Comparative Spermatology in Plecoptera (Insecta)
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Arthropod Structure & Development 31 (2002) 147–156 www.elsevier.com/locate/asd Comparative spermatology in Plecoptera (Insecta). II. An ultrastructural investigation on four species of Systellognatha A.M. Faustoa,*, M. Belardinellia, R. Fochettia, J.M. Tierno de Figueroab, M. Mazzinia aDipartimento di Scienze Ambientali, Universita` della Tuscia, Largo dell’Universita`, 01100 Viterbo, Italy bDepartamento de Biologia Animal y Ecologia, Universidad de Granada, 18071 Granada, Spain Received 22 January 2002; accepted 6 June 2002 Abstract Sperm structure of four Systellognatha species, Dinocras cephalotes (Curtis, 1827) and Perla grandis (Rambur, 1841), family Perlidae, Siphonoperla torrentium (Pictet, 1841), family Chloroperlidae, and Isoperla grammatica (Poda, 1761), family Perlodidae, was investigated by light microscopy, scanning and transmission electron microscopy, and immunofluorescence microscopy. The spermatozoa of all the species are filiform and flagellate; they have an elongated compact nucleus, topped by either a bi-layered or three-layered acrosomal complex, and a ‘9 þ 9 þ 2’ axoneme flanked by two mitochondrial derivatives. However, some variations have been observed, particularly regarding the morphology of the acrosomal complex, the shape of the nucleus, two more or less evident accessory bodies flanking the axoneme and the degree of crystallisation of the mitochondrial derivatives. Our results support the suggestion that Systellognatha is a monophyletic group, showing a single sperm pattern. Chloroperlidae and Perlodidae retain the plesiomorphic condition of some sperm characters while Perlidae show some autoapomorphies. A phylogenetic affinity between Plecoptera and the other orders of Polyneoptera has been confirmed. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Stoneflies; Sperm ultrastructure; Insect phylogeny; Immunofluorescence; Scanning and transmission electron microscopy 1. Introduction phylogenetic evidence has been substantiated for several insect taxa (Baccetti, 1979a, 1996; Burrini et al., 1988; The order Plecoptera is an old insect group (Permian Friedla¨nder, 1983; Carcupino et al., 1995; Fausto et al., fossils) with very ancestral characters. Thus, it has some- 1995; Dallai et al., 1996; Jamieson et al., 1999). In times been proposed as a key group for the understanding of Plecoptera, the current knowledge is scarce and limited to Neoptera evolution (Zwick, 2000). two papers regarding Euholognatha, one of the two groups Although Plecoptera phylogeny has been widely into which the Arctoperlaria (northern hemisphere stone- studied (Nelson, 1984; Zwick, 2000), there are still flies) are traditionally divided. In the first paper the sperm of several points that need to be clarified through the Nemoura cinerea (Retzius, 1783) (family Nemouridae) was acquisition of a wide range of additional characters, as described (Baccetti et al., 1970); later it was hypothesised pointed out by Nelson (1984). Such information would that Plecoptera are related to the Embioptera, especially help us to infer the phylogenetic relationships among the because of the absence of a perforatorium (Baccetti, 1987). families and the relationships between Plecoptera and the Recently, another study concerned some Euholognatha other orders of insects. species: Taeniopteryx kuehtreiberi Aubert, 1950, T. stanko- Sperm structure and functions differ greatly in Insecta vitchi Ikonomov, 1978, Brachyptera risi (Morton, 1896), (Baccetti, 1979a,b, 1996; Dallai and Afzelius, 1993; family Taeniopterygidae; Leuctra fusca (Linneo, 1758), Jamieson et al., 1999). This makes comparative spermato- family Leuctridae (Fausto et al., 2001). The spermatozoa of logical studies potentially useful in resolving systematic these species showed a certain degree of heterogeneity, questions and inferring phylogenies among and within displaying different spermatic patterns in the three families. insect orders. The use of these studies to provide Moreover, a close phylogenetic affinity between Plecoptera and Polyneoptera was suggested, while the previously * Corresponding author. Tel.: þ39-761-357044; fax: þ39-761-357179. suggested relationship between Plecoptera and Embioptera E-mail address: [email protected] (A.M. Fausto). (Baccetti, 1987; Jamieson et al., 1999) was not confirmed 1467-8039/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S1467-8039(02)00024-5 148 A.M. Fausto et al. / Arthropod Structure & Development 31 (2002) 147–156 (Fausto et al., 2001). Thus, it is important to study the sperm facilitate the penetration of the complying agent, then rinsed structure of Systellognatha, the other group of Arctoperlaria three times in PBS. Sperm were stained with the nuclear dye