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Antennal circulatory organ of Apis mellifera L. (Hymenoptera: Apidae) and other Hymenoptera: functional morphology and phylogenetic aspects
\ PERGAMON
International Journal of Insect Morphology 17 "0888# 86Ð098
Antennal circulatory organ of Apis mellifera L[ "Hymenoptera] Apidae# and other Hymenoptera] functional morphology and phylogenetic aspects S[ Matus \ G[ Pass Institut fur Zoologie der Universitat Wien\ Althanstra)e 03\ A!0989 Wien\ Austria Received 09 August 0887^ accepted 09 January 0888
Abstract The antennal circulatory organs of 27 species of Hymenoptera were investigated by means of serial semithin sections\ SEM and TEM[ In Apis\ this organ consists of an unpaired ampulla from which blood vessels that extend into the antennae originate[ The ampulla is a very delicate structure of elastic connective tissue[ Its lumen communicates with the head hemocoel via numerous perforations in its wall[ No speci_c ampulla muscles exist^ contraction of pharynx dilators causes compression of the ampulla\ whereby hemolymph is forced into the antennae[ An absence of directly!acting muscles is common to the functional morphologies of the antennal circulatory organ in all investigated Hymenoptera[ Some anatomical characters of this organ\ however\ vary among taxa] "i# presence of one or two ampullae\ "ii# optional dorsal hemolymph channel\ which connects the ampulla with the dorsal vessel\ "iii# optional ampulla pumping case formed by cuticular apophyses\ and "iv# di}erences in shape and number of the ampulla openings[ The distribution of these characters is discussed along the current views on the phylogeny of Hymenoptera[ Comparison with other insect orders indicates an autapomorphic status for the basic functional morphology of the antennal circulatory organ in Hymenoptera[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Insects^ Accessory pulsatile organs^ Heart^ Hemolymph^ Evolution
0[ Introduction The tubular dorsal heart of insects is apparently not capable of supplying long body appendages with hemo! lymph[ Autonomous auxiliary hearts serve this task in antennae\ legs\ wings\ and cerci "Jones\ 0866^ Miller\ 0874^ Pass\ 0887#[ Most earlier descriptions of those minute organs were based on studies carried out with classical histological techniques and consequently are rather vague[ More recent investigation has revealed that these accessory circulatory organs are surprisingly diverse with respect to their functional morphologies and there! fore are of special interest to phylogenetic studies[ More! Corresponding author[ Tel[] "¦32#!0!202 25:0237^ fax] "¦32#!0! 202 25:667 e!mail] sonja[matusÝunivie[ac[at Abbreviations] Am\ ampulla^ AV\ antennal vessel^ Br\ brain^ FG\ frontal ganglion^ Fr\ frons^ FR\ frontoclypeal ridge^ FS\ frontal sinus^ HC\ hemolymph channel^ He\ hemocyte^ HS\ hypopharyngeal sclerite^ LA\ lateral apophyse^ M0\ frontal pharynx dilators "dilatatores pharyn! gis frontales#^ M1\ postfrontal pharynx dilators "dilatatores pharyngis postfrontales#^ M2\ ring muscle of pharynx^ Os\ ostium^ Ph\ pharynx^ Va\ valve[
over\ due to their relatively simple design\ they are useful model systems for investigation on general aspects of organ evolution "Hantschk\ 0880^ Krenn and Pass\ 0883\ 0884^ Pass\ 0880\ in press# and neurobiological research "Hertel et al[\ 0874^ Pass et al[\ 0877a\b#[ Antennal circulatory organs have been reported to exist in various insect orders and di}er distinctly from each other in structure and functional mechanism "Blat! todea] Pawlowa\ 0784^ Pass\ 0874^ Dermaptera] Pass\ 0877^ Orthoptera] Bayer\ 0857^ Hemiptera] Pinet\ 0853^ Megaloptera] Selman\ 0854^ Coleoptera] Pass\ 0879^ Lepidoptera] Schneider and Kaissling\ 0848\ Diptera] Clements\ 0845^ Dudel\ 0866\ 0867a\b^ comparative investigation and review of non!Holometabola] Pass\ 0880\ 0887#[ In Hymenoptera\ very little is known about these organs[ As early as 0788\ Janet remarked that small ampullary organs to which he ascribed a circulatory func! tion existed in the frontal region of the head of Myrmica[ Brocher "0810# could not _nd a comparable organ in the circulatory system of Vespa crabo in spite of thorough research "Janet\ 0788#[ To date\ the only relatively detailed description of such an organ in a hymenopteran
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 8 # 9 9 9 0 0 ! 1
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is of Apis mellifera[ First notice of the antennal cir! culatory organ of the honeybee was given by Janet "0800#\ followed by a more precise description by Freudenstein "0817#[ In addition to anatomical and histological inves! tigation\ Freudenstein also performed experiments designed to clarify the functional mechanics of this organ[ The aim of the present study is to reinvestigate the functional morphology of the antenna!heart in Apis mel! lifera by means of light and electron microscopy\ to com! pare the antennal circulatory organs within Hymenoptera and to study their phylogenetic devel! opment[ 1[ Materials and methods Serial semithin sections of the antenna!heart region in the head were subjected to a light microscopical evalu! ation in the species listed below[ In some species\ the antennal circulatory organs were examined by means of SEM and in Apis mellifera also by TEM[ Only imagines were considered[ List of investigated species {Symphyta| Xyelidae] Xyela alpi`ena "Strobl\ 0784#^ Xyela sp[ Pamphiliidae] Cephalcia abietis "Linne\ 0647# Siricidae] Urocerus au`ur "Klug\ 0792# Diprionidae] Neodiprion sertifer "Geo}roy\ 0674#^ Diprion sp[ Xiphidriidae] Xiphydria sp[ Tenthredinidae] Allantus sp[^ Pristiphora abietina "Christ\ 0680#^ Tenthredo campestris "Linne\ 0647# Cephidae] Trachelus tabidus "Fabricius\ 0664#^ Janus compressus "Fabricius\ 0682#^ Harti`ia linearis "Sch! rank\ 0670# {Terebrantes| Evaniidae] Evania appendi`aster "Linne\ 0647# Cynipidae] Diplolepis rosae "Linne\ 0647#^ Andricus sp[ Aphelinidae] Encarsia formosa Gahan\ 0813 Leucospididae] Leucospis `i`as Fabricius\ 0682 Ichneumonidae] Amblyteles sp[^ Ophion luteus "Linne\ 0647# Braconidae] Apanteles sp[\ Aphidius sp[ Aculeata Scoliidae] Scolia hirta Schrank\ 0670 Mutillidae] Mutilla europaea Linne\ 0647^ Dasylabris maura "Linne\ 0647# Tiphiidae] Tiphia femorata Fabricius\ 0664 Vespidae] Paravespula `ermanica Fabricius\ 0682^ Paravespula vul`aris "Linne\ 0647#^ Nectarinella championi "Dover\ 0814#
Formicidae] Lasius ni`er "Linne\ 0647#^ Camponotus sp[ Sphecidae] Mellinus arvensis "Linne\ 0647#^ Sce! liphron sp[ Ampulicidae] Ampulex compressa "Fabricius\ 0670# Apidae] Apis mellifera Linne\ 0647^ Bombus hortorum "Linne\ 0650# Megachilidae] Osmia cornuta "Latreille\ 0794# Andrenidae] Andrena humilis Imho}\ 0721 1[0[ Light microscopy As a rule\ the heads of the investigated species were _xed in alcoholic Bouin "{DubosqÐBrasil| mixture\ see Romeis "0878##[ In some cases\ material _xed in ethanol also yielded satisfactory results[ Specimens were embed! ded into Spurr epoxy resin under vacuum impregnation with acetone the in_ltration solvent[ For light microscopical evaluation\ serial semithin sec! tions "0 mm# were made of the antenna!heart region in the head[ The specimens were sectioned with an ultra! microtome with glass or diamond knives "Diatome\ Swit! zerland#[ The sections were stained in a mixture of azure II "0)# and methylene blue "0)# in an aqueous borax solution "0)# at 79>C[ 1[1[ Scanning electron microscopy For microanatomical studies\ the heads of the inves! tigated species were _xed in the aforementioned way[ Specimens were in_ltrated with para.n using methyl! benzoate as intermediate medium[ The embedded heads were then trimmed with a razor blade and the para.n removed in xylol "for 29 min at room temperature#[ After! wards\ the specimens were transferred into acetone and dehydrated using the critical point method[ Finally\ they were sputtered with gold and examined with a Jeol JSM! 24CF microscope[ 1[2[ Transmission electron microscopy Heads of Apis mellifera were _xed in a mixture of glutaraldehyde "1[4)# and paraformaldehyde "1)#\ fol! lowed by 0) osmium tetroxide\ both _xatives being bu}ered with 9[0 M sodium cacodylate "pH 6[1#[ Heads were then embedded into Spurr epoxy resin with acetone the in_ltration solvent[ Ultrathin sections were double! stained in uranyl acetate and lead citrate and examined with a Zeiss EM!891 electron microscope[ 2[ Results 2[0[ Apis mellifera The antennal circulatory organ of Apis mellifera is located in the frontal part of the head between the
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ally diminishes to a diameter of 14 mm before the vessel enters the antenna[ Within the antennae\ the lumen remains equidiametrical all along[ In the distal ~agellar segment\ the antennal vessels open into the antennal hemocoel via a terminal pore[ A minute membrane ~ap exists internally at each an! tennal vessel base "Fig[ 2A and 2B#[ It protrudes in an oblique orientation into the vessel lumen[ Its proximal ends are attached to the vessel wall\ its distal ends\ however\ are loose\ shaping a funnel!like structure acting as back~ow valve between ampulla and antennal vessel[ The valve region at the vessel bases is approximately 34 mm in length[ The vessel wall is about 1[4 mm thick and consists of an epithelium covered by acellular external laminae of 9[1 mm "inner# and 9[2 mm "outer#[
Fig[ 0[ Apis mellifera\ frontal view of antenna bases in the head[ Location of antennal circulatory organ "ampulla\ antennal vessels# indi! cated into the SEM micrograph by broken lines[
antenna bases "Fig[ 0#[ The organ consists of an ampulla with blood vessels originating from either side that extend into their corresponding antenna[ The pharynx and some of its dilator muscles\ which are in close vicinity to the ampulla\ are functionally related to the antennal cir! culatory organ and are therefore included in the following description[ 2[0[0[ Ampulla The ampulla is attached to the frontal cuticle and the frontoclypeal ridge[ It is a somewhat ~at sac measuring about 179 mm in diameter[ The anterior wall of the ampulla is formed by the hypodermis attached to the central part of the frons "Figs 1 and 2#[ Its posterior wall is a very delicate structure whose margins are attached to the integument^ it is also linked to the surrounding organs by numerous thin suspending strands "cross!section about 9[2 mm in diameter#[ The posterior ampulla wall is about 9[5 mm in thick! ness[ It is a lamina consisting of a _lamentous matrix\ into which a few\ ~at cells are embedded "Fig[ 2E#[ Ultra! structurally\ they resemble connective tissue cells and contain no contractile elements[ In its upper part\ the posterior ampulla wall has an area "diameter about 49 mm# with numerous perforations "Fig[ 2C and 2D#[ These are openings about 2Ð4 mm in diameter[ 2[0[1[ Antennal vessels A single blood vessel originates from either side of the ampulla\ arches around the antenna base and continues into the antenna[ The lumen width of their vessel gradu!
