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J. Sci. 12, 287-311 (1973) 287 Printed in Great Britain

THE OF ARTHROPODA

XVIII. THE NON-MOTILE BIFURCATED OF PSYCHODIDAE FLIES

B. BACCETTI, R. DALLAI AND A. G. BURRINI Institute of Zoology of the University of Siena, Via Mattioli 4, Siena, Italy

SUMMARY The spermatozoa of some species of Diptera belonging to the Psychodidae are described. In the 4 species examined, they appear needle-shaped, non-motile, anteriorly flattened, posteriorly bifid. Flagella, and any system, are lacking, and the only encountered are an extremely elongated nucleus, a prominent , a well developed subacrosomal body, a slender, somewhat elongated mitochondrial derivative and a conspicuous multilayered membrane wall, produced by the superimposition of the plasma membrane and the acrosomal membrane. The is devoid of a crystalline axis, while the acrosome contains a longitudinal, cross-striated para-crystalline body. are absent throughout the stage. and are organized in syncytia of 210 elements.

INTRODUCTION So far, a non-motile sperm type among insects has not been reported. In the few instances in which the classic is absent, it is replaced either by a larger number of doublet bundles, as in Protura (Baccetti, Dallai & Fratello, 1973) or the dipteran Sciara (Phillips, 1966; Makielski, 1966), or else by a bundle of simple micro- repeatedly coiled around the sperm, as in coccid insects (Robison, 1966, 1970; Ross & Robison, 1969; Ross, 1971). The sperm morphology of high Isoptera is still unknown, and will be tackled by us in the near future. The present investigation deals with the hitherto undescribed spermatozoon of Diptera belonging to the Psycho- didae which appears to lack an axoneme, to be non-motile and caudally bifid.

MATERIALS AND METHODS Specimens of Telmatoscopus albipunctatus, Psychoda cinerea, Psychoda sp. and Psychoda alternata were supplied in all the instars by Drs M. Sara and G. Salamanna of the Institute of Zoology of the University of Genova. Larvae, pupae and adults were dissected out in cold 5 % glutaraldehyde at pH 7-2 to 7-4 in Hoyle buffer containing 5% sucrose; the testes were excised and fixed in the same fluid for 1-5 h, rinsed overnight in Hoyle buffer with 5 % sucrose, postfixed in 1 % osmium tetroxide for 45 min, dehydrated in a graded series of ethanols and embedded in Epon. Sections, cut with an LKB ultramicrotome, mounted on grids, and stained with uranyl acetate and lead citrate, were observed and photographed under the Siemens Elmiskop IA and Philips EM 300 electron . For study of , thin sections were oxidized for 5 min in H2O2, 12 vol., thoroughly rinsed in distilled and treated with 0-2% pepsin solution in o-i N HC1 for 8-15 min at 37 °C. Control specimens were incubated in o-i N HC1 for the same 288 B. Baccetti, R. Dallai and A. G. Burrini periods as the experimentals. The subcellular localization of polysaccharides was investigated by the periodic acid-thiosemicarbazide-silver proteinate method (Thiery, 1967) with 24-36 h action of TCS. To evaluate sperm , sperm cells, suspended between 2 slides in Gouldin medium, were observed under a Leitz Ortholux phase-contrast . Stereoscopic examination was carried out by the JSM-2 scanning electron microscope on spermatozoa obtained by squeezing the testes, coated with evaporated gold/platinum. For freeze-etching the testes were incubated for 1 h at 40 C in 25 % glycerol in Hoyle buffer, transferred on to small copper disks and rapidly plunged in Freon 23 cooled with liquid nitrogen for 1 min. The disks were then immersed in liquid nitrogen for a period of 10 min to 3 days. Freeze-etching was achieved with a Balzers apparatus, according to the technique devised by Moor, Miihlethaler, Waldner & Frey-Wyssling (1961). The carbon-platinum replica was removed from the specimen holder, treated with chromic acid, thoroughly rinsed in distilled water in order to remove cellular and other debris, and then picked up on copper grids.

