\PERGAMON International Journal of Insect Morphology 17 "0888# 4Ð00

9919!6211:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[PII] S 9 9 1 9 ! 6 2 1 1 " 8 7 # 9 9 9 2 9 ! 9

Intra!follicular visceral musculature in Omor`us freyi "HAAF#"Coleoptera] Trogidae# testes

Michael Friedla�ndera\�\ Rina Eren Jegera\ Clarke H[ Scholtzb

a Department of Life Sciences\ Ben!Gurion University of the Negev\ Beer Sheva 73094\ Israelb Department of Zoology and Entomology\ University of Pretoria\ Pretoria 9991\ South Africa

Received 00 July 0887^ accepted 13 October 0887

Abstract

The testicular follicles of the desert!adapted beetle Omor`us freyi "Haaf# "Coleoptera ] Trogidae# are unusually large\ relative tomale size[ The advance of the sperm!producing cells towards the e}erent duct and\ eventually\ that of spermatozoa to the deferentduct\ is apparently facilitated by the unique structure of these extended follicles[ In contrast to the typical insect follicle\ those of O[freyi and other scarabaeoid beetles have an internalized and elongated e}erent duct[ But\ additionally\ in O[ freyi\ the follicles aresubdivided by longitudinal septa\ radiating from the central e}erent duct[ A net of slow!supercontracting visceral muscles extendsthroughout the septa and e}erent duct[ We hypothesize that this unique structure is an adaptation maximizing reproductive potentialby mobilizing large numbers of spermatozoa throughout the huge testes and transferring them to the female during the irregularshort bouts of reproductive activity following the unpredictable rainfall[ Þ 0888 Elsevier Science Ltd[ All rights reserved[

Keywords] Beetle testes^ Intrafollicular vas e}erens^ Intratesticular sperm mobilization^ Musculature migration during phylogenesis^ Z!line membraneattachment^ Atypical insect follicle^ Muscular slow supercontraction^ Reproductive strategies

0[ Introduction

The insect testis consists of one or several elongatedfollicles ^ each follicle opens individually into an e}erentduct\ connected outside its proximal end[ The e}erentducts gather together into a deferent duct "Snodgrass\0824#[ The follicles contain clusters of synchronouslydi}erentiating spermatogenetic cells[ Each cluster is heldseparately within the lumen of a cyst that is limited by athin somatic epithelium[ The generalized insect folliclehas an undivided lumen containing cysts that are dis!tributed according to the subsequent stages of sper!matogenesis ] early mitotic stages at the distal\ blind endof the follicle and spermatozoa close to the opposite end\leading to the deferent duct "Schwalm\ 0877#[ However\in contrast with this generalized situation\ in the trogidbeetle Omor`us freyi\ the e}erent duct is located insideof the follicle\ at its longitudinal axis[ Additionally\ thelumen of the follicle is longitudinally subdivided intosectors converging towards the e}erent duct[ Within eachsector\ the cysts are distributed centripetally\ according

� Corresponding author[ Fax ] 99 861 65361789^ e!mail ] frdlnÝbgumail[bgu[ac[il

to the subsequent spermatogenetic stages ] early stages atthe periphery of the follicle and later stages close to thecentrally located e}erent duct "Friedla�nder and Scholtz\0882#[

Omor`us freyi is one of the four Kalahari rep!resentatives of the small cosmopolitan keratin!feedingscarabaeoid family Trogidae[ It is ~ightless and endemicto the Kalahari Desert[ The other species are winged andhave more widespread distribution in southern Africa[The trogid species have several unique adaptations to theharsh conditions in the Kalahari where rainfall is bothlow and irregular "mean 119 mm\ range 89Ð459 mm peryear# and temperatures are extreme ^ summer daytimetemperatures may exceed 34>C and winter temperaturesmay drop to below −09>C[ Beetle activity is restricted toperiods after summer rain\ when conditions are favour!able for feeding\ breeding\ and development of immaturestages[ Adults are long lived\ more than 1 years in the_eld\ but immature development is relatively short\ onlyabout 39 days[ The harsh conditions during the oftenlong\ hot dry intervals between rainfall episodes are avo!ided by the adults and larvae entering into a quiescentstate in the soil "Scholtz and Caveney\ 0887\ 0881#[ Notonly does activity cease during quiescence but the adultssuspend ovariole and sperm development until the next

