Structure of ovaries and oogenesis in the snow scorpionfly boreus hyemalis (LINNE)(MECOPTERA : BOREIDAE)

\

Pergamon PII ] S9919Ð6211"87#99915Ð8

Int[ J[ Insect Morphol[ + Embryol[\ Vol[ 16\ No[ 3\ pp[ 222Ð239\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9919!6211:87 ,08[99¦9[99

STRUCTURE OF OVARIES AND OOGENESIS IN THE SNOW SCORPIONFLY BOREUS HYEMALIS "LINNE#"MECOPTERA ] BOREIDAE#

Szczepan M[ Bilinski$ and Jurgen Buning% $ Department of Systematic Zoology\ Institute of Zoology\ Jagiellonian University\ PL!29!959 Krakow\ Poland % Institute of Zoology I\ University of Erlangen!Nurnberg\ D!80!947\ Erlangen\ Germany "Received 13 February 0887 ^ accepted 19 Au`ust 0887# Abstract*The ovaries of the snow scorpion~y\ Boreus hyemalis "Mecoptera ] Boreidae# are panoistic and comprise 6Ð7 ovarioles[ Each ovariole consists of a terminal _lament\ elongated vitellarium\ and ovariole stalk "pedicel# only ^ in adult specimens\ functional germaria are absent[ Five consecutive stages of oogenesis i[e[\ early\ mid! and late previtellogenesis\ vitellogenesis\ and choriogenesis have been distinguished in imagines[ Oocyte nuclei "germinal vesicles# of previtellogenic oocytes contain numerous polymorphic multiple nucleoli "or nucleolar masses#\ endobodies\ and chromatin aggregations[ Next to the nuclear envelope\ large accumulations of nuage material are localized[ The ooplasm of late previtellogenic oocytes is di}erentiated into transparent "perinuclear# and opaque "peripheral# regions[ Ultrastructural investigations have revealed that within the latter\ abundant ribosomes as well as mitochondria\ elements of endoplasmic reticulum\ Golgi complexes\ annulate lamellae\ symbiotic bacteroids\ lipid droplets and distinctive accumulations of membrane!free clathrin!like cages are present[ Early! and mid previtellogenic oocytes are invested with ~at somatic cells that gradually transform into a follicular epithelium[ In the vicinity of 2!cell junctions\ neighbouring follicular cells are joined by narrow intercellular bridges[ During late previtellogenesis\ numerous microvilli develop on the oocyte surface[ They interdigitate with morphologically similar but less frequent microvilli of the follicular cells[ Concurrently\ _rst endocytotic vesicles appear in the cortical ooplasm[ In the context of presented results\ the phylogenetic relationships between mecopterans "boreids# and ~eas are discussed[ Þ 0887 Elsevier Science Ltd[ All rights reserved[ Index descriptors "in addition to those in the title#] secondary panoism\ rDNA ampli_cation\ endobodies\ clathrin\ insect phylogeny[

INTRODUCTION

Two basic morpho!functional categories of insect ovaries "ovarioles# are traditionally recognized\ panoistic and meroistic "see Telfer\ 0864 ^ King and Buning\ 0874 ^ Bun! ing\ 0883\ 0885\ 0886 for reviews#[ In the panoistic ovari! oles\ all oogonia are transformed into functional gametes "oocytes#\ whereas in the meroistic ones some germ cells di}erentiate into synthetically active\ often polyploid nurse cells "trophocytes# that supply the oocytes with macromolecules "e[g[\ rRNA\ mRNA# and organelles during the previtellogenic growth[ The spatial organ! ization of the ovarioles and relationships between the oocytes and nurse cells allow the subdivision of meroistic ovaries into two types ] polytrophic and telotrophic[ In the polytrophic ovarioles\ each oocyte is accompanied by its own group of nurse cells[ Such oocyte!nurse cell complexes are invested with somatic "follicular# cells and constitute the functional units\ termed egg chambers "ovarian follicles#[ In the telotrophic ones\ all tro! phocytes are retained in the apical part of the ovariole thereby forming a trophic chamber "tropharium#[ The contact of growing oocytes with the distant tropharium

 Author to whom correspondence should be addressed[ Tel[] 9937 01 522 52 66 ext[ 398^ Fax] 9937 01 523 26 05^ e!mail] sbiliÝzuk[iz[uj[edu[pl 222

is ensured by elongated cytoplasmic extensions\ referred to as trophic cords[ The distribution of ovary types among various insect groups suggest that ] "0# polytrophic meroistic ovaries evolved twice from panoistic ones ] once in Entognatha s[ str[ "Bilinski\ 0872\ 0882 ^ Bilinski and Szklarzewicz\ 0881#\ once in the common ancestor of Dermaptera and

