Characteristics of the motile spermatozoa in 5 species of gall-midges (Diptera: Cecidomyiidae)

hll..I hTst,(l*loJ7~jtol.~ Iz/~tt~rro/..Vol.9. pp. 383 to 393. ~) PergamonPressLtd. 1980 Printed in Great Britain.

CHARACTERISTICS

0020 7322 80 12(11 I)38~S02.000

OF THE MOTILE SPERMATOZOA

5 SPECIES OF GALL-MIDGES

IN

(DIPTERA: CECIDOMYIIDAE).*

ROMAN() DALLAI a n d MASSIMO M A Z Z I N I Institute of Zoology. University of Siena. 53100 Siena. Italy

(Ac('epted l3 Mar 1980) Abstra,:t The gall midges (Cecidomyiidae) exhibit unusual sperm models and only the species with a giant axoneme have motile spermatozoa. To test if the sperm morphology agrees with the systematics of the family, the spermatozoa of 5 species of gall midges ( Cecidom)'ia pini, Br~luerielht phill3're~e, My('odiplo.~is sp.. Aphido/ctes (Phaenobremia) sp. and Ametr(,diplosis th(dictricol(O have been studied with the transmission electron microscope. C o m m o n features are: absence of an acrosome: absence of accessory elements in the axoneme; mitochondria not fused and so not transformed into mitochondrial derivatives; giant centrioles and axonemes with a large number of doublets: temporary motility. The chromatin condensation, the doublet number, consequent shape of the axoneme, and the location of the mitochondria along the sperm vary in the different species examined, and may serve as taxonomic characters. The possible origin of new doublets in giant axoneme is discussed.

Index descriptors (in addition to those in title): Sperm structure, flagellar axoneme, microtubular doublets, giant centriole, microtubular dynein arms. Cecidomyiidae, transmission electron microscopy. INTRODUCTION

THE GALLMIDC;ES(Cecidomyiidae) exhibit unusual sperm models (Baccetti and Dallai, 1976: Dallai, 1979). In this family only the species belonging to the ~Sciara-like" model have motile spermatozoa, mainly characterized by a giant axoneme. The aim of this paper is to see whether this kind of sperm model is present in a large number of species, and whether the sperm morphology is in agreement with traditional phylogenetic classification of the group. MATERIALS

AND METHODS

The following ,;pecies were studied: adult of Cecidollo,ia pini (De Geer) from larvae found in cocoons of Pitlus .vilvestris (Pinaceae) collected in Sweden (Uppland, Dandcryd, May 1979 and Bogesund, Stensveten, June 1979): Brauer.iella phil!t'~eae Ft. Lw. reared from larvae in galls on leaves of Phillyrea angustg2)/ia (Oleaceae) collected in Elba island, Livorno, Italy, May and September 1976); m)'('odip/osi.~" sp. feeding on rust of Alchemilla leaves. Sweden (Upplanc[, Danderyd, 17.6.1979); Aphidoletes (Phaenobremia) sp., bred from heads of Tr(/olium pratense (Leguminosae), Sweden (Nijucdly, June 1979); Ametro(liphzs'is tha/ictricola (Rubs.) on Thalictrum [telvum (Ranunculaceae), Sweden (Sodermanland, Uto (Kroka), 14.6.1976). In all cases the material was maintained at constant humidity until hatching occurred. Male and female adults were dissected ia Hoyle buffer. Testes, spermiducts and spermathecae were removed and fixed in 4'f,, paraformaldehyde and 5'~;iglutaraldehyde at pH 7.2 in cacodylate buffer for 1 hr at 4°C according to Karnovsky's method (1965). The material was then rinsed overnight in cacodylate buffer, postfixed in 1(',, osmium tetroxide for 1 hr, dehydrated in a graded ethanol series and embedded in Epon 812 and Epon-Araldite. Sections were cut either with an LKB II ul :ratome or with a Reichert OM U2, mounted on grids, stained with uranyl acetate and lead citrate. and examined in a Philips EM 300 and 301. OBSERVATIONS

