J. HYM. RES. Vol. 10(Z), 2001, pp. 119-125 Spermatodesmata of the Sawflies (Hymenoptera: Symphyta): Evidence for Multiple Increases in Sperm Bundle Size NATHAN SCHIFF, ANTHONY J. FLEMMINC, AND DONALD L. J. QUICKE (NS) U.S. Forest Service, Southern Research Station, Center for Bottomland Hardwoods Research, P.O. Box 227, Stoneville, MS 38776, USA; (AJF) Department of Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, U.K.; (DLJQ) Unit of Parasitoid Systematics, Department of Biology, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, UK, and Department of Entomology, The Natural History Museum, London SW7 5BD, UK Abstract.-We present the first survey of spermatodesmata (bundles of spermatozoa connected at the head by an extracellular ‘gelatinous’ matrix) across the sawfly superfamilies. Spermatodes- mata occur in all examined taxa within the sawfly grade (Xyelidae-Orussidae inclusive), but are not found in the Apocrita. Using DAPI staining, the numbers of individual sperm per sperma- todesm were calculated and the values obtained are mapped on to the current phylogenetic hy- pothesis. The plesiomorphic spermatodesm in the Hymenoptera, based on that observed in the putatively basal family Xyelidae, contains relatively few sperm, approximately 16. However, in the Tenthredinoidea and in the Siricidae, far larger numbers are found, reaching up to 256 in the Cimbicidae. In many insects, mature sperm released Until now, spermatodesmata have only from testicular follicles are neither free in- been characterised in a few sawflies, al- dividuals nor packaged into variously most entirely as part of ultrastructural in- complex spermatophores, but are ar- vestigations using transmission electron ranged in organised bundles with their microscopy (Quicke et al. 1992, Newman anterior ends embedded in an extracellu- and Quicke 1999a), but the data obtained lar cap. These structures, called sperma- are not normally easily interpreted in todesmata (spermatodesm singular), oc- terms of the actual size and structure of cur, amongst others, in at least some mem- the spermatodesm. However, it was ap- bers of the Collembola, Orthoptera, Dip- parent that spermatodesmata vary in both tera, Coleoptera, Lepidoptera and size (i.e. number of individual sperm in- Hymenoptera (Jamieson 1987). Within the volved) and shape. For example, in most Hymenoptera, spermatodesmata appear taxa examined the spermatodesmata re- to be limited to the basal sawflies (Quicke semble a tuft of grass with their acrosomes et al. 1992, Quicke 1997), and they have embedded in an extracellular cap and the not been observed in members of the ‘Eva- nuclei and tails splaying out posteriorly. niomorpha’ (Stephanoidea, Megalyroidea, However in the cephid, Cephus pygrnaeus, Evanioidea and Ceraphronoidea exam- the whole spermatodesm is very elongate, ined), proctotrupoid se~zsu lafo (Diapriidae, several times longer than an individual Proctotrupidae, Heloridae and Scelionidae sperm, and sperm are inserted along a examined), chalcidoid, cynipoid, ichneu- thin central extracellular matrix core monoid or aculeate groups (Quicke et al. (Quicke et al. 1992). 1992, Newman and Quicke 1998, 1999a,b, Sperm produced in a given follicle are 2000, Lino-Neto et al. 1999, 2000a,b). all derived from cell divisions from a sin- 120 gle spermatogonial cell (see Quicke 1997). possible. Voucher specimens have there- Because these cell divisions occur synchro- fore been deposited in the United States nously within a follicle and each sperm National Museum (Washington D.C.). mother cell in each follicle undergoes a Light microscopy.-Vas deferentia and fixed number of cell division rounds, the testes were dissected from living sawflies numbers of sperm per spermatodesm are in insect saline (Clark et al. 1979) and expected to be 2” where n is the number teased apart on a clean microscope slide. of rounds of spermatocyte division. After a few minutes to allow the sperm/ Counting the numbers of sperm in each spermatodesmata to swim free of the dis- spermatodesm was not straight-forward rupted tissue, the slides were heat fixed on for most taxa because when stained using an hot plate at approximately 80°C. These traditional dyes, the mass was so opaque slides were stained with a O.O07mg/ml so- with overlapping nuclei and tails that in- lution of 4’,6-diamidino-2-phenylindole dividual cells could not be distinguished dihydrochloride (DAPI) and viewed with and counted. We have therefore employed a Leitz epifluorescence microscope at X a fluorescence staining technique in order 1000 (as in Flemming et nl. 2000). This to measure the total DNA content of the stain specifically binds to double stranded spermatodesm and divided that by the DNA and fluoresces under UV light (Figs. DNA content of an individual sperm nu- 1, 2). Images of stained spermatodesmata cleus. For a few taxa that were no longer were captured using a CV-M300 video available for the current study, we have camera, and a Scion LG3 frame-grabber included some crude estimates of sperm mounted in a Power Macintosh running number obtained from transmission elec- Scion Image 1.62a. Care was taken to en- tron microscopy of transverse sections sure that, in each frame, both a complete (Quicke et al. 1992, Newman and Quicke spermatodesm and an isolated sperm cell 1999a). However, these are likely to be un- was present (This was not possible for derestimated, because more posteriorly in- Acordulecera where only one isolated serted sperm may not have been sec- sperm nucleus could be found). Before tioned. capturing, the image was adjusted to min- imise pixel saturation and these adjust- MATERIALS AND METHODS ments affected the spermatodesm and iso- Mateuiuls.