indirect closing of the elytra in a cockchafer, melolontha...

1836

INTRODUCTIONExperimental studies on the flight of beetles are concentrated ontheir wings: their trajectories (Stellwaag, 1914; Brackenbury, 1994),folding and unfolding (Haas and Beutel, 2001), aerodynamics(Schneider, 1987), elastic structures (Haas et al., 2000),maneuverability and electrical activity of fibrillar muscles (Lestonet al., 1965; Schneider and Krämer, 1974; Bauer and Gewecke, 1985;Burton, 1971) and technical modeling (Syaifuddin et al., 2006).Experimental studies on the elytra were rather scarce. Earlier studiesinvestigated the position of the elytra during the flight, aerodynamicforces acting on the open elytra, coupling between the meso- andmetathoracic flight systems (Nachtigall, 1964; Schneider andMeurer, 1975; Schneider and Hermes, 1976; Schneider, 1986). Wemeasured trajectories and axes of rotation of the elytra duringopening and closing (Frantsevich et al., 2005).

Anatomists have investigated the muscular system and the elytralpivot and sometimes posed hypotheses about the role of certainmuscles (Straus-Duerkheim, 1828; Bauer, 1910; Stellwaag, 1914;Larsén, 1966) but they have not described any experiments inconfirmation. Recent anatomists describe, among others, the musclesin the mesothorax to reconstruct the phylogeny. They pay noattention to functions of the muscles (Beutel and Komarek, 2004;Friedrich et al., 2009). There exist several studies on the structureslocking the elytra together or with other body parts (reviewed inFrantsevich et al., 2005) but they concern immobile elytra. Lastly,we mention my previous observation on a peculiar click of the elytraduring righting in histerids (Frantsevich, 1981).

Here, we elucidate the role of muscles that actuate the elytra using(1) video recordings of tethered flying beetles with or without loadsapplied to the thoracic segments; (2) video recordings of tethered

flying beetles wherein some parts of sclerites were excised or certainmuscles were cut across; and (3) animation, i.e. passive motion ina dead specimen. The model animals were cockchafers, Melolonthahippocastani and Melolontha melolontha. We show below thatclosing is performed indirectly by pressure of the hind edge of thepronotum onto the lateral apophysis of the root of the elytron.Experiments on surgery and animation of mesothoracic muscles willbe described in the next article (L.F., submitted).

MATERIALS AND METHODSInsects

Cockchafers were collected in the field near Kiev, Ukraine.Melolontha hippocastani Fabricius 1801 was stored at 5°C and usedin flight experiments; M. melolontha Linnaeus 1758 was stored at–18°C, together with the former species, for animation experiments.

AnatomyA specimen was killed by freezing. After thawing, we removed theabdomen and, sometimes, cut the thorax down the medial line. Thespecimen was fixed in 70% ethyl alcohol and dissected under thestereomicroscope MBS-9 (Soviet Union). Specimens with themedial cut were halved. Steps of dissection were photographed witha camera Olympus C-2 Zoom (Olympus Corporation, Tokyo,Japan) adjusted to the ocular.

TetheringWe used two sites for tethering: (1) the ventrite behind the middlecoxae, which provided the firm basis for the pterothorax-fixedreference, and (2) the pronotum – only for demonstration of theflight. The holder was glued with the cyanoacrilate glue. Middle

The Journal of Experimental Biology 213, 1836-1843© 2010. Published by The Company of Biologists Ltddoi:10.1242/jeb.041350

Indirect closing of the elytra in a cockchafer, Melolontha hippocastani F. (Coleoptera:Scarabaeidae)

Leonid FrantsevichSchmalhausen-Institute of Zoology, B. Chmielnicki Street, 15, Kiev-30, 01601, Ukraine

[email protected]