Spermatozoa from the 5 species of gall midges examined have many features in common: the absence of an acrosome, a great number of axonemal doublets, each one with outer arms only, and normal mitochondria instead of mitochondrial derivatives: we shall describe only the specific va:'iations of the different components. * Research perform-ned under C.N.R. project ~'Biology of Reproduction". 383

384

ROMANO DALLAI and' MASSIMOMAZZINI

In general, the spermatozoa are filiform, 100-350 pm long (Figs. I, 12), with an elongated extremity, containing the nucleus. In all species, the cell membrane has the typical asymmetrical structure. Ametrmhp/osis thalictricola, however, has a dense 10nm-thick glycocalyx around it (Figs. 6, 7). The anteriorly located nucleus usually shows a uniform condensation of the chromatin material in the form of 2 or 3 masses. Only in Aphidoletes (Phaenohremia) sp. is chromatin organized in peripheral globular formations (0.15 IJm in diameter), surrounding darker homogeneous granular material (Fig. 10). All the cecidomyiid spermatozoa have an unusual giant centriole. This organelle has been carefully studied only in Mycodiplosis sp., because in this species the centriole is smaller than in any other cecidomyiid, so that observation is facilitated at a practical level. In cross section, it consists of an inner cylinder about 0.25 tJm in diameter with a dense wall of fine granular material (Figs. 2, 4), 30 nm-thick. About 18 doublets, instead of triplets, encircle the cylinder in a helical arrangement (Figs, 2, 4). A dense projection from the cylinder wall separates the doublets from one another. Each doublet has a circular A subfiber, about 18 nm in diameter, and C-shaped B subfiber. No arms are present on the doublets, and neither are feet directed toward the cylinder (Figs. 2, 4). In longitudinal sections the cylinder is very short, about 0.1 pro, and the axoneme arises from the electrodense material (Figs. 3,

10. 131. The axoneme is formed by a variable number of longitudinal doublets. Mycodiplosis spermatozoa, in cross section beneath the nucleus, show about 18 doublets, spaced 20 nm, in a regular elliptic alignment (Fig. 5). Farther back, the doublets increase in number to 40. At this level, the crown of doublets is situated near the cell membrane and the mitochondria, external to the axoneme in the anterior sections, are gathered in the centre of the sperm. Just beneath the centriole, along with the axoneme, isolated doublets without arms and singlet microtubules are visible at the periphery of the cell. Serial sections confirm the absence of dynein arms along the doublet (Fig. 5). A few microtubules in a regular row are also found between 2 laminae of the plasma membrane that project from the cell. At the posterior end the doublets are irregularly arranged and reduced in number. B. philO,reae and Aphidoletes (Phaenobremia) sp. have a sperm axoneme with 50-70 doublets located in single row at the cell periphery along the scalloped plasma membrane (Figs. 11, 14). Posteriorly, when the sperm section becomes smaller but the doublet number is nearly the same, 10 15 doublets, always in a row, migrate toward the centre (Fig. 14). The Cecidomyiapini sperm axoneme has at the anterior region a single row of about 70 80 doublets beneath a scalloped plasma membrane (Figs. 8, 9). Along the tail, with the progressive reduction of the sperm diameter, the doublets forma spiral of I 4 layers (Fig. 8). In some sections of the anterior region of the tail, a cluster of 10-15 isolated microtubules breaks the continuity of the doublet row (Fig. 9). The Ametrodiplosis thalictricola sperm tail has about 100 doublets ranged in a few parallel rows. Anteriorly, the axoneme is centrally located and surrounded by several mitochondria (Fig. 6); posteriorly, it occupies the whole cytoplasm (Fig. 7). At this level, the doublets of the peripheral row are nearly parallel to the cell membrane and the B-fibers are linked with the inner membrane leaflet (Fig. 7). The inner doublets are twisted and their arms make contact with the B-fibers of the doublets of the adjacent row (Fig. 7). The mitochondria, which never fuse to form mitochondrial derivatives, always have long, distinct cristae but no inner crystalline material. In Cecidomyia pini, Aphidoletes (Phaenobremia) sp. and Braueriella phillyreae, they are distributed all along the cytoplasmic cylinder and surrounded by the axonemal doublets (Figs. 8, 9, 11, 14). In Mycodiplosis sp. they have a peripheral localization in the subnuclear region (Fig. 5), while they are found