RESULTS The sperm examined under fresh conditions (Fig. 3) by phase-contrast are needle-shaped, about 0-2 mm long. When maintained in fluids which as a rule allow considerable motility in other insect spermatozoa, among Diptera as well, they appear altogether non-motile, nor can they be stimulated in any way by changing the environmental conditions. After coating with gold-platinum, examination in the scanning electron microscope reveals morphological characteristics of particular interest. The anterior portion of the sperm (Fig. 4), about 22-5 /un long, looks markedly flat in all the species, except Telmatoscopus, where it appears keeled lengthwise. In every case, the posterior portion is consistently bifid (Fig. 5) along the terminal 3 /tm of its length. For most of the remaining intermediate portion the sperm is cylindrical. Ultrathin sections make it possible both to confirm these peculiarities and clarify the structure of the sperm. In the 4 species examined here, the sperm is non-flagellated and devoid of any microtubule system whatsoever. The organelles encountered in the mature sperm are the nucleus, a prominent acrosome, a strikingly developed sub- acrosomal body, a slender, somewhat elongated mitochondrial derivative and a con- spicuous multilayered membrane wall surrounding the whole sperm cell and also separating the nucleus from the other organelles. In Telmatoscopus albipunctatus the nucleus lies in a dorsal position beneath the keel of the anterior part of the sperm and along its major cylindrical portion. It is moder- ately compact, its being distributed into filaments which are arranged longitudinally, and always readily identifiable. The nucleus appears as a cylinder (Fig. 10) which gradually flattens out rostralwards, appearing first elliptical (Fig. 9), then C-shaped in cross-section, with dorsally directed convexity (Fig. 8), and finally, at the anterior end of the sperm, presents a thin crescent shape in profile. At this level, the nucleus is seen to expand across almost the whole width. In the posterior regions of the sperm, the nucleus appears as a cylinder with gradually reduced diameter, still lying in a dorsal position (Fig. 11). Ventrally, the sperm is almost entirely occupied by the acrosome, which extends Non-motile bifurcated sperm of Psychodidae 289 distally from the rostral end (Fig. 7), where, reniform in section, it is the only present, to the bifid tail (Fig. 13), filling up the empty spaces left by the other organ- elles (Fig. 11). The acrosome consists of a compact, granular material particularly packed along its axis which is positioned in the centre of the cell. This axis (Fig. 12) exhibits a clear transverse banding, with a period averaging 25 nm. Each period con- sists of a highly electron-dense band and 2 moderately electron-dense bands, which are separated by an interval some 7 nm wide and lie 10 nm from the major band. This striated material is undoubtedly in nature, despite its pronounced pepsin- resistance. In the other region, the acrosome material is not striated, and is pepsin- sensitive, as shown in the posterior segment of the spermatozoon, where it represents almost the whole content (Fig. 15). The nucleus is separated from the acrosome by a conspicuous subacrosomal structure (Fig. 10) simply represented by a furrow about 40—50 nm wide, which extends almost the whole length of the nucleus, assuming the ventral shape of the latter. Along this furrow the nuclear envelope and the acrosomal membrane are likely to fuse. This structure undergoes a marked thickening just in front of the flat anterior region of the sperm (Fig. 9), taking on an ellipsoidal outline first, then an almost circular one in cross-section. At both these levels the subacrosomal structure fills up the sperm almost completely, since dorsally the nucleus is thin and C-shaped, as is the acrosome ventrally. The latter also extends within the 2 wings of the spermato- zoon. In the posterior segment of the spermatozoon the subacrosomal material sur- rounds the nucleus, and proceeds beyond it in caudal direction in a short region, where it appears as a clear sector in the opaque circle constituting the acrosome (Fig. 14). The subacrosomal material is granular, homogeneous, and fairly transparent to the electron beam. It is readily removed by pepsin digestion (Fig. 16), being exclusively protein in nature. It is Thiery-unreactive (Fig. 17) and a-amylase-resistant. Within the acrosome, the mitochondrial derivative is found. It is narrow, elongate, 130 nm wide and some 60 nm thick in sectional view, with its concavity facing the nucleus. In both transverse and longitudinal sections it appears devoid of cristae; its matrix is highly transparent to the electron beam. The mitochondrial derivative is the smallest sperm component. In fact, arising behind the rostral flattened region where the sperm is elliptical in cross-section, it extends only a short distance, i.e. the length of the sperm central portion. The spermatozoon is entirely bounded by a 7-layered wall (Fig. 10) whose overall thickness is 25 nm. The outermost layer, 13 nm thick on average, is made up of 2 osmiophilic laminae divided by a light interspace; farther inwards, after a gap about 5 nm wide, a thinner membrane, averaging 7-5 nm, is encountered. In frozen-etched preparations, the aspect of these membranes stands out sharply (Figs. 18-20). Both the outer and inner aspects of the outermost membrane (13 nm thick) appear smooth, although some irregularity results from the presence of minute, randomly scattered indentations. On the other hand, the thinner membrane (7-5 nm thick) is obliquely striated, with a period of about 30 nm. The striations probably occupy the cleft 5 nm wide between the thick outermost membrane and the inner thinner one; the latter exhibits an even surface on either side, only lightly indented by scanty and widely 19 CE L 12 290 B. Baccetti, R. Dallai and A. G. Burrini spaced shallow pits. The outermost membrane may be interpreted as a rather thick plasmalemma; the inner one as an acrosomal membrane where it surrounds the acro- some, and as a nuclear envelope in the contiguous regions, where it surrounds the nucleus. The outermost membrane is further coated by a tenuous carbohydrate layer, which is reactive to the Thiery method, and selectively a-amylase sensitive. Interpretation of the structure thus far described, all considerably diverging from those of the 'conventional' sperm model, is only possible by insight into spermio- genesis, the consecutive steps of which, as observed by us, are as follows. In