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cycle of favourable conditions when the reproductivecycle repeats "Scholtz and Caveney\ 0877 ^ Friedla�nderand Scholtz\ 0882#[

Testis structure in the Coleoptera is poorly studied butthere appear to be 2 basic follicle types in the order ] "a#Solid slender cylinders in the basal suborder Adephaga\and two types in the derived suborder Polyphaga ^ "b#radial ampoules\ as in most Polyphaga\ and "c# shortfollicles\ as in Scarabaeoidea\ Chrysomeloidea and Cur!culionoidea "Virkki\ 0846#[ Virkki attributed the basicfollicle types to ancestry of the various groups\ implyingrelatedness amongst groups that share similar follicletype[ Although his conclusions do not agree with modernconcepts of relationship among the various groups"Lawrence and Newton\ 0884 ^ Browne and Scholtz\0887#\ he clearly demonstrated that there is considerablevariation in follicle size and shape among and withinmajor beetle groups[ Because the emphasis of his paperwas on the relationship between structure of the testisfollicle and phylogeny of a few randomly selected groupsof Scarabaeoidea\ most of the histological data were pro!vided for these groups[ Virkki "0846# claimed that allscarabaeoids have short follicles with the vas e}erensextended deep into the follicle\ an apparent apomorphiccharacter for the superfamily "our conclusion#[ However\the depth of penetration of the vas e}erens\ its terminalshape and the degree of septal development appear tovary independently at lower taxonomic levels ^ the natureof the variation being phylogenetically determined andthe amount environmentally induced[ Stringer "0889#provided references to the few studies on scarabaeoidtesticular structure undertaken since Virkki|s study[

The family Trogidae is one of the ancient scarabaeoidgroups\ which has evolved numerous unique traits suchas feeding on keratin\ and the ability to survive underextreme conditions in hot deserts around the world "Sch!oltz and Chown\ 0884#[ Some species of the genus Omor!`us are highly adapted to surviving extreme environ!mental conditions in the Kalahari Desert of southernAfrica by using behavioural "Scholtz and Caveney\ 0877\0881# or physiological "Friedla�nder and Scholtz\ 0882 ^Lelagadec et al[\ 0887# mechanisms[ Adult Omor`usbeetles have two reproductive strategies ] short!term\reversible reproductive quiescence during hot\ dry sum!mer periods when conditions are unfavourable for imma!ture development ^ and seasonally irreversible winterdiapause "Friedla�nder and Scholtz\ 0882#[ Males andfemales are capable of breeding within hours of a showerof rain after reproductive quiescence\ thus maximizingreproduction as soon as environmental conditions allow[The present study of Omor`us freyi testes was undertakento clarify whether the highly modi_ed structure oftheir follicles may contribute to the male|s high repro!ductive potential\ adapted to a system of reproductionrestricted to unpredictable\ irregular\ and short bursts ofactivity[

1[ Materials and methods

Omor`us freyi males were collected in the vicinity of TweeRivieren "16> 17|S 19> 26|E# in the Kalahari Gemsbok NationalPark\ South Africa[ The testes were dissected out and _xed in2) glutaraldehyde in 9[1 M cacodylate bu}er\ pH 6[2[ Sub!sequently\ they were post_xed in 0) OsO3 in the same bu}er\dehydrated and embedded in an epoxy resin[ Semithick sections"0Ð1 mm# were stained with toluidine blue "Dawes\ 0860# andstudied by light microscopy[ Thin sections were contrasted inuranyl acetate and lead citrate and studied by electronmicroscopy[

2[ Results

2[0[ Testes design

Omor`us freyi males have two separate\ ellipsoidal andremarkably large testes[ Their long axes measure approxi!mately 4 mm\ while the total length of the male beetle isonly about 09 mm[ Each testis contains six elongated\_nger!like follicles ^ each follicle is enclosed by a basallamina and extends the length of the testis[ The folliclesare held together by loose connective tissue[ The diameterof cross sections through the bulkiest portion of the testesis approximately 0 mm and that through the bulkiestportion of the follicles is about 9[2 mm[ As indicated inthe introduction\ internal architecture of the follicles ofO[ freyi di}ers remarkably from that of the typical insectfollicle[ In O[ freyi\ radial septa subdivide the follicle into01Ð05 longitudinal sectors\ converging towards a centralduct[ Within the sectors\ the cysts containing the suc!cessive spermatogenetic stages are distributed centrip!etally\ rather than in the polarized manner along thefollicular axis\ which characterizes the typical insectfollicle[ Cross sections of Omor`us follicles resemble slicesof an orange cut through the equatorial plane "Fig[ 0#[For additional information see Friedla�nder and Scholtz"0882#[