Characteristics of the Motile Spermatozoa

Fir,;. 1. Living Mycodiplosis sperm photographed with interference contrast microscope, x 500. F1G. 2. Cross section of Mycodiph)sis sperm in subnuclear region showing centriole. A dense ring is encircled by helically arranged microtubular doublets, x 136,000. FI(;. 3. Longitudinal section of centriolar region. Axoneme (ax) originates from dense patches beneath nucleus (N) which correspond to centriolar ring (arrows). x 47,500. FI(;. 4. Cro~s section of centriole in a slightly posterior level compared to Fig. 2: helical doublet arrangement is still evident. × 136,000.

385

386

P, OXl \N~) DAI l \ l and MASSIMO MAZZINI

internal to the axoneme toward the posterior end. Lastly, in Ame;rodil?losis thalictricola numerous mitochondria are concentrated in the postnuclear region, where they encircle the beginning of the axoneme (Fig. 6): none is present along the tail (Fig. 7). It is interesting to point out that the duration of sperm motility seems inversely correlated with the number of doublets in the axoneme. Mycodq?losis spermatozoa which have fewer doublet microtubules with respect to other species, appear to be motile for a longer period (a few minutes) than the previously studied Dildo/ahoncus sperm.

2

FIe;. 5. Cross section of .~l.vcodildo~is sperm tail at anterior lex.el. A x o n e m e (axl is s u r r o u n d e d b> m i t o c h o n d r i a (m). A few m i c r o t u b u l e s (m/) and 2 donblcls without arms are present beneath p l a s m a m e m b r a n e (IIITOWS). [11 c o r n e r is a consecuti\ e cross section showing a doublet still ~,~ithont a r m s (arro\~). × I I(l.000.

DISCUSSION

Data concerning the species described in this work confirm the peculiar features of cecidomyiid sperm: absence of an acrosome, normal mitochondria instead ofmitochondrial derivatives, and absence of classical accessory structures in the axoneme. They also confirm that the spermatozoa belonging to the "Sciara-like" rnodel have a great number of doublet axoneme with only outer arms and are motile in the female spermatheca. The giant axoneme arises from a giant centriole which recalls that of Sciara in many aspects, and once more emphasizes the strong similarity of the odd spermatogenetic behavior of the 2 families: Sciaridae and Cecidomyiidae. As in Sciara (Phillips, 1966a, b: 1967) and in Acerentomidae (Protura) (Baccetti et al.. 1973). the centriole of the cecidomyiids studied here is aberrant, having doublets, instead of triplets, embedded in granular dense material. Doublets in the centriole were also observed by Cotelli ~,z al. (1975) in /he spermatozoa of the isopodan Porce[lio laevis but in this case the feet proiec/from the doublets toward thc center.

Characteristics of the Motile Spermatozoa

FIG. 6. Cross section of .4melrodiplo.;is sperm tail in subnuclear region. Mitochondria (m) encircle giant ax~neme which is formed by doublets having only outer arms (arrows). x 102,000. FIG. 7. Cross section of" dnTetrodil~lo~'i.~' sperm tail in posterior region: plasma membrane shows a thick glycocalyx (gly). Doublets have only outer arms; those of peripheral row contact cell membrane (arrows) while inner ones are twisted, x 170~000.

