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Home , Acordulecera, Anaxyelidae, Cephoidea, Cimbex, Cimbicidae, Evanioidea

J. HYM. RES. Vol. 10(Z), 2001, pp. 119-125

Spermatodesmata of the (: 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 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 superfamilies. Spermatodes- mata occur in all examined taxa within the sawfly grade (- inclusive), but are not found in the . 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 family Xyelidae, contains relatively few sperm, approximately 16. However, in the and in the Siricidae, far larger numbers are found, reaching up to 256 in the .

In many , 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, 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, and exam- the whole spermatodesm is very elongate, ined), proctotrupoid se~zsu lafo (, several times longer than an individual , 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 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 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- 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 : 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; : 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 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; : rrhdonri7znlis sperm nuclei present in the spermato- Say, Illinois; Xiphydria maculata Say, llli- desm. Comparison of this value with that nois; Orussidae: 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, , 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 . 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. vavipicta Norton) suggests that they emerging from the sperm head and in our have the same number of sperm per sper- micrographs the spermatodesmata were matodesm as T. xantha. reasonably isolated so we do not believe Counts of the number of pixels occu- that there is any reason why that sperm pied by isolated sperm nuclei (Fig. 4) al- number will have been over-estimated. low us to estimate nuclear DNA content. For the cephid, Ceyhus, this is 128 (based The distribution is suggestive of a trend on a count of 93 sperm tails), and for the among the sawflies in that Xycla, the most siricid, Trcmcx, it is 512 (based on a count basal genus, has the largest nucleus and of c. 400 transverse sections of sperm in a OUUSSLK, the most derived genus has the largely complete micrograph section). smallest nucleus. Although we have not been able to uti- DISCUSSION lise the present technique to quantify sper- Given that we now have a robust phy- matodesm size in the or Siri- logenetic hypothesis for the superfamilies cidae, inspection of the stained light mi- of sawflies and at least an estimate of fam- crographs and of transmission electron ily level relationships within the tenthre- micrographs for these two superfamilies dinoid lineage (Vilhelmsen 1997, 2000a,b, respectively (Quicke et al. 1992, Newman 2001), we can consider the evolutionary and Quickc 1999a) indicate that both of pattern of spermatodesm size (number of 16 sp. 128 xantha 256 distans 128 tejoniensis 256 americanum 512 triangulum 64 Acordulecera sp. 32 Syntexis libocedrii 32 Xiphydria abdominalis 32 Xiphydria maculata 32 Orussus oeciden talis 32 Orussus thoracicus

16 Xyela sp. 128 Tenthredo xantha 256 Strongylogaster distans 128 Dolerus tejoniensis 256 Cimbex americanum 512 Trichiosoma triangulum 64 Acordulecera sp.- ?128 Cephus 32 Syntexis libecedrii ?512 32 Xiphydria abdominalis 32 Xiphydria maculate. I 32 Orussus occidentalis 56 I 32 Orussus thoracicus Fig. 5. Number of sperm per spermatodcsm shown on the independently obtained cladogram of sawfly relationships (from Vilhelmsen 1997, 2001), showing in (a) only data obtained from DAI’I-staining, and (b) with values for additional taxa based on other estimation techniques incorporated. sperm included). Unfortunately, there is analyses. Therefore we base our interpre- considerable uncertainty about what con- tations on the likely ancestral state in the stitutes a suitable outgroup for the Hy- order on the state shown by the Xyelidae menoptera, and if one accepts a currently which display the most putatively ple- common view that the order is the sister siomorphic character states of any of the group of the remainder of the Holometa- extant Hymenoptera. Visual inspection of bola, then there is too much variation with the DAPI sperm-quantification data this putative to use it as an mapped on to Vilhelmsen’s (20~. cit.) in- outgroup for the purposes of the current dependently derived sawfly phylogeny VOLUME 10, NLJMBEI~ 2, 2001 125