2[0[2[ Functionally related structures Because the ampulla wall is not muscular and because no muscles insert at the ampulla\ its function as a hemo! lymph pump must involve some indirect pumping mech! anism[ Owing to the location of the ampulla between the frons and the pharynx\ movement of the pharynx must a}ect the variable width of the ampulla lumen[ Four pairs of muscles originate at the frontal cuticle in the area above the ampulla] the anterior labral muscle inserts at the inner labrum\ the retractor of the mouth!angle at the anterior part of the hypopharyngeal suspensorium sclerite and the two pairs of the frontal pharynx dilator muscles insert at the pharynx roof[ Contraction of these muscles move the pharynx roof towards the frons and thereby indent the posterior ampulla wall[ 2[1[ Other Hymenoptera 2[1[0[ Symphyta The functional morphology of the antennal circulatory organ varies considerably among high!rank taxa of Sym! phyta s[l[^ however\ fundamental intra!familial di}er! ences were not found[ In Xyelidae\ the antennal circulatory organ is similar to that of the honeybee[ It consists of a median ampulla about 109 mm long and 019 mm wide[ In contrast to Apis\ the ampulla of Xyela alpi`ena has just a single median opening[ It is a relatively large opening of about 34 mm with no valve structures[ Another incongruity to Apis is the presence of lateral cuticular apophyses which are attached to the ampulla wall[ In Tenthredinidae and Diprionidae\ the antennal cir! culatory organ is completely di}erent from those of all other examined Hymenoptera in that it has an ampulla for each antenna base "Fig[ 3A#[ These ampullae lack attached muscles[ The structure of the wall is the same as in all other Hymenoptera[ Ampullae open into a median hemolymph sinus via slit!like\ valved ostia "Fig[ 4#[ This sinus is anteriorly con_ned by the hypodermis\ while
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Fig[ 1[ Apis mellifera\ schematic drawing of the antennal circulatory organ in lateral view "A# and dorsal view "B and C#[ Unpaired ampulla with paired vessels extending into antennae[ Anterior wall of ampulla attached to frontal cuticle above the frontoclypeal ridge[ Posterior wall with numerous perforations "arrow#[ Pharynx behind ampulla\ frontal ganglion in between\ functionally related pharynx dilators M0 and M1 also shown^ other organs omitted[ "B# and "C # show the organ in di}erent phases of action "for explanation see discussion chapter#^ arrows indicate proposed hemolymph ~ow[
its posterior boundary is formed by connective tissue laminae and partly by tracheal sacs[ The ampullae are about 74 mm wide and 049 mm long[ The base of the antennal vessels is located anterio!laterally at the ampul! lae[ Remarkably\ no valve was found at the juncture of ampullae and antennal vessels[ In Pamphiliidae\ Siricidae\ Xiphydridae\ and Cephi! dae\ the antennal circulatory organ is again very di}erent from that of Apis and most other Hymenoptera "Fig[ 3B#[ Cephalcia abietis has a median ampulla between the antennal bases[ Posteriorly\ the ampulla is connected to the anterior end of the dorsal vessel behind the brain via a hemolymph channel which runs along the dorsal side of the pharynx "Fig[ 5#[ Seen from the ampulla\ this channel narrows to about 29 mm in diameter and then keeps a constant width until it fuses with the dorsal vessel[ The channel resembles a tunnel whose upper part is for! med by a thin lamina of connective tissue and the lower part by the pharynx roof[ There are no muscles directly attached to the ampulla or the channel[ A striking feature of Cephalcia abietis is that one pair of the frontal pharynx dilators extends through the entire ampulla lumen^ the second pair lies above the ampulla[ In the siricid Urocerus au`ur and the cephids Janus compressus and Harti`ia linearis\ the antennal circulatory organ also consists of a median ampulla connected to the dorsal vessel via a
hemolymph channel[ In the transitional region between the ampulla and the antennal vessel\ back~ow valves exist similar to those in Apis[ 2[1[1[ {Terebrantes| The investigated taxa of this group show a relatively uniform situation with respect to the functional mor! phology of the antennal circulatory organ which is similar to that in Apis[ While in most investigated hymenopteran taxa\ the antennal circulatory organ as well as the antenna bases are located close to the line between frons and clypeus\ antennae of all terebrant species have a more distant position[ The ampullae\ however\ are found at the frons! clypeus border as be_ts other Hymenoptera[ This gives the proximal antennal vessel portion a notably elongated appearance "Fig[ 3C# unique among Hymenoptera[ In all investigated {Terebrantes|\ the ampulla lumen is connected to the head hemocoel via a perforated area in the posterior ampulla wall^ the perforations have no valve ~aps[ In none of the investigated species of this group\ muscle _bers could be found within the ampulla wall nor directly attached to it[ The functionally associated muscles in the proximity of the ampulla are the dilators of the pharynx and the cibarium[
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Fig[ 2[ Apis mellifera\ antennal circulatory organ[ "A# Cross section[ Ampulla attached to the frontal cuticle along its entire width between the antenna bases[ The delicate posterior ampulla wall is highlighted by several arrowheads[ Paired valves between ampulla and antennal vessels "arrows#[ On the right half the antennal vessel circling around the antennal annule[ "B# Cross section showing valve area of the left antennal vessel in detail[ Arrow indicates direction of hemolymph ~ow[ "C# SEM preparation\ parasagittal cut^ ampulla between frontal cuticle and pharynx roof\ lower part attached to the frontoclypeal ridge[ Arrowheads indicate perforations in the posterior ampulla wall[ Frontal ganglion between pharynx dilator muscle M0 and ampulla[ "D# Sagittal section^ ampulla anteriorly attached to frontal cuticle and partly compressed by the frontal ganglion[ Pharynx dilator M0 can be seen behind the ampulla[ Posterior ampulla wall suspended by connective tissue strands "arrowheads#[ "E# TEM^ sagittal section through posterior ampulla wall[ Cut of wall cell ~anked by two external laminae[
2[1[2[ Aculeata The antennal circulatory organs of all investigated acu! leate species are similar with respect to their overall func! tional morphology which in principle corresponds to the description for Apis given above[ Three characters\ however\ show remarkable di}erences] "i# the type of ampulla opening\ "ii# special associated cuticular struc! tures\ and "iii# the shape of the basal portion of the antennal vessels[ The ampulla of most Aculeata opens through numer! ous perforations in the posterior ampulla wall[ In For! micidae and Vespidae\ however\ hemolymph enters the ampulla through a single median opening[ Camponotus sp[ has a slit!like ostium with a pair of valve ~aps pro!
truding into the ampulla lumen "Figs 6B and 8#[ Para! vespula `ermanica and P[ vul`aris also have a single median opening[ No valve structure could be found "Fig[ 09#[ In Mutillidae and Tiphiidae\ the antennal circulatory organ consists of an ampulla that is clearly attached to distinct cuticular structures "Figs 6A and 7#[ These include the well!developed frontoclypeal ridge just below the ampulla as well as large cuticular apophyses deep in the head capsule boldly framing the ampulla on two sides[ There are no muscles attached to these lateral apophyses\ yet they reinforce the ampulla wall[ The ampulla in these taxa is\ unlike the usual ~at sac of all other Hymenoptera\ almost isometric and framed by these cuticular structures
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Fig[ 3[ Schematic drawings of the antennal circulatory organs in selected species of {Symphyta| "A\B# and {Terebrantes| "C#[ Each species shown in dorsal and lateral view^ arrows indicate ampulla openings[ "A# paired ampullae with median frontal sinus\ "B# unpaired median ampulla with dorsal hemolymph channel\ "C# unpaired median ampulla with elongated antennal vessel bases[
thus forming a kind of pumping case[ Muscles line the ampulla wall where cuticular structures are absent[ Ampulex compressa also has cuticular structures to which the lateral ampulla walls are attached\ as in Mutillidae and Tiphiidae[ Otherwise\ the frontoclypeal ridge and the lateral apophyses are less elongated[ Another anatomical feature\ which di}ers among spe! cies\ is the antennal vessel basis[ While in most Hymeno! ptera\ the antennal vessel has about the same diameter over its entire length\ in Mutillidae and Tiphiidae the antennal vessel bases are markedly widened compared with the rest of the vessel\ thus forming a kind of vesicle^ e[g[\ in Mutilla\ diameter is about 199 mm in the proximal portion and then abruptly diminishes to 69 mm[ In all species\ a funnel!shaped valve was found between ampulla and antennal vessel[ 3[ Discussion 3[0[ Functional morphology 3[0[0[ Apis mellifera The basic structure of the antennal circulatory organ in the honeybee was described by Freudenstein "0817#[ He correctly identi_ed the unpaired ampulla between the antenna bases as well as the point of origin of the antennal vessels\ but did not detect the valve ~aps at the bases of
the antennal vessels[ Freudenstein "0817# was incapable of unequivocally clarifying the histological nature of the ampulla wall\ given the limitations of light microscopy[ His assumption that the wall is not muscular could be substantiated in the present investigation at the electron microscopical level[ The wall is a thin lamina consisting of a delicate _lamentous matrix with a few embedded ~at cells[ The antennal circulatory organs of other insects have so far been studied on an ultrastructural level only in Periplaneta americana "Beattie\ 0865^ Pass\ 0874#[ The structure of the ampulla wall of that species in some way resembles that of Apis\ because it also partly consists of laminae of the same character[ In contrast to Apis\ however\ the wall is a true epithelium\ covered by these external laminae on both sides[ Structures consisting of matrices with unbanded _laments\ comparable to those of the laminae\ are known from some types of connective tissue in insects and said to be elastic "Ashhurst\ 0865#[ They have been found in arthropods in the extracellular sheath covering the dorsal vessel "Bacetti and Bigliardi\ 0858^ Seifert and Rosenberg\ 0867#\ in cell!less strings attaching the dorsal vessel to nearby tissue "Locke and Huie\ 0861\ 0864#\ and in mesenteric connective tissue "Francžois\ 0867#[ Although the chemical nature of these _laments has not yet been identi_ed irrefutably\ they bear a structural similarity to elastin found in the elastic connective tissue of vertebrates "Ashhurst\ 0865#[