Accepted 17 February 2010

SUMMARYActuation of the closing of the elytra was previously ascribed to intrinsic muscles in the mesothorax. We investigated closing(1) by loading or arrest of some thoracic segments in a tethered flying beetle, (2) by animation, i.e. passive motion of preparationsof the thorax simulating the action of some muscle, and (3) by excision of some parts of sclerites or cuts across certain muscles.We found out that depression of the prothorax, necessary to unlock the elytra, precedes their opening but elevation of theprothorax is synchronous with the closing. The closing is retarded if the elevation is retarded by loading; if the elevatingprothorax is clamped, then the closing is also arrested or hindered; animation of the elevation of the prothorax in the dead animalis enough for the closing of the previously spread elytra; the closing is prevented if a piece at the hind edge of the pronotum,positioned in front of the root of an elytron, is excised. This excision also prevents closing in the in vivo experiments. Mechanicalinteraction between the elytron and the prothorax is limited to the contact point between the posterior edge of the pronotum andthe lateral apophysis of the root. Thus, the elevation of the prothorax is the indirect and main mechanism of the closing inMelolontha.

Supplementary material available online at http://jeb.biologists.org/cgi/content/full/213/11/1836/DC1

Key words: insect flight, insect muscles, flight biomechanics, elytra, Melolontha.

THE JOURNAL OF EXPERIMENTAL BIOLOGY

1837Closing of the elytra in a cockchafer

legs were clipped in the first case to prevent grasping to a holder.For indication of the spatial orientation of thoracic segments, thinrods were glued to the scutellum normally to its surface or to thepronotum, normally or tangentially.

Video recordingA beetle was illuminated with a projector containing a halogen50W lamp. The video camera Panasonic NV-A3 (MatsushitaElectric Industrial Co., Osaka, Japan) with a headpiece lensprovided the frame rate of 25s–1 with a frame exposure of 2ms.This rate was enough for rather slow opening or closing of theelytra. Selected episodes were grabbed with the video plate ATIFury Pro (ATI Technologies Inc., Beverley, MA, USA) and thesoftware Video In 6.3 and Video Editor 6.2 supplied with the plate.Films were digitized in the format MS-MPEG4 V3. During threehours of recording, 353 behavioral acts in 53 live specimens wererecorded for the whole project. The quicker method was filmingin the movie mode with the photocamera named above. It wasused for a macro view with a headpiece lens or for filming underthe microscope. 182 behavioral acts in 34 live specimens wererecorded.

AnimationA thawed dead specimen with clipped legs was glued by the ventriteto a pedestal. We used two types of preparations: (1) a whole animalwith the free prothorax actuated with a handle glued tangentially tothe pronotum, or (2) the prothorax was removed; thus, obtainingthe pterothoracic preparation. Elytra were left intact or unlocked.

Image processingFrames for illustrations were grabbed from the films either in the*.mov format replayed by Media Player Classic 6.4.7.5 (GNU, FreeSoftware Foundation, Inc., Boston, MA, USA) or in the *.avi formatwith the software AVIEdit (AM Software, Moscow, Russia). Theywere further processed as Adobe Photoshop 5.5 images (AdobeSystems, Inc., San Jose, CA, USA). Films for coordinatemeasurements were processed frame-by-frame with AVIEdit. Eachframe was pasted in an image window of the Sigma Scan Prosoftware (SPSS Inc., Chicago, IL, USA) for coordinate tracing.Computation of the distance, direction, angle and scaling wasperformed using custom programs in Turbo Basic 1.3 (BorlandInternational, Inc., Austin, TX, USA). Coordinates for the presentarticle were measured in 1325 frames. Demonstrative movies in the

grayscale mode are available as supplementary materials to thepresent article. Colour originals are available at the website:http://izan.kiev.ua/ppages/frantsevich/.

RESULTSAnatomy

Anatomy of M. melolontha was illustrated in Straus-Duerkheim(Straus-Duerkheim, 1828). We mention below only details relevantfor this study. Fig.1A shows the medial section across the skeleton.

Note that: (1) articulations of the prothorax both with the headand the mesothorax are of the ball-and-socket type, the sockets liein the prothorax. No condylar structures exist in these articulations.(2) The endoskeleton includes a sternal apophysis in each thoracicsegment and three transversal folds of the tergal cuticle – theprophragma Ph1 in front of the mesotergite, the mesophragma Ph2in front of the metatergite, and the metaphragma Ph3 at the rearborder of the metatergite. (3) Both the meso- and metasternites arefused together in a hard plate, the ventrite (Friedrich et al., 2009).