(Fig. 5a) suggests that the groundplan Lino-Neto, J., S. N. Bao, and H. Dolder. 1999. Struc- spermatodesm size for sawflies is low (16 ture and ultrastructure of the spermatozoa of Be- phratelloides pornorum (Fabric&) (Hymenoptera: or 32), but that there has been a general ). International Journal of Insect Mor- increase within the Tenthredinoidea phology and Embryology 28: (4) 253-259. (range 64-512) and that particularly large Lino-Neto, J., S. N. Bao, and H. Dolder. 2000a. Struc- numbers (256-512) have evolved at least ture and ultrastructure of the spermatozoa of Tri- twice within this superfamily. However, chogramma pretiosum Riley and Trichogramma ato- incorporating estimates of sperm number poviritia Oatman and Platner (Hymenoptera: Tri- chogrammatidae). Acta Zooloogica, Stockholm 81: for Ccphus pygmctls (Cephoidea) and Tue- 205-211. mex sp. (Siricidae) into Vilhelmsen’s phy- Lino-N&o, J., S. N. Bao, and H. Dolder. 2OOOb. Sperm logeny (lot. cit.) (Fig. 5b) tends to confuse ultrastructure of the honey (Apis melhfera) (L) the picture in that it is equally parsimo- (Hymenoptera, ) with emphasis on the nious that there was a marked increase in nucleus-flagellum transition region. Tissur and Cell 32: 322-327. sperm number per spermatodesm above Newman, T. M. and D. L. J. Quicke. 1998. Sperm de- the Xyelidae, and that there were reversals velopment in the imaginal testes of Aleiodes cox- to lower numbers in the Anaxyelidae and alis (Hymenoptera: : Rogadinae). low- Xiphydriidae + Orussidae as for multiple nal of Hymenoptera Research 7: 25-37. increases from a groundplan of 16 or 32 Newman, T. M. and D. L. J. Quicke. 1999a. Ultrastruc- ture of imaginal spermatozoa of sawflies (Insec- (viz in the Tenthredinoidea, Cephoidea ta: Hymenoptera: Symphyta). fournal of Hyme- and Siricidae). noptera Research 8: 3547. Denser taxon sampling may help to Newman, T. M. and D. L. J. Quicke. 1999b. Ultra- clarify the above issues and may also pro- structure of spermatozoa in Leptopilina (Hyme- vide additional phylogenetic evidence noptera: : Eucoilidae). Journal of Hy- merroptera Research 8: 197-203. within some groups, especially within the Newman, T. M. and D. L. J. Quicke. 2000. Sperm de- Tenthredinoidea. Future work will exam- velopment and ultrastructure of mature sper- ine nuclear DNA content across the Hy- matozoa of Megalyra (Hymenoptera: Megalyro- menoptera in more detail (Schiff, Flem- idea). fournal of Hymenoptrra Research 9: 62-70. ming and Quicke in preparation). Quicke, D. L. J. 1997. Parasitic . Chapman & Hall, London, 470~~. ACKNOWLEDGEMENTS Quicke, D. L. J., S. N. Ingram, I-1. S. Baillie and P. V. Gaitens. 1992. Sperm structure and ultrastructure We would like to thank Virginia Scott for sending in the Hymenoptera (Insecta). Zoologica Scripta us the live male Xyrla, David Smith for comparing 21: 381402. the Tr77thudo voucher specimens with the USNM Vilhelmsen, L. 1997. The phylogeny of lower Hyme- (Washington DC) reference collection, and Larry Be- noptera (Insecta), with a summary of the early zark and Steve Hcydon for helping and providing NS evolutionary history of the order. Journal of Zoo- with company on some of the collecting trips. logic~7l Systematics and Evolutionary Research 35: 49-70. LITERATURE CITED Vilhelmsen L. 2000a. Before the -waist: Com- Clark, R. B., K. A. F. Gration and I’. N. R. Usherwood. parative anatomy and phylogenetic implications 1979. Desensitization of glutamate receptors on of the skeleto-musculature of the thoraco-abdom- innervated and dcnervatcd locust muscle fibres. inal boundary region in basal Hymenoptera (In- Journal of Physiology (London) 290: 551-568. secta). Zoomorphotogy 119: 185-221. Flemming, A. J., 2. 2. Shen, A. Cunha, S. U. Emmons Vilhelmsen L. 2000b. The apparatus of bas- and A. M. Leroi. 2000. Somatic polyploidisation al Hymenoptera (Insecta): phylogenetic implica- and cellular proliferation drive body size evolu- tions and functional morphology. Zoologica Scrip- tion in nematodes. Procerdin,qs qf the National ta 29: 339-345. Academy of Scicnccs, LISA, 97: 5285-5290. Vilhelmsen L. 2001. Phylogeny and classification of Jamieson, R. G. M. 1987. The Ultrnstructure and Phy- the extant basal lineages of the Hymenoptera (In- lqq~rh/ L$ Z~rsrct Spermatozoa. Cambridge Univer- secta). Zoological lournal of the Linnean Society 131: sity Press, Cambridge, 320~~. 393442.