387

388

ROSIANO D,x.i,l,,\l and MASSIMO MAZZIX.I

Fl(;. 8. Cross section of Cecido;;n'ia sperm tails at dilt"erent levels. A x o n e m c doublets form a spiral of a few coils s u r r o u n d i n g m i t o c h o n d r i a (ml. x 62,500.

Characteristics of the Motile Spermatozoa

Fro, 9. Cros~ section of Cecidomvia sperm axoneme in anterior region. Doublets in a single row are orderly localed beneath scallopect plasma membrane, Cluster of isolate microtubules are also visible (arrow). x 69,000. FIG. 10. Longitudinal section of Aphidoletes sperm. Nucleus (N) has densely packed chromatin in center, while globular formations are present at periphery. Axoneme (ax) originates beneath nucleus, x 27,500. FIG. 11. Cross section of Aphidoletes sperm. Axoneme doublets are in a single row at periphery of the cell. x 62,500

389

390

ROMANO DALLAIand MASS1MOMAZZINI

.....

1

F~G. 12. Live Br(,ueriellasperm photographed with interference contrast microscope, x 500. FIG. 13. Longitudinal section of Braueriella sperm showing elongate nucleus (N}, centriolar region (cr) and axoneme (ax). × 29.000. FIG. 14. Cross section of B;'(,ueriella sperm tails showing mitochondria (m) and axoneme doublets beneath scalloped plasma membrane, x 62,500. C o n s i d e r i n g t h e n u m b e r o f d o u b l e t s p r e s e n t in t h e a x o n e m e o f t h e s e s p e r m a t o z o a , it is c l e a r t h a t a p r o g r e s s i v e i n c r e a s e o f t h e d o u b l e t n u m b e r , s t a r t i n g f r o m Asphondylia a n d M vcodiplosis t o w a r d Diploloboncu& exists ( D a l l a i , 1979). I n t h e first 2 species, t h e d o u b l e t

Characteristics of the Motile Spermatozoa

391

number, in f~Lct, is about 40; in Braueriella and Aphidoletes (Phaenobremia) it is 50-70; in Cecidomyia 80 and about 100 in Ametrodiplosis and Clinodiplosis. In Monartropalpus (Baccetti eta,!., 1974) the number is 170, and finally in Diplolaboncus (Baccetti and Dallai, 1976) with about 1000 doublets, we have the highest number of doublets till now discovered in a sperm flagellum. Does this indicate that sperm evolution works in the direction of an increasing do Jblet number or does it simply represent an orthogenetic mechanism leading to a different type of sperm movement? The analysis of sperm movement may provide some useful information. As to the sperm motility, it is known that the sliding displacements between adjacent doublet microtubules of the axoneme result from the direct participation of ATPase dynein, which resides in the 2rows of arms along each doublet microtubule (Gibbons and Gibbons, 1972: Gibbons, 1975; Warner, 1976). In cecidomyiid spermatozoa belonging to the "Sciara-like" model only one arm is present on the A-doublet tubule and, in many cases, it is clearly directed toward the B-tubule of the adjacent doublet row to connect the 2doublets. This single arm, according to Baccetti et al. (1979), contains an enzymatic protein similar to the electrophoretlc A-band of the dynein from the sea urchin spermatozoa. Sperm motility in these spermatozoa is acquired only when the sperm are within the female ducts where they are active only for a short time. Moreover, a reduction of sperm motility seems connected with the increased number of doublets: Mycodiplosis spermatozoa apparently are more active than those of Diplolaboncus. All this suggests that for sperm motility, only one dynein arm is sufficient but a coordinated and prolonged movement needs the presence of the other axonemal components, radial spokes, central sheath and interdoublet links, which are intimately related to the sliding bending mechanism (Warner, 1976). In common flagella, the absence of these components, however, does not permit the formation of a bend even though they can undergo normal sliding (Witman et al., 1976). All the accessory axonemal structures, as we have seen, are absent in the cecidomyiid spermatozoa, but they are able to beat their flagella just the same. Nevertheless, it is extremely difficult for the giant axoneme to maintain or restore an orderly arrangement of their doublets after the first flagellar beats. The observation that sperm motility can be reactivated by pressure on the coverslip is very similar to that described by Mooseker and Tilney (1973) in the axostylar motility of Cryptocercus: it suggests that the flattening of the sperm allows for the association of doublets of adjacent rows and is necessary for the initiation of sperm movement. Another interestin~ point deserving of comment is the presence of isolated doublets and singlet microtubules along the tail at the periphery of the cell. Their presence may explain the increased number of doublets in the tail with respect to the number of doublets in the centriole. It has already been suggested in a previous paper on Mayetiola that there is assembly of ~:ubulin starting from single microtubules (Dallai and Mazzini, 1980)~ thus giving evidence of the possible formation in vivo of new axonemal doublets, commonly obtained in vitro (Binder and Rosenbaum, 1978). As to the organization of the doublet dynein arms in these kinds of spermatozoa which, contrary to Mayetiola, have sperm motility it is to be remembered that in other cells, bulks of major axoneme proteins, tubulin and dynein, pre-exist in relative abundance in the cytoplasm even though they are not appreciably leLbelled (Stephens, 1977; Kobayashi et al., 1978; Pratt, 1980). A similar situation may be suggested for the dynein assembly on the new doublets of the cecidomyiid sperm axoneme. Nevertheless, 2 main factors are necessary: the presence of the protein and the presence of specific sites for its attachment to the tubulin (Shimizu, 1975; Gibbons, 1976: Gibbons and Gibbons, 1976). These 2 conditions may be present in all the "Sciara-like"