Telmatoscopus, as among Diptera in general (see Baccetti & Bairati, 1964; Phillips, 1970) spermatids are usually assembled into a syncytium. In their youngest stages they exhibit a fairly regular shape, a prominent nucleus with rather dispersed chromatin, and their is very scanty, containing only a few free and some regularly patterned mitochondria. These spermatids are interconnected by slender cytoplasmic bridges, forming groups of 1024 (210) units. Later on, as con- densation of the nuclear material and nuclear elongation set in, the spermatids become more closely packed, the limiting membranes between them disappear almost com- pletely, and the syncytium (Fig. 27) becomes a single cytoplasmic mass containing 1024 nuclei. At this moment changes occur in the nuclei, the mitochondria and the . The latter undergoes a striking development (Fig. 27), since a great many Golgi systems, though fewer in number than the spermatids, arise within the syncytial mass; they contain abundant flattened cisternae full of compact electron- opaque material, forming several layers in concentric array. Mitochondria show a tendency to fuse into an elongate cord with longitudinally arranged cristae. Elongation of the nuclei, each of which is surrounded by 14 to 15 (Figs. 21, 22), takes place. Subsequently, the mitochondrion approaches the nucleus, to whose axis it eventually comes to lie parallel (Fig. 21). At this stage the nucleus has elongated further, its chromatin becoming concentrated into thick, increasingly more tightly packed longitudinal threads (Figs. 23, 25, 26). Extended, flattened discrete cisternae are seen to branch out of the Golgi. These cisternae come to lie alongside the nucleus throughout the length of each spermatid, flanking the mitochondrion and partly surrounding it (Figs. 21, 27). The Golgi cisternae appear isolated along most of the spermatid length, except at their rostral ends, at which a single ramified cistern is shared by several spermatids (Fig. 25). Each spermatid is surrounded by two juxta- posed Golgi elements. In fact, between the nucleus and the a vesicle filled with granular material (Figs. 21-25) is seen to form and to flow into other Golgi cisternae winding among the spermatids. This structure, which is interpreted by us as a proacrosomal vesicle, encloses the mitochondrion like a sleeve (Fig. 21). There- after, the peripheral portion of the acrosome seems to accumulate an electron-opaque material, which appears as a dense line in sections (Fig. 25). The interior of the mito- chondrion becomes homogeneous, its cristae disappearing and the matrix shaping itself into a cylinder with a hollow axis. Both the acrosomal vesicle and the mito- chondrion slide into a sort of longitudinal furrow carved along the side of the nucleus facing these 2 organelles (Fig. 26). At the next stage, the 1024 spermatids assemble into an increasingly more compact Non-motile bifurcated sperm of Psychodidae 291 bundle, starting to show up more distinctly within the huge common cytoplasmic mass, and to align in register with one another. The nucleus appears as a bundle of fine electron-dense rodlets; it elongates rapidly, though being rostrally still overlapped by the acrosome. The latter has developed further, attaining its final size; its axial electron-dense strip has concentrated into a striated longitudinal rodlet. The interior of the mitochondrion appears increasingly narrow and more trans- parent. The Golgi cisterna is seen to adhere only to the side of the spermatid where the nucleus is absent (Fig. 25), being separated from the acrosome by a space in which 2 membrane layers take shape, namely, the plasma membrane and the acrosomal membrane. On the opposite side, along which the nucleus extends, a similar stratifica- tion occurs, with the juxtaposition of the plasma membrane and the nuclear envelope. Between the nucleus and the acrosome, deposition of the light material (Figs. 25, 26) which will make up the subacrosomal body takes place. This material is seemingly the result of the fusion of the nuclear envelope and the acrosomal membrane, exhibit- ing the juxtaposition of several membranes. In some zones of the sperm this material becomes increasingly more abundant until it makes up almost the entire sperm volume. Its origin is difficult to define. When its deposition begins the Golgi cisterna is separated from it by the entire width of the acrosome. The newly formed plasma membrane taking shape around the young spermatids isolates them from the common syncytial cytoplasm. At first, this membrane not being complete between sperm (Fig. 32), small sperm clusters are still syncytially connected within the huge initial common cytoplasmic mass. The microtubules involved in the elongation of the sperm are still present. Afterwards, when spermatozoa are sharply individualized (Fig. 27), the undivided cytoplasm degenerates; its microtubules are seen to pack up into con- spicuous bodies with crystalline texture (Fig. 14). Psychoda cinerea sperm (Fig. 28) is characterized by a less-flattened rostral portion than in Telmatoscopus; moreover, the position of the 4 organelles within the cell differs in the 2 species. The nucleus in Psychoda has a cylindrical outline in the median and caudal sperm portions (up to its bifurcation) but it tapers rostrally, becoming filiform. It always lies in the central region of the sperm (Fig. 29), except for a short tract of the anterior sperm portion overlain by the mitochondrial derivative, in which it is displaced ventrally (Fig. 28). The mitochondrion is elliptical, or crescent-shaped in cross-sec- tion. Because of the displacement of the nucleus, the subacrosomal material also becomes cylindrical (Fig. 29), enveloping the nucleus under the form of many concentric membranous rings, and assuming a peripheral position only where the mitochondrion is present. Another consequence of the arrangement of the nucleus is the cylindrical shape of the acrosome, along the axis of which the cylinder of sub- acrosomal material is seen to extend, as does the mitochondrion, for a short distance. The sperm periphery (Fig. 29) is identical to that in Telmatoscopus, although in this case only the acrosomal and the plasma membranes are seen in mutual apposition. The 2 sections of the bifurcated tail are semicircular, and occupied, as in Telmasto- copus, only by the acrosome, which is again found alone at the rostral end; no thick- ening is shown by the subacrosomal material at any level. The structural features of the 4 organelles and of the limiting membranes are not unlike those in Telmatoscopus.

