Phylogenesis of spermatogenesis in Annulipalpia caddisflies: An ultrastructural analysis on philopotamidae spermiogenesis

JOURNAL

OF STRUCTURAL

BIOLOGY

105, 75-79

(1990)

Phylogenesis of Spermatogenesis in Annulipalpia Caddisflies: An Ultrastructural Analysis on Philopotamidae Spermiogenesis MICHAELFRIEDLANDER Department

of Biology, Received

Ben January

Gurion

AND

University

4, 1990,

and

RINA

ERENJEGER

of the Negeu, in revised

form

Beer June

Sheva

84105,

Israel

11, 1990

larvae (Henning, 1981). This lack of consensus in itself indicates that the morphological and behavioural characters of living Trichoptera that have been used for phylogenetic analyses are, evidently, insufficient to determine with certainty the status of several families within the order. To overcome these limitations, it will be necessary to resort to additional characters to complement the conventional ones that have already been scrutinized (Nielsen, 1989). One tool that could serve this purpose is comparative spermatology which has been widely used as a phylogenetic index in several animal taxa (Baccetti, 1979, 1989; Dallai, 1979; Hendelberg, 1977; Jamieson, 1987). In Trichoptera, however, almost nothing has been done in this field. Nevertheless, on the basis of the scarce published data, it has been previously proposed that Integripalpia species have retained the basic type of insect spermatozoa having axonemes with the usual pattern of 9 + 2 pairs of microtubules, while Annulipalpia have developed spermatozoa with different kinds of aberrant axonemes (Friedlander and Morse, 1982; Friedlander, 1983). One extreme case of such an aberration is found in Chimarra florida (Philopotamidae, Annulipalpial that has mature spermatozoa which lack any discernible axoneme at the ultrastructural level (Friedlander, 1983). The present ultrastructural analysis of the spermiogenesis of C. fZorida has been carried out to elucidate the process leading to the appearance of the axoneme-lacking spermatozoa in this species. The resulting data are compared with the corresponding information on other Annulipalpia families and used as a phylogenetic index to evaluate the position of Philopotamidae within the order.

Characters currently used in phylogenetic analyses are insufficient for determining the status of several families of the order Trichoptera (caddisflies). Comparative spermatology can contribute to solving this problem. In the suborder Integripalpia, the sperm axoneme displays the pattern of 9 + 2 pairs of parallel microtubules found in many animal species. In the suborder Annulipalpia, however, axonemes bear remarkable aberrations. Consistently, in Chimarra florida (Philopotamidae, Annulipalpia) we found that the number of central microtubules varies from zero to four in axonemes of the spermatids and that spermatozoa lack axonemes. We propose that, similarly, the axoneme pattern became unstable in the Annulipalpia ancestor, from which branched two phylogenetic lines having different kinds of axoneme aberrations: (a) families in which the number of central microtubules of the axoneme exceeds two (e.g., Philopotamidae, Polycentropodidae) and (b) families in which the number of central microtubules of the axoneme does not exceed two (e.g., Hydropsychidae). 8: Iwo Academic PESS, I~C.

INTRODUCTION

Caddisflies (Trichoptera) form a relatively small order of insects that are closely related to moths and butterflies (Lepidoptera). Most of the authors classify Trichoptera into two suborders, (a) Annulipalpia, which have lost the apical segment of each adult palp and that are retreat makers and (b) Integripalpia, which have retained the apical segment of each adult palp and which are case-tube makers. However, different authors delineate unequally the two systematic groups (e.g. Ross, 1967; Schmid, 1980). Moreover, on the basis of the use of different characters, other alternative phylogenetic schemes have been proposed (Weaver, 1984). One of the most important factors contributing to the disagreement on Trichoptera phylogenesis is that the fossil record, until now scarce and fragmentary (Shields, 19881, lacks many of the constitutive characters of the monophyletic subgroups that either are never visible in the fossilized adults or are present only in the

MATERIALANDMETHODS Chimarra florida (Philopotamidae, Annulipalpia) pupae were collected in the Piedmont region of South Carolina (USA). The testes were dissected out and immediately fixed in 2.5% glutaraldehyde in 0.3 M cacodylate buffer, pH 7.3. Subsequently, they were postfixed in 1% 0~0, in the same buffer. dehydrated through 75 1047.8477/90 $3.00 Copyright ((‘1 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

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FRIEDLANDER

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FIG. 1. Model of the spermatozoon of Chimarru florida showing that the nucleus is horseshoe-shaped in the middle of the cell and ring-shaped in its terminal portions. an ethanol series, and infiltrated trathin sections were contrasted citrate.