392

ROMANO DALLAI and MASSIMOMAZZIN1

cecidomyiid spermatozoa, but one or both of them may be missing in Mayetiola, as well as in the immotile spermatozoa of the family. From taxonomic point of view, our data agree well with the traditional systematics of the group. The 5 species examined belong to the same supertribe Cecidomyiidi (Harris, 1966) and demonstrate nearly the same sperm model. The specific variations we have observed may just represent new systematic characters useful for gall midge identification at generic level. The previously studied Asphondylia sperm (Baccetti and Dallai, 1976) shows an axoneme of the same pattern as the species here described: nevertheless this species belongs to the different supertribe Asphondyliidi. This could indicate a close relationship between the 2groups. Both these supertribes are, however, separate from Oligotrophidi and Lasiopteridi which exhibit an immotile sperm model (Baccetti and Dallai, 1976). Acknowledgements We thank Prof. E. Sylv6n (Swedish Museum of Natural History, Stockholm. Sweden) and Prof. M. Solinas (Istituto di Entomologia Agraria, University of Bari, Italy) for their generous help in classifying the species described in this work.

REFERENCES BACCETTI, B. and R, DALLAI. 1976. The spermatozoon of Arthropoda XXVll. U n c o m m o n axoneme pattern in different species of the Cecidomyiid flies. J. Ultrastruct. Res. 55: 50-69. BACCETTI, B., R. DALLA1and B. FRATELLO. 1973. The spermatozoon of Arthropoda XXlI. The "12 + 0", '" 14 + 0"" or aflagellate sperm of Protura. J. cell Sei. 13:321--35. BA( ('ETTI, B., R. DALLAI, f . GIUSTI and F. BERNINI. 1974. The spermatozoon of Arthropoda XXl I 1. The "9 + 9 + 3" spermatozoon of simuliid Diptera. J. Ullrastruct. Res. 46: 4 2 7 4 0 . BACCETTI. B., A. G. BURRINI, R. DALLAI and V. PALLINI. 1979. The dynein electrophoretic bands in axonemes naturally lacking the inner or the outer arm. J. cell Biol. 80: 33~40. BINDER, L. I. and J. L. ROSENBAUM. 1978. The in vitro assembly of flagellar outer doublet tubulin. J. cell Biol. 79: 500 15, COTELLL F., M. FERRA(;UTI and C. LORA EAMIADONIN. 1975. An unusual centriolar pattern in lsopoda sperm cell. J. Submierose. CvtoL 7:289 92. DALAI, R. 1979. An overview of atypical spermatozoa in Insects, pp. 253 65. h2 D. W. Fawcett and J. M. Bedford (eds.) The Spermatozoon, Urban & Schwarzenberg, Inc. Baltimore, Munich. DALLAi, R. and M. MAZZINI. 1980. Microtubular doublets in a gall midge (Insects, Diptera) and evidence for their assembly. J. Uhrastruet. Res. 70:363 8. GmBONS, B. H. and I. R. GIBBONS. 