19-2 292 B. Baccetti, R. Dallai and A. G. Burrini Psychoda alternata is very similar to Psychoda cynerea in its organelle arrangement, though differing from the latter in the shape of the sperm sections, which at any level are slightly reniform (Figs. 30, 31) instead of circular, and also in that of the nucleus, which is less regularly ellipsoidal and sends out a tenuous fin full of chromatin (Fig. 30) deep inside the subacrosomal material directed towards the concavity of the reniform section of the sperm. In a third Psychoda species, hitherto unidentified, the overall scheme appears the same, although the nucleus lies in a lateral position (Fig. 33) throughout its length, so that the subacrosomal structure appears more similar to that found in Telmato- scopus. The mitochondrial derivative always occupies a central position.

DISCUSSION In Telmatoscopus and Psychoda the sperm is completely non-motile, being devoid of a normal axoneme or any alternative organelle. In this sense it represents a type (Figs. 1,2) of its own, not only among insects but throughout the kingdom as a whole, since non-motile sperms are a fairly rare event. At first sight these sperms might recall the aflagellate sperm of the more highly evolved Myriapoda, though they substantially diverge from the latter in both general shape (which is consistently extremely elongate among Psychodidae) and certain features of the organelles, which in the Diplopoda present as yet ill-defined though rather unusual characteristics among spermatozoa (Horstmann & Breucker, 1969; Reger, 1971; Reger & Cooper, 1968), while among Psychodidae the study of makes it possible to equate them unequivocally with the classical acrosome, the subacrosomal structure and the mitochondrial derivative, despite their peculiar shape and position. Among Myria- poda Diplopoda, the possible maturation of the sperm in the genital tract is still an open question. Here, according to Sokoloff (1914) and to Tuzet & Manier (1955), the sperm might even become tailed and motile. On the contrary, among Psychodidae the pattern described by us is retained also after penetration into the female organism. Mention has already been made here that in these sperm the component organelles are the classic ones to the extent that they bear no relationship with motility. How- ever, they display some peculiarities deserving to be stressed. The acrosome is always a huge structure occupying most of the spermatozoon (which despite its elongate shape is practically a whole head); the same applies to the subacrosomal structure, which starting from the simple ' inner cone' typical of many Orthopteroidea (Kaye, 1962; Baccetti, Rosati & Selmi, 1971) has become a conspicuous furrowed body, or even a cylindrical one, intervening between the acrosome and the nucleus. The membrane surrounding the spermatozoon is very thick and multilayered due to the superimposition of the plasma membrane and acrosomal membrane. By contrast, the mitochondrial derivative is rather thin, embedded in the acrosome in which it lies by the side of the nucleus. The mitochondrial fine structure denotes involution, being wholly devoid of the crystalline axis which is typical of almost all insects (see Favard & Andre, 1970), whereas the acrosome displays a rather dense inner longitudinal para- Non-motile bifurcated sperm of Psychodidae 293