The elytron is inserted into the articulatory membrane, whichdivides the mesotergite from the mesopleurite. It articulates to thethorax with a small process amidst the basal edge, the root of theelytron, encircled with four small sclerites, the axillary platesAx1–Ax4. Ax2 is articulated with the pleural shaft, three othersoffer insertions for the direct elytral muscles. The dorsal side of theroot is formed by a narrow medial apophysis and a broader lateralone.

Two closed elytra are tightly compressed to the body and to eachother with the aid of specific structures, the locks (Stellwaag, 1914):a friction lock between two elytra down the suture, a clamp betweenthe anterior part of the sutural edge and the groove on the metanotum,a clamp between the most anterior part of the sutural edge and thescutellum, a clamp between the antero-costal edge and theepipleuron, etc. The named locks must be clipped off to providefreedom for animated elytra. Closed elytra are also locked by thehind edge of the pronotum fitting into the groove on the basal edgeof the elytron.

Fig.1B schematically shows intersegmental muscles of themesothorax. Labeling of the muscles follows Larsén (Larsén, 1966).We show also the indirect longitudinal wing muscle M61. Directionof fibers in M61 indicates the longitudinal body-fixed axis, universalfor all flying beetles. Prothoracical rotator M13 from the pronotumto the mesepisternum is not depicted. Direct wing muscles are listedin Table1 by earlier publications.

A

HC

Prn Tg3

Sctl

Ph1

Ph2

PH

cv

Ph3

BM2 M4

M8

M11

M30

M32

M28M29

M62

M60

Sa1 Sa2Sa3

Fig.1. The skeleton and muscles in Melolontha melolontha. (A)Skeleton of the head and thorax in a parasagittal section. Medial structures are bold, lateralones are shaded, the articulatory membrane is shown by a dotted line. Abbreviations: cv – cervicale, HC – head capsule, PH – posterior horn of themesotergite, Ph1–Ph3 – fragmata, Prn – pronotum, Sa1–Sa3 – sternal apophysi, Sctl – mesoscutellum, Tg3 – metatergite. An asterisk shows projection ofthe center of rotation of the prothorax about the mesothorax. (B)Intersegmental muscles of the mesothorax. Labeling of the muscles follows Larsén (Larsén,1966). Arrows indicate structures remote from their labels. Scale bar, 1mm.


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Flight behavior in tethered beetlesBeetles from the fridge were put into a transparent box. Positivelyphototropic specimens were selected for tethering. The vast majorityof them were able to fly after some recovery period. Out of 87tethered flying specimens of M. hippocastani, seven flew constantlyand were arrested only by touching their legs with a brush. Mostof beetles flew in bouts. 162 interbout intervals were counted frame-by-frame in 34 undisturbed beetles. Mean interval comprised18.6±14.0s (mean ± s.d., range 3–100s, median 14.5s). Durationof wing oscillation was about 1.5s. Between the flights, a beetledevoid of the ground support always performed righting searchmovements, i.e. lifted the legs above the dorsum and vigorouslymoved its prothorax.

Flight coordinationThe flight includes a preparatory and an accomplishing stage andinvolves the whole body into this activity (see supplementarymaterial Movie1). Preparation for flight starts with a deep depressionof the prothorax followed by lifting of the linked elytra and laterby their abduction and elevation. The abdomen is depressed at thismoment to give space for unfolding wings. The wings unfold andbegin to oscillate. The spread elytra rise and droop in synchronywith the wings, the mesotergite oscillates too. The legs are heldstatic. The abdomen is constantly elevated during the flight. Afterthe cessation of wing strokes, the wing folding begins, which isaccompanied by the depression and adduction of the elytra,depression of the abdomen and obligate elevation of the prothorax.Evidently, during the elevation, the hind edge of the pronotum movesbackwards towards the mesotergite (see supplementary materialMovie2). The legs resume their search. The wings in most casesare not folded entirely; the accomplishing folding includescoordinated movements of the abdomen and elytra which have beenstudied earlier (reviewed in Haas and Beutel, 2001).