tys and Bilinski\ 0889 ^ Buning\ 0883\ 0885\ Eumetabola "S 0886# ^ "1# telotrophic ovaries represent the most advanced and specialized category that evolved inde! pendently four times ] in Hemiptera\ Coleoptera ] Poly! phaga\ Rhaphidioptera and Megaloptera ] Sialidae from a polytrophic background and in Ephemeroptera directly from a panoistic one "Buning\ 0883\ 0885\ 0886# ^ "2# in some advanced taxa "see Discussion for details# sec! ondary panoistic ovaries evolved "by the loss of nurse cells# from meroistic ones "King and Buning\ 0874 ^ Stys and Bilinski\ 0889 ^ Buning\ 0883\ 0886#[ To indicate the evolutionary distinctness of such {{reduced|| ovaries from the primary panoistic ones\ Stys and Bilinski "0889# have proposed the term {{neopanoism[|| Mecoptera are traditionally regarded as one of the oldest and most plesiomorphic taxa among higher\ hol! ometabolous insects "Kaltenbach\ 0867 ^ Willman\ 0876#[ They comprise nine extant families that are grouped into two or three suborders "Penny\ 0864 ^ Kaltenbach\ 0867 ^

223

S[ M[ Bilinski and J[ Buning

Willman\ 0876 ^ Byers\ 0880 ^ Kristensen\ 0884#[ The structure of female reproductive organs was analysed to date in representatives of only three families[ It has been shown that Panorpidae and Bittacidae possess poly! trophic!meroistic ovaries "Ramamurty\ 0869 ^ Ando\ 0862 ^ Simiczyjew\ 0885#\ whereas Boreidae are char! acterized by the secondary panoistic ones "Steiner\ 0826 ^ Buning\ 0883#[ Here\ we present the results of the detailed ultrastructural and histochemical investigations on the ovaries of the snow scorpion~y\ Boreus hyemalis[ MATERIALS AND METHODS Insects Adult specimens of the snow scorpion~y\ Boreus hyemalis were col! lected in StoC for 1 h[ The specimens were then rinsed and post_xed in ] "0# 0) osmium tetroxide in the same bu}er or "1# a mixture of 1) osmium tetroxide and 9[7) potassium ferricyanide in 9[0 M phosphate bu}er "pH 6[3#[ After dehydration in a series of ethanols and acetone\ the material was embedded in Epon 701 "Fullam Inc[\ Latham\ NY\ U[S[A[#[ Ultrathin sections were doubly contrasted with uranyl acetate and lead citrate and examined in a JEM 099SX or Zeiss EM09 electron microscopes at 59 kV[ Semithin epon sections "9[6 mm# were cut in a plane parallel to the longitudinal axis of the ovariole[ The sections were stained in 0) methylene blue in 0) borax and examined in a Jenalumar or Axiophot "Zeiss# microscopes[ Fluorescence microscopy The ovaries were _xed in 3) formaldehyde\ freshly prepared from paraformaldehyde\ in phosphate!bu}ered saline "PBS# for 29 min at room temperature[ The specimens were then rinsed and stained in darkness with rhodamine!conjugated phalloidin "1 mg:ml ^ Sigma Chemical Co[\ St[ Louis\ MO\ U[S[A[#[ After rinsing in PBS\ individual ovarioles were mounted on microscopic slides and examined with a Jenalumar epi~uorescence microscope equipped with appropriate _lters[ RNA and DNA stainin` The ovaries were _xed in 3) formaldehyde in PBS for 29Ð34 min at room temperature[ After dehydration\ the material was in_ltrated and embedded in Histocryl "Agar Scienti_c Ltd[\ Stansted\ Essex\ England ^ for details see manufacturer|s instructions#[ Semithin sections "9[7Ð0[9 mm# were stained with ] "0# 3?\5 Diamidino!1 phenylindole dihy! drochloride "DAPI\ 0 mg:ml ^ Sigma#\ "1# propidium iodide "0 mg:ml\ Sigma# or "2# 0) toluidine blue\ pH 4[9[ The sections were examined with a Jenalumar microscope using epi~uorescence "0\ 1# or bright _eld "2# illumination[ A`NOR technique AgNOR proteins were visualized according to the method of Howell and Black "0879# with some modi_cations proposed by Bilinski and Bilinska "0885#[ Semithin histocryl sections "see above# were stained in a mixture of 1) gelatin in 0) formic acid and 49) silver nitrate "Sigma# "volume proportion 0 ] 1#[ The staining was carried out in a moist chamber at 26>C[ Sections were then rinsed in PBS and mounted in glycerol[ Ultrathin sections were collected on nickel grids and stained in a droplet "about 199 ml# of the same mixture[ Grids were thoroughly rinsed in double!distilled water and examined\ without additional con! trasting\ in a JEM 099SX electron microscope[

RESULTS

Gross anatomy The ovaries of the adult snow scorpion~y\ Boreus hye! malis are panoistic "Fig[ 0"A## and comprise 6Ð7 ovari!