Fig. 1 F Fig. 1. The spermatozoon of Tehnatoscopus albipunctatus. a, acrosome; m, mito- chondrion; n, nucleus; s, subacrosomal material. Fig. 2. The spermatozoon of Psychoda dnerea. a, acrosome; m, mitochondrion; n, nucleus; s, subacrosomal material. 294 B. Baccetti, R. TDallai and A. G. Burrini crystalline body, longitudinally striated. This is a widespread acrosome feature, even of the simpler ones among other Diptera, as recently pointed out by Warner (1971) in Sarcophaga. The lack of an axoneme is correlated with the lack of the , which is absent throughout the spermatid stage. In the spermatid, further peculiarities deserve to be stressed. They constitute the most conspicuous syncytium hitherto reported in spermatozoa, scoring as many as 210 nuclei within each cyst. The syncytium has a real significance here, since spermatozoa become individual- ized only at a very late stage and their arise from Golgi cisternae branching out from a few, enormous Golgi complexes, which are shared by a number of spermatids. To conclude, this spermatozoon type appears to stem directly from the classic one among Diptera, lacking, however, any organelle permitting motility, but displaying very highly developed structures, constituting the greater part of the volume of the sperm, for its entrance into the egg. In Telmatoscopus and Psychoda the sperms are less aberrant than the few other non-motile sperms so far reported, but represent an altogether new type, which characterizes a whole dipteran family. This work was supported by a grant of the Consiglio Nazionale delle Ricerche.