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Figs 4 + 5[ Fig[ 4[ Allanthus sp[\ antennal circulatory organ[ "A# Cross section showing the two ampullae attached to the frontal cuticle and the hemolymph sinus between[ Arrows indicate valves in ampulla openings through which hemolymph can enter from the sinus[ "B# Parasagittal section through an ampulla in the ostium region^ note the long valve ~ap[ Fig[ 5[ Cephalcia abietis\ antennal circulatory organ[ "A# Cross section through ampulla[ The lateral walls fuse with the pharynx[ "B# Sagittal section[ Ampulla with hemolymph channel stretching along the dorsal side of the pharynx and connecting to the dorsal vessel behind the brain[ Note cuts of the pharynx dilator muscles within the ampulla lumen "arrowhead#[
Because direct observation of the ampulla activity in Apis is not possible due to the opaque cuticle of the frons\ the functional mechanics of this organ must be indirectly accessed from its functional morphology and from exper! imental data[ In an initial approach to functional mech! anics\ Freudenstein "0817# examined the direction of hemolymph ~ow in the head of the honeybee[ He injected a mixture of ink and egg albumin into the dorsal vessel and afterwards killed the bees in heated ringer solution[ Heating denatured the protein and _xed the ink[ In his! tological sections made from these specimens\ hemo! lymph ~ow could be tracked[ By doing so\ he clearly showed that hemolymph was being pumped through the ampulla into the antennal vessels[ Freudenstein|s "0817# conclusions are con_rmed by our _nding of the valve ~aps at the antennal vessel bases\ which allow only a one! way ~ow towards the antennae[ But what causes the pumping movements< Because we could not detect any muscle _bers within the ampulla wall\ no active contraction of the ampulla in Apis seems possible[ This holds true for the walls of the antennal circulatory organs of all other insects examined so far
"Pass\ 0880\ 0887#[ Only for two species "Periplaneta] Pawlowa\ 0784\ Rhodinus] Pinet\ 0853# has a muscular ampulla wall yet been reported[ Later elec! tronmicroscopical investigation\ however\ could sub! stantiate these results "Pass\ 0874\ unpublished#[ Despite many structural di}erences\ antennal circulatory organs of most other insects have muscles in common that are either attached to or are very closely associated with the ampullae[ These muscles either operate as dilators or as compressors antagonized by the elasticity of the ampulla wall "Pass\ 0879\ 0874\ 0880#[ Only a few other insects possess antennal circulatory organs without associated muscles "Odonata] Pass\ 0880\ Lepidoptera] Schneider and Kaissling\ 0848\ some Diptera] Dudel\ 0866\ 0867a\b#[ In Apis\ dilatory movements of the pharynx must entail a simultaneous compression of the ampulla\ due to its location between frons and pharynx "Fig[ 1C#[ Hemo! lymph is thereby forced out of the ampulla lumen into the antennal vessels[ After relaxation of the pharynx dilators\ the pharynx roof resumes its original position[ Congruently\ the ampulla lumen is likely to widen due to
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Fig[ 6[ Schematic drawings of the antennal circulatory organs in selected species of Aculeata[ Each species shown in dorsal and lateral view^ arrows indicate ampulla openings[ "A\ B# Large lateral apophyses supporting the ampulla wall and "C# ostium slit with valve ~aps protruding into the ampulla lumen[
the combined elasticities of ampulla wall and suspending strands[ During dilation of the ampulla\ lumen pressure is reduced and hemolymph enters the ampulla via the wall perforations "Fig[ 1B#[ In this phase\ back~ow of hemolymph from the antennal vessels is forestalled by the valve ~aps at the vessel bases[ Dilation of the pharynx occurs through rhythmic con! tractions of the pharynx dilators while the bee is feeding[ How an adequate hemolymph exchange rate can be achieved while the animal is not feeding remains unknown[ Freudenstein|s "0817# observations on the hemolymph meniscus of an apically truncated honeybee antenna are noteworthy in this respect] he described per! iodical\ weak pulses of this meniscus and non!periodical stronger pulses[ He concluded that there was always a certain pharyngeal peristaltic activity causing hemo! lymph circulation in the antennae[ Because a functional connection between circulatory organs and tracheal ventilation has been established in some insects "Wasserthal\ 0871a\b\ 0885#\ ventilatory movement must also be taken into account to explain functional mechanics of the antenna!heart in Hymen! optera[ In all species large tracheal sacs can be found close to the ampulla[ The helical design of their taenidia suggests that certain elastic properties are inherent in these structures[ In any case\ ventilatory movement of
these tracheal sacs need to cause changes in the hem! olymph pressure of the head hemocoel and must therefore somewhat a}ect the function of the antennal pulsatile organ[ To make a major contribution to pumping activity\ however\ these sacs would have to oscillate[ The structure of the antennal vessels in Apis is quite similar to that of most other insects[ The wall of the vessels is formed by a thin epithelium covered on both sides with external laminae[ A modi_cation of the proxi! mal vessel portion\ _tted with a transporting epithelium as in Periplaneta "Pass\ 0874# could not be found in Apis[ 3[0[1[ Other Hymenoptera An absence of muscles attached to the ampullae is common to all investigated species of Hymenoptera[ Some other characters\ however\ can be di}erent among species] "i# the number of ampullae\ "ii# the occurrence of a hemolymph channel\ "iii# the formation of an ampulla opening\ and "iv# associated cuticular structures[ All Hymenoptera possess unpaired ampullae\ with the exception of Tenthredinidae and Diprionidae\ which have paired ones[ In both taxa\ the anatomical situation in the frontal head region suggests that movement of the pharynx roof probably does not strongly compress the ampullae[ In these taxa\ the frontal sinus seems to be the compressible unit\ forcing hemolymph into the ampullae
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Figs[ 7\ 8 + 09[ Fig[ 7[ Mutilla europaea\ antennal circulatory organ[ "A# Cross section[ Ampulla wall attached to lateral apophyses and to pharynx roof[ Pharynx dilator M0 originating at frons[ Widened antennal vessel bases beside apophyses[ "B# Sagittal section of the ampulla surrounded by frontal cuticle\ frontoclypeal ridge\ pharynx dilator M0 and ring muscles of the pharynx roof M2[ "C# Horizontal section showing the cuticular pumping case of the ampulla formed by frontoclypeal ridge\ lateral apophyses and frontal pharynx dilator[ To either side the widened antennal vessel bases and valve ~aps[ "D# Horizontal section showing in detail the long valve ~ap between ampulla and the right antennal vessel[ Fig[ 8[ Camponotus sp[\ antennal circulatory organ[ Sagittal section[ Ampulla attached to frontal cuticle and to frontoclypeal ridge[ Posterior wall close to pharynx ring muscles] frontal pharynx dilators M0 above the ampulla and dilator M1 below[ Ostium with valve ~aps "appearing as a single ~ap in this section#[ Fig[ 09[ Paravespula `ermanica\ antennal circulatory organ[ Sagittal section through ampulla[ Posterior wall slightly indented by frontal ganglion[ Arrow indicates ostium[
via ostia[ The valve ~aps of these ostia prevent back~ow from the ampullae into the sinus[ There is no valve between the ampullae and the antennal vessels[ In Pamphiliidae\ Siricidae\ Xiphydridae\ and Cephi! dae\ the hemolymph channel represents a direct con! nection between the antennal circulatory organ and the
dorsal vessel[ The posterior boundary of the ampulla is the pharynx roof[ In these species\ dilatory movement of the pharynx probably propels hemolymph into the antennal vessels and thereby into the antennae[ Because the antennal circulatory organ is directly connected to the dorsal vessel\ pulses resulting from vessel contraction
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must have a signi_cant impact on circulation in the ante! nnae[ In Hymenoptera\ the most common type of ampullae in~ow openings are wall perforations[ A few species have but a single opening] Xyela alpi`ena has a large opening in its posterior ampulla wall^ a slit!like ostium was found in the investigated formicid and vespid species[ In Cam! ponotus\ this ostium has two valve ~aps protruding into the ampulla lumen[ In many hymenopterans\ the ampulla is attached to the frontoclypeal ridge in an anterior position lending mechanical reinforcement to the ampulla wall[ In the xyelid\ mutillid\ tiphiid\ and ampulicid species\ lateral apophyses provide such reinforcement to an even higher degree[ Together with the frontoclypeal ridge\ these apo! physes build a kind of pumping case for the ampulla[ In Mutillidae and Tiphiidae\ the lateral apophyses and the frontoclypeal ridge are extraordinarily long^ in this way\ the ampulla is tightly surrounded by these cuticular struc! tures and muscles[ 3[1[ Phylogenetic aspects 3[1[0[ Hymenoptera and their sister!group Phylogenetic relationships of Hymenoptera with other insects have not yet been entirely uncovered "Hennig\ 0858^ Konigsmann\ 0865^ Kristensen\ 0884#[ A distinct phylogenetic isolation of this group has long been recog! nized and Ross "0854# has even suggested a sister!group relationship between Hymenoptera and all other Holo! metabola[ A feasible working hypothesis\ for the time being\ is to assume a sister!group relationship between Hymenoptera and Mecopteroidea "Konigsmann\ 0865^ Kristensen 0884#[ Comparing the antennal circulatory organs of Hymen! optera with those in Mecopteroidea\ we _nd distinct di}erences which are not easy to interpret[ Moreover\ within Mecopteroidea the situation is not uniform] in all investigated Amphiesmenoptera\ the antennal vessels are linked to a frontal sac\ which lies dorsally of the pharynx and just in front of the brain "Trichoptera] Pass\ unpub! lished^ Lepidoptera] Schneider and Kaissling\ 0848#[ As in Hymenoptera\ no muscles are directly attached to it[ In Antliophora\ however\ we have pulsatile ampullae connected to the antennal vessels "Diptera] Clements\ 0845^ Dudel\ 0866\ 0867a\ 0867b^ Mecoptera] Pass\ unpublished#[ In Diptera\ there are species with one ampulla and others with two[ The existence of two ampul! lae in the sister!group Mecoptera "Panorpa] Pass\ unpub! lished# suggests that the two!ampullae type is the plesiomorphic character state for Diptera as well[ Strik! ing di}erences exist within Antliophora in number and function of the muscles associated with ampullae[ Only in few taxa of Diptera\ are ampullae devoid of muscles "Stratiomyidae] Dudel\ 0866^ Pipunculidae] Dudel\ 0867a^ Diopsidae] Dudel\ 0867b#[ The investigated spec!