L. Frantsevich

Table 1. Direct wing muscles and some other mesothoracic muscles [labeling by Larsén (Larsén,1966)]No. Origo Insertio Suggested function Reference

M33 Inferior side of anterior horn Distal part of thepleural apodeme

Adductor of the elytron Elevator of the elytron

(Straus-Duerkheim, 1828)(Stellwaag, 1914; Bauer, 1910)

M42 Antero-lateral corner of themesocoxa under the trochantine

Ax1 Extensor of the elytron Anterior extensor of the elytron

(Straus-Duerkheim, 1828)(Stellwaag, 1914)

Anterior extending rotator (Herbst, 1944)

Flexor of the mesocoxa (Bauer, 1910)

M35 Articulatory membrane above thespiracle

Ax4 Flexor of the elytron Wing flexor

(Straus-Duerkheim, 1828)(Snodgrass, 1935)

Posterior extensor of the elytron (Stellwaag, 1914)

Posterior extending rotator (Schneider and Meurer, 1975)

M43 Mesotergite (analog of M35 inAdephaga)

Ax4 Extensor of the mesocoxa (elevatesthe elytron and holds it in its open stateduring the flight)

(Bauer, 1910)

Retractor of the coxa (Larsén, 1966)

M36a Basal part of the pleural apodeme Ax3 Adductor of the elytron (Straus-Duerkheim, 1828)

First adductor of the elytron (Stellwaag, 1914)

Anterior adducting rotator (Schneider and Meurer, 1975)

Depressor of the elytron (Bauer, 1910)

M36b Mesepisternum Ax3 Adductor of the elytron (Straus-Duerkheim, 1828)

Second adductor of the elytron (Stellwaag, 1914)

Posterior adducting rotator (Schneider and Meurer, 1975)

M40 Posterior edge of the prophragma Postero-lateraledge of the coxa

Retractor of the coxaExtensor of the coxa

(Larsén, 1966)(Bauer, 1910)

Long extensor of the coxa (Straus-Duerkheim, 1828)

Ax1–Ax4, first to fourth axillary plates.

Elytron

Abdomen

Length of elytron

Mesotergite

Prothorax

0

80

100

deg.

40 d

eg.

40 d

eg.

40 d

eg.

px

Time (s)0 1 3 42 5

Fig.2. Coordination between body parts in a flying Melolontha hippocastani.Frame-by-frame analysis of a video film. Change of orientation in the pitchplane is shown for the pronotum (elevation upwards), mesotergite(protraction upwards), elytron (elevation upwards) and abdomen (elevationupwards). The projection of the elytral edge measured in pixels (px) is longif the elytron is closed or elevated, and short if depressed. Wing flaps occurbetween the dashed lines. Inset – the pixelization error in 20measurements of the orientation of the prothorax in the same frame.



The time course of these events is plotted in Fig.2. We measuredangles in projection on the pitch plane between the tethering handleand direction markers: rods glued to the prothorax and the scutellum,the edge of the elytron, the line from the base to the tip of theabdomen. We take into account only the relative displacement. Thepixelization error in 20 separate measurements of the prothoracicrod in the same frame yielded the standard deviation of direction±0.41deg. The range of excursions of the prothorax was 24.5deg.,that of the abdomen was 23.5deg. and vibration of the mesonotumwas 14deg.

Strokes of the elytron occur perpendicularly to the pitch plane,they are not recognized by change of direction of the elytron. Weevaluated position of the elytron by the length, in pixels, of theprojection of the elytral edge: long if closed or elevated high, shortif elevated low. Strokes are apparently slowed down due to anoccasional stroboscopic effect of the low frame rate of 25s–1

combined with the short exposure of 2ms. Synchrony between thewings, elytra and mesotergite is evident. The downstroke issynchronized with the protraction of the mesotergite.

Opening and closing in loaded beetlesIf depression of the prothorax before the flight seems obligate inorder to unlock elytra for a free opening, the obligate elevation apriori seems not so necessary, because the open elytra have spaceto turn back. We disturbed the elevation, using loads that sloweddown this act in the upright tethered beetles.