REFERENCES BACCETTI, B. & BAIRATI, A. Jr. (1964). Indagini comparative sull'ultrastruttura delle cellule germinali maschili in Dacus oleae Gmel. ed in melanogaster Meig. (Ins. Diptera). Redia 49, 1-29. BACCETTI, B., DALLAI, R. & FRATELLO, B. (1973). The '12 + 0', '14 + 0' or aflagellate sperm of Protura. (The spermatozoon of Arthropoda, XXII.) In preparation. BACCETTI, B., ROSATI, F. & SELMI, G. (1971). The spermatozoon of Arthropoda. XVI. The acrosoma of Orthoptera. J. Siibmicr. Cytol. 3, 319-337. FAVARD, P. & ANDRE, J. (1970). The mitochondria of spermatozoa. In Comparative Spermato- logy (ed. B. Baccetti), pp. 415-429. Roma: Accademia dei Lincei. New York: Academic Press. HORSTMANN, E. & BREUCKER, H. (1969). Spermatozoen und Spermiohistogenese von Graphi- dostreptus spec. (Myriapoda, Diplopoda). I. Die reifen Spermatozcen. Z. Zellforsch.mikrosk. Anat. 96, 505-520. KAYE, J. S. (1962). Acrosome formation in the house cricket. J. Cell Biol. 12, 411-431. MAKIELSKI, S. K. (1966). The structure and maturation of the spermatozoa of Sciara copro- phila.J. Morph. 118, 11-41. MOOR, H., MUHLETHALER, K., WALDNER, H. & FREY-WYSSLING, A. (1961). A new freezing- ultramicrotome. J. biophys. biochem. Cytol. 10, 1-14. PHILLIPS, D. M. (1966). Fine structure of Sciara coprophila sperm. J. Cell Biol. 30, 499-517. PHILLIPS, D. M. (1970). Insect sperm: their structure and morphogenesis. J. Cell Biol. 44, 243-277- REGER, J. F. (1971). Studies on the fine structure of spermatids and spermatozoa from the millipede Spirobolus sp. J. Submicr. Cytol. 3, 33-44. REGER, J. F. & COOPER, D. P. (1968). Studies on the fine structure of spermatids and spermatozoa from the millipede Polydesnuts sp. J. Ultrastruct. Res. 23, 60-70. ROBISON, W. G. (1966). Microtubules in relation to the motility of a sperm syncytium in an armored scale insect. J. Cell Biol. 29, 251-265. ROBISON, W. G. (1970). Unusual arrangements of microtubules in relation to mechanisms of sperm movement. In Comparative Spermatology (ed. B. Baccetti), pp. 311—320. Roma: Accademia dei Lincei. New York: Academic Press. Non-motile bifurcated sperm of Psychodidae 295 Ross, J. (1971). Microtubules and the development of the corkscrew region of a mealybug sperm bundle. Tissue & Cell 3, 35-56. Ross, J. & ROBISON, W. G. (1969). Unusual microtubular patterns and three dimensional movement of mealybug sperm and sperm bundles. J. Cell Biol. 40, 426-445. SOKOLOFF, J. (1914). Ueber die Spermatogenese bei Polyxenus sp. Zool. Anz. 44, 558-566. THIERY, J. P. (1967). Mise en evidence des polysaccharides sur coupes fines en microscopie electronique. J. Microscopie 6, 987-1018. TUZET, O. & MANIER, J. F. (1955). Achevement de la spermiogenese dans les voies genitales femelles: cas de Polyxenus lucidus Chalande (Myriapode Diplopode). Annls Sci. nat. 17, 351-356. WARNER, F. D. (1971). Spermatid differentiation in the blowfly Sarcophaga bullata with par- ticular reference to flagellar morphogenesis. J. Ultrastruct. Res. 35, 210-232.