oles[ Each ovariole is tightly surrounded by a reticular network of striated muscle _bers "Fig[ 0"B## and consists of a terminal _lament\ elongated vitellarium "Fig[ 0"A## and ovariole stalk[ In adult specimens\ functional ger! maria are absent "Fig[ 0A# ^ however\ in some ovarioles\ 1 or 2 degenerating germ cell occur between terminal _lament and the _rst "youngest# oocyte[ The vit! ellarium is composed of 09Ð03 growing oocytes in a linear arrangement[ To facilitate the description\ the oocytes have been classi_ed into _ve developmental categories ] early\ mid! and late previtellogenic\ vitellogenic\ and cho! riogenic[ In the present paper\ ultrastrutural and his! tochemical investigations on 2 previtellogenic stages are reported[

Early previtello`enesis Early previtellogenic oocytes are localized in the apical "anterior# part of the ovariole "Figs[ 0"A# and "C# and 1"A##[ Each of them is accompanied by squamous somatic cells[ The oocyte nucleus is relatively large\ roughly spherical\ and contains three morphologically distinct structures ] a dense nucleolar mass "an area con! taining numerous nucleolus!like components# "Fig[ 0"C##\ endobodies and chromatin aggregations "Fig[ 0"G##[ Within the latter\ characteristic regularly spaced grains are immersed "Fig[ 0"I##[ An analysis of preparations stained with DAPI indicates that in the nucleolar mass\ numerous DNA!positive foci are present "Fig[ 1"A##\ whereas the endobodies are DNA!negative[ The envelope of the oocyte nucleus is slightly folded and bears numer! ous nuclear pores "Fig[ 0"F##[ The ooplasm appears hom! ogenous "Fig[ 0"C##\ and comprises mitochondria\ elements of endoplasmic reticulum\ individual Golgi complexes\ ribosomes\ and large accumulations of nuage material "Fig[ 0"F##[ The latter structures are located exlusively in the vicinity of the nuclear envelope "Fig[ 0"C##[

Mid previtello`enesis During this stage\ the number of somatic follicular cells investing oocytes gradually increases "Fig[ 0"D##[ The oocyte nucleus becomes slightly elongated or ovoid ^ sim! ultaneously\ the dense nucleolar mass starts to divide into more or less individual\ irregular lobes "Fig[ 0"D##[ Within the mass "or lobes# DNA!positive foci are still present "Fig[ 1"B##[ The ooplasm is homogeneous and resembles that of the early previtellogenic oocytes "Fig[ 0"D##[

Late previtello`enesis During late previtellogenesis the oocyte is encom! passed by cylindrical follicular cells that constitute a typi! cal mono!layered follicular epithelium "Fig[ 0"E##[ The nuclei of these cells are slightly elongated and contain prominent nucleoli "Figs[ 0"E#\ 1"C#\ "D# and "E##[ Their

Structure of Ovaries and Oogenesis in the Snow Scorpion~y B[ hyemalis

224

Fig[ 0[ "A# The anterior part of an ovariole[ Nomarski interference contrast[ Oocyte nuclei "arrows#\ terminal _lament "asterisk#×059[ "B# The network of striated muscle _bers encompassing an ovariole[ Whole mount preparation stained with rhodamine!conjugated phalloidin×319[ "C# "D# and "E# Oocytes in succesive stages of the previtellogenic growth ^ epon semithin sections stained with methylene blue[ "C# Early previtellogenesis ×549[ "D# Mid previtellogenesis×549[ "E# Late previtellogenesis×549[ Follicular epithelium "f#\ accumulations of nuage material "arrowheads#[ Note gradual fragmentation of the nucleolar mass "asterisk in C# into numerous multiple nucleoli "E# and two clearly recognizable regions of the late previtellogenic ooplasm[ "F# Early previtellogenesis ^ oocyte nucleus "n# and surrounding ooplasm[ Accumulations of nuage material "asterisks#\ elements of endoplasmic reticulum "er#[ Note numerous pores perforating the nuclear envelope[ EM\ ×04\999[ "G# and "I# Late previtellogenesis[ Multiple nucleoli "asterisks#\ endobody "e# and chromatin aggregations "arrows#[ Note regularly spaced grains within the chromatin aggregation "arrowheads in "I##[ "G# EM\ ×6\999[ "I# EM\ ×03\999[ "H# Late previtellogenesis[ Note small islands of transparent ooplasm "arrowheads# within opaque ooplasm[ Epon semithin section\ methylene blue×599[

cytoplasm comprises mitochondria\ Golgi complexes and abundant ribosomes "Fig[ 2"B##[ In the vicinity of 2! cell junctions\ neighbouring follicular cells are joined by narrow intercellular bridges "Fig[ 2"B# and "C##[ The internal rim of the bridge is lined with a thick _lamentous coat that consists of inner "dense# and outer "more trans! parent# layers "Fig[ 2"C##[ The cytoplasm adjacent to

the bridge contains numerous microtubules "Fig[ 2"C#\ arrowheads# The volume of the late previtellogenic germinal ves! icles inceases considerably "Fig[ 0"E##[ Concurrently\ the lobes of the nucleolar mass undergo fragmentation "Fig[ 0"E##[ Arising polymorphic aggregations of coarse granular material "Fig[ 0"G#\ "I## are RNA! and