ies of Stratiomyidae and Pipunculidae have a single median ampulla\ which probably functions in a manner similar to the ampullae of Hymenoptera[ Diopsidae have paired ampullae probably compressed via contractions of speci_c antennal muscles lying close to each of the ampullae[ In all other species of Diptera\ muscles dilate the ampullae[ While they are always directly attached to the ampulla wall\ their other attachment site can be very di}erent structures[ Among Mecoptera\ Panorpa also has paired ampullae to which dilator muscles are directly attached^ here the other attachment site is the pharynx roof "Pass\ unpublished#[ The indirect pumping mechanism in the functional anatomy of the antennal circulatory organ in Hymen! optera is only distantly related to the respective ana! tomies of other insects\ both exopterygotes and endopterygotes[ Therefore\ this unique type of antennal circulatory organ of Hymenoptera appears to be an aut! apomorphy of this order which con_rms the isolated position of Hymenoptera within insects[ 3[1[1[ Phylogenetic aspects in Hymenoptera Relationships among major taxa of Hymenoptera that stood _rm in the main phylogenetic branches for a long time have recently begun to be questioned "Konigsmann\ 0866^ Gibson\ 0874^ Whit_eld\ 0881^ Dowton and Austin\ 0883#[ Classical high!rank taxa like Symphyta and Tere! brantes turned out to be paraphyletic when their status was critically examined by means of phylogenetic sys! tematics "Konigsmann\ 0866\ 0867^ Rasnitsyn\ 0879\ 0877#[ Only Aculeata could defend their position as a truly monophyletic taxon "Brothers\ 0864^ Konigsmann\ 0868^ Dowton and Austin\ 0883#[ The evolutionary pathways of the antennal circulatory organ in Hymenoptera are di.cult to trace^ various con! clusions are possible[ Figure 00 gives a survey of the systematic distribution of the di}erent characters present in antennal circulatory organs of investigated Hymeno! ptera[ The most crucial di}erences and the greatest diver! sity can be found among the {Symphyta|\ in which abun! dance of plesiomorphic character states is generally recognized[ Xyelidae\ often considered as those Hymen! optera with the greatest number of plesiomorphic charac! ter states "Rasnitsyn\ 0879#\ have an unpaired antennal circulatory organ similar to that in the derived Aculeata[ In Mecopteroidea\ the probable sister!group of Hymeno! ptera\ the ancestral character state of the antennal cir! culatory organ is paired ampullae^ therefore outgroup comparison suggests that the organs of Tenthredinidae and Diprionidea with their paired ampullae represent the plesiomorphic character state in Hymenoptera[ The similar character states found in Xyelidae and Aculeta have thus to be rather considered a convergence than the plesiomorphic character state in Hymenoptera[ The occurrence of an ampulla connected to the dorsal vessel by a hemolymph channel in most lineages of investigated
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096
Fig[ 00[ Antennal circulatory organ characters mapped onto a cladogram of Hymenoptera combined and modi_ed according to Heraty "0883#[ Superfamilies and families that were not investigated in the present study have been omitted[ Nomenclature follows Huber and Goulet "0882#[ Number of investigated species in parentheses[
{Symphyta| has to be interpreted as a convergence too\ because such a character state does not exist in any non! hymenopteran insect[ In this study\ only few taxa in the so!called Terebrantes were investigated which show a uniform situation with regard to their antennal circulatory organs[ All possess an unpaired median ampulla with one antennal vessel originating from either side[ Unique among Hymeno! ptera are the very long bases of their antennal vessels in the head capsule[ In Aculeata\ the overall morphology of the antennal circulatory organ is also relatively uniform[ Some ana! tomical features\ however\ possess noteworthy phylo! genetic aspects] while in most taxa the ampulla openings are wall perforations\ the ampulla opening to the head
hemocoel in Formicidae and Vespidae represents an ostium[ A valve exists only in Formicidae[ Therefore\ and owing to the fact that in Scoliidae\ the sister!group of the VespidaeÐMasaridaeÐEumenidae\ the ampulla openings are numerous perforations\ as in all other Aculeata\ the ampulla ostium may have developed convergently in Ves! pidae and Formicidae[ Cuticular structures attached to the antenna!hearts\ i[e[\ lateral apophyses are relatively simple to interpret from a phylogenetic point of view[ They appear in both Mutillidae and Tiphiidae\ which supports the idea of the sister!group relationship between these two taxa "Brothers\ 0864#[ This structure\ however\ is absent in Scoliidae\ which further supports the splitting of the Scoleoidea!complex\ as suggested by Brothers "0864#[ Within Sphecoidea\ this character appears to have
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developed convergently in Ampulicidae\ which\ because of other characters assume quite an isolated and pre! carious position in this superfamily "Brothers\ 0864^ Ohl\ 0885#[ In conclusion\ the unique anatomy of the antennal circulatory organ in Hymenoptera with special regard to its indirect pumping mechanism\ is an autapomorphy for this group^ no similarities to the respective anatomies of any other insect order can be found[ Among the taxa of Hymenoptera\ a remarkable variety exists in anatomical detail of the antennal circulatory organ[ On the basis of the currently most widely accepted cladogram "Fig[ 00#\ however\ most of these character states must be inter! preted as mere convergences[ Acknowledgements We are very grateful to E[ Altenhofer\ H[ F[ Paulus\ W[ Schedl and H[ Zettl for providing identi_ed specimens\ to B[ Lorenz and Th[ Micholitsch for correcting the English manuscript and to H[ Grillitsch for help with the illus! trations[ Thanks also go to D[ Gruber and A[ Losert for their technical support and to W[ Klepal for making available laboratory facilities[
References Ashhurst\ D[E[\ 0871[ The structure and development of insect con! nective tissues[ In] King\ R[C[\ Akai\ H[ "Eds[#\ Insect Ultrastructure[ Vol[0[ Plenum Press\ New York\ pp[ 202Ð249[ Baccetti\ B[\ Bigliardi\ E[\ 0858[ Studies on the _ne structure of the dorsal vessel of arthropods[ I[ The {heart| of an orthopteran[ Zeit! schrift fur Zellforschung und Mikroskopische Anatomie 88\ 02Ð13[ Bayer\ R[\ 0857[ Untersuchungen am Kreislaufsystem der Wan! derheuschrecke "Locusta mi`ratoria mi`ratorioide R[ et F[\ Orthop! teroidea#[ Zeitschrift fur Vergleichende Physiologie 47\ 65Ð044[ Beattie\ T[M[\ 0865[ Autolysis in axon terminals of a new neurohaemal organ in the cockroach Periplaneta americana[ Tissue + Cell 7\ 294Ð 209[ Brocher\ F[\ 0810[ E łtude experimentale sur le fonctionnement du vais! seau dorsal et sur la circulation chez les insectes[ IV[ Partie] La Vespa crabo[ Annales de la Societe Entomologique de France 78\ 198Ð121[ Brothers\ D[J[\ 0864[ Phylogeny and classi_cation of the Aculeate Hymenoptera\ with special reference to Mutillidae[ University of Kansas Science Bulletin 49\ 372Ð537[ Clements\ A[N[\ 0845[ The antennal pulsating organs of the mosquitoes and other Diptera[ Quarterly Journal of Microscopical Science 86\ 318Ð322[ Dowton\ M[ and Austin\ A[D[\ 0883[ Molecular phylogeny of the insect order Hymenoptera] Apocritan relationships[ Proceedings of the National Academy of Sciences of the United States of America 80\ 8800Ð8804[ Dudel\ H[\ 0866[ Vergleichende funktionsanatomische Untersuchungen uber die Antennen der Dipteren[ I[ Bibiomorpha\ Homoeodactyla\ Asilomorpha[ Zoologische Jahrbucher\ Abteilung fur Anatomie und Ontogenie der Tiere 87\ 192Ð297[ Dudel\ H[\ 0867a[ Vergleichende funktionsanatomische Unter! suchungen uber die Antennen der Dipteren[ II[ Cyclorrhapha "Asch! iza und Schizophora!Acalyptratae#[ Zoologische Jahrbucher\ Abteilung fur Anatomie und Ontogenie der Tiere 88\ 113Ð187[
Dudel\ H[\ 0867b[ Vergleichende funktionsanatomische Unter! suchungen uber die Antennen der Dipteren[ III[ Calyptratae "I[O[ Cyclorrhapha#[ Zoologische Jahrbucher\ Abteilung fur Anatomie und Ontogenie der Tiere 88\ 290Ð296[ Francžois\ J[\ 0867[ The ultrastructure and histochemistry of the mes! enteric connective tissue in the cockroach Periplaneta americana L[ "Insecta\ Dictyoptera#[ Cell + Tissue Research 078\ 80Ð096[ Freudenstein\ K[\ 0817[ Das Herz und das Circulations system der Honigbiene "Apis melli_ca L[#[ Zeitschrift fur Wissenschaftliche Zoologie 021\ 393Ð364[ Gibson\ G[A[P[\ 0874[ Some pro! and mesothoracic structures impor! tant for phylogenetic analysis of Hymenoptera\ with a review of terms used for the structures[ Canadian Entomologist 006\ 0284Ð 0332[ Hantschk\ A[M[\ 0880[ Functional morphology of accessory pulsatile organs in the legs of Hemiptera[ International Journal of Insect Morphology + Embryology 19\ 148Ð162[ Hennig\ W[\ 0858[ Die Stammesgeschichte der Insekten[ Kramer\ Frankfurt[ Heraty\ J[M[\ Wooley\ J[B[\ Darling\ D[C[\ 0883[ Phylogenetic impli! cations of the Mesofurca and Mesopostnotum in Hymenoptera[ Journal of Hymenoptera Research 2\ 130Ð166[ Hertel\ W[ et al[\ 0874[ Electrophysiological investigation of the ante! nnal heart of Periplaneta americana and its reactions to proctolin[ Journal of Insect Physiology 20\ 452Ð461[ Huber\ J[T[\ Goulet\ H[\ 0882[ Families of Hymenoptera of the World[ Research Branch[ Agriculture Canada[ Publication 0783:E[ Janet\ C[\ 0788[ Essai sur la constitution morphologique de la te¼te de l|insecte[ Carre and Naud\ Paris[ Janet\ C["0800[ Sur l|existence d|un organe chordotoneal et d|une ves! icule pulsatile antennaire chez l|albeille et sur la morphologie de la te¼te de cette espece[ Comptes Rendus de l|Academie Sciences 041\ 009Ð001[ Jones\ J[C[\ 0866[ The Circulatory System of Insects[ Thomas\ Spring! _eld[ Konigsmann\ E[\ 0865[ Das phylogenetische System der Hymenoptera[ Teil 0] Einfuhrung\ Grundbauplanmerkmale\ Schwestergruppe und Fossilfunde[ Deutsche Entomologische Zeitschrift 12\ 142Ð168[ Konigsmann\ E[\ 0866[ Das phylogenetische System der Hymenoptera[ Teil 1] {Symphyta|[ Deutsche Entomologische Zeitschrift 13\ 0Ð39[ Konigsmann\ E[\ 0867[ Das phylogenetische System der Hymenoptera[ Teil 2] {Terebrantes| "Unterordnung Apocrita#[ Deutsche Ento! mologische Zeitschrift 14\ 0Ð44[ Konigsmann\ E[\ 0868[ Das phylogenetische System der Hymenoptera[ Teil 3] Aculeata "Unterordnung Apocrita#[ Deutsche Ento! mologische Zeitschrift 15\ 254Ð324[ Krenn\ H[W[\ Pass\ G[\ 0883[ Morphological diversity and phylogenetic analysis of wing circulatory organs in insects\ part I] Non!Hol! ometabola[ Zoology 87\ 6Ð11[ Krenn\ H[W[\ Pass\ G[\ 0884[ Morphological diversity and phylogenetic analysis of wing circulatory organs in insects\ part II] Holometabola[ Zoology 87\ 036Ð053[ Kristensen\ N[P[\ 0884[ Forty years| insect phylogenetic systematics[ Zoologische Beitrage 25\ 72Ð013[ Locke\ M[\ Huie\ P[\ 0861[ The _ber components of insect connective tissue[ Tissue + Cell 3\ 590Ð501[ Locke\ M[\ Huie\ P[\ 0864[ Staining of the elastic _bers in insect con! nective tissue after tannic acid:glutaraldehyde _xation[ Tissue + Cell 6\ 100Ð105[ Miller\ T[A[\ 0874[ Structure and physiology of the circulatory system[ In] Kerkut\ G[A[\ Gilbert\ L[I[ "Eds[#\ Comprehensive Insect Physi! ology\ Biochemistry and Pharmacology\ Vol[ 2[ Pergamon\ Oxford\ pp[ 178Ð242[ Ohl\ M[\ 0885[ Die phylogenetischen Beziehungen der Sphecinae "Hymenoptera] Apoidea] {Sphecidae|# aufgrund morphologischer Merkmale des Exoskeletts[ Zoologische Beitrage 26 "0#\ 2Ð39[ Pass\ G[\ 0879[ The anatomy and ultrastructure of the antennal cir!