(Received 19 May 1972) 296 B. Baccetti, R. Dallai and A. G. Burrini

Fig. 3. A bundle of spermatozoa of Telmatoscopus albipunctatiis. Phase-contrast, x 320. Fig. 4. The keeled rostral part of the Telmatoscopus spermatozoon, surrounded by the medial portion. Scanning electron microscope, x 10000. Fig. 5. The bifid tail of the same spermatozoon. Scanning electron microscope, x 10000. Non-motile bifurcated sperm of Psychodidae 297

Fig. 6. A cross-sectioned bundle of about 210 spermatozoa in an adult Telmato- scopus. x 13500. 298 B. Baccetti, R. Dallai and A. G. Burrini

Fig. 7. Cross-sections of the extreme rostral portion of Telmatoscopus spermatozoon. In the reniform sections the apex, consisting only of acrosome (a), is cut; in the elongated sections the immediately following region is cut, showing a flattened nucleus (n), a narrow subacrosomal structure (s) and a large acrosome (la). 48000. Fig. 8. Cross-sections of a Telmatoscopus sperm region immediately posterior to that of Fig. 7. The nucleus (n) is almost cylindrical, the acrosome (a) very large, the general outline of the spermatozoon very flattened, x 37500. Fig. 9. Cross-sections of a Telmatoscopus sperm region immediately posterior to that of Fig. 8. Each section appears thicker, with the 3 organelles (acrosome, a; nucleus n; tubacrosomal material, s) of similar size. The lateral portions of the sperm are still occupied only by acrosomal material, x 75 000. Non-motile bifurcated sperm of Psychodidae 299

) 2 . rn

' •«*• 300 B. Baccetti, R. Dallai and A. G. Burrini

Fig. 10. Cross-section of the main part of the Telmatoscopus spermatozoon. The nucleus (n) is ellipsoidal surrounded by a narrow layer of subacrosomal material (s). The acrosome (a) occupies the other part of the section, bounded by the double mem- brane (mb). A small mitochondrion (m) is present, embedded in the acrosomal material, at this level, x 120000. Fig. 11. Different levels of Telmatoscopus spermatozoon, towards the posterior end. A is the same level as Fig. 10; B a more posterior level, lacking the mitochondrion; C is still more posterior, showing a narrower nucleus; in D the nucleus is reduced to a filiform central rod, and the section is occupied by the acrosome (a) and the sub- acrosomal material (s). x 37500. Fig. 12. Longitudinal sections of Telmatoscopus spermatozoon, showing the striation of the central axis of the acrosome (arrow), x 90000. Fig. 13. Posterior end of Telmatoscopus spermatozoa in cross-section. The 2 arms of the bifid tail are indicated by arrows, x 60000. Non-motile bifurcated sperm of Psychodidae 301 3

Fig. 14. Cross-section of the degenerating cytoplasmic region between 2 sperm bundles in Telmatoscopus. A crystalline array of microtubules is evident (cm). x 60000. Fig. 15. Cross-sections of the posterior region of Telmatoscopus spermatozoa after pepsin digestion. A strong action is detectable at the level of the acrosomal material (a). x 60000. Fig. 16. Cross-section of the anterior region of Telmatoscopus spermatozoa after pepsin digestion. The strongest action is detectable on the subacrosomal material (s) whereas the acrosome, here more compact, is resistant, x 60000. Fig. 17. Cross-sections of Telmatoscopus spermatozoa after treatment by Thiery's method. A positive reaction is shown by the outermost membrane, x 60000. Non-motile bifurcated sperm of Psychodidae 3°3