225

S[ M[ Bilinski and J[ Buning

Fig[ 1[ "A# and "B# Oocytes in consecutive stages of the previtellogenic growth[ Histocryl semithin sections stained with DAPI[ "A# Early previtellogenesis×259[ "B# Mid previtellogenesis×259[ Note DNA!positive foci within the oocyte nuclei[ "C# "D# and "E# Late previtellogenesis ^ Histocryl semithin sections stained with propidium iodide "C#\ toluidine blue "D# and AgNOR technique "E#[ Follicular epithelium "f#\ oocyte nuclei "arrows#[ Note RNA and AgNOR positive multiple nucleoli within the nuclei[ "C\ D\ E#×419[ "F# Late previtellogenesis ^ electron micrograph of AgNOR positive multiple nucleoli×19\599[

AgNOR!positive "Fig[ 1"C#\ "D#\ "E# and "F##[ Such results of the histochemical tests suggest that the aggre! gations represent multiple nucleoli[ Throughout the stage\ endobodies become more opaque "compare Figs 0"G# and 2"A##\ whereas their number decreases[ Chromatin clumps get smaller\ less conspicuous and ultimately disintegrate[ The oocyte nucleus is surrounded by relatively trans! parent ooplasm "Fig[ 0"E## ^ it contains accumulations of nuage material\ tiny vesicles of endoplasmic reticulum\ mitochondria and scarce ribosomes only[ In contrast\ peripheral ooplasm appears more opaque "Figs 0"E# and 2"D## and comprises numerous cellular organelles "mito! chondria\ Golgi complexes\ elements of endoplasmic reticulum\ annulate lamellae\ lipid droplets#\ symbiotic bacteroids and abundant ribosomes[ Besides the above!

mentioned constituents\ the dense ooplasm houses also characteristic accumulations of clathrin!like cages "Fig[ 2"D##[ The cages measure 59Ð79 nm in diameter and\ in contrast to coated vesicles\ are devoid of membranes[ Spherical masses of granular material and Golgi com! plexes are often associated with the accumulations "Fig[ 2"D##[ In the _nal phase of the late previtellogenesis the transparent ooplasm disperses into small islands immersed in the opaque ooplasm "Figs 0"H#\ 2"A##[ This process leads to the complete absence of the former in the subsequent stages of oogenesis[ During late previtellogenesis numerous microvilli develop on the oocyte surface[ They interdigitate with morphologically similar but less frequent microvilli of the follicular cells "Fig[ 2"B##[ Simultaneously\ _rst endo! cytotic vesicles arise in the cortical ooplasm[

Structure of Ovaries and Oogenesis in the Snow Scorpion~y B[ hyemalis

226

Fig[ 2[ "A# Late previtellogenesis ^ the oocyte nucleus and surrounding ooplasm[ Endobody "e#\ multiple nucleoli "arrowheads#\ accumulation of nuage material "asterisk#[ Note perinuclear "transparent# ooplasm "arrows#[ EM\ ×3\799[ "B# Late previtellogenesis ^ 1 neighbouring follicular cells and cortical ooplasm[ Oocyte microvilli "m#\ nucleus of the follicular cell "n#\ intercellular bridge "arrow#[ Note microvilli of follicular cells "arrowheads#[ EM\ ×00\499[ "C# Intercellular bridge connecting adjacent follicular cells[ Note the rim of the bridge "arrow# and microtubules "arrowheads#[ EM\ ×11\399[ "D# Late previtellogenesis ^ an accumulation of clathrin like cages in opaque ooplasm[ Endoplasmic reticulum "er#\ vesicles of a Golgi complex "arrow#\ spherical mass of a granular material "asterisk#[ EM\ ×25\799[

DISCUSSION

Gross morpholo`y Among the hexapods secondary panoistic "neo! panoistic# ovarioles evolved independently _ve times ] in proturans "Bilinski\ 0882# and in four taxa of {{true|| ectognathous insects ] Thysanoptera "Pritsch and Buning\ 0878 ^ Tsutsumi et al[\ 0884#\ Megaloptera ] Sia! lidae¦Chauliodidae "Matsuzaki et al[\ 0874 ^ Buning\ 0885 ^ Bilinski unpublished observations#\ Siphonaptera

"Buning and Sohst\ 0877\ 0878# and Mecoptera ] Boreidae "Steiner\ 0826 ^ Buning\ 0883#[ The ontogenetic devel! opment of such {{reduced|| ovarioles has been traced to date only in thysanopterans[ According to Pritsch and Buning "0878#\ germaria of terebrantian thrips\ Par! thenothrips dracenae contain germ cell clusters similar to those characteristic of meroistic ovarioles[ All cells of the clusters develop into oocytes\ become individual and migrate to the vitellarium[ Remarkably similar process has been described by Tsutsumi et al[\ "0884# in tub!