S[ Matus\ G[ Pass : International Journal of Insect Morpholo`y 17 "0888# 86Ð098 culatory organs in the cockchafer beetle Melolontha melolontha L[ "Coleoptera\ Scarabeidae#[ Zoomorphology 85\ 66Ð78[ Pass\ G[\ 0874[ Gross and _ne structure of the antennal circulatory organ in cockroaches "Blattodea\ Insecta#[ Journal of Morphology 074\ 144Ð157[ Pass\ G[\ 0877[ Functional morphology and evolutionary aspects of the unusual antennal circulatory organs in Labidura riparia Pallas "Labiduridae#\ For_cula auricularia L[\ Chelidurella acanthopy`ia Gene "For_culidae[ "Insecta] Dermaptera#[ International Journal of Insect Morphology + Embryology 06\ 092Ð001[ Pass\ G[\ 0880[ Antennal circulatory organs in Onychophora\ Myria! poda and Hexapoda] Functional morphology and evolutionary implications[ Zoomorphology 009\ 034Ð053[ Pass\ G[\ 0887[ Accessory pulsatile organs[ In] Harrison\ F[\ Locke\ M[ "Eds[#\ Microscopic Anatomy of Invertebrates[ Vol 00B] Insecta[ Wiley\ New York\ pp[ 510Ð539[ Pass\ G[\ In press[ Accessory pulsatile organs] evolutionary innovations in insects[ Annual Review of Entomology 34[ Pass\ G[\ Agricola\ H[\ Birkenbeil H[\ Penzlin H[\ 0877a[ Morphology of neurones associated with the antennal heart of Periplaneta americana "Blattodea\ Insecta#[ Cell + Tissue Research 142\ 208Ð215[ Pass\ G[\ Sperk\ G[\ Agricola\ H[\ Baumann\ E[\ Penzlin\ H[\ 0877b[ Octopamine in a neurohemal area within the antennal heart of the American cockroach[ Journal of Experimental Biology 024\ 384Ð 387[ Pawlowa\ M[\ 0784[ U Ý ber ampullenartige Blutcirculationsorgane im Kopfe verschiedener Orthopteren[ Zoologischer Anzeiger 07\ 6Ð02[ Pinet\ J[M[\ 0853[ Les coe'urs accessoires antennaires de Rhodinus prolixus Stal[ "Heteroptera\ Reduviidae#[ Bulletin de la Societe Zoologique de France 78\ 332Ð338[ Rasnitsyn\ A[P[\ 0879[ Origin and evolution of the lower Hymenoptera[
098
Trudy Palaeontologicheskogo Instituta Akademiya Nauk SSSR 063\ pp[ 089[ Rasnitsyn\ A[P[\ 0877[ An outline of evolution of hymenopterous insects "Order] Vespida#[ Oriental Insects 11\ 004Ð034[ Romeis\ B[\ 0878[ Mikroskopische Techniken[ 06th edn[ Urban + Sch! warzenberg\ Wien[ Ross\ H[H[\ 0854[ A Textbook of Entomology[ 2rd edn[ New York\ London\ Sydney\ Tokyo[ Schneider\ D[\ Kaissling\ K[!E[\ 0848[ Der Bau der Antenne des Sei! denspinners Bombyx mori L[ III[ Das Bindegewebe und das Blut! gefa)[ Zoologische Jahrbucher\ Abteilun` fur Anatomie und Onto`enie der Tiere 66\ 000Ð021[ Seifert\ G[\ Rosenberg\ J[\ 0867[ Feinstruktur der Herzwand des Doppel! fussers Oxiolus `racilis "Diplopoda] Paradoxosomatidae# und allgemeine Betrachtungen zum Aufbau der Gefa)e von Tracheata und Onychophora[ Entomologica Germanica 3\ 113Ð122[ Selman\ B[J[\ 0854[ The circulatory system of the alder ~y Sialis lutaria[ Proceedings of the Zoological Society of London 033\ 376Ð424[ Wasserthal\ L[T[\ 0871a[ Antagonism between haemolymph transport and tracheal ventilation in an insect wing "Attacus atlas L[#[ Journal of Comparative Physiology 036\ 16Ð39[ Wasserthal\ L[T[\ 0871b[ Wechselseitige funktionelle und strukturelle Anpassungen von Kreislauf und Tracheensystem bei adulten Insekten[ Verhandlungen der Deutschen Zoologischen Gesellschaft 64\ 094Ð005[ Wasserthal\ L[T[\ 0885[ Interaction of circulation and tracheal ven! tilation in holometabolous insects[ Advances in Insect Physiology 15\ 186Ð240[ Whit_eld\ J[B[\ 0881[ Phylogeny of the non!aculeate Apocrita and the evolution of parasitism in the Hymenoptera[ Journal of Hymen! optera Research 0\ 2Ð03[
International Journal of Insect Morphology 17 "0888# 86Ð098
Antennal circulatory organ of Apis mellifera L[ "Hymenoptera] Apidae# and other Hymenoptera] functional morphology and phylogenetic aspects S[ Matus \ G[ Pass Institut fur Zoologie der Universitat Wien\ Althanstra)e 03\ A!0989 Wien\ Austria Received 09 August 0887^ accepted 09 January 0888
Abstract The antennal circulatory organs of 27 species of Hymenoptera were investigated by means of serial semithin sections\ SEM and TEM[ In Apis\ this organ consists of an unpaired ampulla from which blood vessels that extend into the antennae originate[ The ampulla is a very delicate structure of elastic connective tissue[ Its lumen communicates with the head hemocoel via numerous perforations in its wall[ No speci_c ampulla muscles exist^ contraction of pharynx dilators causes compression of the ampulla\ whereby hemolymph is forced into the antennae[ An absence of directly!acting muscles is common to the functional morphologies of the antennal circulatory organ in all investigated Hymenoptera[ Some anatomical characters of this organ\ however\ vary among taxa] "i# presence of one or two ampullae\ "ii# optional dorsal hemolymph channel\ which connects the ampulla with the dorsal vessel\ "iii# optional ampulla pumping case formed by cuticular apophyses\ and "iv# di}erences in shape and number of the ampulla openings[ The distribution of these characters is discussed along the current views on the phylogeny of Hymenoptera[ Comparison with other insect orders indicates an autapomorphic status for the basic functional morphology of the antennal circulatory organ in Hymenoptera[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Insects^ Accessory pulsatile organs^ Heart^ Hemolymph^ Evolution
0[ Introduction The tubular dorsal heart of insects is apparently not capable of supplying long body appendages with hemo! lymph[ Autonomous auxiliary hearts serve this task in antennae\ legs\ wings\ and cerci "Jones\ 0866^ Miller\ 0874^ Pass\ 0887#[ Most earlier descriptions of those minute organs were based on studies carried out with classical histological techniques and consequently are rather vague[ More recent investigation has revealed that these accessory circulatory organs are surprisingly diverse with respect to their functional morphologies and there! fore are of special interest to phylogenetic studies[ More! Corresponding author[ Tel[] "¦32#!0!202 25:0237^ fax] "¦32#!0! 202 25:667 e!mail] sonja[matusÝunivie[ac[at Abbreviations] Am\ ampulla^ AV\ antennal vessel^ Br\ brain^ FG\ frontal ganglion^ Fr\ frons^ FR\ frontoclypeal ridge^ FS\ frontal sinus^ HC\ hemolymph channel^ He\ hemocyte^ HS\ hypopharyngeal sclerite^ LA\ lateral apophyse^ M0\ frontal pharynx dilators "dilatatores pharyn! gis frontales#^ M1\ postfrontal pharynx dilators "dilatatores pharyngis postfrontales#^ M2\ ring muscle of pharynx^ Os\ ostium^ Ph\ pharynx^ Va\ valve[
over\ due to their relatively simple design\ they are useful model systems for investigation on general aspects of organ evolution "Hantschk\ 0880^ Krenn and Pass\ 0883\ 0884^ Pass\ 0880\ in press# and neurobiological research "Hertel et al[\ 0874^ Pass et al[\ 0877a\b#[ Antennal circulatory organs have been reported to exist in various insect orders and di}er distinctly from each other in structure and functional mechanism "Blat! todea] Pawlowa\ 0784^ Pass\ 0874^ Dermaptera] Pass\ 0877^ Orthoptera] Bayer\ 0857^ Hemiptera] Pinet\ 0853^ Megaloptera] Selman\ 0854^ Coleoptera] Pass\ 0879^ Lepidoptera] Schneider and Kaissling\ 0848\ Diptera] Clements\ 0845^ Dudel\ 0866\ 0867a\b^ comparative investigation and review of non!Holometabola] Pass\ 0880\ 0887#[ In Hymenoptera\ very little is known about these organs[ As early as 0788\ Janet remarked that small ampullary organs to which he ascribed a circulatory func! tion existed in the frontal region of the head of Myrmica[ Brocher "0810# could not _nd a comparable organ in the circulatory system of Vespa crabo in spite of thorough research "Janet\ 0788#[ To date\ the only relatively detailed description of such an organ in a hymenopteran
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 8 # 9 9 9 0 0 ! 1
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is of Apis mellifera[ First notice of the antennal cir! culatory organ of the honeybee was given by Janet "0800#\ followed by a more precise description by Freudenstein "0817#[ In addition to anatomical and histological inves! tigation\ Freudenstein also performed experiments designed to clarify the functional mechanics of this organ[ The aim of the present study is to reinvestigate the functional morphology of the antenna!heart in Apis mel! lifera by means of light and electron microscopy\ to com! pare the antennal circulatory organs within Hymenoptera and to study their phylogenetic devel! opment[ 1[ Materials and methods Serial semithin sections of the antenna!heart region in the head were subjected to a light microscopical evalu! ation in the species listed below[ In some species\ the antennal circulatory organs were examined by means of SEM and in Apis mellifera also by TEM[ Only imagines were considered[ List of investigated species {Symphyta| Xyelidae] Xyela alpi`ena "Strobl\ 0784#^ Xyela sp[ Pamphiliidae] Cephalcia abietis "Linne\ 0647# Siricidae] Urocerus au`ur "Klug\ 0792# Diprionidae] Neodiprion sertifer "Geo}roy\ 0674#^ Diprion sp[ Xiphidriidae] Xiphydria sp[ Tenthredinidae] Allantus sp[^ Pristiphora abietina "Christ\ 0680#^ Tenthredo campestris "Linne\ 0647# Cephidae] Trachelus tabidus "Fabricius\ 0664#^ Janus compressus "Fabricius\ 0682#^ Harti`ia linearis "Sch! rank\ 0670# {Terebrantes| Evaniidae] Evania appendi`aster "Linne\ 0647# Cynipidae] Diplolepis rosae "Linne\ 0647#^ Andricus sp[ Aphelinidae] Encarsia formosa Gahan\ 0813 Leucospididae] Leucospis `i`as Fabricius\ 0682 Ichneumonidae] Amblyteles sp[^ Ophion luteus "Linne\ 0647# Braconidae] Apanteles sp[\ Aphidius sp[ Aculeata Scoliidae] Scolia hirta Schrank\ 0670 Mutillidae] Mutilla europaea Linne\ 0647^ Dasylabris maura "Linne\ 0647# Tiphiidae] Tiphia femorata Fabricius\ 0664 Vespidae] Paravespula `ermanica Fabricius\ 0682^ Paravespula vul`aris "Linne\ 0647#^ Nectarinella championi "Dover\ 0814#