We applied a weight on a lever glued to the prothorax, whichwould bend this segment down in an upright beetle tethered by themesoventrite. The lever was made from an insect pin with a hookat the end. It was glued tangentially to the pronotum. Weights weremade of coins whose mass was 1, 2, 3 or 5g. Upon adding sufficientweight, the prothorax bent down in an upright tethered beetle.Nevertheless, the beetle did not change its flight bout performance.There existed a threshold weight that prevented the flight. Thisthreshold was specific for a tested specimen and ranged from 5 to10g in fresh beetles but only to 2g in aged ones. We analyzedbehavior with the sub-threshold weight.

During interbout search periods, the prothorax in an unloadedbeetle performed rhythmical and equally slow elevations anddepressions. In the loaded upright beetle, elevations were sloweddown and depressions became abrupt. The range of the

elevation–depression of the prothorax was about 25deg. Spatialorientation of the tethered unloaded beetle did not influence its flightperformance. In a beetle turned upside down, the direction of theload was inverted. The intermittent behavior was the same in theupright and overturned unloaded specimens, while the prothorax inthe overturned loaded beetle was almost arrested in its elevatedposition: the range of the elevation–depression was only 8.8deg.

The time course of opening and closing in an upright and upsidedown beetle is plotted in Fig.3. The flight behavior with a negligibleload did not depend on the tilt of a beetle. The elevation of thepronotum was synchronous with the depression of the elytra duringclosing. An additional load did not hinder the opening in an invertedbeetle. It strongly retarded closing in an upright beetle, counteractingelevation of the prothorax whereas in an inverted beetle it facilitatedelevation of the prothorax and closing (see supplementary material

0306090

0306090

0306090

0306090

0306090

0306090

0306090

0306090

0 1–1 0 1–1

010203040

010203040

010203040

010203040

010203040

010203040

010203040

010203040

APronotum Pronotum

ElytronElytronB

C D

E F

G H

Opening Closing Fig.3. Influence of a load applied to thepronotum during the opening (A,C,E,G) andclosing (B,D,F,H) of the elytra in Melolonthahippocastani. The beetle is tethered in anupright position (A–D) or in an upside downposition (E–H). A lever is without additionalweight (A,B,E,F) or with the weight 2g(C,D,G,H). Change of orientation, in deg., inthe pitch plane versus time (abscissa), in s,is shown for the pronotum (right ordinatescale, thin curve, elevation upwards) and theelytron (left ordinate scale, bold curve,elevation upwards). 0 is the moment of thestart (A,C,E,G) or finish (B,D,F,H) of wingvibrations. Frame-by-frame analysis of fourfilms.

0

1

1

Tim

e (s

)

A

B

C

Opening

Closing

Fig.4. Duration of opening and closing of the elytra in the tetheredunloaded or loaded Melolontha hippocastani. Opening – above the zeroline, closing – below the zero line. Blank columns – unloaded beetles, graycolumns – beetles with sub-threshold loads. (A)Upright tethered beetles:39 unloaded flights in six specimens, 37 loaded flights in four specimens.(B)and (C) observations on three specimens tested both in the upright andupside down positions. (B)Upright position (seven and three flights).(C)Upside down position (nine and 20 flights). Error bars show the meanerror.


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Movie3 and Movie 4). Statistics of the time for opening and closingin 105 flight episodes with or without a sub-threshold load is plottedin Fig.4. The load significantly (Student’s t-test, P0<0.1%) retardedonly the closing and only in upright beetles, wherein it also hinderedelevation of the prothorax.

Center of rotation of the prothoraxThe center of rotation of the ball-and-socket joint between the pro-and mesothorax does not coincide with the contact zone betweenthe segments. We found position of the center, filming in profilemovements of the prothorax in a beetle tethered by the ventrite. Apaper stripe was glued to the prothorax in the pitch plane. Twodistant dots on this stripe, above and below the prothorax,circumscribed arcs during movements of the prothorax. Geometryof these arcs provided evaluation of the center location.