-The following taxa were ex- lated sperm cell in each image equivalent- amined. Xyelidae: XycIo sp., Colorado; ly. A densitometric value for both the Tenthredinidae: Tenthrcdo xantha Norton, spermatodesm and the sperm nucleus was N California; Strongylogasfcu distans Nor- determined (the product of area measured ton, S California; Dolcv74s tcjoniclzsis (Nor- in pixels and staining intensity) and the ton), S California; Pergidae: Acordtdccrra number of sperm in the spermatodesm de- sp., Illinois; Cimbicidae: 7’richiosor?ra friar- rived by dividing the two values. On av- glillr/ll (Kirby), NW California; Ciwbex erage four images per species were ana- anrcricanz~m Leach, NW California; Anax- lysed. For one species, Xiphydvia maculafa, yelidae: Syrlfcxis liboccdrii Rohwer, Califor- it was possible to make a direct count of nia; Xiphydriidae: Xiphydria rrhdonri7znlis sperm nuclei present in the spermato- Say, Illinois; Xiphydria maculata Say, llli- desm. Comparison of this value with that nois; Orussidae: Orussus fhoracicus (Ash- obtained densitometrically (35 i- 7 (95% mead), N California; Ortrsms occidentalis confidence interval) and 33 t- 3 (95% con- (Cresson), N California. It should be noted fidence interval) respectively) confirms the that males of many sawflies are taxonom- accuracy of the technique. Basic statistics ically difficult to segregate and at present were calculated using Excel 98 (Microsoft). species-level identifications are not always Making the assumvtion that DNA VOLUMI: 10, NUMBER 2, 2001 121 Fig. 1. Fluorescence images of DAPI-stained sperm nuclei in spermatodesmata of sawflies investigated: A, Acord~tl~~a sp. (Pergidae); B, Trichiosorr~n frimgulum (Cimbicidae); C, Sfron~q&psf~~r disfmrs (Tenthredinidae); D, Dolvrus fc@rimsis (Tenthredinidae); E, Tmfhrdo xmfha (Tenthredinidae); F, Civ~kx mmkmum (Cimbici- dae). (chromatin) is densely packed in sperm the middle. Tenthredinoid spermatodes- nuclei, and since we have no 17 priori rea- mata are far wider and the larger ones (i.e. sons to expect differences in DNA density those with a larger number of sperm; Fig. between taxa, we used sperm nucleus size la,b,f) dry on to the slides as rosette like as a surrogate for haploid DNA content. structures. Although the centres of these appear empty in DAPI-stained material, RESULTS transmission electron micrographs (New- The sperm heads are inserted through- man and Quicke 1999a) suggest that this out the cap of the spermatodesmata, with is the region where the acrosomes are in- those sperm located more centrally being serted in the extracellular matrix of the inserted more anteriorly (Figs. 1, 2). For cap. Xyelidae, Anaxyelidae, Xiphydriidae and Results of numbers of sperm per sper- Orussidae, the spermatodesmata are elon- matodesm are shown graphically in Fig. 3. gate structures, in the case of Ovussz~s, the Visual inspection of numbers (which as sperm appear to be inserted in the cap in explained above are expected to be integer a spiral configuration rather as if a cylin- powers of 2) suggests that for Xycla, the drical roll of paper was ‘pulled out’ from number is 16, for Orussidae, Anaxyelidae 122 :~l’TGIIA RESEARCH Fig. 2. Fluorescence images of DAN-stained sperm nuclei in spermatodesmata of sawflies investigated: A, X~/eln (Xyelidae); B, S!/~ztexis libocedrii (Anaxyelidae); C, Xiphydriu nbdominnlis (Xiphydriidae); D, Xiplzydrin nm ccrlafn (Xiphydriidae); E, OYUSSIE occidrntnlis (Orussidae); F, Orussus thovnciczls (Orussidac). Sperm per Spermatodesm kt 95% Confidence Interval) 700 g 600 OJ z 500 6 400 K k 3oo E 200 &loo 0 Fig. 3. Plot of numbers of sperm per spermatodesm for sawfly taxa ranked according to spermatodesm size. VCXUMI: 10, Nurvim: 2, 2001 123 Mean Nuclear Size (2 95”LConfidence Interval) Fig. 1. Plot of mean nucleus size, a surrogate for DNA content, for individual sperm in each sawfly taxon. and Xiphydriidae, it is 32, for Tenthredi- these have rather large numbers of sperm nidae the number ranges from 128 to 256, per spermatodesm. Our best estimates for Pergidae 64 and for Cimbicidae, 256- were taken as the smallest power of two 512. Visual inspection of toluidine blue larger than the definite minimum number stained slides of two other tenthredinids of individual sperm within a micrograph (Rhoppster californica (Norton) and a Ten- of a spermatodesm. The sperm tails are thredo that was either T. lacticirzcta Cresson relatively straight for a distance after or T.