227

S[ M[ Bilinski and J[ Buning

uliferan thrips\ Bactrothrips brevitubulus and Halothrips yuasai[ In the present paper\ we show that the ovarioles of the adult snow scorpion~y are devoid of functional germaria[ This indicates that initial stages of oogenesis "germ cell mitoses\ cluster formation<\ oocyte deter! mination# take place in pupae or larvae\ and are com! pleted before the reproductive phase[ Similar mode of reproduction has been found also in several insect taxa\ including bugs "Dipsocoridae ] Stys et al[\ 0887 ^ Miridae ] Wightman\ 0862 ^ Ma and Ramaswamy\ 0876# ^ coccids "Orthezidae ] Szklarzewicz and Bilinski\ 0884 ^ Szklar! zewicz\ 0886# ^ dipterans "Sciaridae ] Buning\ 0883 and unpublished observations# ^ and most importantly ~eas "Buning and Sohst\ 0877\ 0878#[

Ultrastructure The selective extrachromosomal replication "ampli! _cation# of ribosomal genes "rDNA# takes place in the oocyte nuclei "germinal vesicles# of numerous species belonging to various animal taxa[ Among insects rDNA ampli_cation has been evidenced in representatives of gryllids "Trendelenburg et al[\ 0862#\ coleopterans "Tren! delenburg\ 0863 ^ Matuszewski and Hoser\ 0864 ^ Matuszewski and Kloc\ 0865 ^ Kloc et al[\ 0884# ^ neu! ropterans "Gruzova et al[\ 0861 ^ Kubrakiewicz and Bilin! ski\ 0884# ^ dipterans "Lima de Faria and Moses\ 0855 ^ Buning\ 0883 and unpublished# ^ and ~eas "Buning and Sohst\ 0877\ 0878#[ Usually\ as a result of this process\ characteristic compact DNA!positive body "referred to as Giardina body or extrachromosomal DNA body# arises in the karyoplasm[ Transcription of ampli_ed rDNA genes\ contained within the body\ starts during the previtellogenic growth\ and as a rule\ is accompanied by the appearance of numerous multiple nucleoli[ We suggest that rDNA ampli_cation takes place also in the oocytes of Boreus[ This assumption is substantiated by the following observations ] "0# the nuclei of early pre! vitellogenic oocytes possess dense nucleolar masses that contain DNA!postive foci ^ "1# during middle and late previtellogensis these masses gradually break up into numerous\ RNA and AgNOR positive units i[e[\ multiple nucleoli[ In our opinion the masses represent classical extrachromosomal DNA bodies\ before fragmentation but after commencement of transcriptional activity[ The formation of the extrachromosomal DNA bodies is pre! sumably completed during earlier stages of oogenesis[ In addition to fragmentating nucleolar masses pre! vitellogenic germinal vesicles of Boreus\ comprise charac! teristic spherical structures termed endobodies[ Similarly as in ~eas "Buning and Sohst\ 0877\ 0878#\ the number of these structures decreases till the end of the pre! vitellogenic growth[ Endobodies\ termed also Binnen! korper\ were for the _rst time described and seriously analysed by Bier and his colleagues "Bier et al[\ 0856#[ Recent immunocytochemical investigations have shown that they contain\ the nucleolar protein _brillarin\ p79! coilin\ snRNPs "Gall et al[\ 0884# as well as Mago and

K09 proteins "Newmark et al[\ 0886 ^ Serano and Cohen\ 0864#[ These results led to the suggestion that at least in some insect species\ within endobodies\ the assembly of snRNP complexes needed for RNA processing takes place "see Gall et al[\ 0884 for details#[ The biochemical composition of Boreus endobodies has not been ana! lysed ^ however\ the correlation between the appearance of these structures and intense rDNA transcription "ulti! mately manifested by the rise of numerous multiple nucle! oli# supports the above hypothesis[ In the ooplasm of the late previtellogenic oocytes of the investigated species a distinctive accumulation of cla! thrin!like cages arises[ Similar accumulations have been previously described in the developing oocytes of mos! quitos "Raikhel\ 0873# and ~eas "Buning and Sohst\ 0877#[ According to Raikhel "0873#\ the morphological characteristics as well as dimensions of the cages indicate that they represent membrane!free aggregates of clathrin molecules that are synthesized and stored in large quan! tities before the onset of endocytotic activity i[e[\ vit! ellogenesis[ The follicular epithelia of insects are usually composed of individual cells[ Narrow intercellular bridges con! necting neighbouring follicular cells have been detected to date in the ovaries of dipterans and hymenopterans only "Meola et al[\ 0866 ^ Ramamurty and Engels\ 0866 ^ Giorgi\ 0867#[ These structures are usually believed to play a role in synchronization of linked cells[ In the pre! sent study we show that morphologically similar bridges join the follicular cells in Boreus ovaries[ In contrast to dipterans and hymenopterans\ in the investigated species the bridges are always localized in the vicinity of 2!cell junctions[