2[1[ Septa

Projections of the septa penetrate among the cysts con!taining the spermatogeneous cells ^ each cyst is wrappedindividually by the projections[ The septa and their pro!jections contain numerous visceral muscle cells\ trachea\tracheoli and their accompanying tracheolar cells[ Allthese components are surrounded by electron!opaquematerial that stains deeply by toluidine blue at the lightmicroscope level "Fig[ 1A#[ The presence of the electron!opaque material considerably obscures the resolution ofthe components of the septa[ Therefore\ to increase res!olution within the septa and to facilitate their scrutiny\in some cases\ we used testes from quiescent individuals[The reason for this is that the dispersion of the com!ponents within the electron!opaque material is more pro!

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Fig[ 0[ "A# Cross section of follicle resembling an orange cut through its equatorial plane[ "B# Higher magni_cation showing sectors separated byradial septa "r# that converge towards a centrally located e}erent duct "d#[ The cysts are distributed centripetally within the follicles\ according thesuccessive spermatogenetic stages ] early cysts at the periphery and mature ones close to the duct[ Primary spermatocytes "sp# ^ spermatozoa "s#[A×119 ^ B×239[

nounced in quiescent individuals than in active ones[ Inturn\ the increased dispersion results from a considerableincrease in the volume of the electron!opaque material[Otherwise\ structure\ dimensions and interrelations of thecomponents appear to be similar in both quiescent andactive individuals[

2[2[ Efferent duct

The e}erent duct forms the long axis of the follicle andconsists of two concentric layers of cells[ The inner layer iscomposed of large cuboidal epithelial cells having horse!shoe or goblet!shaped nuclei that are located at the base\close to the apposed outer cell layer "Fig[ 1B#[ In theinner layer\ cell boundaries are rather obscure at the lightmicroscope level "Fig[ 1B# but\ at the electron microscopelevel\ they are always evident and characteristically tor!tuous showing deep interdigitations[ The membranes ofadjacent cells are connected by electron!opaque\extended separate junctions "Fig[ 2C#[ The outer layercontains visceral muscle cells "Fig[ 1B#\ similar to thosefound in the septa "Fig[ 1C#\ as reported below[

2[3[ Visceral muscle cells

The muscle cells form a continuous network showingidentical structure throughout septa\ septal projectionsand e}erent duct "Fig[ 1E and F#[ The cells are relativelylong and have irregular\ oval or elongate!~at pro_les intransverse sections[ Their nuclei are round or oval andare surrounded by a thin layer of cytoplasm[ The nucleishow no particular location in either the septa or duct[

The larger portion of the muscle cells is within the septa\diverging at diverse angles from the axis of the duct "Fig[1E#[ Longitudinal sections of the duct wall display short\quasi!transversal sections of these cells[ The cytoplasmstains lightly in light microscope sections[ At the ultra!structural level\ the cytoplasm shows low electron opac!ity\ myo_brils and few mitochondria "Fig[ 2A# ^ othercellular structures are inconspicuous[ The myo_brils ofthe septal portion of the cells form a loose network amongthe cysts and attach to the basal lamina of the follicleenvelope "Fig[ 1D and F#[ The myo_brils may fuse in thegap between the duct and the nearest cysts\ forming short\thicker structures[ Subsequently\ these structures disperseagain forming funnel!like nets of thinner myo_brils thatsurround the cysts\ crisscrossing the electron!opaquematerial "Figs 1E and F\ 2A and D\ 3A#[ In cross sections\the clear A bands display a pattern of 5Ð00 actin micro!_laments surrounding each myosin _lament "Fig[ 2E#[No distinct H!zone or M!line could be found ^ I!bands\however\ are sometimes visible[ The Z!lines are con!spicuous but incomplete or perforated\ leaving gapsthrough which the _laments are seen crossing the borderbetween contiguous sarcomeres "Figs 2B and 3C#[ TheZ!lines attach to periodic iris!like invaginations of thecell membrane "Figs 2B and 3C#[ Owing to these inva!ginations\ longitudinal sections of the muscle cellsresemble {{string of beads|| con_gurations at the lightmicroscope level "Fig[ 3B#[ Frequently\ the sections dis!play series of wavy lines connected outside the cell mem!brane\ at the iris!like invaginations to which Z!lines areattached\ inside the membrane "Figs 1A and E#[ Thesewavy lines\ probably of membranous nature\ may extend