0-3>rn,j 16 I 17 304 B. Baccetti, R. Dallai and A. G. Burrini

Figs. 18, 19. Frozen-etched preparations of Telmatoscopus spermatozoa. The inner (ip) and outer (op) aspects of the smooth, indented plasma membrane are shown, as well as the inner (iam) and outer (oam) aspects of the acrosome membrane, x 60000. Fig. 20. Frozen-etched preparation of Telmatoscopus spermatozoa. The 5-nm wide cleft between plasma and acrosome membrane, showing the striations of the surface of the second one, is represented, x 120000. Non-motile bifurcated sperm of Psychodidae 3°5 306 B. Baccetti, R. Dallai and A. G. Burrini

Fig. 21. Section of the same spermatid stage, in which a mitochondrion (tn) is embedded in the acrosomal material (a) between nucleus (n) and Golgi cisterna (g). x 61 500. Fig. 22. Section of a multinucleate region of the spermatid cytoplasm in Telmatoscopus. At this level the nucleus (n) is surrounded by a Golgi cisterna (g), and an acrosomal body (a) interposed between the 2 organelles. x 61 500. Figs. 23-26. Stages of the condensation of chromatin (n) and acrosome (a) material in Telmatoscopus . In the cleft between nucleus and acrosome, the subacrosomal material {s) differentiates in the last stages. In Fig. 23, the Golgi origin of the acrosome is evident (arrow). Figs. 23-25, x 75000; Fig. 26, x 48000. Non-motile bifurcated sperm of Psychodidae 307

21

23 308 B. Baccetti, R. Dallai and A. G. Burrini

Fig. 27. The common Golgi complex from which the cisternae surrounding each nucleus of Telmatoscopus spermatids arise, x 48000. Non-motile bifurcated sperm of Psychodidae 310 B. Baccetti, R. Dallai and A. G. Burrini

Fig. 28. Cross-sections of different levels of Psychoda cinerea sperm bundles. A, an anterior level, showing only the acrosome (a); B, a more posterior level showing the filiform nucleus («), the mitochondrion (m) and the acrosome (a); C, the nucleus («) occupies the sperm central axis, surrounded by concentric layers of subacrosomal material (s) and acrosome (a), x 48000. Fig. 29. Cross-sections of Psychoda cinerea spermatozoa. Concentric layers of nuclear (n), subacrosomal (s) and acrosomal (a) material, x 75000. Fig. 30. Cross-sections of Psychoda alternata spermatozoa. The nucleus (n) is irregular in shape, and sends out a tenuous fin full of chromatin (arrow) deep inside the sub- acrosomal material (s). The acrosome (a) occupies the other part of the section, x 75000. Fig. 31. Cross-sections of Psychoda alternata spermatozoa after pepsin digestion. The subacrosomal material (s) and part of the acrosome (a) have been removed, x 96000. Fig. 32. Cross-sections of Psychoda sp. immature sperm. The nucleus («) lies in lateral position, flanked and partially surrounded by the acrosome (a). The new plasma membranes (p) appear still interconnected, and the microtubules (mi) are concentrated in the old spermatid cytoplasm, outside the sperm bodies. 60000. Fig. 33. Cross-sections of Psychoda sp. spermatozoa, in a stage immediately following that of Fig. 32. The section shape is reniform, and shows the nucleus (ri) on one side, and the large acrosome (a) on the other. The new plasma membranes are formed, and the microtubules (mi) lie outside the spermatozoa, x 120000. Non-motile bifurcated sperm of Psychodidae 311