

I am indebted to Dr. R. W. Dunbar of this Department for providing material and initially discover- ing the fact that s. salopiense will mate in glass tubes, and to Mr. J. A. Downes,Entomology Research Institute, Ottawa, for drawing my attention to the paper by Pomerantzev.

REPBRENCEs

Nematocera (order Diptera: suborder Nematocera). Phil. Trans. R. SOC. (B).234 : 339-96. ABUL-NASR, S. E., 1950, Structure and development of the reproductive system of some species of

CHRISTOPHERS, S. R., 1960, Aedes aegypti (L.), The yellow fever mosquito. Cambndge. DAVEY, K. G., 1960, The evolution of spermatophores in insects. Proc. R. ent. Soc. Lond. (A) 35 :

FREEMAN, P., 1950, The external genitalia of male Simuliidae. Ann. trop. Med. Parasit. 44 : 146-52. GILLIES, M. T., 1956, A new character for the recognition of nulliparous females of Anopheles gambiae.

Bull. Wld. Hlth. Org. 15 : 451-9. HAMON, J., 1963, Etude de l’age physiologique des femelles d’anophefes dans les zones traitbs au DDT,

et non traitbs, de la region de Bobo-Dioulasso, Haute-Volta. Zbid. 28 : 83-109. KHALIFA, A., 1949, Spermatophore production in Trichoptera and some other insects. Trans. R. ent.

Lewrs, D. J., 1960, Observations on Simulium damnosum in the Southern Cameroons and Liberia.

NmeN, E. TETENS, 1959, Copulation of Glyptotendipes (Phytotendipes) paripes Edwards. Nature,

POMERANTZEV, B. I., 1932, Beitrage zur Morphologie und Anatomie der Genitalien von Culicoides

RUBTZOV, I. A,, 1956, Blackflies (fam. Simuiiidae). Fauna of U.S.S.R. Insects (Diptera). (N.S. 64)

107-13.

SOC. Lo&. 100 : 449-71.

Ann. rrop. Med. Parasit. 54 : 208-23.

LO^. 184 : 1252-3.

(Diptera, Nematocera). Mag. Parasir. Leningr. 3 : 183-214.

6 (6) : 1-860. (In Russian.)

349 : 1-144.

- 1959, Simuliidae (Melusinidae), in Lmdner, Flieg. Pal. Reg. 14 : 1-48. STOM, A. & JAMNBACK, H. A., 1955, The black flies of New York State. Bull. N.Y. State Mus.

Proc. R. ent. Soc. Land. ( A ) Vol. 40. PLATE 1

Simulium salopiense Edw. FIG. 1 .--Intact spermatophore showing dark,i.e. opaque, contents. Anterior end towards top of

figure. Fixed in Carnoy’s solution. ( ).. 500 approx.) FIG. 2.-Spermatophore contents, photographed without fixation, in 0.7 per cent saline. Split

anterior end of spermatophore at top of figure, sperm at middle right and bottom, granular material accompanying sperm in middle left. ( x 500 approx.)

L. Davies