The center of rotation marked with an asterisk in Fig.1 is situatedat the level of the costal edge of the closed elytron, 4–5mm behindthe front butt of the mesothorax, far below the prophragma andinsertions of M4 above this phragma.

Arrest of the prothoraxA beetle tethered by the ventrite was allowed to rise and droop thelever glued to the pronotum. Small pliers were fixed in the openstate within the inferior area of the lever excursions. Theexperimenter could clamp the lever manually in its inferior positionduring the flight of the insect; thus, fixing the prothorax depressed.95 episodes of the free or clamped flight were recorded in ninebeetles.

Only one specimen demonstrated the complete arrest of theelytra in the raised state after cessation of the flight (seesupplementary material Movie5). Other specimens were able topartially close their elytra in the clamped state, followed by recoilafter the release. In order to elucidate coordination of body parts,

L. Frantsevich

we recorded three beetles with the straw rods glued normally tothe scutellum. The lever and the rod indicated the spatialorientation of the pronotum and mesotergite. Fig.5 illustratesbehavior during the clamp and recoil. Indeed, the mesotergite isnot arrested by the clamp but slowly moves forwards whereasduring the recoil it promptly returns back.

Mean parameters were compared for seven free and sevenclamped flights in the same three specimens. All angles weremeasured in a projection onto the pitch plane. The range ofexcursions of the prothorax was 24.8±7.5deg. (mean ± s.e.m.) and26.9±4.5deg. for free and clamped flights, respectively. Before thefirst stop at 0.43s after the beginning of the closing, elytra of clampedbeetles moved on average about –37.1±6.4deg., range from –6deg.to –53deg., while during the release they moved on average by–26.6±7.4deg. Duration of release was 0.24±0.02s. On thebackground of the immobile prothorax during the partial closing,the mesotergite protracted by 4.2±0.6deg. The release of themesotergite was retraction about –7.0±1.5deg.; despite this, the turnof the elytra was performed by the strong elevation of the prothorax.

Animation of the closingA frozen and thawed beetle was glued to a pedestal so that theprothorax and the head of the specimen had freedom to move. Thearticulatory membrane and several intersegmental muscles were cutin order to avoid hindrance to induced movements from the side ofstiff contracted muscles. A handle pointing forward was glued tothe pronotum.

The prothorax was depressed with this handle and the elytra werepassively open. Elevation of the prothorax by the handle caused theclosing of the elytra (see supplementary material Movie6). Thisperformance was recorded in 17 episodes in three specimens. Thesame manipulation was successfully tested in a dozen coleopteranspecies as well (L.F., unpublished).

Elytron

Mesotergite

Prothorax

100

deg.

40 d

eg.

40 d

eg.

Time (s)0 1–1 2

Fig.5. Arrest of the prothorax and partial closing of elytra in a tethered Melolontha hippocastani. Left panel – frames from a video film. Relative time, in s, isindicated in each frame. (A)A lever is clamped. (B)The flight continues only for a short time (0.04–0.08s). (C)Cessation of the flight, the partial closing ofelytra and wing folding begins. (D)Incomplete closing lasts during 0.84s. Meanwhile, the mesotergite protracts slowly, its previous orientation in C is printedinto D as a short bar behind an indicator rod. This posture lasts until the release of the clamp in (E). (F)During 0.2s both the prothorax and mesotergiterecoil upwards and backwards, respectively. The previous orientation of the mesotergite in D is printed into F as a short bar in front of the indicator rod.Right panel – angular orientation in the pitch plane of the lever on the prothorax, elevation upwards, of the rod on the mesotergite, protraction upwards, andthe edge of the elytron, elevation upwards. Time of wing vibrations is indicated with the open rectangle, that of the arrest is indicated with the blackrectangle. The gray vertical band indicates omission of 69 frames (2.76s) wherein the relevant body parts did not move.



This result suggests that it is an interaction between the hind edgeof the pronotum and the open elytron, which closes this last. Inorder to locate the interaction site, we excised pieces on the hindedge in several specimens and tested whether the elevation was ableto close the elytron. We excised areas in front of the scutellum(Fig.6A), the root of the elytron (Fig.6B) and the anepisternum.These experiments were video recorded (see supplementary materialMovie8), each prothorax was fixed, cleared of muscles andphotographed. We found out that the first and the last excision didnot impair the animated closing whereas the excision in front of theroot prevented it.