Formicidae] Lasius ni`er "Linne\ 0647#^ Camponotus sp[ Sphecidae] Mellinus arvensis "Linne\ 0647#^ Sce! liphron sp[ Ampulicidae] Ampulex compressa "Fabricius\ 0670# Apidae] Apis mellifera Linne\ 0647^ Bombus hortorum "Linne\ 0650# Megachilidae] Osmia cornuta "Latreille\ 0794# Andrenidae] Andrena humilis Imho}\ 0721 1[0[ Light microscopy As a rule\ the heads of the investigated species were _xed in alcoholic Bouin "{DubosqÐBrasil| mixture\ see Romeis "0878##[ In some cases\ material _xed in ethanol also yielded satisfactory results[ Specimens were embed! ded into Spurr epoxy resin under vacuum impregnation with acetone the in_ltration solvent[ For light microscopical evaluation\ serial semithin sec! tions "0 mm# were made of the antenna!heart region in the head[ The specimens were sectioned with an ultra! microtome with glass or diamond knives "Diatome\ Swit! zerland#[ The sections were stained in a mixture of azure II "0)# and methylene blue "0)# in an aqueous borax solution "0)# at 79>C[ 1[1[ Scanning electron microscopy For microanatomical studies\ the heads of the inves! tigated species were _xed in the aforementioned way[ Specimens were in_ltrated with para.n using methyl! benzoate as intermediate medium[ The embedded heads were then trimmed with a razor blade and the para.n removed in xylol "for 29 min at room temperature#[ After! wards\ the specimens were transferred into acetone and dehydrated using the critical point method[ Finally\ they were sputtered with gold and examined with a Jeol JSM! 24CF microscope[ 1[2[ Transmission electron microscopy Heads of Apis mellifera were _xed in a mixture of glutaraldehyde "1[4)# and paraformaldehyde "1)#\ fol! lowed by 0) osmium tetroxide\ both _xatives being bu}ered with 9[0 M sodium cacodylate "pH 6[1#[ Heads were then embedded into Spurr epoxy resin with acetone the in_ltration solvent[ Ultrathin sections were double! stained in uranyl acetate and lead citrate and examined with a Zeiss EM!891 electron microscope[ 2[ Results 2[0[ Apis mellifera The antennal circulatory organ of Apis mellifera is located in the frontal part of the head between the
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ally diminishes to a diameter of 14 mm before the vessel enters the antenna[ Within the antennae\ the lumen remains equidiametrical all along[ In the distal ~agellar segment\ the antennal vessels open into the antennal hemocoel via a terminal pore[ A minute membrane ~ap exists internally at each an! tennal vessel base "Fig[ 2A and 2B#[ It protrudes in an oblique orientation into the vessel lumen[ Its proximal ends are attached to the vessel wall\ its distal ends\ however\ are loose\ shaping a funnel!like structure acting as back~ow valve between ampulla and antennal vessel[ The valve region at the vessel bases is approximately 34 mm in length[ The vessel wall is about 1[4 mm thick and consists of an epithelium covered by acellular external laminae of 9[1 mm "inner# and 9[2 mm "outer#[
Fig[ 0[ Apis mellifera\ frontal view of antenna bases in the head[ Location of antennal circulatory organ "ampulla\ antennal vessels# indi! cated into the SEM micrograph by broken lines[
antenna bases "Fig[ 0#[ The organ consists of an ampulla with blood vessels originating from either side that extend into their corresponding antenna[ The pharynx and some of its dilator muscles\ which are in close vicinity to the ampulla\ are functionally related to the antennal cir! culatory organ and are therefore included in the following description[ 2[0[0[ Ampulla The ampulla is attached to the frontal cuticle and the frontoclypeal ridge[ It is a somewhat ~at sac measuring about 179 mm in diameter[ The anterior wall of the ampulla is formed by the hypodermis attached to the central part of the frons "Figs 1 and 2#[ Its posterior wall is a very delicate structure whose margins are attached to the integument^ it is also linked to the surrounding organs by numerous thin suspending strands "cross!section about 9[2 mm in diameter#[ The posterior ampulla wall is about 9[5 mm in thick! ness[ It is a lamina consisting of a _lamentous matrix\ into which a few\ ~at cells are embedded "Fig[ 2E#[ Ultra! structurally\ they resemble connective tissue cells and contain no contractile elements[ In its upper part\ the posterior ampulla wall has an area "diameter about 49 mm# with numerous perforations "Fig[ 2C and 2D#[ These are openings about 2Ð4 mm in diameter[ 2[0[1[ Antennal vessels A single blood vessel originates from either side of the ampulla\ arches around the antenna base and continues into the antenna[ The lumen width of their vessel gradu!
2[0[2[ Functionally related structures Because the ampulla wall is not muscular and because no muscles insert at the ampulla\ its function as a hemo! lymph pump must involve some indirect pumping mech! anism[ Owing to the location of the ampulla between the frons and the pharynx\ movement of the pharynx must a}ect the variable width of the ampulla lumen[ Four pairs of muscles originate at the frontal cuticle in the area above the ampulla] the anterior labral muscle inserts at the inner labrum\ the retractor of the mouth!angle at the anterior part of the hypopharyngeal suspensorium sclerite and the two pairs of the frontal pharynx dilator muscles insert at the pharynx roof[ Contraction of these muscles move the pharynx roof towards the frons and thereby indent the posterior ampulla wall[ 2[1[ Other Hymenoptera 2[1[0[ Symphyta The functional morphology of the antennal circulatory organ varies considerably among high!rank taxa of Sym! phyta s[l[^ however\ fundamental intra!familial di}er! ences were not found[ In Xyelidae\ the antennal circulatory organ is similar to that of the honeybee[ It consists of a median ampulla about 109 mm long and 019 mm wide[ In contrast to Apis\ the ampulla of Xyela alpi`ena has just a single median opening[ It is a relatively large opening of about 34 mm with no valve structures[ Another incongruity to Apis is the presence of lateral cuticular apophyses which are attached to the ampulla wall[ In Tenthredinidae and Diprionidae\ the antennal cir! culatory organ is completely di}erent from those of all other examined Hymenoptera in that it has an ampulla for each antenna base "Fig[ 3A#[ These ampullae lack attached muscles[ The structure of the wall is the same as in all other Hymenoptera[ Ampullae open into a median hemolymph sinus via slit!like\ valved ostia "Fig[ 4#[ This sinus is anteriorly con_ned by the hypodermis\ while
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Fig[ 1[ Apis mellifera\ schematic drawing of the antennal circulatory organ in lateral view "A# and dorsal view "B and C#[ Unpaired ampulla with paired vessels extending into antennae[ Anterior wall of ampulla attached to frontal cuticle above the frontoclypeal ridge[ Posterior wall with numerous perforations "arrow#[ Pharynx behind ampulla\ frontal ganglion in between\ functionally related pharynx dilators M0 and M1 also shown^ other organs omitted[ "B# and "C # show the organ in di}erent phases of action "for explanation see discussion chapter#^ arrows indicate proposed hemolymph ~ow[
its posterior boundary is formed by connective tissue laminae and partly by tracheal sacs[ The ampullae are about 74 mm wide and 049 mm long[ The base of the antennal vessels is located anterio!laterally at the ampul! lae[ Remarkably\ no valve was found at the juncture of ampullae and antennal vessels[ In Pamphiliidae\ Siricidae\ Xiphydridae\ and Cephi! dae\ the antennal circulatory organ is again very di}erent from that of Apis and most other Hymenoptera "Fig[ 3B#[ Cephalcia abietis has a median ampulla between the antennal bases[ Posteriorly\ the ampulla is connected to the anterior end of the dorsal vessel behind the brain via a hemolymph channel which runs along the dorsal side of the pharynx "Fig[ 5#[ Seen from the ampulla\ this channel narrows to about 29 mm in diameter and then keeps a constant width until it fuses with the dorsal vessel[ The channel resembles a tunnel whose upper part is for! med by a thin lamina of connective tissue and the lower part by the pharynx roof[ There are no muscles directly attached to the ampulla or the channel[ A striking feature of Cephalcia abietis is that one pair of the frontal pharynx dilators extends through the entire ampulla lumen^ the second pair lies above the ampulla[ In the siricid Urocerus au`ur and the cephids Janus compressus and Harti`ia linearis\ the antennal circulatory organ also consists of a median ampulla connected to the dorsal vessel via a
hemolymph channel[ In the transitional region between the ampulla and the antennal vessel\ back~ow valves exist similar to those in Apis[ 2[1[1[ {Terebrantes| The investigated taxa of this group show a relatively uniform situation with respect to the functional mor! phology of the antennal circulatory organ which is similar to that in Apis[ While in most investigated hymenopteran taxa\ the antennal circulatory organ as well as the antenna bases are located close to the line between frons and clypeus\ antennae of all terebrant species have a more distant position[ The ampullae\ however\ are found at the frons! clypeus border as be_ts other Hymenoptera[ This gives the proximal antennal vessel portion a notably elongated appearance "Fig[ 3C# unique among Hymenoptera[ In all investigated {Terebrantes|\ the ampulla lumen is connected to the head hemocoel via a perforated area in the posterior ampulla wall^ the perforations have no valve ~aps[ In none of the investigated species of this group\ muscle _bers could be found within the ampulla wall nor directly attached to it[ The functionally associated muscles in the proximity of the ampulla are the dilators of the pharynx and the cibarium[
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Fig[ 2[ Apis mellifera\ antennal circulatory organ[ "A# Cross section[ Ampulla attached to the frontal cuticle along its entire width between the antenna bases[ The delicate posterior ampulla wall is highlighted by several arrowheads[ Paired valves between ampulla and antennal vessels "arrows#[ On the right half the antennal vessel circling around the antennal annule[ "B# Cross section showing valve area of the left antennal vessel in detail[ Arrow indicates direction of hemolymph ~ow[ "C# SEM preparation\ parasagittal cut^ ampulla between frontal cuticle and pharynx roof\ lower part attached to the frontoclypeal ridge[ Arrowheads indicate perforations in the posterior ampulla wall[ Frontal ganglion between pharynx dilator muscle M0 and ampulla[ "D# Sagittal section^ ampulla anteriorly attached to frontal cuticle and partly compressed by the frontal ganglion[ Pharynx dilator M0 can be seen behind the ampulla[ Posterior ampulla wall suspended by connective tissue strands "arrowheads#[ "E# TEM^ sagittal section through posterior ampulla wall[ Cut of wall cell ~anked by two external laminae[
2[1[2[ Aculeata The antennal circulatory organs of all investigated acu! leate species are similar with respect to their overall func! tional morphology which in principle corresponds to the description for Apis given above[ Three characters\ however\ show remarkable di}erences] "i# the type of ampulla opening\ "ii# special associated cuticular struc! tures\ and "iii# the shape of the basal portion of the antennal vessels[ The ampulla of most Aculeata opens through numer! ous perforations in the posterior ampulla wall[ In For! micidae and Vespidae\ however\ hemolymph enters the ampulla through a single median opening[ Camponotus sp[ has a slit!like ostium with a pair of valve ~aps pro!