Phylo`enetical considerations According to modern phylogenetisists\ Mecoptera are close relatives of Siphonaptera "e[g[\ Hinton\ 0847 ^ Kri! stensen\ 0884#[ This assumption is supported by the mor! phological evidence as well as the analysis of 07S rDNA sequence "Carmean\ 0881 ^ Whiting et al[\ 0886#[ Some authors have hypothesized also that ~eas could have evol! ved from a Boreus!like ancestor "Hinton\ 0847 ^ Buning and Sohst\ 0877# and are "consequently# phylogenetically subordinate within Mecoptera "Kristensen\ 0884 ^ Whit! ing et al[\ 0886#[ Our cytological investigations have revealed that the ovaries of boreids and ~eas "for review of ~ea oogenesis see King and Teasley\ 0879 ^ Buning and Sohst\ 0877# share the following important characters ] "0# secondary lack of nurse cells ^ "1# completion of initial stages of oogenesis during postembryonic development i[e[\ before the reproductive phase ^ "2# occurrence of rDNA ampli_cation and resulting rise of multiple nucle! oli ^ "3# di}erentiation of the late previtellogenic ooplasm into two clearly distinguishable zones ^ "4# presence of the accumulations of clathrin!like cages[ Therefore\ our studies give an extra support for the both above!men! tioned genealogical hypotheses[

Structure of Ovaries and Oogenesis in the Snow Scorpion~y B[ hyemalis Acknowled`ements*We are greatly indebted to Mrs W[ Jankowska and E[ Kisiel for excellent technical assistance[ We are also grateful to Professor W[ Kilarski "Institute of Zoology\ Jagiellonian University# for electron microscope facilities[ This work was supported by research grant 302:PO3:85:09 from the State Committee for Scienti_c Research "KBN ^ to S[M[B# and DFG "to J[B#[