The site on the root, which must be actuated for the closing, wastested manually with a thin acupuncture pin in an unlockedpreparation of the pterothorax with the passively open elytron. Wefound out that the backward-directed pricking amidst the lateralapophysis readily turned the elytron into its closed position. Thisperformance was recorded in 20 video films (see Fig.7 andsupplementary material Movie7).

Upon the passive opening, the lateral apophysis rotated forwardand protruded ahead relative to its resting position by 0.75mm atthe pinpoint, by 1mm at the base in a specimen of M. melolontha28mm long. We conclude that the pressure of the hind edge of the

Fig.6. Prevention of the closing of an elytron after the excision of the counter-root area in the hind edge of the pronotum in Melolontha hippocastani.(A)View from below at the skeleton of the prothorax with an excision in front of the scutellum (does not prevent the animated closing); (B) same view at theprothorax with the excision in front of the root of the left elytron (prevents closing of this elytron); (C) half-profile view at the pterothoracic preparation, whichshows position of the left root relative to striae 1–5 on the elytron. Scale bar, 5mm. (D–H) Flight behavior in five live specimens with the excisions in front ofthe right root (frames from video films). (D)Stages of flight in one specimen (see supplementary material Movie 9): D1 – start of the opening, D2 – flight, D3– intermediate state of the closing, 0.16s since cessation of wing vibrations, D4 – final stage of the closing, 0.32s since the adduction of the left elytron.E–H – the final stage of the closing in another four operated specimens.

Fig.7. Animation of the elytronclosing by the manual pricking ofthe right lateral apophysis with anacupuncture needle in Melolonthamelolontha. Frames from two videofilms. (A)General view, (B) viewunder a microscope. Stages ofanimation in columns: 1 – beforethe contact of the needle with theapophysis, 2 – the moment of thecontact, 3 – intermediate stage ofclosing, 4 – final stage. Scale bar,5mm.


1842

pronoum onto the lateral apophysis turns the apophysis back,certainly together with the elytron itself. Excursion of the pronotumrelative to the mesotergite in live beetles was in the range1.3–1.5mm. It is possible to animate the opening of the unlockedelytra by manipulations with the mesotergite or the mesepisternabut reverse motions do not close the spread elytra.

Excision of the counter-root area of the pronotum in vivoA deep excision in the hind edge of the pronotum was made in acooled live beetle. The beetle was tethered by the ventrite and testedfor the ability to fly and close the elytra. Preliminary experimentson five specimens were only partly successful: some specimenscould normally close their elytra. An anatomical inspection revealedthat the excision in these specimens was not precise with respectto the root position: either shifted sideward or left residual piecesof the invaginated pronotal edge. The root is covert in an intactbeetle. Therefore, we searched for reliable landmarks on the elytra,which indicated the site of operation (Fig.6C). The root is situatedin front of the area between the second and the third stria(longitudinal ribs on the elytron).

Further operation on five beetles was successful in all specimens(see supplementary material Movie9). The excision was done infront of the root of the right elytron. The beetles were able to openboth forcedly closed elytra, flew quite normally but after cessationof the flight only the left elytron returned to the rest position, theright one remained abducted (Fig.6D–H). Anatomical checksrevealed that the space in front of the right root was clear.

DISCUSSIONDirect wing muscles in the mesothorax of M. melolontha and someother beetles were investigated long ago. We list them in Table1together with some other muscles originating from the mesotergiteand mentioned below. Most probably, judgement on their functionhas been derived from their position and from some undescribedanimations. The opinion on functions of M36, M40 and M42 (inPolyphaga) was unanimous whereas opinions on the function ofM33 and M35 or its analog M43 in Adephaga were diverse.Anyhow, previous authors located actuators of closing exclusivelywithin the mesothorax.