and embedded in Epon. with many1 acetate and

Ullead

RESULTS

The elongating spermatids of C. florida are fusiform, being bulky at the middle and tapering towards both ends. They show no division into the head and tail regions characteristic of the typical spermatozoa of insects and many other animals as well (Baccetti and Afzelius, 1976). The axoneme extends from the anterior tip, where the basal body is located, to the posterior end of the cell. Most of the axoneme is enfolded by the nucleus, except for a small portion, posteriorly, and the basal body, anteriorly. (See Fig 1). Therefore, transverse sections of the spermatids display nucleus and axoneme together, excepting those through both ends of the cell, where the nucleus is absent. Already in the early elongating spermatid (Figs. 2, 5, and 6) the nucleus surrounds the axoneme which remains connected only by a narrow cytoplasmic “corridor” to the bulk of the cytoplasm that surrounds the nucleus. The corridor may contain two to three microtubules running parallel to the axoneme. A likewise parallel row of microtubules is found adjacent to the nucleus, in the region diametrically opposite the corridor. The nuclear envelope is poorly defined and most of the chromatin displays low electron-opacity. However, close to the row of microtubules, the axoneme, and the corridor, the contour of the nucleus is clearly delineated by peripheral lumps of electron-opaque, condensed chromatin

EREN

JEGER

(Fig. 2). Although large portions of the nuclear envelope appear blurred in transverse sections, the annuli of the envelope are clearly seen in longitudinal, tangential sections of the nuclei (Fig. 6). A short electron-opaque spoke connects each peripheral microtubule doublet with the contiguous nuclear contour. The periphery of the axoneme is made of the customary nine pairs of parallel microtubules (doublets) characteristic of the basic model of this structure, as it is found in many animal species (Baccetti and Afzelius, 1976). Atypically, however, the number of central microtubules of the axoneme is inconstant and varies from 0 to 4 among spermatids which develop synchronously within the same cyst. In slightly later stages (Fig. 3), the enclosure of the axoneme by the nucleus becomes tighter, resulting in a keyhole configuration in transverse sections. The row of microtubules diametrically opposed to the corridor divides into two similar portions which remain separated by a continuously increasing gap. All the central microtubules have disappeared from the axoneme, which displays an “empty” lumen within the wall of nine microtubule doublets. Transverse sections through the posterior end of the spermatids are relatively small and contain lower numbers of microtubule doublets, identical to those of the axoneme. In the late spermatids (Fig. 4) the chromatin is mostly condensed, electron-opaque and located, as at earlier stages, mainly close to the row of microtubules, the axoneme, and the corridor. Electronopaque grains or spicules cover the surface of the cell. The nucleus of the mature spermatozoon (Fig. 7) contains only highly condensed, electron-opaque chromatin and is surrounded by a thin layer of cytoplasm displaying no visible organelles. The corridor remains open in the middle portion of the cell but closes up towards both ends. In transverse sections, therefore, the nucleus appears as a “V” where the corridor remains open, or as a ring where the corridor is closed. The interior of the “V” and the ring-shaped nuclei contains a floccular material. Neither a modified axoneme, nor any remains of vanishing axonemes could be found in sections of the mature spermatozoa (Fig. 8) despite a very careful search for them. The cell surface is covered by short

FIG. 2. Transverse sections of early elongating spermatids. Axoneme displaying either zero (01, three (31, or four (4) central microtubules. The nucleus (N) covers the axoneme except for a narrow “corridor” (C) containing a few microtubules. A row of microtubules (M) is adjacent to the nucleus. Lumps of electron-opaque chromatin delineate the nuclear envelope close to the axoneme, corridor, and row of microtubules; other portions of the envelope are indistinct. Spokes connect the axoneme doublets to the nuclear envelope (arrows). Sections through the terminal portions of spermatids (T) display a few doublets of incomplete axonemes that are associated with nuclear envelope-like material. x 36 000. FIG. 3. Transverse sections through spermatids, later than in Fig. 2. Axonemes lack central microtubules. The row of microtubules (Ml is subdivided into two portions. x 36 000. FIG. 4. Transverse sections through late spermatids. Axonemes lack central microtubules and nuclei (N) contain mainly condensed chromatin. The cell is covered by short spicules. Note connections between axonemes doublets and nuclear envelope. T-sections through terminal portions of the spermatids. x 36 000.

ANNULIPALPIA

CADDISFLIES

SPERMIOGENESIS

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FIGS. 5 AND 6. Longitudinal sections through early spermatids, as in Fig. 2. The basal body (B) is located at the anterior tip of the cell and the nucleus (N) covers the axoneme. (Figure 5.) Axonemes showing central microtubules either present (Xl) or absent (X0). x 12 000. (Figure 6.) A tangential section of a nuclear envelope shows nuclear annuli. x 18 000. FIG. 7. Transverse sections through different levels of spermatozoa. Middle section shows horseshoe-shaped nuclei and terminal sections show annular nuclei. x 23 000. FIG. 8. Transverse sections through terminal portions of spermatozoa. Axonemes are absent and short spicules cover the cell. x 36 000.

radial projections in transverse sections (Fig. 8) which most probably form ridges in longitudinal sections (Fig. 1). DISCUSSION

In the present work, we found that the spermatozoa lacking axonemes of C. florida develop from spermatids which do have axonemes. The axonemes, however, are aberrant, displaying in cells developing synchronously a variable number of centrally located microtubules (up to four). This is in accordance with the earlier proposition that Annulipalpia spermatozoa have aberrant axonemes, in contrast with those of Integripalpia which display the cus-

tomary 9 + 2 pairs of microtubules found in many animal species (Friedlander and Morse, 1982). In other words, axoneme aberration appears to be a synapomorphous character of Annulipalpia. The aberrations, however, differ remarkably among different families of the order, a situation that could be explained as follows. The genetical apparatus controlling development of the axoneme, and in particular that of its central microtubules, became unstable in the common ancestor, at the ba’se of the Annulipalpia branching, in a manner similar to that which we found in early spermatids of Philopotamidae. From this common ancestor showing axoneme lability, two phylogenetic lines may have branched.