1972. Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with triton X-100. J. cell Biol. 54:75 97. GIBBONS, B. H. and 1. R. GIBBONS. 1976. Functional recombination of dynein 1 with demembranated sea urchin sperm partially extracted with Kcl. Biochem. Biophys. Res. Commun. 73:1 6. GIBBONS, I. R. 1975. The molecular basis of flagellar motility in sea urchin spermatozoa, pp. 207 31. h7 S. lnoue and R. E. Stephens (eds.) Molecules and Cell Movement, Raven Press, New York. GIBBONS, 1. R. 1976. Structure and function offlagellar microtubules, pp. 348-57. In B. R. Brinkley and K. R. Porter (eds.) h2ternational Cell Biology. Rockefeller University Press, New York. HARRlS, K. M. 1966. Gall-midge genera of economic importance (Diptera: Cecidomyiidae). Part 1: Introduction and subfamily Cecidomyiidae; supertribe Cecidomyiidi. Trans. R. Entomol. Soc. Lond. 118:313 58. KARNOVSKY,M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. cell BioL 27: 137A-38A. KOBAYASm, Y.. K. OGAWA and H. MOrIRI. 1978. Evidence that the Mg-ATPase in the cortical layer of sea urchin egg is dynein. Exp. Cell Res. 114:185 92. MOOSEKIim M, S. and L. G. TILNf~Y. 1973. Isolation and reactivation of the axostyle. Evidence for a dynein-like ATPase in the axostyle. J. cell Biol. 56:13-26. Pnu LIPS, D. M. 1966a. Obsetlvations on spermiogenesis in the fungus gnat Seiara eoprophila. J. cell Biol. 30: 477--97. Pnlt HPS. D. M. 1966b. Fine structure ofSciara coprophila sperm. J. cell Biol. 30:499 5t7, PHII.LIPS, D. M. 1967. Giant centriole formation in Sciara. J. cell Biol. 33:73 92. PRATT, M. M. 1980. The identification o f a dynein ATPase in unfertilized sea urchin eggs. Dev. Biol. 74:364 78. SmMlZt!, T. 1975. Recombination of ciliary dynein of Tetrahymena with the outer fibers. J. Bioehem. 78: 41--9. SIEPnEr
Characteristics of the Motile Spermatozoa

393

WARNER, F. D. 1!)76. Cross-bridge mechanisms in ciliary motility: the sliding-bending conversion, pp. 891 914. hl R. Goldman, T. Pollard and J. R o s e n b a u m (eds,) Cell Motility, Microtubules and related proteins. Cold Spring Harbor Laboratory Press, New York. WITMAN, G. B., R. FAY and J. PLUMMER. 1976. Chlamydomonas mutants: evidence for the roles of specific axonemal components in flagellar movement, pp. 969-986. In R. Goldman. T. Pollard and J. Rosenbaum (eds.) Cell Motili 0 . Microtubules and related proteins. Cold Spring Harbor Laboratory Press, New York.