REFERENCES Ando\ H[ "0862# Old oocytes and newly laid eggs of scorpion! ~ies and hanging!~ies "Mecoptera ] Panorpidae and Bittacidae#[ Science Reports of the Tokyo Kyoiku Dai`aku 04\ 052Ð076[ Bier\ K[\ Kunz\ W[ and Ribbert\ D[ "0856# Struktur und Funktion der Oocytenchromosomen und Nucleolen sowie der Extra!DNS wahrend der Oogenese panoistischer und meroistischer Insekten[ Chromosoma 12\ 103Ð187[ Bilinski\ S[ M[ "0872# Di}erentiation of the oocyte and nurse cells in an apterygote insect "Campodea#[ Tissue + Cell 04\ 854Ð862[ Bilinski\ S[ M[ "0882# Structure of ovaries and oogenesis in entog! nathans "Apterygota#[ International Journal of Insect Morpholo`y + Embryolo`y 11\ 144Ð158[ Bilinski\ S[ M[ and Bilinska\ B[ "0885# A new version of the Ag!NOR technique[ A combination with DAPI staining[ Histochememical Journal 17\ 540Ð545[ Bilinski\ S[ M[ and Szklarzewicz\ T[ "0881# The ovary of Catajapyx aquilonaris "Insecta\ Entognatha# ] ultrastructure of germarium and terminal _lament[ Zoomorpholo`y 001\ 136Ð140[ Buning\ J[ "0883# The Insect Ovary[ Ultrastructure\ Previtello`enic Growth and Evolution[ Chapman + Hall\ London[ Buning\ J[ "0885# Germ cell cluster variety creates diversity of ovary types in insects[ Verhandlun`en der Deutschen Zoolo`ischen Gesellschaft 78\ 012Ð026[ Buning\ J[ "0886# Ovariole ] structure\ type and phylogeny[ In Micro! scopical Anatomy of Invertebrates\ eds[ M[ Locke and H[ Harrison\ Vol[ XI[ "in press# Buning\ J[ and Sohst\ S[ "0877# The ~ea ovary ] ultrastructure and analysis of cell clusters[ Tissue + Cell 19\ 672Ð684[ Buning\ J[ and Sohst\ S[ "0878# The ovary of Hystrichopsylla ] Structure and previtellogenic growth[ In Re`ulation of Insect Reproduction IV[ Proceedin`s of a Symposium\ Zinkovy\ 0876\ eds[ M[ Tonner\ T[ Soldan and B[ Bennetova\ pp[ 002Ð013[ Publishing House\ Czechos! lovak Academy of Sciences\ Praha[ Byers\ G[ W[ "0880# Mecoptera "scorpion!~ies\ hanging!~ies#[ In The Insects of Australia[ A Textbook for students and Research Workers\ ed[ CSIRO\ pp[ 585Ð693[ Melbourne University Press\ Carlton[ Carmean\ D[\ Kimsey\ L[ S[ and Berbee\ M[ L[ "0881# 07S rDNA sequence and the holometabolous insects[ Molecular Phylo`enetics and Evolution 0\ 169Ð167[ Gall\ J[ G[\ Tsvetkov\ A[\ Wu\ Z[ and Murphy\ C[ "0884# Is the sphere organelle:coiled body a universal nuclear component[ Devel! opmental Genetics 05\ 14Ð24[ Giorgi\ F[ "0867# Intercellular bridges in ovarian follicle cells of Dro! sophila melano`aster[ Cell and Tissue Research 075\ 302Ð311[ Gruzova\ M[ N[\ Zaichikova\ Z[ P[ and Sokolov\ I[ Z[ "0861# Functional organization of the nucleus in the oogenesis of Chrysopa perla L[ "Insecta\ Neuroptera#[ Chromosoma 26\ 242Ð275[ Hinton\ H[ E[ "0847# The phylogeny of the panorpoid orders[ Annual Review of Entomolo`y 2\ 070Ð195[ Howell\ W[ M[ and Black\ D[ A[ "0879# Controlled silver! staining of nucleolus organizer regions with a protective colloidal developer ] a 0!step method[ Experientia 25\ 0903[ Kaltenbach\ A[ "0867# Mecoptera "Schnabelhafte\ Schnabel~iegen#[ In Handbuch der Zoologie\ ed[ M[ Beier\ Vol[ IV\ part 1Ð1:17[ ed[ M[ Beier[ Walter de Gruyter\ Berlin[ King\ R[ C[ and Buning\ J[ "0874# The origin and functioning of insect oocytes and nurse cells[ In Comprehensive Insect Physiolo`y\ Bio! chemistry and Pharmacolo`y\ eds[ G[ A[ Kerkut and L[ I[ Gilbert\ Vol[ 0\ pp[ 26Ð71[ Pergamon Press\ Oxford[ King\ R[ C[ and Teasley\ M[ "0879# Insect oogenesis ] some generalities and their bearing on the ovarian development of ~eas[ In Fleas\ eds[ R[ Traub and H[ Starcke\ pp[ 226Ð239[ A[A[ Balkema\ Rotterdam[ Kloc\ M[\ Matuszewski\ B[ and Nurkowska\ B[ "0884# Ribosomal gene ampli_cation in the oocytes of Creophilus maxillosus "Staphylinidae\ Coleoptera!Polyphaga#!!!an insect with telotrophic ovaries[ Folia Histochemica et Cytobiolo`ica 22\ 156Ð165[ Kristensen\ N[ P[ "0884# Forty years| insect phylogenetic systematics[

228

Hennig|s {{Kritische Bemerkungen[[[||\ and subsequent devel! opment[ Zoolo`ische Beitra`e 25\ 72Ð013[ Kubrakiewicz\ J[ and Bilinski\ S[ M[ "0884# Extrachromosomal ampli! _cation of rDNA in oocytes of Hemerobius spp[ "Insecta\ Neur! optera#[ Chromosoma 092\ 595Ð501[ Lima de Faria\ A[ and Moses\ M[ J[ "0855# Ultrastructure and cyto! chemistry of metabolic DNA in Tipula[ Journal of Cell Biolo`y 29\ 066Ð081[ Ma\ W[ K[ and Ramaswamy\ S[ B[ "0876# Histological changes during ovarian maturation in the tarnished plant bug\ Ly`us lineolaris "Palisot de Beauvois# "Hemiptera ] Miridae#[ International Journal of Insect Morpholo`y + Embryolo`y 05\ 298Ð211[ Matsuzaki\ M[\ Sasaki\ A and Komuro\ M[ "0874# Panoistic ovarioles of the dobson~y\ Protohermes `randis "Megaloptera\ Corydalidae#[ In Recent Advances in Insect Embryolo`y in Japan\ ed[ H[ Ando and K[ Miya\ pp[ 02Ð12[ ISEBU\ Tsukuba[ Matuszewski\ B[ and Hoser\ P[ "0864# Gene ampli_cation and its e}ect on the structure and function of the oocyte nucleus in the whirligig beetle\ Gyrinus natator "Gyrinidae\ Coleoptera!Adephaga#[ Exper! ientia 20\ 320! 321[ Matuszewski\ B[ and Kloc\ M[ "0865# Gene ampli_cation in oocytes of the rove beetle Creophilus maxillosus Staphylinidae\ "Coleoptera! Polyphaga#[ Experientia 21\ 23Ð25[ Meola\ S[ M[\ Mollenhauer\ H[ H[ and Thompson\ J[ M[ "0866# Cyto! plasmic bridges within the follicular epithelium of the ovarioles of two Diptera\ Aedes ae`ypti and Stomoxys calcitrans[ Journal of Morpholo`y 042\ 70Ð75[ Newmark\ P[ A[\ Mohr\ S[ A[\ Gong\ L[ and Boswell\ R[ E[ "0886# Mago nashi mediates the posterior follicle cell! to!oocyte signal to organize axis formation in Drosophila[ Development 013\ 2086Ð2196[ Penny\ N[ D[ "0864# Evolution of extant Mecoptera[ Journal of the Kansas Entomolo`ical Society 37\ 220Ð249[ Pritsch\ M[ and Buning\ J[ "0878# Germ cell cluster in the panoistic ovary of Thysanoptera "Insecta#[ Zoomorpholo`y 097\ 298Ð202[ Raikhel\ A[ S[ "0873# Accumulations of membrane!free clathrin! like lattices in mosquito oocyte[ European Journal of Cell Biolo`y 24\ 168Ð172[ Ramamurty\ P[ S[ "0869# Growth and di}erentiation of the follicle epithelium in relation to vitellogenesis in Panorpa communis L[ "Mecoptera\ Insecta#[ La Cellule 57\ 67Ð83[ Ramamurty\ P[ S[ and Engels\ W[ "0866# Occurrence of intercellular bridges between follicle epithelial cells in the ovary of Apis melli_ca queens[ Journal of Cell Science 3\ 084Ð191[ Serano\ T[ S[ and Cohen\ R[ S[ "0884# Gratuitous mRNA localization in the Drosophila oocyte[ Development 010\ 2902Ð2910[ Simiczyjew\ B[ "0885# Cyto! and histochemical studies of the ovariole of Panorpa communis "Insecta\ Mecoptera#[ Folia Histochemica et Cytobiolo`ica 23\ 040Ð043[ Steiner\ P[ "0826# Beitrag zur Fortp~anzungsbiologie und Morphologie des Genitalapparates von Boreus hiemalis L[ Zeitschrift fur Mor!  kolo`ie der Tiere 21\ 165Ð177[ pholo`ie und O