ANNULIPALPIA

CADDISFLIES

In one of them, the number of central microtubules has increased from two to three or four. This type of axoneme is found today in Polycentropodidae (Friedlander, 1983) which have three central microtubules in the axoneme and in the Philopotamidae spermatids which show up to four central microtubules, as reported here. In the Philopotamidae, therefore, the disappearance, first of the central microtubules in late spermatids and eventually of the peripheral microtubules of the mature spermatozoa is an autapomorphy derived from Annulipalpia having axonemes with more than two central microtubules. This type of axoneme evolution is not restricted to Annulipalpia caddisflies. Another similar case of convergent phylogenesis has been reported for homopterous insects. Baccetti and Dallai (1977) found out (1) that the lower homopteran families display the motile plesiomorph spermatozoon characterizing insects with the conventional 9 + 2 axoneme, (2) that Coccidae, the highest family of the suborder, has aberrant spermatozoa which lack any axoneme but which, in spite of that, move by an alternative autapomorphous mechanism involving microtubules, and (3) that Coccidae motile spermatozoa apparently derive from the immotile spermatozoa of Aleyrodidae, which is the family immediately preceding Coccidae in homopteran phylogenesis. Aleyrodidae spermatozoa are aflagellate but, nevertheless, derive ontogenetically from spermatids which do have a transient axoneme. The other line of Annulipalpia evolution is represented by Hydropsychidae, in which there is no increment in the number of the central microtubules of the axoneme (Friedlander and Morse, 1982). In Hydropsychidae, although no microtubules could be found in the lumen of the axoneme throughout spermiogenesis, the central microtubules do exist. However, they are displaced from their original central position and intermixed with the peripheral doublets within the wall of a funnel-like structure which projects backwards from the basal body

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79

(Friedlander and Morse, 1982, their Fig. 12). The disappearance of the central microtubules, therefore, has followed in Philopotamidae a very different pattern from that occurring in Hydropsychidae. Therefore, our data might indicate a more close phylogenetic relationship between Philopotamidae and Polycentropodidae than that previously suggested (Ross, 1967; Schmid, 1980; Weaver, 1984). We thank Dr. John C. Morse (Department of Entomology, Clemson University) for identifying and supplying the insects, and Mr. Gideon Raziel for the skillful printing of the micrographs. This work was partially funded by a Clemson University Alumni Visiting Professorship to M.F. REFERENCES BACCETTI, B. (1979) in GUPTA, A. P. (Ed.), Arthropod Phylogeny, pp. 609-644, Van Nostrand Reinhold Co., New York. BACCE~I, B. (1989) J. Suhmicrosc. C’ytol. Pafhol. 21, 397-398. BACCEWI, B., AND AFZELIUS, B. A. (1976) Monogr. Dev. Biol. 10, l-254. BACCETTI, B., AND DALLAI, R. (1977) J. IJltras/ruct. Res. 61, 260270. DALLAI, R. (1979) in FAWCEW, D. M., AND BEDFORD, J. M. (Eds.), The Spermatozoon, Urban and Schwarzenberg. Baltimore. FRIEDLANDER, M. (1983) J. Ultrustruct. Res. x3, 141-147. FRIEDLANDER, M., AND MORSE, J. C I 1982) J. liltrastruct. Res. 78, 84-94. HENDELBERG, J. (1977) Acta 2001. Fenn. 154, 149-162. HENNING. W. (1981) Insect Phylogeny, pp. 407, Wiley, Chichester. JAMIESON, B. G. M. (1987) Ultrastructure and Phylogeny of Insect Spermatozoa, University Press, Cambridge. NIELSEN, E. S. (1989) in. FERNHOLM, B., BREMER, K., AND JORNVALL, H. (Eds.), The Herarchy of Life. pp. 281-294, Elsevier Science Publishers, Amsterdam. ROSS, H. H. (1967) Annu. Rev. Entomol. 12, 169-206. SCHMID, F. (1980) Genera des Trichopteres du Canada et des Etats Adjacents. Les Insects et Arachnides du Canada, pt. 7, Agriculture Canada, Ottawa. SHIELDS, 0. (1988) J. Paleontol. 62, 251-258. WEAVER, J. S. III (1984) in MORSE, J. C. (Ed.,. Proc. 4th Int. Symp. Trichoptera. pp. 407419, Dr. W. ,Junk Publishers, The Hague.