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Fig[ 1[ "A# Section of septum between cysts "c# containing visceral muscles "v#\ tracheoli "t# and tracheolar cells "tr#[ They are surrounded by deeplystained material showing wavy lines "arrow head#\ attached to muscle cell membrane[ Follicle envelope "f#[ ×0799[ "B# Section through e}erent duct"d#[ The wall is made of two concentric layers ] the inner one "i# is epithelial and have horse!shoe or goblet shaped nuclei "n# and ill de_ned cellboundaries ^ the outer layer "o# contains visceral muscles "v#[ Intrafollicular cysts "c#[ ×1099[ "C# Transverse section through follicle of quiescentindividual[ Cysts are absent from the sectors "�#\ close to the e}erent duct "d#[ Duct contains no spermatozoa "compare with Fig[ 0#[ Nuclei "n# ofvisceral muscles bulge from the septa "compare with "E##×749[ "D# Attachment of intraseptal visceral muscle "arrow heads# to basal lamina "f# offollicle envelope[ ×1099[ "E# Longitudinal section of e}erent duct showing inner layer "i# made of large cuboidal epithelial cells and outer layer "o#\from which visceral muscles "v# diverge "compare with "C##[ Wavy lines extend laterally from muscle cells "arrow heads#[ Visceral muscle nuclei "n#[×749[ "F# Longitudinal section through e}erent duct "d# showing myo_brils that fuse and branch forming a loose network that crisscross amongcysts "c#[ ×249[

deeply within the surrounding electron!opaque materialand are particularly conspicuous in micrographs con!taining longitudinal sections of muscle cells[

3[ Discussion

Omor`us testes display an extended intra!follicular netof visceral muscles\ a type of muscle that is associatedwith slow speed of contraction and characterized by ] "a#contractile material restricted to reduced portions of thesarcoplasm ^ "b# lack of T!tubule invaginations and SR

cisterns ^ "c# scarcity of mitochondria ^ "d# A!bands hav!ing higher actin!myosin ratios than those found in skel!etal muscles ^ "e# variable number of the actin _lamentsin the orbitals surrounding the myosin ones ^ "f# frequentincomplete or imperfect actin orbitals ^ "g# no clear H!zone or M!line\ as actin and myosin _laments are in poorlateral register "Osborne\ 0856 ^ Rice\ 0869 ^ Huddart\0874#[

In addition\ the visceral intrafollicular muscles ofOmor`us have perforated or incomplete Z!lines\ a charac!ter that is related to supercontraction of both visceral

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Fig[ 2[ "A# Section of visceral muscle cell displaying cross "cr# and oblique "ob# sections of branching myo_brils "compare with Fig[ 1E and F#[Nucleus "n#[ ×25\999[ "B# Longitudinal section of visceral muscle cell showing deep iris!like invaginations of the cell membrane encircling the Z!lines"z#[ Z!lines are incomplete leaving gaps through which _brils pass from one to the next sarcomere[ No distinct H!zone or M!lines are present[×25\499[ "C# E}erent duct "d#[ Cells of inner layer "i# have tortuous boundaries displaying deep interdigitations[ Electron!opaque septate junctionsoccur between adjacent cells "arrow heads#[ Outer layer contains visceral muscles "v#[ ×04\699[ "D# Cross section through myo_bril subdivided bydeep membranous invaginations[ ×12\999[ "E# Cross section through A! band that shows orbitals of 5Ð00 actin _laments surrounding each myosin_lament[ ×59\999[

and skeletal muscles "Smith\ 0873#[ We are unaware ofany previous reports on intra!follicular musculature inany insect species[ The only relevant record we could traceis a very good light microscope photograph of {{cords [ [ [resembling muscle _bres||\ found in testes of a cetoniinescarabaeoid beetle belonging to the genus Potosia"Virkki\ 0846#[ We are also unaware of any previousreports on the bizarre attachment of the Z!lines of indi!vidual myo_brils to invaginations of the cell membrane[