Stys\ P[ and Bilinski\ S[ M[ "0889# Ovariole types and the phylogeny of hexapods[ Biolo`ical Reviews 54\ 390Ð318[

Stys\ P[\ Buning\ J[ and Bilinski S[ M[ "0887# Organization of the tropharia in the telotrophic ovaries of the dipsocoromorphan bugs Cryptostemma alienum Herrich! Schae}er and C[ carpaticum Josifov "Heteroptera ] Dipsocoridae#[ International Journal of Insect Mor! pholo`y + Embryolo`y 16\ 018Ð022[ Szklarzewicz\ T[ "0886# Structure and development of the telotrophic ovariole in ensign scale insects "Hemiptera\ Coccomorpha ] Orthezi! idae#[ Tissue + Cell 18\ 20Ð27[ Szklarzewicz\ T[ and Bilinski\ S[ M[ "0884# Structure of ovaries in ensign scale insects\ the most primitive representatives of Coccomorpha "Insecta\ Hemiptera#[ Journal of Morpholo`y 113\ 12Ð18[ Telfer\ W[ H[ "0864# Development and physiology of the oocyte! nurse cell syncytium[ In Advances in Insect Physiolo`y\ eds[ J[ E[ Treherne\ M[ J[ Berrige and V[ B[ Wigglesworth\ Vol[ 00\ pp[ 112Ð208[ Aca! demic Press\ London[ Tsutsumi\ T[\ Matsuzaki\ M[ and Haga\ K[ "0884# Formation of germ cell cluster in tubuliferan thrips "Thysanoptera#[ International Jour! nal of Insect Morpholo`y + Embryolo`y 13\ 176Ð185[ Trendelenburg\ M[ F[ "0863# Morphology of ribosomal RNA cistrons in oocytes of the water beetle\ Dytiscus mar`inalis L[ Chromosoma 37\ 008Ð024[ Trendelenburg\ M[ F[\ Scheer\ U[ and Franke\ W[ W[ "0862# Structural organization of ribosomal DNA in oocytes of the house cricket[ Nature New Biolo`y 134\ 056Ð069[

239

S[ M[ Bilinski and J[ Buning

Whiting\ M[ F[\ Carpenter\ J[ C[\ Wheeler\ Q[ D[ and Wheeler W[ C[ "0886# The Strepsiptera problem ] phylogeny of the holometabolous insect orders inferred from 07S and 17S ribosomal DNA sequence and morphology[ Systematic Biolo`y 35\ 0Ð57[ Wightman\ J[ A[ "0862# Ovariole microstructure and vitellogenesis in

Ly`ocoris pabulinus "L[# and other mirids[ "Hemiptera ] Miridae#[ Journal of Entomolo`y 37\ 092Ð004[ Willman\ R[ "0876# The phylogenetic system of the Mecoptera[ Sys! tematic Entomolo`y 01\ 408Ð413[