stoneflies, to shed light on the phylogenetic relationship Hoechst 33258 at 10 mg/ml in PBS for 5 min. F-actin between Euholognatha and Systellognatha and to infer a filaments of perforatorium were stained for 30 min at general Arctoperlaria model within Plecoptera spermatozoa. room temperature with Rh–phalloidin (Molecular probes, Moreover, the phylogenetic relationships within the super- Eugene, OR, USA) diluted 1:100 with PBS. Sperm were family Perloidea (a subgroup of Systellognatha including then rinsed in PBS, mounted in 90% glycerol containing the three families Perlodidae, Perlidae and Chloroperlidae) 2.5% n-propylgallate to reduce photobleaching and observed are still not clear (Zwick, 2000). with a Zeiss Axiophot epifluorescence microscope. The aim of the present research is to describe the sperm ultrastructure of representatives of Systellognatha stonefly 2.3. Scanning electron microscopy species in order to acquire further information about the systematics and phylogenetic relationships within the order For scanning electron microscopy (SEM), reproductive and in relation to other orders of insects. To achieve this apparatus from adult males were dissected and free goal, we analysed the spermatozoa of four species spermatozoa were removed and fixed in 4% paraformalde- representing the three European families of this group: hyde 5% glutaraldehyde buffered with 0.1 M sodium Dinocras cephalotes (Curtis, 1827) and Perla grandis cacodylate (pH 7.2) for 2 h at 4 8C, rinsed overnight in the (Rambur, 1841), family Perlidae, Siphonoperla torrentium same buffer, post-fixed in 1% OsO4 in the same buffer for (Pictet, 1841), family Chloroperlidae, and Isoperla gram- 1 h at 4 8C, dehydrated in a graded ethanol series (50– matica (Poda, 1761), family Perlodidae, by means of light 100%) critical-point dried with CO2, gold–palladium microscopy, scanning and transmission electron micro- coated and observed with a JEOL JSM 5200 scanning scopy, and immunofluorescence microscopy. electron microscope. 2.4. Transmission electron microscopy 2. Materials and methods For transmission electron microscopy (TEM), specimens The specimens used in this study were collected by net. fixed and dehydrated as above were embedded in Epon– Samples were collected from November 1996 to May 2000. Araldite mixture at 60 8C for 2 days. Thin sections were cut Hereafter we report sites and dates of sampling. with the LKB Nova and Reichert Ultracut ultramicrotomes, Siphonoperla torrentium—Italy, Umbria, River Nera, stained with uranyl acetate and lead citrate and observed Castel Sant’Angelo sul Nera (MC), 22.VI.2000. with a JEOL 1200 EX II transmission electron microscope. Isoperla grammatica—Italy, Umbria, River Nera, Castel Semi-thin sections were stained with toluidine blue and Sant’Angelo sul Nera (MC), 22.VI.2000. observed with an Axiophot Zeiss light microscope. Dinocras cephalotes—Italy, Latium, River Aniene, Trevi (RM), 30.V.2000. Italy, Umbria, River Nera, Arrone (TR), 30.VI.2000. 3. Results Perla grandis—Italy, Umbria, River Nera, Arrone (TR), 30.VI.2000. Italy, Umbria, River Nera, Arrone (TR), The testes of the Plecoptera species consist of a number 23.X.2000. of separate follicles (Fig. 1(A)). Each follicle is enclosed by Testes and vasa deferentia were dissected from mature its epithelium and opens into the vas deferens through a males of the four species in a saline solution prior to short duct. Within the follicle, there are a number of cysts, treatment. each consisting of germ cells at the same stage of maturation. Observed at LM, thin sections of the follicles 2.1. Interference light microscopy in adults of D. cephalotes (Fig. 1(B)) and P. grandis show that all the cysts contain only spermatozoa ready to enter the For light microscopy (LM), mature sperm obtained from ducts. the vasa deferentia dissected in a drop of saline solution In contrast, the follicular cysts of S. torrentium and I. were immediately observed in a Zeiss Axiophot interference grammatica (Fig. 1(C)) are heterogeneous in maturation, light microscope. with germ cells at different stages of spermatogenesis. 2.2. Fluorescence microscopy 3.1. Sperm ultrastructure For fluorescence microscopy, mature sperm were fixed in At SEM and interferential light microscopy level, the a freshly prepared solution of 4% paraformaldehyde in spermatozoa of all four species are filiform and indis- 0.14 M PBS buffer for 15 min at 4 8C, transferred to the tinguishable (Fig. 2(A) and (B)). The fluorescent nuclear same buffer containing 0.1% Triton X-100 for 5 min to dye Hoechst shows that the nucleus, about 12 mm long and A.M. Fausto et al. / Arthropod Structure & Development 31 (2002) 147–156 149 Fig. 1. SEM (A) and LM (B) images of the follicles in adult male of Dinocras cephalotes in which all cysts contain only mature spermatozoa.