truding into the ampulla lumen "Figs 6B and 8#[ Para! vespula `ermanica and P[ vul`aris also have a single median opening[ No valve structure could be found "Fig[ 09#[ In Mutillidae and Tiphiidae\ the antennal circulatory organ consists of an ampulla that is clearly attached to distinct cuticular structures "Figs 6A and 7#[ These include the well!developed frontoclypeal ridge just below the ampulla as well as large cuticular apophyses deep in the head capsule boldly framing the ampulla on two sides[ There are no muscles attached to these lateral apophyses\ yet they reinforce the ampulla wall[ The ampulla in these taxa is\ unlike the usual ~at sac of all other Hymenoptera\ almost isometric and framed by these cuticular structures
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Fig[ 3[ Schematic drawings of the antennal circulatory organs in selected species of {Symphyta| "A\B# and {Terebrantes| "C#[ Each species shown in dorsal and lateral view^ arrows indicate ampulla openings[ "A# paired ampullae with median frontal sinus\ "B# unpaired median ampulla with dorsal hemolymph channel\ "C# unpaired median ampulla with elongated antennal vessel bases[
thus forming a kind of pumping case[ Muscles line the ampulla wall where cuticular structures are absent[ Ampulex compressa also has cuticular structures to which the lateral ampulla walls are attached\ as in Mutillidae and Tiphiidae[ Otherwise\ the frontoclypeal ridge and the lateral apophyses are less elongated[ Another anatomical feature\ which di}ers among spe! cies\ is the antennal vessel basis[ While in most Hymeno! ptera\ the antennal vessel has about the same diameter over its entire length\ in Mutillidae and Tiphiidae the antennal vessel bases are markedly widened compared with the rest of the vessel\ thus forming a kind of vesicle^ e[g[\ in Mutilla\ diameter is about 199 mm in the proximal portion and then abruptly diminishes to 69 mm[ In all species\ a funnel!shaped valve was found between ampulla and antennal vessel[ 3[ Discussion 3[0[ Functional morphology 3[0[0[ Apis mellifera The basic structure of the antennal circulatory organ in the honeybee was described by Freudenstein "0817#[ He correctly identi_ed the unpaired ampulla between the antenna bases as well as the point of origin of the antennal vessels\ but did not detect the valve ~aps at the bases of
the antennal vessels[ Freudenstein "0817# was incapable of unequivocally clarifying the histological nature of the ampulla wall\ given the limitations of light microscopy[ His assumption that the wall is not muscular could be substantiated in the present investigation at the electron microscopical level[ The wall is a thin lamina consisting of a delicate _lamentous matrix with a few embedded ~at cells[ The antennal circulatory organs of other insects have so far been studied on an ultrastructural level only in Periplaneta americana "Beattie\ 0865^ Pass\ 0874#[ The structure of the ampulla wall of that species in some way resembles that of Apis\ because it also partly consists of laminae of the same character[ In contrast to Apis\ however\ the wall is a true epithelium\ covered by these external laminae on both sides[ Structures consisting of matrices with unbanded _laments\ comparable to those of the laminae\ are known from some types of connective tissue in insects and said to be elastic "Ashhurst\ 0865#[ They have been found in arthropods in the extracellular sheath covering the dorsal vessel "Bacetti and Bigliardi\ 0858^ Seifert and Rosenberg\ 0867#\ in cell!less strings attaching the dorsal vessel to nearby tissue "Locke and Huie\ 0861\ 0864#\ and in mesenteric connective tissue "Francžois\ 0867#[ Although the chemical nature of these _laments has not yet been identi_ed irrefutably\ they bear a structural similarity to elastin found in the elastic connective tissue of vertebrates "Ashhurst\ 0865#[
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Figs 4 + 5[ Fig[ 4[ Allanthus sp[\ antennal circulatory organ[ "A# Cross section showing the two ampullae attached to the frontal cuticle and the hemolymph sinus between[ Arrows indicate valves in ampulla openings through which hemolymph can enter from the sinus[ "B# Parasagittal section through an ampulla in the ostium region^ note the long valve ~ap[ Fig[ 5[ Cephalcia abietis\ antennal circulatory organ[ "A# Cross section through ampulla[ The lateral walls fuse with the pharynx[ "B# Sagittal section[ Ampulla with hemolymph channel stretching along the dorsal side of the pharynx and connecting to the dorsal vessel behind the brain[ Note cuts of the pharynx dilator muscles within the ampulla lumen "arrowhead#[
Because direct observation of the ampulla activity in Apis is not possible due to the opaque cuticle of the frons\ the functional mechanics of this organ must be indirectly accessed from its functional morphology and from exper! imental data[ In an initial approach to functional mech! anics\ Freudenstein "0817# examined the direction of hemolymph ~ow in the head of the honeybee[ He injected a mixture of ink and egg albumin into the dorsal vessel and afterwards killed the bees in heated ringer solution[ Heating denatured the protein and _xed the ink[ In his! tological sections made from these specimens\ hemo! lymph ~ow could be tracked[ By doing so\ he clearly showed that hemolymph was being pumped through the ampulla into the antennal vessels[ Freudenstein|s "0817# conclusions are con_rmed by our _nding of the valve ~aps at the antennal vessel bases\ which allow only a one! way ~ow towards the antennae[ But what causes the pumping movements< Because we could not detect any muscle _bers within the ampulla wall\ no active contraction of the ampulla in Apis seems possible[ This holds true for the walls of the antennal circulatory organs of all other insects examined so far
"Pass\ 0880\ 0887#[ Only for two species "Periplaneta] Pawlowa\ 0784\ Rhodinus] Pinet\ 0853# has a muscular ampulla wall yet been reported[ Later elec! tronmicroscopical investigation\ however\ could sub! stantiate these results "Pass\ 0874\ unpublished#[ Despite many structural di}erences\ antennal circulatory organs of most other insects have muscles in common that are either attached to or are very closely associated with the ampullae[ These muscles either operate as dilators or as compressors antagonized by the elasticity of the ampulla wall "Pass\ 0879\ 0874\ 0880#[ Only a few other insects possess antennal circulatory organs without associated muscles "Odonata] Pass\ 0880\ Lepidoptera] Schneider and Kaissling\ 0848\ some Diptera] Dudel\ 0866\ 0867a\b#[ In Apis\ dilatory movements of the pharynx must entail a simultaneous compression of the ampulla\ due to its location between frons and pharynx "Fig[ 1C#[ Hemo! lymph is thereby forced out of the ampulla lumen into the antennal vessels[ After relaxation of the pharynx dilators\ the pharynx roof resumes its original position[ Congruently\ the ampulla lumen is likely to widen due to
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Fig[ 6[ Schematic drawings of the antennal circulatory organs in selected species of Aculeata[ Each species shown in dorsal and lateral view^ arrows indicate ampulla openings[ "A\ B# Large lateral apophyses supporting the ampulla wall and "C# ostium slit with valve ~aps protruding into the ampulla lumen[
the combined elasticities of ampulla wall and suspending strands[ During dilation of the ampulla\ lumen pressure is reduced and hemolymph enters the ampulla via the wall perforations "Fig[ 1B#[ In this phase\ back~ow of hemolymph from the antennal vessels is forestalled by the valve ~aps at the vessel bases[ Dilation of the pharynx occurs through rhythmic con! tractions of the pharynx dilators while the bee is feeding[ How an adequate hemolymph exchange rate can be achieved while the animal is not feeding remains unknown[ Freudenstein|s "0817# observations on the hemolymph meniscus of an apically truncated honeybee antenna are noteworthy in this respect] he described per! iodical\ weak pulses of this meniscus and non!periodical stronger pulses[ He concluded that there was always a certain pharyngeal peristaltic activity causing hemo! lymph circulation in the antennae[ Because a functional connection between circulatory organs and tracheal ventilation has been established in some insects "Wasserthal\ 0871a\b\ 0885#\ ventilatory movement must also be taken into account to explain functional mechanics of the antenna!heart in Hymen! optera[ In all species large tracheal sacs can be found close to the ampulla[ The helical design of their taenidia suggests that certain elastic properties are inherent in these structures[ In any case\ ventilatory movement of
these tracheal sacs need to cause changes in the hem! olymph pressure of the head hemocoel and must therefore somewhat a}ect the function of the antennal pulsatile organ[ To make a major contribution to pumping activity\ however\ these sacs would have to oscillate[ The structure of the antennal vessels in Apis is quite similar to that of most other insects[ The wall of the vessels is formed by a thin epithelium covered on both sides with external laminae[ A modi_cation of the proxi! mal vessel portion\ _tted with a transporting epithelium as in Periplaneta "Pass\ 0874# could not be found in Apis[ 3[0[1[ Other Hymenoptera An absence of muscles attached to the ampullae is common to all investigated species of Hymenoptera[ Some other characters\ however\ can be di}erent among species] "i# the number of ampullae\ "ii# the occurrence of a hemolymph channel\ "iii# the formation of an ampulla opening\ and "iv# associated cuticular structures[ All Hymenoptera possess unpaired ampullae\ with the exception of Tenthredinidae and Diprionidae\ which have paired ones[ In both taxa\ the anatomical situation in the frontal head region suggests that movement of the pharynx roof probably does not strongly compress the ampullae[ In these taxa\ the frontal sinus seems to be the compressible unit\ forcing hemolymph into the ampullae
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Figs[ 7\ 8 + 09[ Fig[ 7[ Mutilla europaea\ antennal circulatory organ[ "A# Cross section[ Ampulla wall attached to lateral apophyses and to pharynx roof[ Pharynx dilator M0 originating at frons[ Widened antennal vessel bases beside apophyses[ "B# Sagittal section of the ampulla surrounded by frontal cuticle\ frontoclypeal ridge\ pharynx dilator M0 and ring muscles of the pharynx roof M2[ "C# Horizontal section showing the cuticular pumping case of the ampulla formed by frontoclypeal ridge\ lateral apophyses and frontal pharynx dilator[ To either side the widened antennal vessel bases and valve ~aps[ "D# Horizontal section showing in detail the long valve ~ap between ampulla and the right antennal vessel[ Fig[ 8[ Camponotus sp[\ antennal circulatory organ[ Sagittal section[ Ampulla attached to frontal cuticle and to frontoclypeal ridge[ Posterior wall close to pharynx ring muscles] frontal pharynx dilators M0 above the ampulla and dilator M1 below[ Ostium with valve ~aps "appearing as a single ~ap in this section#[ Fig[ 09[ Paravespula `ermanica\ antennal circulatory organ[ Sagittal section through ampulla[ Posterior wall slightly indented by frontal ganglion[ Arrow indicates ostium[
via ostia[ The valve ~aps of these ostia prevent back~ow from the ampullae into the sinus[ There is no valve between the ampullae and the antennal vessels[ In Pamphiliidae\ Siricidae\ Xiphydridae\ and Cephi! dae\ the hemolymph channel represents a direct con! nection between the antennal circulatory organ and the
dorsal vessel[ The posterior boundary of the ampulla is the pharynx roof[ In these species\ dilatory movement of the pharynx probably propels hemolymph into the antennal vessels and thereby into the antennae[ Because the antennal circulatory organ is directly connected to the dorsal vessel\ pulses resulting from vessel contraction
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must have a signi_cant impact on circulation in the ante! nnae[ In Hymenoptera\ the most common type of ampullae in~ow openings are wall perforations[ A few species have but a single opening] Xyela alpi`ena has a large opening in its posterior ampulla wall^ a slit!like ostium was found in the investigated formicid and vespid species[ In Cam! ponotus\ this ostium has two valve ~aps protruding into the ampulla lumen[ In many hymenopterans\ the ampulla is attached to the frontoclypeal ridge in an anterior position lending mechanical reinforcement to the ampulla wall[ In the xyelid\ mutillid\ tiphiid\ and ampulicid species\ lateral apophyses provide such reinforcement to an even higher degree[ Together with the frontoclypeal ridge\ these apo! physes build a kind of pumping case for the ampulla[ In Mutillidae and Tiphiidae\ the lateral apophyses and the frontoclypeal ridge are extraordinarily long^ in this way\ the ampulla is tightly surrounded by these cuticular struc! tures and muscles[ 3[1[ Phylogenetic aspects 3[1[0[ Hymenoptera and their sister!group Phylogenetic relationships of Hymenoptera with other insects have not yet been entirely uncovered "Hennig\ 0858^ Konigsmann\ 0865^ Kristensen\ 0884#[ A distinct phylogenetic isolation of this group has long been recog! nized and Ross "0854# has even suggested a sister!group relationship between Hymenoptera and all other Holo! metabola[ A feasible working hypothesis\ for the time being\ is to assume a sister!group relationship between Hymenoptera and Mecopteroidea "Konigsmann\ 0865^ Kristensen 0884#[ Comparing the antennal circulatory organs of Hymen! optera with those in Mecopteroidea\ we _nd distinct di}erences which are not easy to interpret[ Moreover\ within Mecopteroidea the situation is not uniform] in all investigated Amphiesmenoptera\ the antennal vessels are linked to a frontal sac\ which lies dorsally of the pharynx and just in front of the brain "Trichoptera] Pass\ unpub! lished^ Lepidoptera] Schneider and Kaissling\ 0848#[ As in Hymenoptera\ no muscles are directly attached to it[ In Antliophora\ however\ we have pulsatile ampullae connected to the antennal vessels "Diptera] Clements\ 0845^ Dudel\ 0866\ 0867a\ 0867b^ Mecoptera] Pass\ unpublished#[ In Diptera\ there are species with one ampulla and others with two[ The existence of two ampul! lae in the sister!group Mecoptera "Panorpa] Pass\ unpub! lished# suggests that the two!ampullae type is the plesiomorphic character state for Diptera as well[ Strik! ing di}erences exist within Antliophora in number and function of the muscles associated with ampullae[ Only in few taxa of Diptera\ are ampullae devoid of muscles "Stratiomyidae] Dudel\ 0866^ Pipunculidae] Dudel\ 0867a^ Diopsidae] Dudel\ 0867b#[ The investigated spec!