The intra!follicular musculature of Omor`us probablyderives from that of the e}erent duct\ migrating during

phylogenesis from its original external position\ at theproximal end of the follicle\ to a newer internal andcentrally located one[ This idea is supported by the fol!lowing ] "a# the common mesodermal origin of both fol!licular wall and e}erent duct "Imms\ 0853 ^ Johannsenand Butt\ 0830# ^ "b# the coherence and continuity of thecommon musculature throughout the septa and duct ^ "c#the correlation between the presence of the intrafollicularsepta containing the muscles and the intrafollicularlocation of the vas e}erens\ characters that are lackingfrom the typical insect testes ^ "d# the unusual attachment

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Fig[ 3[ "A# Branching myo_brils crisscrossing among cysts "c#[ ×0299[ "B# Longitudinal section of visceral muscle _bril resembling {{string of beads||due to cell membrane iris!like invaginations encircling the Z!lines[ ×0099[ "C# Longitudinal section through visceral muscle cell showing iris!likeinvaginations encircling Z!lines[ The Z!lines show perforations through which myo_brils pass from one sarcomere to the next one[ H!zones or M!lines are absent[ Septal electron!opaque material "e#[ ×8799[

of the Z!lines to cell membrane invaginations\ an organ!ization that may make possible functioning of the isolatedmyo_brils\ after they branch from the e}erent duct andpenetrate the septa and their projections[

It may well be that intrafollicular musculature\ as _rstreported here\ is not restricted to Omor`us[ It probablyoccurs in other beetle species too\ since internal locationof the e}erent duct has also been reported in other scara!baeoid species "Virkki\ 0846 ^ Stringer\ 0889#[

The typical insect follicle is relatively short in com!parison to body dimensions[ In such follicles\ the pressureproduced by the newer cysts formed at the folliculardistal\ blind end is enough to push the older cysts towardsthe opposite end\ leading to the extra!follicular duct"Blum\ 0874#[ The _nal part of the cyst displacementwithin the follicle has been related to synchronously beat!

ing of the ~agellae of the late di}erentiating spermatids"Hinton\ 0853#[ Eventually\ extra!testicular muscle layersdisplace the spermatozoa through the deferent duct"Romoser\ 0862#[ However\ such a system may be un_tfor transferring and discharging enough spermatozoafrom the huge Omor`us follicle that extends about halfthe length of the male beetle\ during the relatively shortperiods of summer activity[ The innovative appearanceand persistence of the intra!follicular septal muscles mayhave a selective advantage related to this problem[ The{{Omor`us solution|| brought the musculature\ which wasoriginally located outside the follicle\ to its axis[ Thiswould solve the problem of mobilizing and transferringthe cysts through the length of the follicle by using mainlythe relatively weak pressure exerted by the newly formedcysts[ The original mechanism\ found in relatively small

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follicles\ would most probably be ine.cient for trans!ferring the cysts of the huge Omor`us follicle from thedistal end to the opposite one[ In Omor`us follicles\ themuscular network extends uninterruptedly throughoutsepta and duct and its myo_brils are of the type able toproduce slow supercontraction[ This system portrays acoordinated unit able to work intensively for the rela!tively short intervals of beetle activity[ During these shortintervals\ mobilization of large amounts of spermatozoathroughout the length of a huge testis\ and their transferto the female\ should be maximized[ This mode of oper!ation may _t very well for an insect such as Omor`us\which is active only during the very short\ sporadic andunpredictable rainfall periods\ which characterize theKalahari Desert[ Consequently\ Omor`us freyi testesstructure clearly contains a phylogenetic component asillustrated by the deep penetration of the vas e}erensin the follicle\ something shared with all other studiedscarabaeoids[ On the other hand\ the extraordinary sizeof the follicles and the presence of the septal musculaturemay be adaptations to the extreme conditions in whichthe species occurs[

Acknowledgements

We thank Gideon Raziel for the skilful printing ofthe micrographs and the South African Foundation forResearch and Development for funding a visit by M[F[to South Africa[

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