Straus-Duerkheim considered M33 in M. melolontha to be theadductor of the elytron, i.e. the closer muscle (Straus-Duerkheim,1828). Stellwaag suggested that closing in a stag beetle Lucanuscervus demanded relaxation of M42, M35 and then contraction ofM36a, M36b (Stellwaag, 1914). This idea was corroborated bySchneider and Meurer for a dynastine beetle Oryctes boas (Schneiderand Meurer, 1975). According to Herbst, closing in chafersdemanded relaxation of M33and M40 (Herbst, 1944). Elytra returnback driven by gravity and elasticity of the mesonotum, supportedby contraction of M36a. This last folds the area of Ax3 and pullsthe sutural edge of the elytron mesad.

The animation experiment clearly demonstrated that the elevationof the prothorax in a dead animal is enough for closing of thepreviously spread elytra. The question is whether this mechanismdoes work in vivo. Measurements of coordination between bodyparts during the flight reveal that, during slow opening, thedepression of the prothorax precedes the divergence of the elytra.On the contrary, during closing, both convergence and elevationare synchronous. Experimental retardation of the elevation retardedthe closing. An arrest of the depressed prothorax in a flying beetleeither hindered or prevented the closing.

Mechanical interaction between the elytron and the prothorax maybe limited to the contact point between the posterior edge of the

L. Frantsevich

pronotum and the lateral apophysis of the root, which rotates forwardduring the opening. The pronotum presses on the root and turns itbackwards. Deprivation of this contact by excision on the pronotaledge prevented closing in animation experiments as well as in vivo.Thus, we can consider the elevation of the prothorax as the indirectand main mechanism of closing in Melolontha and perhaps in otherbeetles.

The hypothesis about closing by pressure of the pronotal edgeupon the root of the elytron was proposed by the present author(Frantsevich, 1981) in his early work on histerid beetles. Despitesome errors in reconstruction of the musculature, we believe thatwe properly described the mechanism of the momentary click ofthe closing elytra in these beetles: the closing lasted 2–3ms inthe larger Hister unicolor and only 0.5–1ms in the small Atholusduodecimstriatus. The direct elytral muscles pretending to beclosers are so feeble in histerids that M36 and M35 wereomitted by me (Frantsevich, 1981), M35 [labeling by Larsén,(Larsén 1966)] was not identified by Beutel and Komarek andannounced as probably absent, while their description of M36corresponds not to M36 but to M35 (Beutel and Komarek,2004). It is clear that such fast movement needs previous storageof energy, presumably in the isometrically contracting largeelevators of the prothorax, and its momentary release afterunlocking, the mechanism resembling the jump of a click beetle(Evans, 1973).

The muscles which elevate the prothorax in Melolontha areproposed below judging by their arrangement relative to the centerof rotation of the prothorax about the mesothorax: these are M11and M4, maybe the transsegmental M2 and M8. M4 was definedas the depressor in an elaterid Athous haemorrhoidalis (Evans,1973). The difference between Athous and Melolontha with respectto the drive of this muscle is in the shape of the prophragma (straightin Melolontha, curved down in Athous) and in the type of the jointbetween the pro- and mesothorax. It is a ball-and-socket joint inMelolontha with the center of rotation situated in the metathorax,behind and below the insertion of M4 above the prophragma. It isa bicondylic monoaxial joint in Athous, pits for condyles lie at theanterior edge of the mesotergite above the prophragma and theinsertion of M4.

The proposed mechanism of the indirect closing does not excludecontribution of other mesothoracic muscles. Partial closing of theelytra after cessation of the flight in a clamped beetle may beexplained by the limited protraction of the mesotergite: whichtogether with the bases of the elytra approaches the hind edge ofthe pronotum. This protraction may be caused either by elevatorsnamed above or by M29, a presumable antagonist to M28. To date,we have no experimental evidence about the role of some directelytral muscles in the closing. Direct M36 and M35 are feeblecomparing with large and strong elevators. These last provideenough power for the broad rotation of the elytron during closing,sometimes very prompt, and for compression of wings under theelytron during wing folding after the flight.

ACKNOWLEDGEMENTSI am indebted to Dmytro Gladun for collection of M. melolontha.

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indirect closing of the elytra in a cockchafer, melolontha...

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