ies of Stratiomyidae and Pipunculidae have a single median ampulla\ which probably functions in a manner similar to the ampullae of Hymenoptera[ Diopsidae have paired ampullae probably compressed via contractions of speci_c antennal muscles lying close to each of the ampullae[ In all other species of Diptera\ muscles dilate the ampullae[ While they are always directly attached to the ampulla wall\ their other attachment site can be very di}erent structures[ Among Mecoptera\ Panorpa also has paired ampullae to which dilator muscles are directly attached^ here the other attachment site is the pharynx roof "Pass\ unpublished#[ The indirect pumping mechanism in the functional anatomy of the antennal circulatory organ in Hymen! optera is only distantly related to the respective ana! tomies of other insects\ both exopterygotes and endopterygotes[ Therefore\ this unique type of antennal circulatory organ of Hymenoptera appears to be an aut! apomorphy of this order which con_rms the isolated position of Hymenoptera within insects[ 3[1[1[ Phylogenetic aspects in Hymenoptera Relationships among major taxa of Hymenoptera that stood _rm in the main phylogenetic branches for a long time have recently begun to be questioned "Konigsmann\ 0866^ Gibson\ 0874^ Whit_eld\ 0881^ Dowton and Austin\ 0883#[ Classical high!rank taxa like Symphyta and Tere! brantes turned out to be paraphyletic when their status was critically examined by means of phylogenetic sys! tematics "Konigsmann\ 0866\ 0867^ Rasnitsyn\ 0879\ 0877#[ Only Aculeata could defend their position as a truly monophyletic taxon "Brothers\ 0864^ Konigsmann\ 0868^ Dowton and Austin\ 0883#[ The evolutionary pathways of the antennal circulatory organ in Hymenoptera are di.cult to trace^ various con! clusions are possible[ Figure 00 gives a survey of the systematic distribution of the di}erent characters present in antennal circulatory organs of investigated Hymeno! ptera[ The most crucial di}erences and the greatest diver! sity can be found among the {Symphyta|\ in which abun! dance of plesiomorphic character states is generally recognized[ Xyelidae\ often considered as those Hymen! optera with the greatest number of plesiomorphic charac! ter states "Rasnitsyn\ 0879#\ have an unpaired antennal circulatory organ similar to that in the derived Aculeata[ In Mecopteroidea\ the probable sister!group of Hymeno! ptera\ the ancestral character state of the antennal cir! culatory organ is paired ampullae^ therefore outgroup comparison suggests that the organs of Tenthredinidae and Diprionidea with their paired ampullae represent the plesiomorphic character state in Hymenoptera[ The similar character states found in Xyelidae and Aculeta have thus to be rather considered a convergence than the plesiomorphic character state in Hymenoptera[ The occurrence of an ampulla connected to the dorsal vessel by a hemolymph channel in most lineages of investigated
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Fig[ 00[ Antennal circulatory organ characters mapped onto a cladogram of Hymenoptera combined and modi_ed according to Heraty "0883#[ Superfamilies and families that were not investigated in the present study have been omitted[ Nomenclature follows Huber and Goulet "0882#[ Number of investigated species in parentheses[
{Symphyta| has to be interpreted as a convergence too\ because such a character state does not exist in any non! hymenopteran insect[ In this study\ only few taxa in the so!called Terebrantes were investigated which show a uniform situation with regard to their antennal circulatory organs[ All possess an unpaired median ampulla with one antennal vessel originating from either side[ Unique among Hymeno! ptera are the very long bases of their antennal vessels in the head capsule[ In Aculeata\ the overall morphology of the antennal circulatory organ is also relatively uniform[ Some ana! tomical features\ however\ possess noteworthy phylo! genetic aspects] while in most taxa the ampulla openings are wall perforations\ the ampulla opening to the head
hemocoel in Formicidae and Vespidae represents an ostium[ A valve exists only in Formicidae[ Therefore\ and owing to the fact that in Scoliidae\ the sister!group of the VespidaeÐMasaridaeÐEumenidae\ the ampulla openings are numerous perforations\ as in all other Aculeata\ the ampulla ostium may have developed convergently in Ves! pidae and Formicidae[ Cuticular structures attached to the antenna!hearts\ i[e[\ lateral apophyses are relatively simple to interpret from a phylogenetic point of view[ They appear in both Mutillidae and Tiphiidae\ which supports the idea of the sister!group relationship between these two taxa "Brothers\ 0864#[ This structure\ however\ is absent in Scoliidae\ which further supports the splitting of the Scoleoidea!complex\ as suggested by Brothers "0864#[ Within Sphecoidea\ this character appears to have
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S[ Matus\ G[ Pass : International Journal of Insect Morpholo`y 17 "0888# 86Ð098
developed convergently in Ampulicidae\ which\ because of other characters assume quite an isolated and pre! carious position in this superfamily "Brothers\ 0864^ Ohl\ 0885#[ In conclusion\ the unique anatomy of the antennal circulatory organ in Hymenoptera with special regard to its indirect pumping mechanism\ is an autapomorphy for this group^ no similarities to the respective anatomies of any other insect order can be found[ Among the taxa of Hymenoptera\ a remarkable variety exists in anatomical detail of the antennal circulatory organ[ On the basis of the currently most widely accepted cladogram "Fig[ 00#\ however\ most of these character states must be inter! preted as mere convergences[ Acknowledgements We are very grateful to E[ Altenhofer\ H[ F[ Paulus\ W[ Schedl and H[ Zettl for providing identi_ed specimens\ to B[ Lorenz and Th[ Micholitsch for correcting the English manuscript and to H[ Grillitsch for help with the illus! trations[ Thanks also go to D[ Gruber and A[ Losert for their technical support and to W[ Klepal for making available laboratory facilities[
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S[ Matus\ G[ Pass : International Journal of Insect Morpholo`y 17 "0888# 86Ð098 culatory organs in the cockchafer beetle Melolontha melolontha L[ "Coleoptera\ Scarabeidae#[ Zoomorphology 85\ 66Ð78[ Pass\ G[\ 0874[ Gross and _ne structure of the antennal circulatory organ in cockroaches "Blattodea\ Insecta#[ Journal of Morphology 074\ 144Ð157[ Pass\ G[\ 0877[ Functional morphology and evolutionary aspects of the unusual antennal circulatory organs in Labidura riparia Pallas "Labiduridae#\ For_cula auricularia L[\ Chelidurella acanthopy`ia Gene "For_culidae[ "Insecta] Dermaptera#[ International Journal of Insect Morphology + Embryology 06\ 092Ð001[ Pass\ G[\ 0880[ Antennal circulatory organs in Onychophora\ Myria! poda and Hexapoda] Functional morphology and evolutionary implications[ Zoomorphology 009\ 034Ð053[ Pass\ G[\ 0887[ Accessory pulsatile organs[ In] Harrison\ F[\ Locke\ M[ "Eds[#\ Microscopic Anatomy of Invertebrates[ Vol 00B] Insecta[ Wiley\ New York\ pp[ 510Ð539[ Pass\ G[\ In press[ Accessory pulsatile organs] evolutionary innovations in insects[ Annual Review of Entomology 34[ Pass\ G[\ Agricola\ H[\ Birkenbeil H[\ Penzlin H[\ 0877a[ Morphology of neurones associated with the antennal heart of Periplaneta americana "Blattodea\ Insecta#[ Cell + Tissue Research 142\ 208Ð215[ Pass\ G[\ Sperk\ G[\ Agricola\ H[\ Baumann\ E[\ Penzlin\ H[\ 0877b[ Octopamine in a neurohemal area within the antennal heart of the American cockroach[ Journal of Experimental Biology 024\ 384Ð 387[ Pawlowa\ M[\ 0784[ U Ý ber ampullenartige Blutcirculationsorgane im Kopfe verschiedener Orthopteren[ Zoologischer Anzeiger 07\ 6Ð02[ Pinet\ J[M[\ 0853[ Les coe'urs accessoires antennaires de Rhodinus prolixus Stal[ "Heteroptera\ Reduviidae#[ Bulletin de la Societe Zoologique de France 78\ 332Ð338[ Rasnitsyn\ A[P[\ 0879[ Origin and evolution of the lower Hymenoptera[
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